The Effect of High Fat Diet and H2S Donor GYY-4137 on Vascular Function in Spontaneously Hypertensive Rats

The Effect of High Fat Diet and H2S Donor GYY-4137 on Vascular Function in Spontaneously Hypertensive Rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The Effect of High Fat Diet and H2S Donor GYY-4137 on Vascular Function in Spontaneously Hypertensive Rats Basak G. Aydemir, Andrea Berenyiova, Martina Cebova, John D. Henderson, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6030564/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Metabolic syndrome is a growing global health burden, resulting in an urgent need for new therapeutic strategies. We evaluated the effects of 3 weeks of treatment with slow-releasing H 2 S donor GYY-4137 on adiposity, systolic blood pressure (sBP), plasma biochemical indices, vascular function and nitric oxide (NO) and hydrogen sulfide (H 2 S) pathways in the isolated thoracic aortas (TAs) and mesenteric arteries (MAs) from spontaneously hypertensive rats (SHRs) fed a high-fat diet (HFD) for 8 weeks. Although the HFD increased TAs relaxation, it induced cardiac remodeling, decreased adrenergic contraction, reduced NO participation in vasoactive responses, decreased NO-synthase (NOS) activity, altered the expression of the endothelial NOS (reduced), inducible NOS, and tumor necrosis factor alpha (TNFα) (both increased) proteins and increased adiposity and plasma chemerin levels. Treatment with GYY-4137 reduced sBP, improved relaxation of the MA, partially restored the contractility of the TA, generally restored NO signaling, and decreased the expression of the inducible NOS and TNFα proteins and plasma chemerin. Thus, a slow H 2 S-releasing donor could partially ameliorate metabolic changes induced by increased fat intake during essential hypertension and trigger beneficial vasoactive effects associated with the restoration of NO signaling and suppression of inflammation. Biological sciences/Physiology Health sciences/Diseases Health sciences/Pathogenesis hydrogen sulfide GYY-4137 high-fat diet spontaneously hypertensive rat vascular Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 1. Introduction Characterized by a combination of symptoms, including insulin resistance, hypertension, and obesity, metabolic syndrome is one of the leading causes of mortality worldwide, and a high-fat diet (HFD) is the main driver. The global obesity epidemic directly contributes to cardiovascular disease risk factors, including dyslipidemia, type 2 diabetes, and hypertension, as highlighted by the American Heart Association, which reported that in 2021, 39–49% of the global population (2.8–3.5 billion people) were overweight or obese [ 1 ]. Metabolic syndrome initiated by chronic HFD consumption increases circulating levels of free fatty acids and inflammatory markers, causing chronic, systemic, low-grade inflammation and an unbalanced oxidative status in several organs [ 2 ]. New interventions that improve cardiovascular and metabolic health in these patients are essential to address the rising health problem that metabolic syndrome represents. Targeting gasotransmitter signaling is a promising area of ​​treatment for both cardiovascular and metabolic diseases. Hydrogen sulfide (H 2 S) is a gaseous transmitter that regulates intracellular signaling. The colonic microbiota represents the largest source of H 2 S in the body and results from recent years show that gut-derived H 2 S can induce systemic effects and changes in H 2 S homeostasis in the colon H 2 S can be associated with various pathological stages such as hypertension or atherosclerosis [ 3 ]. At the same time, many studies have been conducted on the effects of H 2 S produced enzymatically by various tissues of the cardiovascular system. However, the physiological and pathological role of H 2 S remains much less well understood than that of nitric oxide (NO), despite promising work from our group suggesting its potential antihypertensive function [ 4 ]. Research shows that strategies that increase the bioavailability of H 2 S are promising therapeutic approaches to treat diseases. The inorganic sulfide donors such as NaHS and Na 2 S provide fast extracellular release of H 2 S and therefore have restricted therapeutic potential. Among the natural sources of H 2 S, there are polysulfides, which include garlic-derived compounds or isothiocyanates, including sulforaphane and erucin [ 5 ] Synthetic H 2 S donating compounds have also been developed. A very promising modern approach consists in designing multi-target molecular hybrids where some of the H 2 S-releasing moieties have been largely combined with different drugs already used in the clinic to obtain novel multi-target molecular hybrids [ 6 ]. A slow-releasing H 2 S donor with vasodilatory and antihypertensive properties, GYY-4137, has been shown to be effective in models of cardiovascular disease [ 7 ]. GYY-4137 reduced vascular inflammation and oxidative stress, improved endothelial function, and reduced atherosclerotic plaque formation in apolipoprotein E-/- mice fed a high-fat diet [ 8 ]. Moreover, the long-term application of this donor led to a significant decrease in systolic blood pressure and alleviated myocardial fibrosis in spontaneously hypertensive rats (SHRs) [ 9 ]. In our recent work, we induced metabolic syndrome-like pathologies in SHRs via long-term fructose administration and reported that treatment with GYY-4137 alleviated fructose-induced structural malformations associated with tissue inflammation while also restoring the impaired vascular contractile ability [ 4 ]. In this study we investigated the effects of GYY-4137 on blood pressure, biometric parameters, markers of inflammation, cardiac fibrosis and both H 2 S and NO signaling in the vascular function of SHRs fed a HFD to provide further information on the therapeutic potential of exogenous H 2 S donor in metabolic disorders. An original contribution is also the comparison of two types of arteries - the thoracic aorta (elastic type) and the mesenteric artery (muscular type). We hypothesized that chronic consumption of an HFD would induce metabolic syndrome-like pathologies in SHRs, impair vascular function, increase blood pressure, and interfere with vascular NO and H 2 S signaling; however, these effects would be attenuated by GYY-4137. 2. Materials and Methods 2.1 Experimental animals All the rats were maintained on a 12-h light/dark cycle in a temperature (20–22°C)- and humidity (45–65%)-controlled room with access to standard laboratory rat chow (Altromin 1324P, Lage, Germany) or a high-fat obesity-inducing diet (Altromin, type C 1090-45, Germany) and drinking water ad libitum. The contents of the standard laboratory rat chow were 11% fat, 24% protein, and 65% carbohydrates, while the carbohydrate contents were as follows: 0 mg/kg monosaccharides, 47.8 mg/kg disaccharides, and 391.2 mg/kg polysaccharides. The remainder of the diet contained minerals and trace elements. The content of the high-fat diet was 45% fat, 18% protein, and 37% carbohydrates, and the carbohydrate content was 101.6 mg/kg monosaccharides, 52.1 mg/kg disaccharides, and 238.9 mg/kg polysaccharides; the remainder of the diet contained minerals and trace elements. 2.2 Experimental design An animal model of primary hypertension, SHRs, was used in this study. Twenty-four male SHRs aged 9 weeks and ranging in weight from 180–220 grams were subsequently divided into three groups. One rat died during the systolic blood pressure measurements; thus, 23 SHRs were used for the experiments. The control SHR group (n = 8) received a standard diet for 8 weeks, the SHR + HFD group (n = 7) received a high-fat diet (HFD) for 8 weeks, and the SHR + HFD + GYY group (n = 8) received an HFD for 8 weeks. The SHR + HFD + GYY group also received GYY-4137 (266 µmol/kg/day dissolved in 1% dimethyl sulfoxide (DMSO) in physiological solution), which was injected intraperitoneally (i.p.) daily for three weeks at the age of 14–17 weeks. The rats in the SHR and SHR + HFD groups received vehicle via i.p. injection (1% DMSO in physiological solution; volumes were determined according to the actual body weight). 2.3 Blood pressure measurement and biometric parameters Systolic blood pressure (sBP) was measured using tail-cuff plethysmography (MRBP; IITC Life Science Inc., Los Angeles, CA, USA). Animals were trained for the tail-cuff method for measuring blood pressure for two consecutive days before the evaluation of basal sBP levels (at the age of 9 weeks, the beginning of the HFD treatment). In all groups, sBP was subsequently measured at the end of the 14th week (the beginning of the treatment with GYY-4137) and at the end of the experiment (at the age of 17 weeks). sBP was calculated as the average of the five measurements completed for each rat. 2.4 Body weights and diet consumption Rat body weight (BW) and food intake were monitored daily during the treatments. At the end of the experiment, the rats were euthanized by decapitation after brief CO 2 anesthetization. CO₂ was used to render the rats unconscious at a concentration of 30–40%, which was applied gradually to minimize stress and discomfort to the animal. Heart weight (HW), retroperitoneal adipose tissue weight (RTW), and the length of the tibia (TL) were measured. The ratios of HW to TL or BW and RTW to TL or BW were calculated to determine the degree of cardiac hypertrophy and body adiposity, respectively. 2.5 Blood sample analysis Blood samples from the trunks of all the animals were collected at the end of the treatment day into preprepared heparinized tubes (140 UI/5 mL) and then centrifuged (850× g, 10 min, 4°C, Centrifuge 5430 R, Eppendorf, Hamburg, Germany). The resulting plasma fraction was then analyzed to determine the levels of glucose (GLU), cholesterol (CHOL), triacylglycerol (TAG), urea (UREA) and creatine (CRE) using a biochemical analyzer and auxiliary reagent discs (Celercare, MNCHIP Technologies Co., Ltd., Tianjin, China). According to the manufacturer’s instructions for analysis, 100 µL of plasma was pipetted into the sample chamber, and 430 µL of distilled water was then added to the diluent chamber of the test-specific reagent disk. 2.6 ELISA Chemerin levels were measured in plasma samples using a commercially available Rat CHEM ELISA Kit (ELK Biotechnology, Denver, CO, USA) according to the manufacturer's protocol. The samples of the trunk blood of the animals were collected at the end of the treatment into pre-prepared heparinized tubes (140 UI/5 mL) and were then centrifuged (850× g, 10 min, 4°C) and the plasma samples were frozen immediately at -80ºC until the assay. After thawing on ice, serum samples were diluted 20-fold in the kit dilution buffer. 100ul of the diluted samples and the kit standards were then added to the ELISA plate and incubated at 37°C for 80 minutes, after washing 3 times with 100ul of wash buffer. 100ul of the biotinylated antibody working solution was added to each well and the plate was incubated at 37°C for 50 minutes. The wells were then washed 3 times with 100ul of wash buffer, followed by the addition of 100ul of the Streptavidin-HRP working solution and incubated at 37°C for 50 minutes. The plate was then washed 5 times in the wash buffer, before 90ul of TMB solution was added leave in the dark for 20 minutes at 37°C, after which 50ul of the stop reagent was added and the plate was then read in a spectrophotometer at 450nm. 2.7 Vasoactive responses of isolated arteries The descending part of the thoracic aorta (TA), beginning under the arcus aorta, and the superior mesenteric artery (MA) were isolated under a binocular microscope, cleaned of connective tissue and cut into 5 mm long rings with a preserved endothelium. The rings were set vertically between 2 stainless wire triangles and placed in a 20 ml incubation organ bath with Krebs solution (118 mmol/L NaCl; 5 mmol/L KCl; 25 mmol/L NaHCO 3 ; 1.2 mmol/L MgSO 4 ×7 H 2 O; 1.2 mmol/L KH 2 PO 4 ; 2.5 mmol/L CaCl 2 ; 11 mmol/L glucose; and 0.032 mmol/L CaNa 2 EDTA). The solution was oxygenated (95% O 2 and 5% CO 2 ) and maintained at 37°C throughout the experiment. Vasoactive changes in the TAs and MAs were determined using isometric tension sensors (FSG-01, MDE, Budapest, Hungary) connected to the upper triangle (the lower triangle was firmly fixed). The NI USB-6221 AD converter (MDE, Budapest, Hungary) and S.P.E.L. Advanced Kymograph software (MDE, Budapest, Hungary) were used to record vascular changes. Before the function of the arteries was tested, resting tension was applied to the rings, which were maintained for a stabilization period (45–60 minutes). A series of preliminary experiments was conducted to set the optimal value of resting tension. Different values of resting tension, from the lowest to the highest, were applied to rings of both arteries isolated from SHRs, and the rings were exposed to increasing concentrations of noradrenaline, which induced a concentration-dependent contractile response. The value of resting tension (1 g) at which the largest contractile response was repeatedly recorded was applied to the arterial rings used in this study. The arteries were then precontracted by adding noradrenaline (NA, 10 − 6 mol, Zentiva, Czech Republic) before a single dose of acetylcholine (Ach, 10 − 5 mol/L) was added, thus controlling the integrity of the endothelium and the contractile properties of smooth muscle cells. After 3 washes with physiological Krebs solution and an equilibration period, the experiment was started by applying cumulative doses of NA (10 − 10 –10 − 5 mol/L) to determine the adrenergic contractions in TAs and MAs. Vasocontractile responses are presented as the measured changes in isometric tension in g. Cumulative doses of Ach (10 − 10 –10 − 5 mol/L) were applied to NA-precontracted arteries to evaluate endothelium-dependent relaxation. The relaxation responses were determined as a percentage of the maximal NA-stimulated contraction. Individual inhibitors were used to analyze the involvement of the endogenous NO and H 2 S pathways in both contractile and vasorelaxant responses. Nonspecific inhibitors of NO synthase (NOS), N G -nitro-L-arginine methyl ester (LN, 10 − 4 mol/L) or H 2 S-producing enzymes, such as cystathionine-γ-lyase (CSE) and DL-propargylglycine (PPG, 10 mmol/L), were added for 20 minutes to the organ bath, and the concentration‒response curves for NA and Ach were generated again. Since we prepared two rings of the thoracic aorta and two rings of the mesenteric artery from each animal, individual inhibitors (LN and PPG) were not applied to the same ring. Areas under the curve (AUCs, in arbitrary units) were calculated and compared before and after the incubation with the inhibitors to assess the involvement of NO and H 2 S in the vasoactive response in each group. 2.8 Analysis of protein expression The protein expression levels of endothelial and inducible NOS isoforms, cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS), and tumor necrosis factor alpha (TNFα), which are inflammatory factors, in the abdominal aorta and transforming growth factor-beta (TGF-β) in the left ventricle were determined via Western blot analysis. Briefly, aortic tissue lysates were prepared by homogenization in protease inhibitor-supplemented 0.05 mM Tris buffer (protease inhibitor cocktail), followed by centrifugation and a measurement of the protein concentration using the Lowry assay. The supernatants were then subjected to SDS‒PAGE on 12% or 15% gels for protein separation and transferred to nitrocellulose membranes. Immunoblotting was performed using primary antibodies against endothelial eNOS (1:1000 dilution) (Abcam, Cambridge, UK), inducible iNOS (1:1000 dilution), CSE (1:5000 dilution), CBS (1:3000 dilution) and TNF-α (1:3000 dilution) (Proteintech, Manchester, UK), and β-actin (1:5000 dilution) (Abcam, Cambridge, UK) was used as a loading control. The membrane was incubated with secondary peroxidase-conjugated antibodies; eNOS (1:5000 dilution), iNOS (1:5000 dilution), CBS (1:5000 dilution), β-actin (1:5000 dilution) anti-rabbit (Abcam, Cambridge, UK), CSE (1:3000 dilution), TNF-α (1:3000 dilution) anti-mouse (Cell Signaling, Danvers, MA, USA), anti-TGF β (1:1000 dilution), anti-rabbit, polyclonal (Sigma- Aldrich, Germany) at room temperature for 2 h followed by enhanced chemiluminescence (ECL) reagent (BioRad, Inc., Hercules, CA, USA) which facilitated band visualization and subsequent quantification using standardized software (Image LabTM Touch software 2.4 BioRad, Inc., Hercules, CA, USA). The results were normalized to β-actin expression for accurate representation. 2.9 Total NO Synthase Activity NOS activity was determined in the aortic arch and tissue remnants from the thoracic aorta. The isolated aortic tissue samples were homogenized, and nitric oxide synthase (NOS) activity was quantified by measuring the conversion of [3H]-L-arginine (obtained from MP Biochemicals) to [3H]-L-citrulline, according to a previously established method [ 10 ]. [3H]-L-citrulline levels were measured using the Quanta Smart TriCarb Liquid Scintillation Analyzer (TriCarb, Packard, UK). NOS activity was then normalized to the protein content and reported as picokatal per gram of protein (pkat/g protein). 2.10. Collagen amount The amount of collagen in tissue was quantified by measuring hydroxyproline (hyp), an amino acid that is a major component of collagen. To determine the amount of hyp in the left ventricle, we used a modified method according to Reddy and Enwemeka [ 11 ]. Briefly, we used the same homogenate as used for measurement of NOS activity. 25 µl of sample was placed into a test tube and subjected to hydrolysis by 50 µl of 2 mol/l NaOH. The samples were then heated to 110°C 60 min, which breaks down the tissue and converts the collagen’s hydroxyproline into a free form that can be measured. After hydrolysis, the tissue samples were neutralized with 450 µl of chloramine-T for 25 min in room temperature. Following chloramine-T treatment, 500 µl of p-dimethylaminobenzaldehyde (DMAB) was added to the sample. Incubation lasted 20min in 65°C. The absorbance of the resulting solution was measured at 550 nm using a spectrophotometer. The collagen content in the tissue samples was calculated using a hydroxyproline standard calibration curve. The samples were repeated in triplicate to ensure consistency in results. It is assumed that 12.5% of collagen is composed of hydroxyproline. A standard curve was prepared by creating a series of hydroxyproline standards with known concentrations. 2.11 Statistical Analysis The data are presented as the means ± S.E.Ms. For the statistical comparison of sBP and vasoactive responses between groups, two-way analysis of variance (ANOVA) and the Bonferroni post hoc correction were used, or paired Student´s t tests (AUCs before and after inhibitor treatments). One-way ANOVA was used to evaluate general cardiovascular parameters, plasma parameters, NO synthase activity, collagen content and protein expression. Differences in means between groups were considered significant at p < 0.05. The data were analyzed with OriginPro 2019b (OriginLab Corporation, Northampton, MA, USA). 2.10 Drugs All the chemicals used in this study were purchased from Sigma‒Aldrich (Germany), unless stated otherwise. 3. Results 3.1 General parameters of the experimental animals A total of 23 rats (8 in the SHR group, 7 in the SHR+HFD group, and 8 in the SHR+HFD+GYY group, Table 1) were included in the study and divided into 3 groups according to whether they received the HFD and were treated with the H 2 S donor GYY-4137. At the beginning of the experiment, no difference in basal body weight (BW) was observed between the groups: SHR—244.12±6.98 g, SHR+HFD—238.50±4.90 g, and SHR+HFD+GYY—230.13±3.68 g. From the beginning to the end of the experiment, we noticed a significant increase in BW in all groups (F (2,65) = 83.14; p = 9.25x10 -18 ), and the greatest change was observed in the HFD group. The HFD induced significant increases in BW, retroperitoneal adipose tissue weight (RTW), and tibia length (TL). Additionally, chronic consumption of the HFD increased the ratios of RTW to BW (RTW/BW), and RTW to TL (RTW/TL) (Table 1). These results confirmed increased adiposity in the HFD group. However, treatment with GYY-4137 did not affect these parameters. Neither the HFD nor treatment with GYY-4137 significantly altered the plasma glucose (GLU) levels. A significant increase in the plasma triacylglycerol (TAG) levels was observed in the HFD group, as expected. However, the plasma cholesterol (CHOL) level was lower in the HFD group than in the SHR group. The urea (URE) level and the ratio of URE to creatinine (URE/CRE) were significantly altered in the groups that received the HFD compared with those in the control diet (SHR) group; however, treatment with GYY-4137 partially attenuated this effect. HFD intake significantly increased chemerin levels in plasma, on the other hand, GYY-4137 administration returned the level of chemerin to control level. Food intake was significantly lower in the HFD group than in the control diet (SHR) group, and this effect was increased by treatment with GYY-4137 (Table 1). Table 1: General parameters of the experimental animals Parameters SHR n SHR+HFD n SHR+HFD+GYY n BW (g) 282.1±13.3 8 332.4±6.3*** 7 327.3±7.3 ** 8 HW (g) 1.047±0.05 8 1.335±0.03 *** 7 1.372±0.05*** 8 RTW (g) 2.07±0.24 8 3.40±0.20** 7 3.44±0.24** 8 TL (mm) 34.42±0.27 8 37.51±0.9** 7 37.05±0.55** 8 RTW/BW (mg/g) 7.21±0.6 8 10.21±0.46** 7 10.48±0.55*** 8 RTW/TL (mg/mm) 60.38±7.14 8 90.59±4.38** 7 93.24±6.71** 8 GLU (mmol/L) 8.26±0.4 8 8.68±0.4 7 8.09±0.3 8 TAG (mmol/L) 1.20±0.07 8 2.42±0.20*** 7 2.07±0.24** 8 CHOL (mmol/L) 2 .67±0.19 8 2.32±0.07* 7 2.53±0.10 8 UREA (mmol/L) 7.86±0.11 8 7.09±0.13** 7 7.26±0.26 8 CRE (umol/L) 23.57±3.40 8 33.14±4.38 7 34±11.4 8 URE/CRE 361.72±32.76 8 237.27±31.90* 7 305.05±031.06 8 Chemerin (ng/ml) 158.25±3.28 6 220.94±7.66* 7 161.31±5.91 + 8 Food intake (g/day) 20.87±0.45 8 15.56±0.15*** 7 14.90±0.16*** + 8 Abbreviations: n - number of rats; SHR - spontaneously hypertensive rats; SHR+HFD - spontaneously hypertensive rats receiving a high-fat diet; SHR+HFD+GYY - spontaneously hypertensive rats receiving an HFD and the H 2 S donor GYY-4137; BW - body weight; RTW - retroperitoneal adipose tissue weight; TL - tibia length; RTW/BW - ratio of retroperitoneal adipose tissue weight to body weight; RTW/TL - ratio of retroperitoneal adipose tissue weight to tibia length; GLU - glucose; TAG - triacylglycerol; CHOL - cholesterol; CRE - creatinine; URE/CRE - ratio of urea to creatinine. The values are presented as the means ± S.E.Ms.; * p<0.05, ** p<0.01 and *** p<0.001 compared with the SHR group, + p<0.05 and ++ p<0.01 compared with the SHR+HFD group. Statistical analyses were performed by one-way ANOVA with the Bonferroni post hoc correction. 3.2. Cardiac parameters of the experimental animals Chronic consumption of the HFD increased heart weight (HW) as well as the ratios of HW to BW and HW to TL suggesting the occurrence of cardiac hypertrophy. Moreover, HFD increased the protein expression of central regulator in the development of fibrosis TGF-β as well as collagen content in the left ventricle. The treatment with GYY-4137 returned TGF-β to control levels, and collagen levels decreased, but not significantly (Table 2). Table 2: Cardiac parameters of the experimental animals Parameters SHR n SHR+HFD n SHR+HFD+GYY n HW (g) 1.047±0.05 8 1.335±0.03 *** 7 1.372±0.05*** 8 HW/BW (mg/g) 3.72±0.78 8 4.02±0.07* 7 4.19±0.13* 8 HW/TL (mg/mm) 30.43±1.52 8 35.69±0.85* 7 37.08±1.44** 8 TGF-β (Density TGF-β/ β-actin) 0.11±0,01 6 2.45 ± 0.16*** 6 1.41±0.17 +++ 6 Collagen ( m g/mg) 6.65±0.77 6 9.14±0.57* 6 6.79±0.46* 6 Abbreviations: n - number of rats; SHR - spontaneously hypertensive rats; SHR+HFD - spontaneously hypertensive rats receiving a high-fat diet; SHR+HFD+GYY - spontaneously hypertensive rats receiving an HFD and the H 2 S donor GYY-4137; HW- heart weight; HW/BW - ratio of heart weight to body weight; HW/TL- ratio of heart weight to tibia length; TGF-β - protein expression of transforming growth factor-β in the left ventricle; Collagen – the amount of collagen in the left ventricle. The values are presented as the means ± S.E.Ms.; * p<0.05, ** p<0.01 and *** p<0.001 compared with the SHR group, +++ p<0.01 compared with the SHR+HFD group. Statistical analyses were performed by one-way ANOVA with the Bonferroni post hoc correction. 3.3. Blood pressure of the experimental animals sBP measurements for all groups were recorded at week 9, week 14 (after 5 weeks of HFD consumption), and week 17 (after 8 weeks of HFD consumption and 3 weeks of GYY-4137 treatment). Two-way ANOVA confirmed that sBP differed not only depending on the administration of a high-fat diet or GYY (F (2,61) = 12.81; p ˂ 0.0001) but also depending on time (F (2,61) = 14.62; p ˂ 0.0001). Although the Bonferroni post hoc test confirmed a significant increase in sBP over time only in the control SHR group (p ˂ 0.001), at the end of the experiment, a significant reduction in sBP was observed in the group treated with GYY-4137 compared with both the SHR and HFD groups (p<0.001 and p<0.01) (Figure 1). 3.4 Endothelial Function and Contractility of the Thoracic Aorta and Mesenteric Artery Contractile responses were induced by the activation of adrenergic receptors through the successive application of exogenous noradrenaline (NA) at concentrations ranging from 10 -10 to 3x10 -6 mol/L (TA) or 3x10 -5 mol/L (MA). HFD intake induced a significant decrease in NA-induced contraction of both the TA (F (2,218) = 12.71; p = 6.59x10 -6 ) (Figure 2a) and MA (F (2,238) = 70.20; p<6.35x10 -24 ) (Figure 2b), which indicates deterioration of the contractile properties. On the other hand, the administration of GYY-4137 partially improved the inhibited contractile response in the TA only (p<0.05, Figure 2a). Endothelial function was subsequently investigated by applying cumulative doses of acetylcholine (10 -10 to 3x10 -6 mol/L in the TA or 3x10 -5 mol/L in the MA) to the NA-precontracted arterial rings. Compared with the SHR group, both the HFD and the GYY-4137 treatment significantly increased endothelium-dependent vasorelaxation in the TA, although the maximum response remained unchanged (F (2,208) = 14.03; p = 2.17x10 -6 ) (Figure 2c). The vasorelaxant response of the MA remained unaffected by the HFD, but following treatment with GYY-4137, a significant increase was observed (F (2,238) = 8.52; p = 2.79x10 -4 ) (Figure 2d). Maximum reached responses and molar concentrations of noradrenaline and acetylcholine that induced the half-maximum response (EC 50 ) are shown in Table 3 (thoracic aorta) and Table 4 (mesenteric artery). Table 3: Characterization of noradrenaline-induced contraction and acetylcholine-induced relaxation in the thoracic aorta Thoracic aorta SHR n SHR+HFD n SHR+HFD+GYY n NA max (g) 0.58±0.03 7 0.37±0.07*** 7 0.44±0.03*+ 8 NA EC 50 (-log mol/L) 8.39±0.25 7 9.08±0.26 7 9.53±0.07*** 8 Ach max (%) 78.59±4.75 7 76.95±5.54 6 78.74±5.79 7 Ach EC 50 (-log mol/L) 8.70±0.19 7 9.30±0.17* 6 9.37±0.09** 7 Abbreviations: n—number of rats; SHR—spontaneously hypertensive rats SHR+HFD—spontaneously hypertensive rat receiving the high-fat diet; SHR+HFD+GYY—spontaneously hypertensive rat receiving the HFD and H 2 S donor GYY-4137, NA—noradrenaline, NA max —maximum noradrenaline-induced contraction, NA EC 50 —the negative logarithm of the NA molar concentration inducing the half-maximal response, Ach—acetylcholine, Ach max —maximum acetylcholine-induced relaxation, Ach EC 50 —the negative logarithm of the Ach molar concentration inducing the half-maximal response. Values are presented as the means ± S.E.Ms. * p<0.05, ** p<0.01 and *** p<0.001 compared with the SHR group, + p<0.05 compared with the SHR+HFD group. The statistical analysis was performed by one-way or two-way ANOVA with the Bonferroni post hoc correction. Mesenteric artery SHR n SHR+HFD n SHR+HFD+GYY n NA max (g) 1.18±0.06 7 0.78±0.06*** 6 0.74±0.04*** 7 NA EC 50 (-log mol/L) 7.43±0.07 7 7.41±0.13 6 7.42±0.10 7 Ach max (%) 43.97±3.97 7 40.68±2.80 6 47.72±3.12*+ 6 Ach EC 50 (-log mol/L) 7.56±0.18 7 7.74±0.29 6 8.18±0.43 6 Table 4: Characterization of noradrenaline-induced contraction and acetylcholine-induced relaxation in the mesenteric artery Abbreviations: n—number of rats; SHR—spontaneously hypertensive rats SHR+HFD—spontaneously hypertensive rat receiving the high-fat diet; SHR+HFD+GYY—spontaneously hypertensive rat receiving the HFD and H 2 S donor GYY-4137, NA—noradrenaline, NA max —maximum noradrenaline-induced contraction, NA EC 50 —the negative logarithm of the NA molar concentration inducing the half-maximal response, Ach—acetylcholine, Ach max —maximum acetylcholine-induced relaxation, Ach EC 50 —the negative logarithm of the Ach molar concentration inducing the half-maximal response. Values are presented as means ± S.E.Ms. * p<0.05 and *** p<0.001 compared with the SHR group, + p<0.05 compared with the SHR+HFD group. The statistical analysis was performed by one-way or two-way ANOVA with the Bonferroni post hoc correction. 3.5 Evaluation of NO/NOS System Participation The arterial rings were incubated with N G -nitro-L-arginine methyl ester (LN; 10 −4 mol/L) for 20 minutes to analyze the participation of NO signaling in vasoactive responses. The NA-induced contractile response of the TA of the SHR group was significantly increased after treatment with LN (F (1,118) = 147; p<0.0001; Figure 3a). However, treatment with LN had no effect on the contractile response of the SHR+HFD group (F (1,78) = 1.98; p = 0.16) (Figure 3b). The contractile response of the TA of the SHR+HFD+GYY group was again significantly increased after treatment with LN (F (1,138) = 5.08; p = 0.03; Figure 3c). The results of the AUC evaluation revealed that after the LN incubation, the contractile response of the TAs was significantly increased in the control group (p<0.01); however, both HFD consumption and treatment with GYY-4137 reduced the influence of NO (both p<0.001, Figure 3d). In terms of endothelial function, treatment with LN had a significant effect on acetylcholine-induced relaxation in the TA, and the vasorelaxation responses were significantly reduced in the SHR (F (1,118) = 69.7; p = 4.03x10 -13 ), SHR+HFD (F (1,58) = 17.1; p = 1.74x10 -4 ) and SHR+HFD+GYY (F (1,118) = 63.1; p = 2.96x10 -12 ) groups (Figure 4 a, b, c). The AUC analysis revealed that the HFD decreased the participation of NO in the relaxation response of TAs, which was restored after treatment with GYY-4137 (p<0.05, Figure 4d). In the MA, in contrast to the control SHR group and HFD group, only the chronic administration of GYY-4137 significantly increased the participation of NO in the contractile response induced by NA (p<0.05, Figure 5 a, b, c, d). With respect to endothelial function, we observed a significant reduction in the relaxation response in the MA of the SHR control (p<0.05) and GYY-4137 groups (p<0.001) after LN treatment (Figure 6 a, d), which was confirmed by the AUC analysis (both p<0.01, Figure 6d). 3.6 Evaluation of H 2 S/CSE System Participation Acute treatment with the specific CSE inhibitor DL-propargylglycine (PPG, 10 mmoL/L) was used to analyze the contribution of H 2 S to both relaxant and contractile responses. The direct application of PPG evoked distinct effects on the TA and MA. PPG administration to the TA evoked a slight increase in basal tone (0.127±0.04 g). This effect was reversed by HFD consumption (-0.145±0.041 g; p<0.001 vs. the SHR group), but GYY administration returned it to control levels (0.043±0.051 g; p<0.05 vs. the HFD group). PPG application in MA hardly affected the basal tone, indicating minimal basal production of H 2 S (0.033±0.026 g). The consumption of the HFD induced a slight decrease in basal tone (-0.113±0.026; p<0.001 vs. the SHR group), which was partially alleviated by the administration of GYY (-0.032±0.018 g; p<0.05 vs. the HFD group; p<0.01 vs. the SHR group). Regarding the contractions induced by NA, the inhibitor was applied to the organ bath 20 minutes before the implementation of concentration-dependent responses. The TA exhibited significantly reduced contractile responses after treatment with PPG in the SHR (F (1,138) = 11.5; p = 9.28x10 -4 ), SHR+HFD (F (1,118) = 46.1; p = 8.27x10 -10 ) and SHR+HFD+GYY (F (1,157) = 120.3; p<0.0001) groups (Figure 7 a, b, c). The levels of H 2 S participation determined by calculating the AUC revealed that endogenously produced H 2 S stimulated a pro-contractile effect, and this effect on the TA was not changed by either HFD consumption or treatment with GYY-4137 (Figure 7d). In terms of endothelial function, after the TA was incubated with PPG, a significant increase in vasorelaxation occurred in the SHR (F (1,78) = 15.5; p = 2.14x10 -4 ), SHR+HFD (F (1,58) = 18.4; p = 1.11x10 -4 ) and SHR+HFD+GYY groups (F (1,98) = 27.3; p = 1.35x10 -6 ) (Figure 8 a, b, c). However, AUC analyses revealed that in the TA, the significant antirelaxant effect of endogenous H 2 S was confirmed only in the SHR+HFD+GYY group (p<0.05, Figure 8d). In the MA, the procontractile effect of H 2 S was confirmed in the control SHR group (p<0.05), with PPG having no effect on either the SHR+HFD or SHR+HFD+GYY groups (Figure 9 a, b, c), which was also confirmed by the AUC evaluation (p<0.01, Figure 9d). With respect to endothelial function, PPG had no effect on the vasorelaxation response in the MA in any of the groups (Figure 10). 3.7 Total NOS Activity and Protein Expression Levels Total NO synthase (NOS) activity in the aorta was significantly lower in the high-fat diet group than in the control SHR group (p<0.05) (Figure 11). This effect was confirmed by a decreased level of eNOS expression (p<0.001) (Figure 12a). GYY-4137 administration increased the level of total NOS activity almost to the control level (Figure 11); however, the eNOS expression level decreased to an even greater extent than those in both the control and HFD groups (p<0.001 and p<0.01, respectively) (Figure 12a). On the other hand, chronic consumption of the HFD increased iNOS expression in the HFD group only (p<0.001), and GYY-4137 administration decreased this value almost to the control level (Figure 12b). Only simultaneous HFD and GYY-4137 administration increased the CSE protein level (p<0.01), whereas the CBS protein level decreased after high-fat diet consumption (p<0.05) (Figure 12 c, d). In addition, the HFD significantly increased the TNFα expression level (p<0.001); however, the administration of GYY-4137 partially reduced the levels of inflammatory markers (p<0.05) (Figure 12e). Full scans of the entire original blots can be found in the Supplementary material. 4. Discussion The first goal of our study was to determine the dysfunction triggered in the cardiovascular system, especially in arteries, by the long-term intake of an HFD in rats with hypertension. Second, we aimed to investigate the possible beneficial effects of the slow-releasing H 2 S donor GYY-4137 on treating the pathology in this model. 4.1 Effects of the High-Fat Diet We confirmed that adiposity increased along with TAG levels in HFD-treated SHRs; however, unexpectedly, cholesterol levels decreased (Table 1). On the other hand, we previously showed that the administration of 10% fructose did not change the level of cholesterol [4] and Gaspárová et al [12] confirmed increased serum levels of total cholesterol and TAG after administration of 60% fructose to SHR. Although HFD-induced dyslipidemia typically involves elevated cholesterol levels, the finding of decreased plasma cholesterol may be related to strain-specific differences in plasma lipid metabolism in SHR. Several authors have confirmed that the lower plasma cholesterol observed in hypertensive SHR was paralleled by specific differences in hepatic catalase and glutathione redox antioxidant enzyme activities and that increased cholesterol excretion associated with defects in molecular transport may also play a role [13, 14]. Similar reduction in plasma cholesterol levels accompanied by an increase in plasma TAG levels has also been confirmed in stroke-prone SHRs compared with control normotensive Wistar-Kyoto (WKYs), where a notable decrease in the cholesterol synthesis pathway was verified [13]. Furthermore, the effect of HFD on ectopic lipid deposition should also be considered. Hojná et al. [15] similarly to us found an increase in TAG and decrease in total cholesterol in plasma of SHR fed a HFD which was associated with substantial ectopic accumulation of both cholesterol and TAG in the liver. We suggest that the presence of abnormal lipid metabolism in SHRs could persist or even be strengthened by increased lipid intake. Although high-fat diets and high fructose intake are metabolically analogous, their effect in SHR may differ at the level of heart and BP regulation. Our previous results showed that fructose administration did not affect relative heart weight but led to an increase in BP due to, among others, increased fluid retention and stimulation of the aldosterone renin angiotensin system [16]. Shiou et al. [17] reported that after the consumption of an HFD, a significant decrease in BP occurred, along with cardiac dysfunction in both SHRs and WKYs, indicating lipotoxicity related to atrial and ventricular remodeling. In our study, excessive dietary fat intake did not affect BP but promoted the development of cardiac hypertrophy. Moreover, we also confirmed increased protein expression of transforming growth factor-beta (TGF-β) and the amount of total collagen in the left ventricle (Table 2). Upon activation of TGF-β fibroblasts differentiate into myofibroblasts, which are responsible for producing excessive extracellular matrix components, including collagen types I and III. This process is critical in tissue remodeling during fibrosis [18]. Concurrently, TGF-β upregulates the expression of tissue inhibitors of metalloproteinases resulting in a net increase in ECM deposition and the progression of fibrosis [19]. Moreover, we found that, the urea levels and urea-to-creatinine ratio in the HFD group were lower than those in the SHR group. The urea-to-creatinine ratio plays a role in monitoring and identifying acute kidney injury, and its decline could indicate potential intrinsic renal damage [20]. Since we measured only the urea-to-creatinine ratio, further verification of HFD-associated kidney injury is needed. Nevertheless, the combination of an HFD and pre-existing hypertension appears to significantly affect the structure and function of the heart, emphasizing the need for a comprehensive understanding of all interactions to target therapeutic strategies. Obesity and hypertension are comorbid conditions that act as independent risk factors for the onset of endothelial dysfunction. We found that the HFD did not affect endothelium-dependent vasorelaxation in the MA (Figure 2c). This result contrasts with our previous results where improved endothelial function of MA associated with stimulation of the NO signaling pathway was confirmed in fructose-treated SHRs. This result also contrasts with the findings of a Bosse et al. [21], which reported an improvement in acetylcholine-stimulated vasorelaxation associated with an increase in eNOS phosphorylation in the MA of SHRs. In contrast to our study, where the proportion of carbohydrates was 36% and that of fats was 45%, in that study, the proportion of carbohydrates was 20%, and that of the fats was 60%. Hence, the different ratios of carbohydrate and fat intake could be a limiting factor for the ability to initiate compensatory mechanisms. Another explanation could be tissue specificity, since our results confirmed increased endothelium-dependent relaxation of the TA in the HFD group (Figure 2d). Nevertheless, the evaluation of the NO component by the AUC revealed that, in contrast to the other experimental groups, NO did not significantly participate in vasorelaxant responses in either artery in the HFD group (Figure 4d, 6d). A similar result was observed in our previous study, where SHRs receiving a fructose diet for 8 weeks presented a reduced NO contribution to the vasorelaxant response in the TA [4]. Moreover, we also found that high-fat diet consumption decreased NOS activity and the expression of the endothelial NOS protein in aortic tissue (Figures 11 and 12a), while the expression of inducible NOS (iNOS) was significantly increased. iNOS is expressed in response to stress, and elevated levels of iNOS protein expression, together with increased expression of TNFα, trigger the inflammatory response [22]. The reduced eNOS protein production could be explained by the increased caveolin-1 expression that occurs in vascular tissue under HFD conditions [23]. Taken together, the activation of the NO signaling pathway was not responsible for the improved vasorelaxation of TA. Nevertheless, while NO, a key vasodilator produced by endothelial cells, is essential for maintaining vascular health and regulating blood pressure, other vasorelaxant agents, such as prostacyclin and endothelium-derived hyperpolarizing factor (EDHF), which may compensate for reduced NO availability, are also involved in the TA [24]. Thus, the activity of vasorelaxants other than NO, which may serve as a backup mechanism, could be responsible for the increase in vasorelaxation in the TA. In addition, NO may function as important negative feedback regulator of the catalytic activity of its effector, soluble guanylate cyclase (sGC); hence, any reduction in the NO level may lead to an increase in the sensitivity of sGC to NO. A study by Jebelovszki et al. [25] confirmed that the administration of a HFD led to increased NO sensitivity in rat coronary arterioles because of sGC activation, which is also consistent with our findings, as we recorded an unchanged maximal response but increased sensitivity to acetylcholine. Next, we assumed that H 2 S is the next relevant mediator, which would be consistent with it previously being identified as EDHF [26]. However, our results showed that the application of PPG, an inhibitor of the H 2 S-producing enzyme CSE, shifted the relaxation response curves to the left in the TAs of both the SHR and HFD groups (Figure 8 a,b), indicating the antirelaxation effect of endogenously released H 2 S, although, the evaluation of the AUC did not reveal any significant alterations in either the TAs or the MAs (Figure 8d, 10d). Moreover, we found that the HFD had no significant effect on the CSE protein levels, and that CBS expression was slightly reduced. Similar results were reported in SHRs fed fructose, where endogenously produced H 2 S did not participate in endothelium-dependent relaxation [4] and in nonobese, hypertriglyceridemic rats, where endogenous H 2 S participated in the inhibition of endothelium-dependent vasorelaxation of the TA [27]. It seems that in metabolic disorders, endogenously produced H 2 S generally has an antirelaxant effect rather than helping to maintain endothelial function. With respect to contractility, we observed a significantly reduced contractile response to NA in both examined arteries following the consumption of an HFD. We observed similar impairment of contractile abilities in both TA and MA in SHR after fructose administration. We proposed that the reduced contraction was not associated with stimulated NO/NOS participation, since decreased and unchanged NO involvement were observed in the TA and MA, respectively (Figure 3b, d, 5 b,d). Previously, Li et al. [28] confirmed that under a HFD regimen, vascular matrix remodeling, including thickening of the vessel wall, fibrosis and apoptosis, was evident in the aortas of obese rats and was accompanied by increased arterial inflammation and oxidative stress. Moreover, Panchal et al. [29] observed reduced NA-induced contraction in the TA of HFD-fed rats, indicating smooth muscle dysfunction. Thus, we consider that the pathological conditions caused by an HFD might lead to a decreased contractile response through the induction of vascular remodeling of the arterial wall. TA, an elastic type of artery, is predisposed to further harmful remodeling induced by a HFD in SHRs. In our previous study, we confirmed a reduced vasocontractile response to NA in SHRs compared with that in Wistar rats, which was related to the fact that the highest component involved in TA hypertrophy in adult SHRs was the extracellular matrix and not smooth muscle cells [30]. Regarding the MA, similar to our findings, a diminished adrenergic contractile response induced by phenylephrine was observed by Bosse et al. [21] in SHR fed a low-carbohydrate/high-fat diet. Although the authors consider this finding to be an improvement in vascular function, since the MA of SHRs is characterized by hypercontractility compared with that of controls, we assume that the observed compromised contractile function in both vessels is associated with a deteriorating (rather than beneficial) effect of the HFD on the contractile apparatus. Indeed, in our study, we detected increased expression of the iNOS and TNFα proteins in arterial tissue (Figure 12 b,f), indicating the development of an inflammatory process, which could also lead to impaired contractility in both arteries. Moreover, we also confirmed reduced plasmatic level of the adipokine chemerin, which has been proposed as a possible link between metabolic, vascular disorders and inflammation. Chemerin affects vascular function, which is mediated by the production of reactive oxygen species and redox signaling [31]. In addition, serum chemerin levels correlated with markers of inflammation, insulin resistance and an unfavorable lipid profile and have been proposed as a biomarker linking inflammation and cardiovascular diseases [32, 33]. Similarly, Trovato et al. [34] showed that a high-fat Western diet could impair muscle metabolism, leading to muscle damage, which was associated with increased levels of inflammatory factors. Thus, we posit that contractile function was impaired by increased systemic inflammation under conditions of increased fat intake. With respect to the sulfide signaling pathway, we found that, unlike in the TA, the HFD eliminated the pro-contractile effect of endogenous H 2 S observed in the control MA (Figure 6a, c). Although the anticontractile effect of endogenous sulfide signaling is usually considered compensatory and beneficial, in our experiments the loss of the pro-contractile effect of endogenously produced H 2 S contributed to the inhibition of contractile responses, at least in MA. On the other hand, after fructose intake, where we observed similar impairment of contractile abilities in both TA and MA, this H 2 S action was not observed in any of the arteries and structural remodeling and inflammation were responsible for the impaired contractility. Taken together, in SHRs, high fat intake led to increased adiposity, elevated plasma triglyceride levels, and cardiac hypertrophy, as well as to generally decreased adrenergic contraction and reduced NO participation in vasoactive responses associated with decreased NOS activity and reduced expression of the eNOS protein. The increased expression of iNOS and TNFα indicates that the initiation of the inflammatory process is likely responsible for the impairment of vascular function. Furthermore, comparison with previous experiments showed that while with fructose intake, a backup mechanism for maintaining endothelial function was confirmed in both TA (NO-dependent) and MA (NO-independent), with a high-fat diet, improved vasorelaxation capacity was confirmed only in TA (NO-independent). Moreover, although both dietary regimens resulted in impaired contractility of both arteries likely due to inflammation and structural remodeling, H 2 S produced by MA in SHR fed a high-fat diet contributed to the blunted contractility. 4.2 Effects of the GYY-4137 Treatment As a next step, we investigated the effect of the slow-releasing H 2 S donor GYY-4137 on HFD-induced metabolic and vasoactive changes. We found that body adiposity was not altered after GYY-4137 treatment, similar to that in fructose-fed SHR [4]. On the other hand, while GYY-4137 significantly reduced TAG levels in fructose-fed SHR, it had no effect on cholesterol and TAG levels in rats fed a high-fat diet (Table 1). Literature data suggest that H 2 S administration with GYY-4137 could reverse the chain of events leading to lipid accumulation in vitro and in vivo. Casili et al. [35] confirmed the suppressed lipid accumulation in adipocyte-like cells treated with GYY-4137 (6 mmol/l) in vitro. Zhao et al. (2020) [36] showed in LDLr-/- mice treated with streptozotocin and a HFD that 4 weeks of GYY-4137 treatment (133 μmol/L) attenuated tissue lipid deposition. Geng et al. [37] demonstrated that HFD consumption for 13 weeks resulted in downregulation of the CSE-H 2 S system in adipose tissue of HFD-fed mice, while GYY-4137 treatment (200 μmol/kg/day) reduced lipolysis by inhibiting the phosphorylation of hormone-sensitive lipase. Based on these findings, it is surprising that the dose of GYY-4137 we used (266 μmol/l) had no significant effect on the lipid profile or other plasma markers in HFD-fed rats. On the other hand, Qabazard et al. [38] and Alshahwan et al. [39], who administered GYY-4137 at doses of 25 and 50 mg/kg for 28 days to streptozotocin-induced diabetic SD rats, showed that the efficacy of the donor is not proportional to its dose. Indeed, the regulation of adipose tissue lipolysis by H 2 S remains a matter of debate. The different metabolic and endocrine stages of pathological conditions may lead to contradictory regulation of lipolysis, differences in the involvement of the sulfide signaling pathway, and inconsistent effects of H 2 S donors. GYY-4137 treatment for 3 weeks profoundly reduced sBP compared with that in both HFD-fed and control SHRs (Figure 1). This finding is consistent with the findings of multiple studies that have shown that administering H 2 S donors can reduce BP in SHR. Zhu et al. [40] reported that treatment with GYY-4137 decreased sBP in SHRs for two weeks, and Li et al. [7] reported that the reduction in sBP persisted for 14 days after the end of the treatment period. Another study in which SHRs were treated with GYY-4137 for 4 weeks reported reduced sBP, the inhibition of angiotensin II and a reduced occurrence of myocardial fibrosis [9]. In our study, we did not observe significant effect of GYY-4137 on myocardial remodeling associated with HFD administration, only the amount of collagen in the left ventricle decreased. However, changes in cardiac trophicity may not be fully dependent on BP, as has been confirmed during the ontogenesis of SHR [40]. Similarly, we recently showed that 3 weeks of treatment with GYY-4137 in fructose-fed SHRs resulted in a reduction in sBP but without an effect on cardiac parameters [4]. Although multiple mechanisms may be responsible for the GYY-4137-induced decrease in BP, the interaction with the renin‒angiotensin‒aldosterone system (RAAS) may play an important role. Despite SHRs being a normal-to-low renin and normal-to-low angiotensin/aldosterone model of hypertension [41], increased RAAS activity along with elevated levels of angiotensin II have been observed in HFD-fed animals [42]. Laggner et al. [43] showed that NaHS-generated H 2 S inhibited the activity of angiotensin-converting enzyme (ACE) in endothelial cells. In our previous study we confirmed that an in vivo bolus administration of the ACE inhibitor captopril reduced the H 2 S donor-induced decrease in BP, suggesting that captopril disabled and masked the inhibitory effect of H 2 S on the RAAS [44]. We posit that the inhibition of the RAAS could be responsible for the decrease in sBP induced by the administration of GYY-4137. Treatment with GYY-4137 increased the endothelium-dependent relaxation of both arteries, although some differences were observed between the TA and MA. Similarly increased vasorelaxation of the TA was found in both the HFD and GYY groups; however, only the GYY-4137 treatment increased endothelium-dependent vasorelaxation in the MA compared with both the control SHR and the HFD-treated rats (Figure 2c, d). Furthermore, we observed that NO participation in vasorelaxant responses in both the TA and MA was restored after the administration of GYY-4137, although the anti-relaxation action of endogenous H 2 S was confirmed in TA. On the other hand, after fructose intake the endothelium-dependent relaxation of both arteries was similarly increased after treatment with GYY-4137, while in MA this effect was mediated by increased participation of NO, in TA the pro-relaxant action of endogenous H 2 S was responsible. These findings suggest that GYY-4137 administration leads to activation of one of the pathways (nitrate or sulfide), which could be tissue-specific and influenced by the origin of the metabolic disorder. We hypothesize that GYY, which acts as an H 2 S donor, could in high fat fed rats stimulate the production of endogenous H 2 S, which was confirmed by the increased expression of CSE in aortic tissue. An increased supply of H 2 S could subsequently sulfhydrate eNOS [45] and restart its activity. This result was also supported by the finding that, unlike in the HFD group, NOS activity did not remain reduced after GYY administration, and a trend toward increasing NOS activity was noted; thus, no difference was observed compared with the control group. Surprisingly, the decrease in endothelial NOS expression observed after HFD consumption was further exacerbated by GYY-4137 administration. We assume that a negative feedback mechanism may have been behind this effect. eNOS expression is known to be activated by the proinflammatory factor NF-kB as part of a negative feedback loop, such that increases in NO levels (and NOS activity) that repress NF-kB might lead to decreased levels of eNOS and vice versa [46]. Taken together, these findings indicate that GYY-4137 administration improved endothelial function, which was likely related to the restoration of the NO signaling pathway, and endogenously produced H 2 S did not directly contribute to this effect, as it was either not involved (MA) or involved in the antirelaxation (TA) response. Nevertheless, it appears that administration of exogenous H 2 S donor helps maintain the balance between nitrous and sulfide signaling disrupted due to modifications in both lipid and saccharide metabolism. HFD-induced impairments in contraction were partially ameliorated by GYY-4137 treatment only in the TA, highlighting the tissue-specific effects of this donor, which could also be related to the different biomechanical properties of the MA and TA or smooth muscle phenotypic differences. Soares et al. [47] confirmed greater smooth muscle cell damage in the small MA than in the TA in the obese mice fed a HFD, which was associated with higher expression of genes required for maintaining contractile capacity in the TA than in the MA and increased collagen deposition in the MA but not in the TA. Moreover, phenotypic differences between aortic and mesenteric perivascular adipose tissue (PVAT) could also take place. Aortic PVAT generates much less pro-inflammatory cytokines and thus could be more resistant to diet-induced inflammation. Mesenteric PVAT is more sensitive to the high-fat diet challenge, where the adipose “browning” genes are dramatically down-regulated [48]. With respect to the endogenous nitrous and sulfide signaling pathways, both NO and H 2 S likely did not contribute to the action of GYY-4137 in the TA; compared with those in the HFD group, the proportion of NO or H 2 S involvement in the contractile response did not change (Figure 3d, 7d). We posit that the improvement in the contractile response in the GYY group could be related to the anti-inflammatory effect of H 2 S donor. Indeed, H 2 S has notable anti-inflammatory effects, such as reducing the levels of proinflammatory cytokines, chemokines and enzymes by inhibiting the activation of NF-kB [49]. The anti-inflammatory role of H 2 S is further supported by our finding that the GYY-4137 treatment attenuated HFD-induced increases in iNOS and TNFα levels (Figure 12b, d), which is consistent with the finding that GYY-4137 has anti-inflammatory activity in disease states. Li et al. [50] showed that in LPS-stimulated human synoviocytes, GYY-4137 decreased TNFα and IL-6 production, reduced iNOS levels, and inhibited NF-kB activation. Similarly, GYY-4137 significantly reduced the serum levels of TNFα and interleukin-6 (IL-6), as well as iNOS expression, in mice with sepsis [51]. In conclusion, based on the results of this and previous study [4], different regimens, namely, fructose and high-fat diets, induce similar metabolic changes, such as increased adiposity and dyslipidemia. Although the treatment did not have the same effect on BP, vascular function was impaired in both models. Nevertheless, the fibrotic changes in the heart, tissue-specific limitation of compensatory vasoactive capacities, and impaired contractility associated with the action of endogenous H 2 S represent the original finding that high-fat consumption appears to impair the cardiovascular system of SHR more severely than fructose. A slow-releasing H 2 S donor triggered beneficial vasoactive effects, regardless of the origin of the metabolic disorder, although the mechanisms involved differ. Fructose-related disorders predominantly stimulate the beneficial pro-relaxant action of endogenous H 2 S. On the other hand, under high-fat diet conditions, endogenous H 2 S did not participate in the improvement of vascular function. GYY-4137 partially improved relaxation in the MA and restored the contractility of the TA, which was associated with restored NO signaling, and decreased the expression of the iNOS and TNFα proteins, suggesting that slow-releasing H 2 S donors could partially ameliorate metabolic changes and trigger beneficial vasoactive effects associated with the recovery of NO signaling and the suppression of inflammation. Study limitations After both the high-fat diet and GYY-4137 treatment, we observed tissue-specific vascular changes depending on the type of artery, elastic vs. muscular. For their deeper analysis, future research will need to focus on detailed specification of signaling pathways and regulatory mechanisms using in vivo (e.g. pulse wave velocity measurement) and in vitro (e.g. Western blot, Real-time PCR analysis) methods. Also, isolation and cultivation of endothelial cells and smooth muscle cells from specific types of arteries will allow the study of their individual properties and responses to different stimuli. Regarding the effects of GYY-4137, although GYY-4137 treatment generally reduced BP, the limiting factor could be the insufficient effect of GYY-4137 on adiposity and dyslipidemia which may be related to the method (single dose) and duration of administration. Therefore, it should be assessed whether GYY-4137 can improve long-term outcomes in disorders associated with HFD in the future. The metabolic changes induced by high fat intake are complex and multifactorial, therefore, long-term studies should be considered when evaluating its potential therapeutic effects. Finally, understanding the physiological roles of H 2 S compared to NO, as well as the stability of H 2 S donors, poses a challenge for effective treatment. Declarations Author Contributions Conceptualization, S.C. and B.G.A.; methodology, S.C., B.G.A., A.B., A.Ba. and M.C.; validation, S.C., B.G.A., and M.C.; formal analysis, A.B., B.G.A., and M.C.; investigation, A.B., B.G.A., J.D.H., and M.C.; writing—original draft preparation, B.G.A. and S.C.; writing—review and editing, S.C. and M.C.; visualization, S.C., B.G.A., and M.C.; funding acquisition, S.C. All authors have read and agreed to the published version of the manuscript. Data availability statement The raw data supporting the conclusions of this study are available from the corresponding author upon request, without undue reservation. Ethics statement The animals were bred in accordance with the institutional guidelines of the State Veterinary and Food Administration of the Slovak Republic and the Committee on the Ethics of Procedures in Animal, Clinical and other Biomedical Experiments (Permit Number: 2652/2021-220, 19/03/2021) of the Centre of Experimental Medicine. The Committee on the Ethics of Procedures verified the justification of the experiments from a scientific and benefit perspective and assessed the compliance of the experiments with the requirements for animal protection and concluded that the experiments meet and respect the requirements for the protection of animals used for experimental and other scientific purposes in accordance with the Decree of the Ministry of Education and Research of the Slovak Republic No. 436/2012 Coll. and the Regulation of the Government of the Slovak Republic No. 377/2012 Coll. The animals were bred also in accordance with the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes, Directive 2010/63/EU of the European Parliament. Animals were imported from the accredited breeding facility of the Center of Experimental Medicine, Slovak Academy of Sciences, Dobrá Voda, Slovak Republic, and were housed by the Institute of Normal and Pathological Physiology, Center of Experimental Medicine, Slovak Academy of Sciences (INPP CEM SAS). Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest. Supplementary material Full scans of the entire original blots for eNOS, iNOS, CSE, CBS and TNFα can be found in the Supplementary material. References Powell-Wiley, T. M. et al. Obesity and cardiovascular disease: a scientific statement from the American Heart Association. Circulation 143 (21), e984–e1010. https://doi:10.1161/CIR.0000000000000973 (2021). Duan, Y. et al. Inflammatory links between high fat diets and diseases. Front. immunol. 9 , 2649. https://doi:10.3389/fimmu.2018.02649 (2018). Gui, D. D. et al. Effects of gut microbiota on atherosclerosis through hydrogen sulfide. Eur. J. Pharmacol. 896 , 173916. https://doi:10.1016/j.ejphar.2021.173916 (2021). Berenyiova, A. et al. Vasoactive Effects of Chronic Treatment with Fructose and Slow-Releasing H2S Donor GYY-4137 in Spontaneously Hypertensive Rats: The Role of Nitroso and Sulfide Signalization. Int. J. Mol. Sci. 23 (16), 9215. https://doi:10.3390/ijms23169215 (2022). Bello, I. et al. Erucin, an H 2 S-Releasing Isothiocyanate, Exerts Anticancer Effects in Human Triple-Negative Breast Cancer Cells Triggering Autophagy-Dependent Apoptotic Cell Death. Int. J. Mol. Sci. 24 (7), 6764. http://doi:10.3390/ijms24076764 (2023). Corvino, A. et al. Pills of Multi-Target H 2 S Donating Molecules for Complex Diseases. Int. J. Mol. Sci. 25 (13), 7014. http://doi:10.3390/ijms25137014 (2024). Li, L. et al. Characterization of a novel, water-soluble hydrogen sulfide–releasing molecule (GYY4137) new insights into the biology of hydrogen sulfide. Circulation 117 (18), 2351–2360. https://doi:10.1161/CIRCULATIONAHA.107.753467 (2008). Liu, Z. et al. The hydrogen sulfide donor, GYY 4137, exhibits anti-atherosclerotic activity in high fat fed apolipoprotein E–/– mice. Br. J. Pharmacol. 169 (8), 1795–1809. https://doi:10.1111/bph.12246 (2013). Meng, G. et al. Hydrogen sulfide donor GYY4137 protects against myocardial fibrosis. Oxid Med Cell Longev 2015:691070. (2015). https://doi:10.1155/2015/691070 Pechanova, O., Bernatova, I., Pelouch, V. & Simko, F. Protein remodelling of the heart in NO-deficient hypertension: the effect of captopril. J. Mol. Cell. Cardiol. 29 (12), 3365–3374. https://doi:10.1006/jmcc.1997.0566 (1997). Reddy, G. K. & Enwemeka, C. S. A simplified method for the analysis of hydroxyproline in biological tissues. Clin Biochem 29(3):225-9. (1996). http://doi:10.1016/0009-9120(96)00003-6 . PMID: 8740508. Gasparova, Z. et al. High-fructose intake-induced dyslipidemia and oxidative stress accompanied by hippocampal dysfunctions in hypertensive but not hypertriacylglycerolemic rats. Gen. Physiol. Biophys. 42 , 25–36. http://doi:10.4149/gpb_2022053 (2023). Kitts, D. D., Yuan, Y. V. & Godin, D. V. Plasma and lipoprotein lipid composition and hepatic antioxidant status in spontaneously hypertensive (SHR) and normotensive (WKY) rats. Can. J. Physiol. Pharmacol. 76 (2), 202–209 (1998). Yuan, Y. & Kitts, D. D. Dietary fat source and cholesterol interactions alter plasma lipids and tissue susceptibility to oxidation in spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats. Mol. Cell. Biochem. 232 (1–2), 33–47. https://doi:10.1023/a:1014837131439 (2002). Hojna, S. et al. Hepatoprotective and cardioprotective effects of empagliflozin in spontaneously hypertensive rats fed a high-fat diet. Biomed. Pharm. 174 , 116520. https://doi.org/10.1016/j.biopha.2024.116520 (2024). Kurbel, S. Arterial Hypertension Due to Fructose Ingestion: Model Based on Intermittent Osmotic Fluid Trapping in the Small Bowel. Theor. Biol. Med. Model. 7 , 27. http://doi:10.1186/1742-4682-7-27 (2010). Shiou, Y. L., Huang, I. C., Lin, H. T. & Lee, H. C. High fat diet aggravates atrial and ventricular remodeling of hypertensive heart disease in aging rats. J. Formos. Med. Assoc. 117 (7), 621–631. https://doi:10.1016/j.jfma.2017.08.008 (2018). Gabbiani, G. The myofibroblast in wound healing and fibrocontractive diseases. J. Pathol. 200 (4), 500–503. http://doi:10.1002/path.1427 (2003). Frangogiannis, N. G. Transforming growth factor-β in myocardial disease. Nat. Rev. Cardiol. 19 (7), 435–455. http://doi:10.1038/s41569-021-00646-w (2022). Přeček, J., Hutyra, M., Kováčik, F., Orság, J. & Táborský, M. Biomarkers of renal function in prognostic stratification of patients with acute coronary syndrome. Cor et Vasa . 60 (2), e148–154. https://doi:10.1016/j.crvasa.2017.05.018 (2018). Bosse, J. D. et al. A low-carbohydrate/high-fat diet reduces blood pressure in spontaneously hypertensive rats without deleterious changes in insulin resistance. Am. J. Physiol. Heart Circ. Physiol. 304 (12), H1733–H1742. https://doi:10.1152/ajpheart.00631.2012 (2013). Haddad, Y. & Couture, R. Interplay between the kinin B1 receptor and inducible nitric oxide synthase in insulin resistance. Br. J. Pharmacol. 173 (12), 1988–2000. 10.1111/bph.13491 (2016). https:// Yang, N. et al. High-fat diet up-regulates caveolin-1 expression in aorta of diet-induced obese but not in diet-resistant rats. Cardiovasc. Res. 76 (1), 167–174. https://doi:10.1016/j.cardiores.2007.05.028 (2007). Félétou, M. Endothelium-dependent hyperpolarization and endothelial dysfunction. J. Cardiovasc. Pharmacol. 67 (5), 373–387. https://doi:10.1097/FJC.0000000000000346 (2016). Jebelovszki, E. et al. High-fat diet-induced obesity leads to increased NO sensitivity of rat coronary arterioles: role of soluble guanylate cyclase activation. Am. J. Physiol. Heart Circ. Physiol. 294 (6), H2558–H2564. https://doi:10.1152/ajpheart.01198.2007 (2008). Tang, G. et al. H 2 S is an endothelium-derived hyperpolarizing factor. Antiox Red Sig . 19 (14), 1634–1646. https://doi:10.1089/ars.2012.4805 (2013). Cacanyiova, S. et al. The vasoactive role of perivascular adipose tissue and the sulfide signaling pathway in a nonobese model of metabolic syndrome. Biomolecules 11 (1), 108. https://doi:10.3390/biom11010108 (2021). Li, W. et al. Inhibition of ROS and inflammation by an imidazopyridine derivative X22 attenuate high fat diet-induced arterial injuries. Vascul Pharmacol. 72 , 153–162. https://doi:10.1016/j.vph.2015.05.006 (2015). Panchal, S. K. et al. High-carbohydrate, high-fat diet–induced metabolic syndrome and cardiovascular remodeling in rats. J. Cardiovasc. Pharmacol. 57 (5), 611–624. https://doi:10.1097/FJC.0b013e31821b1379 (2011). Berenyiova, A., Drobna, M., Cebova, M., Kristek, F. & Cacanyiova, S. Changes in the vasoactive effects of nitric oxide, hydrogen sulfide and the structure of the rat thoracic aorta: the role of age and essential hypertension. J. Physiol. Pharmacol. 69 (4). https://doi:10.26402/jpp.2018.4.05 (2018). Neves, K. B. et al. Chemerin reduces vascular nitric oxide/cGMP signalling in rat aorta: a link to vascular dysfunction in obesity? Clin. Sci. 127 (2), 111–122. http://doi:10.1042/CS20130286 (2014). Szpakowicz, A. et al. Serum Chemerin Concentration Is Associated with Proinflammatory Status in Chronic Coronary Syndrome Biomolecules 11 : 1149. (2021). https://doi.org/10.3390/biom11081149 Eichelmann, F. et al. Chemerin as a Biomarker Linking Inflammation and Cardiovascular Diseases. J. Am. Coll. Cardiol. 73 (3), 378–379. 10.1016/j.jacc.2018.10.058 (2019). Trovato, F. M., Castrogiovanni, P., Szychlinska, M. A., Purrello, F. & Musumeci, G. Impact of Western and Mediterranean diets and vitamin D on muscle fibers of sedentary rats. Nutrients 10 (2), 231. https://doi:10.3390/nu10020231 (2018). Casili, G. et al. Inhibition of the 3–mercaptopyruvate sulfurtransferase—hydrogen sulfide system promotes cellular lipid accumulation. GeroScience 44 , 2271–2289. https://doi.org/10.1007/s11357-022-00600-9 (2022). Zhao, S. et al. Hydrogen sulfide alleviates liver injury via S-sulfhydrated-Keap1/Nrf2/LRP1 pathway. Hepatol 73 (1), 282–302. http://doi:10.1002/hep.31247 (2021). Geng, B. et al. Increase or decrease hydrogen sulfide exert opposite lipolysis, but reduce global insulin resistance in high fatty diet induced obese mice. PLoS One . 8 (9), e73892. https://doi:10.1371/journal.pone.0073892 (2013). Qabazard, B., Yousif, M., Mousa, A. & Phillips, O. A. GYY4137 attenuates functional impairment of corpus cavernosum and reduces fibrosis in rats with STZ-induced diabetes by inhibiting the TGF-β1/Smad/CTGF pathway. Biomed. Pharm. 138 , 111486. https://doi.org/10.1016/j.biopha.2021.111486 (2021). Alshahwan, H. et al. Hydrogen sulfide donor GYY4137 attenuates vascular complications in mesenteric bed of streptozotocin-induced diabetic rats. Eur. J. Pharmacol. 933 , 175265. http://doi:10.1016/j.ejphar.2022.175265 (2022). Epub 2022 Sep 12. Cebova, M. & Kristek, F. Age-dependent ultrastructural changes of coronary artery in spontaneously hypertensive rats. Gen. Physiol. Biophys. 30 (4), 364–372. 10.4149/gpb_2011_04_364 (2011). http:// Simko, F. et al. Sacubitril/Valsartan and Ivabradine Attenuate Left Ventricular Remodelling and Dysfunction in Spontaneously Hypertensive Rats: Different Interactions with the Renin–Angiotensin–Aldosterone System. Biomedicines 10 (8), 1844. https://doi:10.3390/biomedicines10081844 (2022). Nag, S., Patel, S., Mani, S. & Hussain, T. Role of angiotensin type 2 receptor in improving lipid metabolism and preventing adiposity. Mol. Cell. Biochem. 461 (1–2), 195–204. 10.1007/s11010-019-03602-y (2019). https:// Laggner, H. et al. The novel gaseous vasorelaxant hydrogen sulfide inhibits angiotensin-converting enzyme activity of endothelial cells. J. Hypertens. 25 (10), 2100–2104. https://doi:10.1097/HJH.0b013e32829b8fd0 (2007). Drobna, M. et al. Captopril Partially Decreases the Effect of H 2 S on Rat Blood Pressure and Inhibits H 2 S-Induced Nitric Oxide Release From S-Nitrosoglutathione. Physiol. Res. 64 (4), 479–486. https://doi:10.33549/physiolres.932772 (2015). Altaany, Z., Ju, Y., Yang, G. & Wang, R. The coordination of S-sulfhydration, S-nitrosylation, and phosphorylation of endothelial nitric oxide synthase by hydrogen sulfide. Sci. Signal. 7 (342), ra87. https://doi:10.1126/scisignal.2005478 (2014). Grumbach, I. M., Chen, W., Mertens, S. A. & Harrison, D. G. A negative feedback mechanism involving nitric oxide and nuclear factor kappa-B modulates endothelial nitric oxide synthase transcription. J. Mol. Cell. Cardiol. 39 (4), 595–603. https://doi:10.1016/j.yjmcc.2005.06.012 (2005). Soares, A. G., Catelli de Carvalho, M. H. & Akamine, E. Obesity Induces Artery-Specific Alterations: Evaluation of Vascular Function and Inflammatory and Smooth Muscle Phenotypic Markers. BioMed. Res. Int. 17: Article ID . 5038602 , 10pages. https://doi.org/10.1155/2017/5038602 (2017). Li, X., Ma, Z. & Zhu, Y. Z. Regional Heterogeneity of Perivascular Adipose Tissue: Morphology, Origin, and Secretome. Front Pharmacol 12: Article 697720. (2021). http://doi:10.3389/fphar.2021.697720 Bhatia, M. & Gaddam, R. R. Hydrogen sulfide in inflammation: a novel mediator and therapeutic target. ARS 34 (17), 1368–1377. https://doi:10.1089/ars.2020.8211 (2021). Li, L. et al. The complex effects of the slow‐releasing hydrogen sulfide donor GYY 4137 in a model of acute joint inflammation and in human cartilage cells. J. Cell. Mol. Med. 17 (3), 365–376. https://doi:10.1111/jcmm.12016 (2013). Cui, W., Chen, J., Yu, F., Liu, W. & He, M. GYY4137 protected the integrity of the blood-brain barrier via activation of the Nrf2/ARE pathway in mice with sepsis. FASEB J. 35 (9), e21902. https://doi:10.1096/fj.202100074R (2021). Additional Declarations No competing interests reported. Supplementary Files SupplemetaryMaterial.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6030564","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":443532462,"identity":"bb0bc90e-fb7b-437c-a44d-fa428f03434a","order_by":0,"name":"Basak G. Aydemir","email":"","orcid":"","institution":"Slovak Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Basak","middleName":"G.","lastName":"Aydemir","suffix":""},{"id":443532463,"identity":"afc3d333-8479-46c7-b8a3-caae23e1b81e","order_by":1,"name":"Andrea Berenyiova","email":"","orcid":"","institution":"Slovak Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Andrea","middleName":"","lastName":"Berenyiova","suffix":""},{"id":443532464,"identity":"d73ce288-ee5a-4e16-9fca-b2178d4b66fc","order_by":2,"name":"Martina Cebova","email":"","orcid":"","institution":"Slovak Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Martina","middleName":"","lastName":"Cebova","suffix":""},{"id":443532465,"identity":"78e8d5b0-f574-441d-889b-5776fcc6300a","order_by":3,"name":"John D. Henderson","email":"","orcid":"","institution":"University of Copenhagen","correspondingAuthor":false,"prefix":"","firstName":"John","middleName":"D.","lastName":"Henderson","suffix":""},{"id":443532467,"identity":"7afdcb3a-3cf1-4486-8186-ff09cb65d123","order_by":4,"name":"Andrej Barta","email":"","orcid":"","institution":"Slovak Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Andrej","middleName":"","lastName":"Barta","suffix":""},{"id":443532468,"identity":"68b7bc88-37f9-4c89-bbe1-7b5d3717a365","order_by":5,"name":"Sona Cacanyiova","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAUlEQVRIiWNgGAWjYLCDA0AsB8RsII4B0VqMSdMCAokNhLTIt/ce/MBQYyfPP7vH8MDHNrv0+e1nzB78YKgzxqXF4My5ZAmGY8mGM+6cMTg4sy05d8OZHHPDHobDZji1SOQYSDA2MCcw3MgxOMxzhjl3A0NamgQPwwEbnA6bkWP8g7GhPkEeoqU+Xb7/WZrkH4Y6nFqAhpsBbTmcYADWUnEYaF3yMWkeBmbcDjtzxswi4dhxw4030goOzqg4brjhxuPjxjIGh3F6X769x/jGh5pqebkbyZs/fDColpfvT2x7+KaizrABp8uAIAFMciDHBHERyf6AKGWjYBSMglEw8gAA+f1XmTW7dOYAAAAASUVORK5CYII=","orcid":"","institution":"Slovak Academy of Sciences","correspondingAuthor":true,"prefix":"","firstName":"Sona","middleName":"","lastName":"Cacanyiova","suffix":""}],"badges":[],"createdAt":"2025-02-14 12:23:14","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-6030564/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6030564/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":80855452,"identity":"c7c23804-a343-4bdf-9724-6143ea10c263","added_by":"auto","created_at":"2025-04-17 20:59:00","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":250410,"visible":true,"origin":"","legend":"\u003cp\u003eThe systolic blood pressure of the control SHR (SHR, n=8), SHR receiving HFD (SHR+HFD, n=7), and SHR receiving HFD and 266 µmol/kg/day GYY-4137 (SHR+HFD+GYY, n=8) groups. The data are shown as the means ± SEMs. The statistical analysis was performed by two-way ANOVA with the Bonferroni post hoc correction. N—number of rats. ***p\u0026lt;0.001 compared with the SHR group (17\u003csup\u003eth\u003c/sup\u003e week), ++p\u0026lt;0.01 compared with the SHR+HFD group (17\u003csup\u003eth\u003c/sup\u003e week), \u0026lt;\u0026lt; p\u0026lt;0.001 compared with the SHR group (14\u003csup\u003eth\u003c/sup\u003e week).\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6030564/v1/57805cee09e09ee1f5fd41a2.jpeg"},{"id":80855633,"identity":"7c79113e-295a-4cb2-a1a5-acedb56270c1","added_by":"auto","created_at":"2025-04-17 21:07:00","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":570059,"visible":true,"origin":"","legend":"\u003cp\u003eContractile responses induced by noradrenaline in the thoracic aorta (A) and mesenteric artery (B) and endothelium-dependent vasorelaxant responses induced by acetylcholine in the thoracic aorta (C) and mesenteric artery (D). Arteries were isolated from control rats (SHRs), SHRs fed the HFD (SHR+HFD), and SHRs fed the HFD and treated with GYY-4137 (SHR+HFD+GYY). Number of rats: thoracic aorta—SHR group n=6, SHR+HFD group n=6–7, and SHR+HFD+GYY group n=7–8; mesenteric artery—SHR group n=7, SHR+HFD group n=6, and SHR+HFD+GYY group n=6–7. The results are presented as the means ± S.E.Ms.. Statistical analyses were performed by two-way ANOVA with the Bonferroni post hoc correction. *p\u0026lt;0.05, ** p\u0026lt;0.01 and *** p\u0026lt;0.001 compared with the SHR group, and + p\u0026lt;0.05 compared with the SHR+HFD group.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6030564/v1/dc39fbadf906827173913db1.jpeg"},{"id":80855636,"identity":"f3654897-c4e8-4cdb-ab8d-45e7e94bc1b9","added_by":"auto","created_at":"2025-04-17 21:07:00","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":542944,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of N\u003csup\u003eG\u003c/sup\u003e-nitro-L-arginine methyl ester (LN) on the contractile responses of the thoracic aorta induced by noradrenaline in control SHRs (A), SHRs fed the HFD (B), and SHRs fed the HFD and treated with GYY-4137 (C). The level of NO participation was determined via an AUC evaluation (D). White column—before the addition of the inhibitor; hatched column—after the addition of the inhibitor. The number of arterial segments per group was as follows: SHR n=6; SHR+HFD n=5; and SHR+HFD+GYY n=7. The results are presented as the means ± S.E.Ms. The statistical analysis was performed by two-way ANOVA (line graphs) or paired Student´s t test/one-way ANOVA (AUC) with the Bonferroni post hoc correction. * p\u0026lt;0.05, ** p\u0026lt;0.01, *** p\u0026lt;0.001 vs LN- within the relevant group, + p\u0026lt;0.05 vs SHR LN+.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6030564/v1/3a6eac6cda2e39a43ac04e75.jpeg"},{"id":80855455,"identity":"9496b83b-af76-43d9-8f25-026ae46012b1","added_by":"auto","created_at":"2025-04-17 20:59:00","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":503113,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of N\u003csup\u003eG\u003c/sup\u003e-nitro-L-arginine methyl ester (LN) on the relaxation responses of the thoracic aorta induced by acetylcholine in control SHRs (A), SHRs fed the HFD (B), and SHRs fed the HFD and treated with GYY-4137 (C). The level of NO participation was determined via an AUC evaluation (D). White column—before the addition of the inhibitor; hatched column—after the addition of the inhibitor. The number of arterial segments per group was as follows: SHR n=6; SHR+HFD n=6; and SHR+HFD+GYY n=6. The results are presented as the means ± S.E.Ms. The statistical analysis was performed by two-way ANOVA (line graphs) or paired Student’s t test/one-way ANOVA (AUC) with the Bonferroni post hoc correction. * p\u0026lt;0.05, *** p\u0026lt;0.001 vs LN- within the relevant group, + p\u0026lt;0.05, +++ p\u0026lt;0.001 vs SHR LN+.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6030564/v1/df73488ad4aa5bd0b2780881.jpeg"},{"id":80855962,"identity":"0a790e0f-0da2-42d7-808d-4cf5b753adbf","added_by":"auto","created_at":"2025-04-17 21:15:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":243691,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of N\u003csup\u003eG\u003c/sup\u003e-nitro-L-arginine methyl ester (LN) on the contractile responses of the mesenteric artery induced by noradrenaline in control SHRs (A), SHRs fed the HFD (B), and SHRs fed the HFD and treated with GYY-4137 (C). The level of NO participation was determined via an AUC evaluation (D). White column—before the addition of the inhibitor; hatched column—after the addition of the inhibitor. The number of arterial segments per group was as follows: SHR n=6; SHR+HFD n=6; and SHR+HFD+GYY n=7. The results are presented as the means ± S.E.Ms. The statistical analysis was performed by two-way ANOVA (line graphs) or a paired Student’s t test/one-way ANOVA (AUC) with the Bonferroni post hoc correction. * p\u0026lt;0.05 vs LN- within the relevant group, + p\u0026lt;0.05 vs SHR LN+.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6030564/v1/62eaf6dabf38c3fdbd03ce3c.png"},{"id":80855224,"identity":"b0de0cbd-22c7-4681-97f8-2533ccc23f94","added_by":"auto","created_at":"2025-04-17 20:51:00","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":529528,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of N\u003csup\u003eG\u003c/sup\u003e-nitro-L-arginine methyl ester (LN) on relaxation responses of the mesenteric artery induced by acetylcholine in control SHRs (A), SHRs fed the HFD (B), and SHRs fed the HFD and treated with GYY-4137 (C). The level of NO participation was determined via an AUC evaluation (D). White column—before the addition of the inhibitor; hatched column—after the addition of the inhibitor. The number of arterial segments per group was as follows: SHR n=7; SHR+HFD n=6; and SHR+HFD+GYY n=6. The results are presented as the means ± S.E.Ms. The statistical analysis was performed by two-way ANOVA (line graphs) or a paired Student’s t test/one-way ANOVA (AUC) with the Bonferroni post hoc correction. * p\u0026lt;0.05, ** p\u0026lt;0.01, and *** p\u0026lt;0.001 compared with the relevant group without LN treatment.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6030564/v1/1e15b07dc6d15f96f7e545a5.jpeg"},{"id":80855235,"identity":"890c2c1f-97c9-4746-ab33-03b19c7290e3","added_by":"auto","created_at":"2025-04-17 20:51:00","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":304682,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of DL-propargylglycine (PPG) on the contractile response of the thoracic aorta induced by noradrenaline in control SHRs (A), SHRs fed the HFD (B), and SHRs fed the HFD and treated with GYY-4137 (C). The level of H\u003csub\u003e2\u003c/sub\u003eS participation was evaluated via the AUC (D). White column—before the addition of the inhibitor; hatched column—after the addition of the inhibitor. The number of arterial segments per group was as follows: SHR n=7; SHR+HFD n=6; and SHR+HFD+GYY n=7. The results are presented as the means ± S.E.Ms. The statistical analysi\u003cstrong\u003es\u003c/strong\u003e was performed by two-way ANOVA (line graphs) or paired Student´s t test/one-way ANOVA (AUC) with the Bonferroni post hoc correction. * p\u0026lt;0.05 and *** p\u0026lt;0.001 compared within the relevant group without PPG treatment.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6030564/v1/4f3f1a35642ba13f437933fd.png"},{"id":80855233,"identity":"d0bb3c62-9990-41db-a716-6699112e80e6","added_by":"auto","created_at":"2025-04-17 20:51:00","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":519690,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of DL-propargylglycine (PPG) on the relaxation responses of the thoracic aorta induced by acetylcholine in control SHRs (A), SHRs fed the HFD (B), and SHRs fed the HFD and treated with GYY-4137 (C). The level of H\u003csub\u003e2\u003c/sub\u003eS participation was evaluated via the AUC (D). White column—before the addition of the inhibitor; hatched column—after the addition of the inhibitor. The number of arterial segments per group was as follows: SHR n=6; SHR+HFD n=6; and SHR+HFD+GYY n=6. The results are presented as the means ± S.E.Ms. The statistical analysi\u003cstrong\u003es\u003c/strong\u003e was performed by two-way ANOVA (line graphs) or paired Student´s t test/one-way ANOVA (AUC) with the Bonferroni post hoc correction. * p\u0026lt;0.05 and *** p\u0026lt;0.001 compared within the relevant group without PPG treatment, + p\u0026lt;0.05 compared with the SHR+PPG group.\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6030564/v1/3441ef47b8429c0c29e58107.jpeg"},{"id":80855228,"identity":"68a1a9b5-cd7b-4528-96f0-33cda15efab9","added_by":"auto","created_at":"2025-04-17 20:51:00","extension":"jpeg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":586959,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of DL-propargylglycine (PPG) on the contractile response of the mesenteric artery induced by noradrenaline in control SHRs (A), SHRs fed the HFD (B), and SHRs fed the HFD and treated with GYY-4137 (C). The level of H\u003csub\u003e2\u003c/sub\u003eS participation was evaluated via the AUC (D). White column—before the addition of the inhibitor; hatched column—after the addition of the inhibitor. The number of arterial segments per group was as follows: SHR n=7; SHR+HFD n=6; and SHR+HFD+GYY n=7. The results are presented as the means ± S.E.Ms. The statistical analysi\u003cstrong\u003es\u003c/strong\u003e was performed by two-way ANOVA (line graphs) or paired Student’s t test/one-way ANOVA (AUC) with the Bonferroni post hoc correction. * p\u0026lt;0.05 and ** p\u0026lt;0.01 compared within the relevant group without PPG treatment.\u003c/p\u003e","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6030564/v1/f8dbc2eb79d6084f45bc264e.jpeg"},{"id":80855247,"identity":"4451cb3f-18b4-455d-9709-7d05752c2664","added_by":"auto","created_at":"2025-04-17 20:51:01","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":213128,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of DL-propargylglycine (PPG) on the relaxation responses of the thoracic aorta induced by acetylcholine in control SHRs (A), SHRs fed the HFD (B), and SHRs fed the HFD and treated with GYY-4137 (C). The level of H\u003csub\u003e2\u003c/sub\u003eS participation was evaluated via the AUC (D). White column—before the addition of the inhibitor; hatched column—after the addition of the inhibitor. The number of arterial segments per group was as follows: SHR n=6; SHR+HFD n=6; and SHR+HFD+GYY n=6. The results are presented as the means ± S.E.Ms. The statistical analysi\u003cstrong\u003es\u003c/strong\u003e was performed by two-way ANOVA (line graphs) or paired Student’s t test/one-way ANOVA (AUC) with Bonferroni post-hoc test.\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-6030564/v1/a7d4c59e6ce8a8d422e27311.png"},{"id":80855463,"identity":"f0034f3d-0c73-4f09-ae95-b0edbb3beb72","added_by":"auto","created_at":"2025-04-17 20:59:01","extension":"jpeg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":119738,"visible":true,"origin":"","legend":"\u003cp\u003eTotal NOS activity in aortic tissue. Arteries were isolated from control rats (SHRs), SHRs fed the HFD (SHR+HFD), and SHRs fed the HFD and treated with GYY-4137 (SHR+HFD+GYY). The numbers of samples from the rats in each group were as follows: SHR n=8, SHR+HFD n=6, and SHR+HFD+GYY n=6. The data are presented as the means ± S.E.Ms. The statistical analysis was performed by one-way ANOVA. * p\u0026lt;0.05 compared with SHRs.\u003c/p\u003e","description":"","filename":"floatimage11.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6030564/v1/8d49604fa0f455a5b03f86b7.jpeg"},{"id":80855465,"identity":"8f460204-968c-4616-b36f-33aba7e93b43","added_by":"auto","created_at":"2025-04-17 20:59:01","extension":"jpeg","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":465631,"visible":true,"origin":"","legend":"\u003cp\u003eProtein expression of endothelial NOS (A), inducible NOS (B), the H\u003csub\u003e2\u003c/sub\u003eS-producing enzyme CSE (C), CBS (D) and TNFα (E) in aortic tissue. Arteries were isolated from control rats (SHRs), SHRs fed the HFD (SHR+HFD), and SHRs fed the HFD and treated with GYY-4137 (SHR+HFD+GYY). Endothelial NOS—eNOS; inducible NOS—iNOS; CSE—cystathionine γ-lyase; CBS—cystathionine β-synthase; TNFα—tumor necrosis factor alpha. The numbers of samples from rats in each group (for each protein) were as follows: SHR n=6, SHR+HFD n=6, and SHR+HFD+GYY n=6. The data are presented as the means ± S.E.Ms. The statistical analysis was performed by one-way ANOVA. * p\u0026lt;0.05, ** p\u0026lt;0.01, and *** p\u0026lt;0.001 compared with the SHR group; + p\u0026lt;0.05, ++ p\u0026lt;0.01, and +++ p\u0026lt;0.001 compared with the SHR+HFD group.\u003c/p\u003e","description":"","filename":"floatimage12.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6030564/v1/b0546deb175d63f2265dfda9.jpeg"},{"id":81010410,"identity":"2b3b65ef-8c6b-4d8e-b3e1-d47746be4bdd","added_by":"auto","created_at":"2025-04-21 08:16:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5901018,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6030564/v1/df0dc39a-0bf7-4274-a6ee-d49e0b7f4786.pdf"},{"id":80855451,"identity":"a98f296c-6b8c-4014-b30a-a0a7996a06c5","added_by":"auto","created_at":"2025-04-17 20:59:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":426981,"visible":true,"origin":"","legend":"","description":"","filename":"SupplemetaryMaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6030564/v1/a20785b5985ef7b11fb3227e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Effect of High Fat Diet and H2S Donor GYY-4137 on Vascular Function in Spontaneously Hypertensive Rats","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCharacterized by a combination of symptoms, including insulin resistance, hypertension, and obesity, metabolic syndrome is one of the leading causes of mortality worldwide, and a high-fat diet (HFD) is the main driver. The global obesity epidemic directly contributes to cardiovascular disease risk factors, including dyslipidemia, type 2 diabetes, and hypertension, as highlighted by the American Heart Association, which reported that in 2021, 39\u0026ndash;49% of the global population (2.8\u0026ndash;3.5\u0026nbsp;billion people) were overweight or obese [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Metabolic syndrome initiated by chronic HFD consumption increases circulating levels of free fatty acids and inflammatory markers, causing chronic, systemic, low-grade inflammation and an unbalanced oxidative status in several organs [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. New interventions that improve cardiovascular and metabolic health in these patients are essential to address the rising health problem that metabolic syndrome represents.\u003c/p\u003e \u003cp\u003eTargeting gasotransmitter signaling is a promising area of ​​treatment for both cardiovascular and metabolic diseases. Hydrogen sulfide (H\u003csub\u003e2\u003c/sub\u003eS) is a gaseous transmitter that regulates intracellular signaling. The colonic microbiota represents the largest source of H\u003csub\u003e2\u003c/sub\u003eS in the body and results from recent years show that gut-derived H\u003csub\u003e2\u003c/sub\u003eS can induce systemic effects and changes in H\u003csub\u003e2\u003c/sub\u003eS homeostasis in the colon H\u003csub\u003e2\u003c/sub\u003eS can be associated with various pathological stages such as hypertension or atherosclerosis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. At the same time, many studies have been conducted on the effects of H\u003csub\u003e2\u003c/sub\u003eS produced enzymatically by various tissues of the cardiovascular system. However, the physiological and pathological role of H\u003csub\u003e2\u003c/sub\u003eS remains much less well understood than that of nitric oxide (NO), despite promising work from our group suggesting its potential antihypertensive function [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Research shows that strategies that increase the bioavailability of H\u003csub\u003e2\u003c/sub\u003eS are promising therapeutic approaches to treat diseases. The inorganic sulfide donors such as NaHS and Na\u003csub\u003e2\u003c/sub\u003eS provide fast extracellular release of H\u003csub\u003e2\u003c/sub\u003eS and therefore have restricted therapeutic potential. Among the natural sources of H\u003csub\u003e2\u003c/sub\u003eS, there are polysulfides, which include garlic-derived compounds or isothiocyanates, including sulforaphane and erucin [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] Synthetic H\u003csub\u003e2\u003c/sub\u003eS donating compounds have also been developed. A very promising modern approach consists in designing multi-target molecular hybrids where some of the H\u003csub\u003e2\u003c/sub\u003eS-releasing moieties have been largely combined with different drugs already used in the clinic to obtain novel multi-target molecular hybrids [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA slow-releasing H\u003csub\u003e2\u003c/sub\u003eS donor with vasodilatory and antihypertensive properties, GYY-4137, has been shown to be effective in models of cardiovascular disease [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. GYY-4137 reduced vascular inflammation and oxidative stress, improved endothelial function, and reduced atherosclerotic plaque formation in apolipoprotein E-/- mice fed a high-fat diet [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Moreover, the long-term application of this donor led to a significant decrease in systolic blood pressure and alleviated myocardial fibrosis in spontaneously hypertensive rats (SHRs) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In our recent work, we induced metabolic syndrome-like pathologies in SHRs via long-term fructose administration and reported that treatment with GYY-4137 alleviated fructose-induced structural malformations associated with tissue inflammation while also restoring the impaired vascular contractile ability [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In this study we investigated the effects of GYY-4137 on blood pressure, biometric parameters, markers of inflammation, cardiac fibrosis and both H\u003csub\u003e2\u003c/sub\u003eS and NO signaling in the vascular function of SHRs fed a HFD to provide further information on the therapeutic potential of exogenous H\u003csub\u003e2\u003c/sub\u003eS donor in metabolic disorders. An original contribution is also the comparison of two types of arteries - the thoracic aorta (elastic type) and the mesenteric artery (muscular type). We hypothesized that chronic consumption of an HFD would induce metabolic syndrome-like pathologies in SHRs, impair vascular function, increase blood pressure, and interfere with vascular NO and H\u003csub\u003e2\u003c/sub\u003eS signaling; however, these effects would be attenuated by GYY-4137.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Experimental animals\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAll the rats were maintained on a 12-h light/dark cycle in a temperature (20\u0026ndash;22\u0026deg;C)- and humidity (45\u0026ndash;65%)-controlled room with access to standard laboratory rat chow (Altromin 1324P, Lage, Germany) or a high-fat obesity-inducing diet (Altromin, type C 1090-45, Germany) and drinking water ad libitum. The contents of the standard laboratory rat chow were 11% fat, 24% protein, and 65% carbohydrates, while the carbohydrate contents were as follows: 0 mg/kg monosaccharides, 47.8 mg/kg disaccharides, and 391.2 mg/kg polysaccharides. The remainder of the diet contained minerals and trace elements. The content of the high-fat diet was 45% fat, 18% protein, and 37% carbohydrates, and the carbohydrate content was 101.6 mg/kg monosaccharides, 52.1 mg/kg disaccharides, and 238.9 mg/kg polysaccharides; the remainder of the diet contained minerals and trace elements.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Experimental design\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAn animal model of primary hypertension, SHRs, was used in this study. Twenty-four male SHRs aged 9 weeks and ranging in weight from 180\u0026ndash;220 grams were subsequently divided into three groups. One rat died during the systolic blood pressure measurements; thus, 23 SHRs were used for the experiments. The control SHR group (n\u0026thinsp;=\u0026thinsp;8) received a standard diet for 8 weeks, the SHR\u0026thinsp;+\u0026thinsp;HFD group (n\u0026thinsp;=\u0026thinsp;7) received a high-fat diet (HFD) for 8 weeks, and the SHR\u0026thinsp;+\u0026thinsp;HFD\u0026thinsp;+\u0026thinsp;GYY group (n\u0026thinsp;=\u0026thinsp;8) received an HFD for 8 weeks. The SHR\u0026thinsp;+\u0026thinsp;HFD\u0026thinsp;+\u0026thinsp;GYY group also received GYY-4137 (266 \u0026micro;mol/kg/day dissolved in 1% dimethyl sulfoxide (DMSO) in physiological solution), which was injected intraperitoneally (i.p.) daily for three weeks at the age of 14\u0026ndash;17 weeks. The rats in the SHR and SHR\u0026thinsp;+\u0026thinsp;HFD groups received vehicle via i.p. injection (1% DMSO in physiological solution; volumes were determined according to the actual body weight).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Blood pressure measurement and biometric parameters\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eSystolic blood pressure (sBP) was measured using tail-cuff plethysmography (MRBP; IITC Life Science Inc., Los Angeles, CA, USA). Animals were trained for the tail-cuff method for measuring blood pressure for two consecutive days before the evaluation of basal sBP levels (at the age of 9 weeks, the beginning of the HFD treatment). In all groups, sBP was subsequently measured at the end of the 14th week (the beginning of the treatment with GYY-4137) and at the end of the experiment (at the age of 17 weeks). sBP was calculated as the average of the five measurements completed for each rat.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Body weights and diet consumption\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eRat body weight (BW) and food intake were monitored daily during the treatments. At the end of the experiment, the rats were euthanized by decapitation after brief CO\u003csub\u003e2\u003c/sub\u003e anesthetization. CO₂ was used to render the rats unconscious at a concentration of 30\u0026ndash;40%, which was applied gradually to minimize stress and discomfort to the animal. Heart weight (HW), retroperitoneal adipose tissue weight (RTW), and the length of the tibia (TL) were measured. The ratios of HW to TL or BW and RTW to TL or BW were calculated to determine the degree of cardiac hypertrophy and body adiposity, respectively.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Blood sample analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eBlood samples from the trunks of all the animals were collected at the end of the treatment day into preprepared heparinized tubes (140 UI/5 mL) and then centrifuged (850\u0026times; g, 10 min, 4\u0026deg;C, Centrifuge 5430 R, Eppendorf, Hamburg, Germany). The resulting plasma fraction was then analyzed to determine the levels of glucose (GLU), cholesterol (CHOL), triacylglycerol (TAG), urea (UREA) and creatine (CRE) using a biochemical analyzer and auxiliary reagent discs (Celercare, MNCHIP Technologies Co., Ltd., Tianjin, China). According to the manufacturer\u0026rsquo;s instructions for analysis, 100 \u0026micro;L of plasma was pipetted into the sample chamber, and 430 \u0026micro;L of distilled water was then added to the diluent chamber of the test-specific reagent disk.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 ELISA\u003c/h2\u003e \u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e Chemerin levels were measured in plasma samples using a commercially available Rat CHEM ELISA Kit (ELK Biotechnology, Denver, CO, USA) according to the manufacturer's protocol. The samples of the trunk blood of the animals were collected at the end of the treatment into pre-prepared heparinized tubes (140 UI/5 mL) and were then centrifuged (850\u0026times; g, 10 min, 4\u0026deg;C) and the plasma samples were frozen immediately at -80\u0026ordm;C until the assay. After thawing on ice, serum samples were diluted 20-fold in the kit dilution buffer. 100ul of the diluted samples and the kit standards were then added to the ELISA plate and incubated at 37\u0026deg;C for 80 minutes, after washing 3 times with 100ul of wash buffer. 100ul of the biotinylated antibody working solution was added to each well and the plate was incubated at 37\u0026deg;C for 50 minutes. The wells were then washed 3 times with 100ul of wash buffer, followed by the addition of 100ul of the Streptavidin-HRP working solution and incubated at 37\u0026deg;C for 50 minutes. The plate was then washed 5 times in the wash buffer, before 90ul of TMB solution was added leave in the dark for 20 minutes at 37\u0026deg;C, after which 50ul of the stop reagent was added and the plate was then read in a spectrophotometer at 450nm.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Vasoactive responses of isolated arteries\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe descending part of the thoracic aorta (TA), beginning under the arcus aorta, and the superior mesenteric artery (MA) were isolated under a binocular microscope, cleaned of connective tissue and cut into 5 mm long rings with a preserved endothelium. The rings were set vertically between 2 stainless wire triangles and placed in a 20 ml incubation organ bath with Krebs solution (118 mmol/L NaCl; 5 mmol/L KCl; 25 mmol/L NaHCO\u003csub\u003e3\u003c/sub\u003e; 1.2 mmol/L MgSO\u003csub\u003e4\u003c/sub\u003e \u0026times;7 H\u003csub\u003e2\u003c/sub\u003eO; 1.2 mmol/L KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e; 2.5 mmol/L CaCl\u003csub\u003e2\u003c/sub\u003e; 11 mmol/L glucose; and 0.032 mmol/L CaNa\u003csub\u003e2\u003c/sub\u003eEDTA). The solution was oxygenated (95% O\u003csub\u003e2\u003c/sub\u003e and 5% CO\u003csub\u003e2\u003c/sub\u003e) and maintained at 37\u0026deg;C throughout the experiment.\u003c/p\u003e \u003cp\u003eVasoactive changes in the TAs and MAs were determined using isometric tension sensors (FSG-01, MDE, Budapest, Hungary) connected to the upper triangle (the lower triangle was firmly fixed). The NI USB-6221 AD converter (MDE, Budapest, Hungary) and S.P.E.L. Advanced Kymograph software (MDE, Budapest, Hungary) were used to record vascular changes.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eBefore the function of the arteries was tested, resting tension was applied to the rings, which were maintained for a stabilization period (45\u0026ndash;60 minutes). A series of preliminary experiments was conducted to set the optimal value of resting tension. Different values of resting tension, from the lowest to the highest, were applied to rings of both arteries isolated from SHRs, and the rings were exposed to increasing concentrations of noradrenaline, which induced a concentration-dependent contractile response. The value of resting tension (1 g) at which the largest contractile response was repeatedly recorded was applied to the arterial rings used in this study. The arteries were then precontracted by adding noradrenaline (NA, 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e mol, Zentiva, Czech Republic) before a single dose of acetylcholine (Ach, 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e mol/L) was added, thus controlling the integrity of the endothelium and the contractile properties of smooth muscle cells. After 3 washes with physiological Krebs solution and an equilibration period, the experiment was started by applying cumulative doses of NA (10\u003csup\u003e\u0026minus;\u0026thinsp;10\u003c/sup\u003e\u0026ndash;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e mol/L) to determine the adrenergic contractions in TAs and MAs. Vasocontractile responses are presented as the measured changes in isometric tension in g. Cumulative doses of Ach (10\u003csup\u003e\u0026minus;\u0026thinsp;10\u003c/sup\u003e\u0026ndash;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e mol/L) were applied to NA-precontracted arteries to evaluate endothelium-dependent relaxation. The relaxation responses were determined as a percentage of the maximal NA-stimulated contraction. Individual inhibitors were used to analyze the involvement of the endogenous NO and H\u003csub\u003e2\u003c/sub\u003eS pathways in both contractile and vasorelaxant responses. Nonspecific inhibitors of NO synthase (NOS), N\u003csup\u003eG\u003c/sup\u003e-nitro-L-arginine methyl ester (LN, 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e mol/L) or H\u003csub\u003e2\u003c/sub\u003eS-producing enzymes, such as cystathionine-γ-lyase (CSE) and DL-propargylglycine (PPG, 10 mmol/L), were added for 20 minutes to the organ bath, and the concentration‒response curves for NA and Ach were generated again. Since we prepared two rings of the thoracic aorta and two rings of the mesenteric artery from each animal, individual inhibitors (LN and PPG) were not applied to the same ring. Areas under the curve (AUCs, in arbitrary units) were calculated and compared before and after the incubation with the inhibitors to assess the involvement of NO and H\u003csub\u003e2\u003c/sub\u003eS in the vasoactive response in each group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Analysis of protein expression\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe protein expression levels of endothelial and inducible NOS isoforms, cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS), and tumor necrosis factor alpha (TNFα), which are inflammatory factors, in the abdominal aorta and transforming growth factor-beta (TGF-β) in the left ventricle were determined via Western blot analysis. Briefly, aortic tissue lysates were prepared by homogenization in protease inhibitor-supplemented 0.05 mM Tris buffer (protease inhibitor cocktail), followed by centrifugation and a measurement of the protein concentration using the Lowry assay. The supernatants were then subjected to SDS‒PAGE on 12% or 15% gels for protein separation and transferred to nitrocellulose membranes. Immunoblotting was performed using primary antibodies against endothelial eNOS (1:1000 dilution) (Abcam, Cambridge, UK), inducible iNOS (1:1000 dilution), CSE (1:5000 dilution), CBS (1:3000 dilution) and TNF-α (1:3000 dilution) (Proteintech, Manchester, UK), and β-actin (1:5000 dilution) (Abcam, Cambridge, UK) was used as a loading control. The membrane was incubated with secondary peroxidase-conjugated antibodies; eNOS (1:5000 dilution), iNOS (1:5000 dilution), CBS (1:5000 dilution), β-actin (1:5000 dilution) anti-rabbit (Abcam, Cambridge, UK), CSE (1:3000 dilution), TNF-α (1:3000 dilution) anti-mouse (Cell Signaling, Danvers, MA, USA), anti-TGF β (1:1000 dilution), anti-rabbit, polyclonal (Sigma- Aldrich, Germany) at room temperature for 2 h followed by enhanced chemiluminescence (ECL) reagent (BioRad, Inc., Hercules, CA, USA) which facilitated band visualization and subsequent quantification using standardized software (Image LabTM Touch software 2.4 BioRad, Inc., Hercules, CA, USA). The results were normalized to β-actin expression for accurate representation.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Total NO Synthase Activity\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eNOS activity was determined in the aortic arch and tissue remnants from the thoracic aorta. The isolated aortic tissue samples were homogenized, and nitric oxide synthase (NOS) activity was quantified by measuring the conversion of [3H]-L-arginine (obtained from MP Biochemicals) to [3H]-L-citrulline, according to a previously established method [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. [3H]-L-citrulline levels were measured using the Quanta Smart TriCarb Liquid Scintillation Analyzer (TriCarb, Packard, UK). NOS activity was then normalized to the protein content and reported as picokatal per gram of protein (pkat/g protein).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Collagen amount\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe amount of collagen in tissue was quantified by measuring hydroxyproline (hyp), an amino acid that is a major component of collagen. To determine the amount of hyp in the left ventricle, we used a modified method according to Reddy and Enwemeka [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Briefly, we used the same homogenate as used for measurement of NOS activity. 25 \u0026micro;l of sample was placed into a test tube and subjected to hydrolysis by 50 \u0026micro;l of 2 mol/l NaOH. The samples were then heated to 110\u0026deg;C 60 min, which breaks down the tissue and converts the collagen\u0026rsquo;s hydroxyproline into a free form that can be measured. After hydrolysis, the tissue samples were neutralized with 450 \u0026micro;l of chloramine-T for 25 min in room temperature. Following chloramine-T treatment, 500 \u0026micro;l of p-dimethylaminobenzaldehyde (DMAB) was added to the sample. Incubation lasted 20min in 65\u0026deg;C. The absorbance of the resulting solution was measured at 550 nm using a spectrophotometer. The collagen content in the tissue samples was calculated using a hydroxyproline standard calibration curve. The samples were repeated in triplicate to ensure consistency in results. It is assumed that 12.5% of collagen is composed of hydroxyproline. A standard curve was prepared by creating a series of hydroxyproline standards with known concentrations.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Statistical Analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe data are presented as the means \u0026plusmn; S.E.Ms. For the statistical comparison of sBP and vasoactive responses between groups, two-way analysis of variance (ANOVA) and the Bonferroni post hoc correction were used, or paired Student\u0026acute;s t tests (AUCs before and after inhibitor treatments). One-way ANOVA was used to evaluate general cardiovascular parameters, plasma parameters, NO synthase activity, collagen content and protein expression. Differences in means between groups were considered significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The data were analyzed with OriginPro 2019b (OriginLab Corporation, Northampton, MA, USA).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Drugs\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAll the chemicals used in this study were purchased from Sigma‒Aldrich (Germany), unless stated otherwise.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cem\u003e3.1 General parameters of the experimental animals\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp; A total of 23 rats (8 in the SHR group, 7 in the SHR+HFD group, and 8 in the SHR+HFD+GYY group, Table 1) were included in the study and divided into 3 groups according to whether they received the HFD and were treated with the H\u003csub\u003e2\u003c/sub\u003eS donor GYY-4137. At the beginning of the experiment, no difference in basal body weight (BW) was observed between the groups: SHR\u0026mdash;244.12\u0026plusmn;6.98 g, SHR+HFD\u0026mdash;238.50\u0026plusmn;4.90 g, and SHR+HFD+GYY\u0026mdash;230.13\u0026plusmn;3.68 g. From the beginning to the end of the experiment, we noticed a significant increase in BW in all groups (F\u003csub\u003e(2,65)\u003c/sub\u003e = 83.14; p = 9.25x10\u003csup\u003e-18\u003c/sup\u003e), and the greatest change was observed in the HFD group. The HFD induced significant increases in BW, retroperitoneal adipose tissue weight (RTW), and tibia length (TL). Additionally, chronic consumption of the HFD increased the ratios of RTW to BW (RTW/BW), and RTW to TL (RTW/TL) (Table 1). These results confirmed increased adiposity in the HFD group. However, treatment with GYY-4137 did not affect these parameters.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp; Neither the HFD nor treatment with GYY-4137 significantly altered the plasma glucose (GLU) levels. A significant increase in the plasma triacylglycerol (TAG) levels was observed in the HFD group, as expected. However, the plasma cholesterol (CHOL) level was lower in the HFD group than in the SHR group. The urea (URE) level and the ratio of URE to creatinine (URE/CRE) were significantly altered in the groups that received the HFD compared with those in the control diet (SHR) group; however, treatment with GYY-4137 partially attenuated this effect. HFD intake significantly increased chemerin levels in plasma, on the other hand, GYY-4137 administration returned the level of chemerin to control level. Food intake was significantly lower in the HFD group than in the control diet (SHR) group, and this effect was increased by treatment with GYY-4137 (Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1: \u003c/strong\u003eGeneral parameters of the experimental animals\u003c/p\u003e\n\u003ctable width=\"671\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"148\"\u003e\n\u003cp\u003e\u003cstrong\u003eParameters\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003e\u003cstrong\u003eSHR\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e\u003cstrong\u003en\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"138\"\u003e\n\u003cp\u003e\u003cstrong\u003eSHR+HFD\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e\u003cstrong\u003en\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e\u003cstrong\u003eSHR+HFD+GYY\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"36\"\u003e\n\u003cp\u003e\u003cstrong\u003en\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"148\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"138\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"36\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"148\"\u003e\n\u003cp\u003e\u003cstrong\u003eBW (g)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003e282.1\u0026plusmn;13.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"138\"\u003e\n\u003cp\u003e332.4\u0026plusmn;6.3***\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e327.3\u0026plusmn;7.3 **\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"36\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"148\"\u003e\n\u003cp\u003e\u003cstrong\u003eHW (g)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003e1.047\u0026plusmn;0.05\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"138\"\u003e\n\u003cp\u003e1.335\u0026plusmn;0.03 ***\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e1.372\u0026plusmn;0.05***\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"36\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"148\"\u003e\n\u003cp\u003e\u003cstrong\u003eRTW (g)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003e2.07\u0026plusmn;0.24\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"138\"\u003e\n\u003cp\u003e3.40\u0026plusmn;0.20**\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e3.44\u0026plusmn;0.24**\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"36\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"148\"\u003e\n\u003cp\u003e\u003cstrong\u003eTL (mm)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003e34.42\u0026plusmn;0.27\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"138\"\u003e\n\u003cp\u003e37.51\u0026plusmn;0.9**\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e37.05\u0026plusmn;0.55**\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"36\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"148\"\u003e\n\u003cp\u003e\u003cstrong\u003eRTW/BW (mg/g)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003e7.21\u0026plusmn;0.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"138\"\u003e\n\u003cp\u003e10.21\u0026plusmn;0.46**\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e10.48\u0026plusmn;0.55***\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"36\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"148\"\u003e\n\u003cp\u003e\u003cstrong\u003eRTW/TL (mg/mm)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003e60.38\u0026plusmn;7.14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"138\"\u003e\n\u003cp\u003e90.59\u0026plusmn;4.38**\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e93.24\u0026plusmn;6.71**\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"36\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"148\"\u003e\n\u003cp\u003e\u003cstrong\u003eGLU (mmol/L)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003e8.26\u0026plusmn;0.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"138\"\u003e\n\u003cp\u003e8.68\u0026plusmn;0.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e8.09\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"36\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"148\"\u003e\n\u003cp\u003e\u003cstrong\u003eTAG (mmol/L)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003e1.20\u0026plusmn;0.07\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"138\"\u003e\n\u003cp\u003e2.42\u0026plusmn;0.20***\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e2.07\u0026plusmn;0.24**\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"36\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"148\"\u003e\n\u003cp\u003e\u003cstrong\u003eCHOL (mmol/L)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003e2 .67\u0026plusmn;0.19\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"138\"\u003e\n\u003cp\u003e2.32\u0026plusmn;0.07*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e2.53\u0026plusmn;0.10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"36\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"148\"\u003e\n\u003cp\u003e\u003cstrong\u003eUREA (mmol/L)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003e7.86\u0026plusmn;0.11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"138\"\u003e\n\u003cp\u003e7.09\u0026plusmn;0.13**\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e7.26\u0026plusmn;0.26\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"36\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"148\"\u003e\n\u003cp\u003e\u003cstrong\u003eCRE (umol/L)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003e23.57\u0026plusmn;3.40\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"138\"\u003e\n\u003cp\u003e33.14\u0026plusmn;4.38\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e34\u0026plusmn;11.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"36\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"148\"\u003e\n\u003cp\u003e\u003cstrong\u003eURE/CRE\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003e361.72\u0026plusmn;32.76\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"138\"\u003e\n\u003cp\u003e237.27\u0026plusmn;31.90*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e305.05\u0026plusmn;031.06\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"36\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"148\"\u003e\n\u003cp\u003e\u003cstrong\u003eChemerin (ng/ml)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003e158.25\u0026plusmn;3.28\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"138\"\u003e\n\u003cp\u003e220.94\u0026plusmn;7.66*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e161.31\u0026plusmn;5.91\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"36\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"148\"\u003e\n\u003cp\u003e\u003cstrong\u003eFood intake (g/day)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003e20.87\u0026plusmn;0.45\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"138\"\u003e\n\u003cp\u003e15.56\u0026plusmn;0.15***\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e14.90\u0026plusmn;0.16***\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"36\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAbbreviations: n - number of rats; SHR - spontaneously hypertensive rats; SHR+HFD - spontaneously hypertensive rats receiving a high-fat diet; SHR+HFD+GYY - spontaneously hypertensive rats receiving an HFD and the H\u003csub\u003e2\u003c/sub\u003eS donor GYY-4137; BW - body weight; RTW - retroperitoneal adipose tissue weight; TL - tibia length; RTW/BW - ratio of retroperitoneal adipose tissue weight to body weight; RTW/TL - ratio of retroperitoneal adipose tissue weight to tibia length; GLU - glucose; TAG - triacylglycerol; CHOL - cholesterol; CRE - creatinine; URE/CRE - ratio of urea to creatinine. The values are presented as the means \u0026plusmn; S.E.Ms.; * p\u0026lt;0.05, ** p\u0026lt;0.01 and *** p\u0026lt;0.001 compared with the SHR group, + p\u0026lt;0.05 and ++ p\u0026lt;0.01 compared with the SHR+HFD group. Statistical analyses were performed by one-way ANOVA with the Bonferroni post hoc correction.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.2. Cardiac parameters of the experimental animals\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; Chronic consumption of the HFD increased heart weight (HW) as well as the ratios of HW to BW and HW to TL suggesting the occurrence of cardiac hypertrophy. Moreover, HFD increased the protein expression of central regulator in the development of fibrosis TGF-\u0026beta; as well as collagen content in the left ventricle. The treatment with GYY-4137 returned TGF-\u0026beta; to control levels, and collagen levels decreased, but not significantly (Table 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2: \u003c/strong\u003eCardiac parameters of the experimental animals\u003c/p\u003e\n\u003ctable width=\"671\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"148\"\u003e\n\u003cp\u003e\u003cstrong\u003eParameters\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003e\u003cstrong\u003eSHR\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e\u003cstrong\u003en\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"138\"\u003e\n\u003cp\u003e\u003cstrong\u003eSHR+HFD\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e\u003cstrong\u003en\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e\u003cstrong\u003eSHR+HFD+GYY\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"36\"\u003e\n\u003cp\u003e\u003cstrong\u003en\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"148\"\u003e\n\u003cp\u003e\u003cstrong\u003eHW (g)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003e1.047\u0026plusmn;0.05\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"138\"\u003e\n\u003cp\u003e1.335\u0026plusmn;0.03 ***\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e1.372\u0026plusmn;0.05***\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"36\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"148\"\u003e\n\u003cp\u003e\u003cstrong\u003eHW/BW (mg/g)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003e3.72\u0026plusmn;0.78\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"138\"\u003e\n\u003cp\u003e4.02\u0026plusmn;0.07*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e4.19\u0026plusmn;0.13*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"36\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"148\"\u003e\n\u003cp\u003e\u003cstrong\u003eHW/TL (mg/mm)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003e30.43\u0026plusmn;1.52\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"138\"\u003e\n\u003cp\u003e35.69\u0026plusmn;0.85*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e37.08\u0026plusmn;1.44**\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"36\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"148\"\u003e\n\u003cp\u003e\u003cstrong\u003eTGF-\u0026beta;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; (Density TGF-\u0026beta;/ \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026beta;-actin)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003e0.11\u0026plusmn;0,01\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"138\"\u003e\n\u003cp\u003e2.45\u003cstrong\u003e \u0026plusmn;\u003c/strong\u003e0.16***\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e1.41\u0026plusmn;0.17\u003csup\u003e+++\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"36\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"148\"\u003e\n\u003cp\u003e\u003cstrong\u003eCollagen (\u003c/strong\u003e\u003cstrong\u003em\u003c/strong\u003e\u003cstrong\u003eg/mg)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003e6.65\u0026plusmn;0.77\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"138\"\u003e\n\u003cp\u003e9.14\u0026plusmn;0.57*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"41\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e6.79\u0026plusmn;0.46*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"36\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAbbreviations: n - number of rats; SHR - spontaneously hypertensive rats; SHR+HFD - spontaneously hypertensive rats receiving a high-fat diet; SHR+HFD+GYY - spontaneously hypertensive rats receiving an HFD and the H\u003csub\u003e2\u003c/sub\u003eS donor GYY-4137; HW- heart weight; HW/BW - ratio of heart weight to body weight; HW/TL- ratio of heart weight to tibia length; TGF-\u0026beta; - protein expression of transforming growth factor-\u0026beta; in the left ventricle; Collagen \u0026ndash; the amount of collagen in the left ventricle. The values are presented as the means \u0026plusmn; S.E.Ms.; * p\u0026lt;0.05, ** p\u0026lt;0.01 and *** p\u0026lt;0.001 compared with the SHR group, +++ p\u0026lt;0.01 compared with the SHR+HFD group. Statistical analyses were performed by one-way ANOVA with the Bonferroni post hoc correction.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.3. Blood pressure of the experimental animals\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp; sBP measurements for all groups were recorded at week 9, week 14 (after 5 weeks of HFD consumption), and week 17 (after 8 weeks of HFD consumption and 3 weeks of GYY-4137 treatment). Two-way ANOVA confirmed that sBP differed not only depending on the administration of a high-fat diet or GYY (F\u003csub\u003e(2,61) \u003c/sub\u003e= 12.81; p ˂ 0.0001) but also depending on time (F\u003csub\u003e(2,61) \u003c/sub\u003e= 14.62; p ˂ 0.0001). Although the Bonferroni post hoc test confirmed a significant increase in sBP over time only in the control SHR group (p ˂ 0.001), at the end of the experiment, a significant reduction in sBP was observed in the group treated with GYY-4137 compared with both the SHR and HFD groups (p\u0026lt;0.001 and p\u0026lt;0.01) (Figure 1).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.4 Endothelial Function and Contractility of the Thoracic Aorta and Mesenteric Artery\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp; Contractile responses were induced by the activation of adrenergic receptors through the successive application of exogenous noradrenaline (NA) at concentrations ranging from 10\u003csup\u003e-10\u003c/sup\u003e to 3x10\u003csup\u003e-6\u003c/sup\u003e mol/L (TA) or 3x10\u003csup\u003e-5\u003c/sup\u003e mol/L (MA). HFD intake induced a significant decrease in NA-induced contraction of both the TA (F\u003csub\u003e(2,218) \u003c/sub\u003e= 12.71; p = 6.59x10\u003csup\u003e-6\u003c/sup\u003e) (Figure 2a) and MA (F\u003csub\u003e(2,238) \u003c/sub\u003e= 70.20; p\u0026lt;6.35x10\u003csup\u003e-24\u003c/sup\u003e) (Figure 2b), which indicates deterioration of the contractile properties. On the other hand, the administration of GYY-4137 partially improved the inhibited contractile response in the TA only (p\u0026lt;0.05, Figure 2a). Endothelial function was subsequently investigated by applying cumulative doses of acetylcholine (10\u003csup\u003e-10\u003c/sup\u003e to 3x10\u003csup\u003e-6\u003c/sup\u003e mol/L in the TA or 3x10\u003csup\u003e-5\u003c/sup\u003e mol/L in the MA) to the NA-precontracted arterial rings. Compared with the SHR group, both the HFD and the GYY-4137 treatment significantly increased endothelium-dependent vasorelaxation in the TA, although the maximum response remained unchanged (F\u003csub\u003e(2,208) \u003c/sub\u003e= 14.03; p = 2.17x10\u003csup\u003e-6\u003c/sup\u003e) (Figure 2c). The vasorelaxant response of the MA remained unaffected by the HFD, but following treatment with GYY-4137, a significant increase was observed (F\u003csub\u003e(2,238) \u003c/sub\u003e= 8.52; p = 2.79x10\u003csup\u003e-4\u003c/sup\u003e) (Figure 2d). Maximum reached responses and molar concentrations of noradrenaline and acetylcholine that induced the half-maximum response (EC\u003csub\u003e50\u003c/sub\u003e) are shown in Table 3 (thoracic aorta) and Table 4 (mesenteric artery).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3: \u003c/strong\u003eCharacterization of noradrenaline-induced contraction and acetylcholine-induced relaxation in the thoracic aorta\u003c/p\u003e\n\u003ctable width=\"680\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"120\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"6\" width=\"560\"\u003e\n\u003cp\u003e\u003cstrong\u003eThoracic aorta\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"154\"\u003e\n\u003cp\u003e\u003cstrong\u003eSHR\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"33\"\u003e\n\u003cp\u003e\u003cstrong\u003en\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003e\u003cstrong\u003eSHR+HFD\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"31\"\u003e\n\u003cp\u003e\u003cstrong\u003en\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"149\"\u003e\n\u003cp\u003e\u003cstrong\u003eSHR+HFD+GYY\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"38\"\u003e\n\u003cp\u003e\u003cstrong\u003en\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"120\"\u003e\n\u003cp\u003eNA\u003csub\u003emax\u003c/sub\u003e (g)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"154\"\u003e\n\u003cp\u003e0.58\u0026plusmn;0.03\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"33\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003e0.37\u0026plusmn;0.07***\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"31\"\u003e\n\u003cp\u003e\u0026nbsp;7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"149\"\u003e\n\u003cp\u003e0.44\u0026plusmn;0.03*+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"38\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"120\"\u003e\n\u003cp\u003eNA EC\u003csub\u003e50\u003c/sub\u003e \u003cbr /\u003e (-log mol/L)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"154\"\u003e\n\u003cp\u003e8.39\u0026plusmn;0.25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"33\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003e9.08\u0026plusmn;0.26\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"31\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"149\"\u003e\n\u003cp\u003e9.53\u0026plusmn;0.07***\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"38\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"120\"\u003e\n\u003cp\u003eAch\u003csub\u003emax\u003c/sub\u003e (%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"154\"\u003e\n\u003cp\u003e78.59\u0026plusmn;4.75\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"33\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003e76.95\u0026plusmn;5.54\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"31\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"149\"\u003e\n\u003cp\u003e78.74\u0026plusmn;5.79\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"38\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"120\"\u003e\n\u003cp\u003eAch EC\u003csub\u003e50\u003c/sub\u003e \u003cbr /\u003e (-log mol/L)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"154\"\u003e\n\u003cp\u003e8.70\u0026plusmn;0.19\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"33\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003e9.30\u0026plusmn;0.17*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"31\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"149\"\u003e\n\u003cp\u003e9.37\u0026plusmn;0.09**\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"38\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAbbreviations: n\u0026mdash;number of rats; SHR\u0026mdash;spontaneously hypertensive rats SHR+HFD\u0026mdash;spontaneously hypertensive rat receiving the high-fat diet; SHR+HFD+GYY\u0026mdash;spontaneously hypertensive rat receiving the HFD and H\u003csub\u003e2\u003c/sub\u003eS donor GYY-4137, NA\u0026mdash;noradrenaline, NA\u003csub\u003emax\u003c/sub\u003e\u0026mdash;maximum noradrenaline-induced contraction, NA EC\u003csub\u003e50\u003c/sub\u003e\u0026mdash;the negative logarithm of the NA molar concentration inducing the half-maximal response, Ach\u0026mdash;acetylcholine, Ach\u003csub\u003emax\u003c/sub\u003e\u0026mdash;maximum acetylcholine-induced relaxation, Ach EC\u003csub\u003e50\u003c/sub\u003e\u0026mdash;the negative logarithm of the Ach molar concentration inducing the half-maximal response. Values are presented as the means \u0026plusmn; S.E.Ms. * p\u0026lt;0.05, ** p\u0026lt;0.01 and *** p\u0026lt;0.001 compared with the SHR group, + p\u0026lt;0.05 compared with the SHR+HFD group. The statistical analysis was performed by one-way or two-way ANOVA with the Bonferroni post hoc correction.\u003c/p\u003e\n\u003ctable width=\"680\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"120\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"6\" width=\"560\"\u003e\n\u003cp\u003e\u003cstrong\u003eMesenteric artery\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"154\"\u003e\n\u003cp\u003e\u003cstrong\u003eSHR\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"33\"\u003e\n\u003cp\u003e\u003cstrong\u003en\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003e\u003cstrong\u003eSHR+HFD\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"31\"\u003e\n\u003cp\u003e\u003cstrong\u003en\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"149\"\u003e\n\u003cp\u003e\u003cstrong\u003eSHR+HFD+GYY\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"38\"\u003e\n\u003cp\u003e\u003cstrong\u003en\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"120\"\u003e\n\u003cp\u003eNA\u003csub\u003emax\u003c/sub\u003e (g)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"154\"\u003e\n\u003cp\u003e1.18\u0026plusmn;0.06\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"33\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003e0.78\u0026plusmn;0.06***\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"31\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"149\"\u003e\n\u003cp\u003e0.74\u0026plusmn;0.04***\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"38\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"120\"\u003e\n\u003cp\u003eNA EC\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003e(-log mol/L)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"154\"\u003e\n\u003cp\u003e7.43\u0026plusmn;0.07\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"33\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003e7.41\u0026plusmn;0.13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"31\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"149\"\u003e\n\u003cp\u003e7.42\u0026plusmn;0.10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"38\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"120\"\u003e\n\u003cp\u003eAch\u003csub\u003emax\u003c/sub\u003e (%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"154\"\u003e\n\u003cp\u003e43.97\u0026plusmn;3.97\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"33\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003e40.68\u0026plusmn;2.80\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"31\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"149\"\u003e\n\u003cp\u003e47.72\u0026plusmn;3.12*+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"38\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"120\"\u003e\n\u003cp\u003eAch EC\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003e(-log mol/L)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"154\"\u003e\n\u003cp\u003e7.56\u0026plusmn;0.18\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"33\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003e7.74\u0026plusmn;0.29\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"31\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"149\"\u003e\n\u003cp\u003e8.18\u0026plusmn;0.43\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"38\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4: \u003c/strong\u003eCharacterization of noradrenaline-induced contraction and acetylcholine-induced relaxation in the mesenteric artery\u003c/p\u003e\n\u003cp\u003eAbbreviations: n\u0026mdash;number of rats; SHR\u0026mdash;spontaneously hypertensive rats SHR+HFD\u0026mdash;spontaneously hypertensive rat receiving the high-fat diet; SHR+HFD+GYY\u0026mdash;spontaneously hypertensive rat receiving the HFD and H\u003csub\u003e2\u003c/sub\u003eS donor GYY-4137, NA\u0026mdash;noradrenaline, NA\u003csub\u003emax\u003c/sub\u003e\u0026mdash;maximum noradrenaline-induced contraction, NA EC\u003csub\u003e50\u003c/sub\u003e\u0026mdash;the negative logarithm of the NA molar concentration inducing the half-maximal response, Ach\u0026mdash;acetylcholine, Ach\u003csub\u003emax\u003c/sub\u003e\u0026mdash;maximum acetylcholine-induced relaxation, Ach EC\u003csub\u003e50\u003c/sub\u003e\u0026mdash;the negative logarithm of the Ach molar concentration inducing the half-maximal response. Values are presented as means \u0026plusmn; S.E.Ms. * p\u0026lt;0.05 and *** p\u0026lt;0.001 compared with the SHR group, + p\u0026lt;0.05 compared with the SHR+HFD group. The statistical analysis was performed by one-way or two-way ANOVA with the Bonferroni post hoc correction.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.5 Evaluation of NO/NOS System Participation\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp; The arterial rings were incubated with N\u003csup\u003eG\u003c/sup\u003e-nitro-L-arginine methyl ester (LN; 10\u003csup\u003e\u0026minus;4\u003c/sup\u003e mol/L) for 20 minutes to analyze the participation of NO signaling in vasoactive responses. The NA-induced contractile response of the TA of the SHR group was significantly increased after treatment with LN (F\u003csub\u003e(1,118)\u003c/sub\u003e = 147; p\u0026lt;0.0001; Figure 3a). However, treatment with LN had no effect on the contractile response of the SHR+HFD group (F\u003csub\u003e(1,78) \u003c/sub\u003e= 1.98; p = 0.16) (Figure 3b). The contractile response of the TA of the SHR+HFD+GYY group was again significantly increased after treatment with LN (F\u003csub\u003e(1,138)\u003c/sub\u003e = 5.08; p = 0.03; Figure 3c). The results of the AUC evaluation revealed that after the LN incubation, the contractile response of the TAs was significantly increased in the control group (p\u0026lt;0.01); however, both HFD consumption and treatment with GYY-4137 reduced the influence of NO (both p\u0026lt;0.001, Figure 3d). In terms of endothelial function, treatment with LN had a significant effect on acetylcholine-induced relaxation in the TA, and the vasorelaxation responses were significantly reduced in the SHR (F\u003csub\u003e(1,118)\u003c/sub\u003e = 69.7; p = 4.03x10\u003csup\u003e-13\u003c/sup\u003e), SHR+HFD (F\u003csub\u003e(1,58)\u003c/sub\u003e = 17.1; p = 1.74x10\u003csup\u003e-4\u003c/sup\u003e) and SHR+HFD+GYY (F\u003csub\u003e(1,118)\u003c/sub\u003e = 63.1; p = 2.96x10\u003csup\u003e-12\u003c/sup\u003e) groups (Figure 4 a, b, c). The AUC analysis revealed that the HFD decreased the participation of NO in the relaxation response of TAs, which was restored after treatment with GYY-4137 (p\u0026lt;0.05, Figure 4d). In the MA, in contrast to the control SHR group and HFD group, only the chronic administration of GYY-4137 significantly increased the participation of NO in the contractile response induced by NA (p\u0026lt;0.05, Figure 5 a, b, c, d). With respect to endothelial function, we observed a significant reduction in the relaxation response in the MA of the SHR control (p\u0026lt;0.05) and GYY-4137 groups (p\u0026lt;0.001) after LN treatment (Figure 6 a, d), which was confirmed by the AUC analysis (both p\u0026lt;0.01, Figure 6d).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.6 Evaluation of H\u003csub\u003e2\u003c/sub\u003eS/CSE System Participation\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp; Acute treatment with the specific CSE inhibitor DL-propargylglycine (PPG, 10 mmoL/L) was used to analyze the contribution of H\u003csub\u003e2\u003c/sub\u003eS to both relaxant and contractile responses. The direct application of PPG evoked distinct effects on the TA and MA. PPG administration to the TA evoked a slight increase in basal tone (0.127\u0026plusmn;0.04 g). This effect was reversed by HFD consumption (-0.145\u0026plusmn;0.041 g; p\u0026lt;0.001 vs. the SHR group), but GYY administration returned it to control levels (0.043\u0026plusmn;0.051 g; p\u0026lt;0.05 vs. the HFD group). PPG application in MA hardly affected the basal tone, indicating minimal basal production of H\u003csub\u003e2\u003c/sub\u003eS (0.033\u0026plusmn;0.026 g). The consumption of the HFD induced a slight decrease in basal tone (-0.113\u0026plusmn;0.026; p\u0026lt;0.001 vs. the SHR group), which was partially alleviated by the administration of GYY (-0.032\u0026plusmn;0.018 g; p\u0026lt;0.05 vs. the HFD group; p\u0026lt;0.01 vs. the SHR group).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp; Regarding the contractions induced by NA, the inhibitor was applied to the organ bath 20 minutes before the implementation of concentration-dependent responses. The TA exhibited significantly reduced contractile responses after treatment with PPG in the SHR (F\u003csub\u003e(1,138)\u003c/sub\u003e = 11.5; p = 9.28x10\u003csup\u003e-4\u003c/sup\u003e), SHR+HFD (F\u003csub\u003e(1,118)\u003c/sub\u003e = 46.1; p = 8.27x10\u003csup\u003e-10\u003c/sup\u003e) and SHR+HFD+GYY (F\u003csub\u003e(1,157)\u003c/sub\u003e = 120.3; p\u0026lt;0.0001) groups (Figure 7 a, b, c). The levels of H\u003csub\u003e2\u003c/sub\u003eS participation determined by calculating the AUC revealed that endogenously produced H\u003csub\u003e2\u003c/sub\u003eS stimulated a pro-contractile effect, and this effect on the TA was not changed by either HFD consumption or treatment with GYY-4137 (Figure 7d). In terms of endothelial function, after the TA was incubated with PPG, a significant increase in vasorelaxation occurred in the SHR (F\u003csub\u003e(1,78)\u003c/sub\u003e = 15.5; p = 2.14x10\u003csup\u003e-4\u003c/sup\u003e), SHR+HFD (F\u003csub\u003e(1,58)\u003c/sub\u003e = 18.4; p = 1.11x10\u003csup\u003e-4\u003c/sup\u003e) and SHR+HFD+GYY groups (F\u003csub\u003e(1,98)\u003c/sub\u003e = 27.3; p = 1.35x10\u003csup\u003e-6\u003c/sup\u003e) (Figure 8 a, b, c). However, AUC analyses revealed that in the TA, the significant antirelaxant effect of endogenous H\u003csub\u003e2\u003c/sub\u003eS was confirmed only in the SHR+HFD+GYY group (p\u0026lt;0.05, Figure 8d). In the MA, the procontractile effect of H\u003csub\u003e2\u003c/sub\u003eS was confirmed in the control SHR group (p\u0026lt;0.05), with PPG having no effect on either the SHR+HFD or SHR+HFD+GYY groups (Figure 9 a, b, c), which was also confirmed by the AUC evaluation (p\u0026lt;0.01, Figure 9d). With respect to endothelial function, PPG had no effect on the vasorelaxation response in the MA in any of the groups (Figure 10).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.7 Total NOS Activity and Protein Expression Levels\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp; Total NO synthase (NOS) activity in the aorta was significantly lower in the high-fat diet group than in the control SHR group (p\u0026lt;0.05) (Figure 11). This effect was confirmed by a decreased level of eNOS expression (p\u0026lt;0.001) (Figure 12a). GYY-4137 administration increased the level of total NOS activity almost to the control level (Figure 11); however, the eNOS expression level decreased to an even greater extent than those in both the control and HFD groups (p\u0026lt;0.001 and p\u0026lt;0.01, respectively) (Figure 12a). On the other hand, chronic consumption of the HFD increased iNOS expression in the HFD group only (p\u0026lt;0.001), and GYY-4137 administration decreased this value almost to the control level (Figure 12b).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp; Only simultaneous HFD and GYY-4137 administration increased the CSE protein level (p\u0026lt;0.01), whereas the CBS protein level decreased after high-fat diet consumption (p\u0026lt;0.05) (Figure 12 c, d). In addition, the HFD significantly increased the TNF\u0026alpha; expression level (p\u0026lt;0.001); however, the administration of GYY-4137 partially reduced the levels of inflammatory markers (p\u0026lt;0.05) (Figure 12e). Full scans of the entire original blots can be found in the Supplementary material.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe first goal of our study was to determine the dysfunction triggered in the cardiovascular system, especially in arteries, by the long-term intake of an HFD in rats with hypertension. Second, we aimed to investigate the possible beneficial effects of the slow-releasing H\u003csub\u003e2\u003c/sub\u003eS donor GYY-4137 on treating the pathology in this model.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.1 Effects of the High-Fat Diet\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp;We confirmed that adiposity increased along with TAG levels in HFD-treated SHRs; however, unexpectedly, cholesterol levels decreased (Table 1). On the other hand, we previously showed that the administration of 10% fructose did not change the level of cholesterol [4] and Gaspárová et al [12] confirmed increased serum levels of total cholesterol and TAG after administration of 60% fructose to SHR. Although HFD-induced dyslipidemia typically involves elevated cholesterol levels, the finding of decreased plasma cholesterol may be related to strain-specific differences in plasma lipid metabolism in SHR. Several authors have confirmed that the lower plasma cholesterol observed in hypertensive SHR was paralleled by specific differences in hepatic catalase and glutathione redox antioxidant enzyme activities and that increased cholesterol excretion associated with defects in molecular transport may also play a role [13, 14]. Similar reduction in plasma cholesterol levels accompanied by an increase in plasma TAG levels has also been confirmed in stroke-prone SHRs compared with control normotensive Wistar-Kyoto (WKYs), where a notable decrease in the cholesterol synthesis pathway was verified\u0026nbsp;[13].\u0026nbsp;Furthermore, the effect of HFD on ectopic lipid deposition should also be considered. Hojná et al. [15] similarly to us found an increase in TAG and decrease in total cholesterol in plasma of SHR fed a HFD which was associated with substantial ectopic accumulation of both cholesterol and TAG in the liver. We suggest that the presence of abnormal lipid metabolism in SHRs could persist or even be strengthened by increased lipid intake.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp;Although high-fat diets and high fructose intake are metabolically analogous, their effect in SHR may differ at the level of heart and BP regulation. Our previous results showed that fructose administration did not affect relative heart weight but led to an increase in BP due to, among others, increased fluid retention and stimulation of the aldosterone renin angiotensin system [16].\u0026nbsp;Shiou et al. [17] reported that after the consumption of an HFD, a significant decrease in BP occurred, along with cardiac dysfunction in both SHRs and WKYs, indicating lipotoxicity related to atrial and ventricular remodeling. In our study, excessive dietary fat intake did not affect BP but promoted the development of cardiac hypertrophy. Moreover, we also confirmed\u0026nbsp;increased protein expression of transforming growth factor-beta (TGF-β) and\u0026nbsp;the amount of total collagen in the left ventricle (Table 2). Upon activation of\u0026nbsp;TGF-β fibroblasts differentiate into myofibroblasts, which are responsible for producing excessive extracellular matrix components, including collagen types I and III. This process is critical in tissue remodeling during fibrosis\u0026nbsp;[18].\u0026nbsp;Concurrently, TGF-β upregulates the expression of tissue inhibitors of metalloproteinases resulting in a net increase in ECM deposition and the progression of fibrosis\u0026nbsp;[19].\u0026nbsp;Moreover, we found that, the urea levels and urea-to-creatinine ratio in the HFD group were lower than those in the SHR group. The urea-to-creatinine ratio plays a role in monitoring and identifying acute kidney injury, and its decline could indicate potential intrinsic renal damage [20]. Since we measured only the urea-to-creatinine ratio, further verification of HFD-associated kidney injury is needed. Nevertheless, the combination of an HFD and pre-existing hypertension appears to significantly affect the structure and function of the heart, emphasizing the need for a comprehensive understanding of all interactions to target therapeutic strategies.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; Obesity and hypertension are comorbid conditions that act as independent risk factors for the onset of endothelial dysfunction. We found that the HFD did not affect endothelium-dependent vasorelaxation in the MA (Figure 2c). This result contrasts with our previous results where improved endothelial function of MA associated with stimulation of the NO signaling pathway was confirmed in fructose-treated SHRs. \u0026nbsp; This result also contrasts with the findings of a Bosse et al.\u0026nbsp;[21], which reported an improvement in acetylcholine-stimulated vasorelaxation associated with an increase in eNOS phosphorylation in the MA of SHRs. In contrast to our study, where the proportion of carbohydrates was 36% and that of fats was 45%, in that study, the proportion of carbohydrates was 20%, and that of the fats was 60%. Hence, the different ratios of carbohydrate and fat intake could be a limiting factor for the ability to initiate compensatory mechanisms. Another explanation could be tissue specificity, since our results confirmed increased endothelium-dependent relaxation of the TA in the HFD group (Figure 2d). Nevertheless, the evaluation of the NO component by the AUC revealed that, in contrast to the other experimental groups, NO did not significantly participate in vasorelaxant responses in either artery in the HFD group (Figure 4d, 6d). A similar result was observed in our previous study, where SHRs receiving a fructose diet for 8 weeks presented a reduced NO contribution to the vasorelaxant response in the TA [4]. Moreover, we also found that high-fat diet consumption decreased NOS activity and the expression of the endothelial NOS protein in aortic tissue (Figures 11 and 12a), while the expression of inducible NOS (iNOS) was significantly increased.\u0026nbsp;iNOS is expressed in response to stress, and elevated levels of iNOS protein expression, together with increased expression of TNFα, trigger the inflammatory response\u0026nbsp;[22]. The reduced eNOS protein production could be explained by the increased caveolin-1 expression that occurs in vascular tissue under HFD conditions\u0026nbsp;[23]. \u0026nbsp;Taken together, the activation of the NO signaling pathway was not responsible for the improved vasorelaxation of TA. Nevertheless, while NO, a key vasodilator produced by endothelial cells, is essential for maintaining vascular health and regulating blood pressure, other vasorelaxant agents, such as prostacyclin and endothelium-derived hyperpolarizing factor (EDHF), which may compensate for reduced NO availability, are also involved in the TA\u0026nbsp;[24]. Thus, the activity of vasorelaxants other than NO, which may serve as a backup mechanism, could be responsible for the increase in vasorelaxation in the TA. In addition, NO may function as important negative feedback regulator of the catalytic activity of its effector, soluble guanylate cyclase (sGC); hence, any reduction in the NO level may lead to an increase in the sensitivity of sGC to NO. A study by Jebelovszki et al.\u0026nbsp;[25]\u0026nbsp;confirmed that the administration of a HFD led to increased NO sensitivity in rat coronary arterioles because of sGC activation, which is also consistent with our findings, as we recorded an unchanged maximal response but increased sensitivity to acetylcholine.\u0026nbsp;Next,\u0026nbsp;we assumed that H\u003csub\u003e2\u003c/sub\u003eS is the next relevant mediator, which would be consistent with it previously being identified as EDHF\u0026nbsp;[26]. However, our results showed that the application of PPG, an inhibitor of the H\u003csub\u003e2\u003c/sub\u003eS-producing enzyme CSE, shifted the relaxation response curves to the left in the TAs of both the SHR and HFD groups (Figure 8 a,b), indicating\u0026nbsp;the antirelaxation effect of endogenously released H\u003csub\u003e2\u003c/sub\u003eS, although,\u0026nbsp;the\u0026nbsp;evaluation of\u0026nbsp;the\u0026nbsp;AUC did not reveal any significant alterations in either the\u0026nbsp;TAs\u0026nbsp;or\u0026nbsp;the MAs\u0026nbsp;(Figure 8d, 10d). Moreover, we found that the\u0026nbsp;HFD had no significant effect on the CSE protein levels, and\u0026nbsp;that\u0026nbsp;CBS expression was slightly reduced. Similar results\u0026nbsp;were reported in SHRs fed\u0026nbsp;fructose,\u0026nbsp;where endogenously produced H\u003csub\u003e2\u003c/sub\u003eS did not participate in endothelium-dependent relaxation\u0026nbsp;[4] and in nonobese, hypertriglyceridemic rats,\u0026nbsp;where endogenous H\u003csub\u003e2\u003c/sub\u003eS participated in the inhibition of endothelium-dependent vasorelaxation of\u0026nbsp;the\u0026nbsp;TA\u0026nbsp;[27]. It seems that in\u0026nbsp;metabolic disorders, endogenously produced H\u003csub\u003e2\u003c/sub\u003eS\u0026nbsp;generally has\u0026nbsp;an antirelaxant effect rather than helping to maintain endothelial function.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp;With respect to contractility, we observed a significantly reduced contractile response to NA in both examined arteries following the consumption of an HFD. We observed similar impairment of contractile abilities in both TA and MA in SHR after fructose administration. We proposed that the reduced contraction was not associated with stimulated NO/NOS participation, since decreased and unchanged NO involvement were observed in the TA and MA, respectively (Figure 3b, d, 5 b,d). Previously, Li et al.\u0026nbsp;[28]\u0026nbsp;confirmed that under a HFD regimen, vascular matrix remodeling, including thickening of the vessel wall, fibrosis and apoptosis, was evident in the aortas of obese rats and was accompanied by increased arterial inflammation and oxidative stress. Moreover, Panchal et al.\u0026nbsp;[29]\u0026nbsp;observed reduced NA-induced contraction in the TA of HFD-fed rats, indicating smooth muscle dysfunction. Thus, we consider that the pathological conditions caused by an HFD might lead to a decreased contractile response through the induction of vascular remodeling of the arterial wall. TA, an elastic type of artery, is predisposed to further harmful remodeling induced by a HFD in SHRs. In our previous study, we confirmed a reduced vasocontractile response to NA in SHRs compared with that in Wistar rats, which was related to the fact that the highest component involved in TA hypertrophy in adult SHRs was the extracellular matrix and not smooth muscle cells\u0026nbsp;[30]. Regarding the MA, similar to our findings, a diminished adrenergic contractile response induced by phenylephrine was observed by Bosse et al.\u0026nbsp;[21]\u0026nbsp;in SHR fed a low-carbohydrate/high-fat diet. Although the authors consider this finding to be an improvement in vascular function, since the MA of SHRs is characterized by hypercontractility compared with that of controls, we assume that the observed compromised contractile function in both vessels is associated with a deteriorating (rather than beneficial) effect of the HFD on the contractile apparatus. Indeed, in our study, we detected increased expression of the iNOS and TNFα proteins\u0026nbsp;in arterial tissue (Figure 12 b,f), indicating the development of an inflammatory process, which could also lead to impaired contractility in both arteries. Moreover, we also confirmed reduced plasmatic level of the adipokine chemerin, which has been proposed as a possible link between metabolic, vascular disorders and inflammation. Chemerin affects vascular function, which is mediated by the production of reactive oxygen species and redox signaling\u0026nbsp;[31]. In addition, serum chemerin levels correlated with markers of inflammation, insulin resistance and an unfavorable lipid profile and have been proposed as a biomarker linking inflammation and cardiovascular diseases\u0026nbsp;[32, 33]. Similarly, Trovato et al.\u0026nbsp;[34]\u0026nbsp;showed that a high-fat Western diet could impair muscle metabolism, leading to muscle damage, which was associated with increased levels of inflammatory factors. Thus, we posit that contractile function was impaired by increased systemic inflammation under conditions of increased fat intake. With respect to the sulfide signaling pathway, we found that, unlike in the TA, the HFD eliminated the pro-contractile effect of endogenous H\u003csub\u003e2\u003c/sub\u003eS observed in the control MA (Figure 6a, c). Although the anticontractile effect of endogenous sulfide signaling is usually considered compensatory and beneficial, in our experiments the loss of the pro-contractile effect of endogenously produced H\u003csub\u003e2\u003c/sub\u003eS contributed to the inhibition of contractile responses, at least in MA. On the other hand, after fructose intake, where we observed similar impairment of contractile abilities in both TA and MA, this H\u003csub\u003e2\u003c/sub\u003eS action was not observed in any of the arteries and structural remodeling and inflammation were responsible for the impaired contractility.\u003c/p\u003e\n\u003cp\u003eTaken together, in\u0026nbsp;SHRs, high fat intake led to increased adiposity, elevated plasma triglyceride levels,\u0026nbsp;and\u0026nbsp;cardiac hypertrophy,\u0026nbsp;as well as to generally decreased adrenergic contraction and reduced NO participation in vasoactive responses associated with decreased NOS activity and reduced expression of the eNOS protein. The increased expression of iNOS and TNFα\u0026nbsp;indicates\u0026nbsp;that the initiation of the inflammatory process is\u0026nbsp;likely\u0026nbsp;responsible for the impairment of vascular function. Furthermore, comparison with previous experiments showed that while with fructose intake, a backup mechanism for maintaining endothelial function was confirmed in both TA (NO-dependent) and MA (NO-independent), with a high-fat diet, improved vasorelaxation capacity was confirmed only in TA (NO-independent).\u0026nbsp;Moreover, although both dietary regimens resulted in impaired contractility of both arteries likely due to inflammation and structural remodeling, H\u003csub\u003e2\u003c/sub\u003eS produced by MA in SHR fed a high-fat diet contributed to the blunted contractility.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.2 Effects of the GYY-4137 Treatment\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAs a next step, we investigated the effect of the slow-releasing H\u003csub\u003e2\u003c/sub\u003eS donor GYY-4137 on HFD-induced metabolic and vasoactive changes. We found that body adiposity was not altered after GYY-4137 treatment, similar to that in fructose-fed SHR [4]. On the other hand, while GYY-4137 significantly reduced TAG levels in fructose-fed SHR, it had no effect on cholesterol and TAG levels in rats fed a high-fat diet (Table 1). Literature data suggest that H\u003csub\u003e2\u003c/sub\u003eS administration with GYY-4137 could reverse the chain of events leading to lipid accumulation in vitro and in vivo. Casili et al. [35] confirmed the suppressed lipid accumulation in adipocyte-like cells treated with GYY-4137 (6 mmol/l) in vitro. Zhao et al. (2020) [36] showed in LDLr-/- mice treated with streptozotocin and a HFD that 4 weeks of GYY-4137 treatment (133 μmol/L) attenuated tissue lipid deposition. Geng et al. [37] demonstrated that HFD consumption for 13 weeks resulted in downregulation of the CSE-H\u003csub\u003e2\u003c/sub\u003eS system in adipose tissue of HFD-fed mice, while GYY-4137 treatment (200 μmol/kg/day) reduced lipolysis by inhibiting the phosphorylation of hormone-sensitive lipase. Based on these findings, it is surprising that the dose of GYY-4137 we used (266 μmol/l) had no significant effect on the lipid profile or other plasma markers in HFD-fed rats. On the other hand, Qabazard et al. [38] and Alshahwan et al. [39], who administered GYY-4137 at doses of 25 and 50 mg/kg for 28 days to streptozotocin-induced diabetic SD rats, showed that the efficacy of the donor is not proportional to its dose. Indeed, the regulation of adipose tissue lipolysis by H\u003csub\u003e2\u003c/sub\u003eS remains a matter of debate. The different metabolic and endocrine stages of pathological conditions may lead to contradictory regulation of lipolysis, differences in the involvement of the sulfide signaling pathway, and inconsistent effects of H\u003csub\u003e2\u003c/sub\u003eS donors. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp;GYY-4137 treatment for 3 weeks profoundly reduced sBP compared with that in both HFD-fed and control SHRs (Figure 1). This finding is consistent with the findings of multiple studies that have shown that administering H\u003csub\u003e2\u003c/sub\u003eS donors can reduce BP in SHR. Zhu et al. [40] reported that treatment with GYY-4137 decreased sBP in SHRs for two weeks, and Li et al. [7] reported that the reduction in sBP persisted for 14 days after the end of the treatment period. Another study in which SHRs were treated with GYY-4137 for 4 weeks reported reduced sBP, the inhibition of angiotensin II and a reduced occurrence of myocardial fibrosis [9]. In our study, we did not observe significant effect of GYY-4137 on myocardial remodeling associated with HFD administration, only the amount of collagen in the left ventricle decreased. However, changes in cardiac trophicity may not be fully dependent on BP, as has been confirmed during the ontogenesis of SHR [40]. Similarly, we recently showed that 3 weeks of treatment with GYY-4137 in fructose-fed SHRs resulted in a reduction in sBP but without an effect on cardiac parameters\u0026nbsp;[4]. Although multiple mechanisms may be responsible for the GYY-4137-induced decrease in BP, the interaction with the renin‒angiotensin‒aldosterone system (RAAS) may play an important role. Despite SHRs being a normal-to-low renin and normal-to-low angiotensin/aldosterone model of hypertension [41], increased RAAS activity along with elevated levels of angiotensin II have been observed in HFD-fed animals [42]. Laggner et al. [43] showed that NaHS-generated H\u003csub\u003e2\u003c/sub\u003eS inhibited the activity of angiotensin-converting enzyme (ACE) in endothelial cells. In our previous study we confirmed that an \u003cem\u003ein vivo\u003c/em\u003e bolus administration of the ACE inhibitor captopril reduced the H\u003csub\u003e2\u003c/sub\u003eS donor-induced decrease in BP, suggesting that captopril disabled and masked the inhibitory effect of H\u003csub\u003e2\u003c/sub\u003eS on the RAAS [44]. We posit that the inhibition of the RAAS could be responsible for the decrease in sBP induced by the administration of GYY-4137.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp;Treatment with GYY-4137 increased the endothelium-dependent relaxation of both arteries, although some differences were observed between the TA and MA. Similarly increased vasorelaxation of the TA was found in both the HFD and GYY groups; however, only the GYY-4137 treatment increased endothelium-dependent vasorelaxation in the MA compared with both the control SHR and the HFD-treated rats (Figure 2c, d). Furthermore, we observed that NO participation in vasorelaxant responses in both the TA and MA was restored after the administration of GYY-4137, although the anti-relaxation action of endogenous H\u003csub\u003e2\u003c/sub\u003eS was confirmed in TA. On the other hand, after fructose intake the endothelium-dependent relaxation of both arteries was similarly increased after treatment with GYY-4137, while in MA this effect was mediated by increased participation of NO, in TA the pro-relaxant action of endogenous H\u003csub\u003e2\u003c/sub\u003eS was responsible. These findings suggest that GYY-4137 administration leads to activation of one of the pathways (nitrate or sulfide), which could be tissue-specific and influenced by the origin of the metabolic disorder. We hypothesize that GYY, which acts as an H\u003csub\u003e2\u003c/sub\u003eS donor, could in high fat fed rats stimulate the production of endogenous H\u003csub\u003e2\u003c/sub\u003eS, which was confirmed by the increased expression of CSE in aortic tissue. An increased supply of H\u003csub\u003e2\u003c/sub\u003eS could subsequently sulfhydrate eNOS [45] and restart its activity. This result was also supported by the finding that, unlike in the HFD group, NOS activity did not remain reduced after GYY administration, and a trend toward increasing NOS activity was noted; thus, no difference was observed compared with the control group. Surprisingly, the decrease in endothelial NOS expression observed after HFD consumption was further exacerbated by GYY-4137 administration. We assume that a negative feedback mechanism may have been behind this effect. eNOS expression is known to be activated by the proinflammatory factor NF-kB as part of a negative feedback loop, such that increases in NO levels (and NOS activity) that repress NF-kB might lead to decreased levels of eNOS and vice versa\u0026nbsp;[46]. Taken together, these findings indicate that GYY-4137 administration improved endothelial function, which was likely related to the restoration of the NO signaling pathway, and endogenously produced H\u003csub\u003e2\u003c/sub\u003eS did not directly contribute to this effect, as it was either not involved (MA) or involved in the antirelaxation (TA) response. Nevertheless, it appears that administration of exogenous H\u003csub\u003e2\u003c/sub\u003eS donor helps maintain the balance between nitrous and sulfide signaling disrupted due to modifications in both lipid and saccharide metabolism.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp;HFD-induced impairments in contraction were partially ameliorated by GYY-4137 treatment only in the TA, highlighting the tissue-specific effects of this donor, which could also be related to the different biomechanical properties of the MA and TA or smooth muscle phenotypic differences. Soares et al.\u0026nbsp;[47]\u0026nbsp;confirmed greater smooth muscle cell damage in the small MA than in the TA in the obese mice fed a HFD, which was associated with higher expression of genes required for maintaining contractile capacity in the TA than in the MA and increased collagen deposition in the MA but not in the TA.\u0026nbsp;Moreover, phenotypic differences between aortic and mesenteric perivascular adipose tissue (PVAT) could also take place. Aortic PVAT generates much less pro-inflammatory cytokines and thus could be more resistant to diet-induced inflammation. Mesenteric PVAT is more sensitive to the high-fat diet challenge, where the adipose “browning” genes are dramatically down-regulated\u0026nbsp;[48]. With respect to the\u0026nbsp;endogenous nitrous and sulfide\u0026nbsp;signaling\u0026nbsp;pathways, both NO and H\u003csub\u003e2\u003c/sub\u003eS\u0026nbsp;likely\u0026nbsp;did not contribute to the\u0026nbsp;action of\u0026nbsp;GYY-4137 in\u0026nbsp;the\u0026nbsp;TA;\u0026nbsp;compared\u0026nbsp;with those in\u0026nbsp;the HFD group, the proportion of NO or H\u003csub\u003e2\u003c/sub\u003eS\u0026nbsp;involvement in\u0026nbsp;the contractile response\u0026nbsp;did\u0026nbsp;not\u0026nbsp;change\u0026nbsp;(Figure 3d, 7d). We posit that the improvement in the contractile response in the GYY group could be related to the anti-inflammatory effect of H\u003csub\u003e2\u003c/sub\u003eS donor. Indeed, H\u003csub\u003e2\u003c/sub\u003eS has notable anti-inflammatory effects,\u0026nbsp;such as reducing the\u0026nbsp;levels of proinflammatory\u0026nbsp;cytokines, chemokines and enzymes by inhibiting\u0026nbsp;the\u0026nbsp;activation of NF-kB\u0026nbsp;[49]. The anti-inflammatory role of H\u003csub\u003e2\u003c/sub\u003eS is further supported by our finding that the GYY-4137 treatment attenuated HFD-induced increases in iNOS and TNFα levels (Figure 12b, d), which is\u0026nbsp;consistent with\u0026nbsp;the finding that\u0026nbsp;GYY-4137\u0026nbsp;has\u0026nbsp;anti-inflammatory activity in disease states. Li et al.\u0026nbsp;[50]\u0026nbsp;showed that in LPS-stimulated human synoviocytes, GYY-4137 decreased TNFα and IL-6 production, reduced iNOS levels, and inhibited NF-kB activation. Similarly, GYY-4137 significantly reduced\u0026nbsp;the\u0026nbsp;serum levels of TNFα and interleukin-6 (IL-6), as well as iNOS expression, in mice with sepsis\u0026nbsp;[51].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp;In conclusion, based on the results of this and previous\u0026nbsp;study [4], different regimens, namely, fructose and high-fat diets, induce similar metabolic changes, such as increased adiposity and dyslipidemia. Although the treatment did not have the same effect on BP, vascular function was impaired in both models. Nevertheless, the fibrotic changes in the heart, tissue-specific limitation of compensatory vasoactive capacities, and impaired contractility associated with the action of endogenous H\u003csub\u003e2\u003c/sub\u003eS represent the original finding that high-fat consumption appears to impair the cardiovascular system of SHR more severely than fructose. A slow-releasing H\u003csub\u003e2\u003c/sub\u003eS donor triggered beneficial vasoactive effects, regardless of the origin of the metabolic disorder, although the mechanisms involved differ. Fructose-related disorders predominantly stimulate the beneficial pro-relaxant action of endogenous H\u003csub\u003e2\u003c/sub\u003eS. On the other hand, under high-fat diet conditions, endogenous H\u003csub\u003e2\u003c/sub\u003eS did not participate in the improvement of vascular function. GYY-4137 partially improved relaxation in the MA and restored the contractility of the TA, which was associated with restored NO signaling, and decreased the expression of the iNOS and TNFα proteins, suggesting that slow-releasing H\u003csub\u003e2\u003c/sub\u003eS\u0026nbsp;donors\u0026nbsp;could partially\u0026nbsp;ameliorate\u0026nbsp;metabolic changes and trigger beneficial vasoactive effects associated with the recovery of NO signaling and the\u0026nbsp;suppression of\u0026nbsp;inflammation.\u003c/p\u003e\n\u003cp\u003eStudy limitations\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp;After both the high-fat diet and GYY-4137 treatment, we observed tissue-specific vascular changes depending on the type of artery, elastic vs. muscular. For their deeper analysis, future research will need to focus on detailed specification of signaling pathways and regulatory mechanisms using in vivo (e.g. pulse wave velocity measurement) and in vitro (e.g. Western blot, Real-time PCR analysis) methods. Also, isolation and cultivation of endothelial cells and smooth muscle cells from specific types of arteries will allow the study of their individual properties and responses to different stimuli. Regarding the effects of GYY-4137, although GYY-4137 treatment generally reduced BP, the limiting factor could be the insufficient effect of GYY-4137 on adiposity and dyslipidemia\u0026nbsp;which may be related to the method (single dose) and duration of administration. Therefore, it should be assessed whether GYY-4137 can improve long-term outcomes in disorders associated with HFD in the future. The metabolic changes induced by high fat intake are complex and multifactorial, therefore, long-term studies should be considered when evaluating its potential therapeutic effects. Finally, understanding the physiological roles of H\u003csub\u003e2\u003c/sub\u003eS compared to NO, as well as the stability of H\u003csub\u003e2\u003c/sub\u003eS donors, poses a challenge for effective treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, S.C. and B.G.A.; methodology, S.C., B.G.A., A.B., A.Ba. and M.C.; validation, S.C., B.G.A., and M.C.; formal analysis, A.B., B.G.A., and M.C.; investigation, A.B., B.G.A., J.D.H., and M.C.; writing\u0026mdash;original draft preparation, B.G.A. and S.C.; writing\u0026mdash;review and editing, S.C. and M.C.; visualization, S.C., B.G.A., and M.C.; funding acquisition, S.C. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp;The raw data supporting the conclusions of this study are available from the corresponding author upon request, without undue reservation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp;The animals were bred in accordance with the institutional guidelines of the State Veterinary and Food Administration of the Slovak Republic and the Committee on the Ethics of Procedures in Animal, Clinical and other Biomedical Experiments (Permit Number: 2652/2021-220, 19/03/2021) of the Centre of Experimental Medicine. The Committee on the Ethics of Procedures verified the justification of the experiments from a scientific and benefit perspective and assessed the compliance of the experiments with the requirements for animal protection and concluded that the experiments meet and respect the requirements for the protection of animals used for experimental and other scientific purposes in accordance with the Decree of the Ministry of Education and Research of the Slovak Republic No. 436/2012 Coll. and the Regulation of the Government of the Slovak Republic No. 377/2012 Coll. The animals were bred also in accordance with the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes, Directive 2010/63/EU of the European Parliament. Animals were imported from the accredited breeding facility of the Center of Experimental Medicine, Slovak Academy of Sciences, Dobr\u0026aacute; Voda, Slovak Republic, and were housed by the Institute of Normal and Pathological Physiology, Center of Experimental Medicine, Slovak Academy of Sciences (INPP CEM SAS).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp;The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFull scans of the entire original blots for eNOS, iNOS, CSE, CBS and TNF\u0026alpha; can be found in the Supplementary material.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePowell-Wiley, T. M. et al. Obesity and cardiovascular disease: a scientific statement from the American Heart Association. \u003cem\u003eCirculation\u003c/em\u003e \u003cb\u003e143\u003c/b\u003e (21), e984\u0026ndash;e1010. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1161/CIR.0000000000000973\u003c/span\u003e\u003cspan address=\"https://doi:10.1161/CIR.0000000000000973\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuan, Y. et al. Inflammatory links between high fat diets and diseases. \u003cem\u003eFront. immunol.\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, 2649. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.3389/fimmu.2018.02649\u003c/span\u003e\u003cspan address=\"https://doi:10.3389/fimmu.2018.02649\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGui, D. D. et al. Effects of gut microbiota on atherosclerosis through hydrogen sulfide. \u003cem\u003eEur. J. Pharmacol.\u003c/em\u003e \u003cb\u003e896\u003c/b\u003e, 173916. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1016/j.ejphar.2021.173916\u003c/span\u003e\u003cspan address=\"https://doi:10.1016/j.ejphar.2021.173916\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBerenyiova, A. et al. Vasoactive Effects of Chronic Treatment with Fructose and Slow-Releasing H2S Donor GYY-4137 in Spontaneously Hypertensive Rats: The Role of Nitroso and Sulfide Signalization. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e \u003cb\u003e23\u003c/b\u003e (16), 9215. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.3390/ijms23169215\u003c/span\u003e\u003cspan address=\"https://doi:10.3390/ijms23169215\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBello, I. et al. Erucin, an H\u003csub\u003e2\u003c/sub\u003eS-Releasing Isothiocyanate, Exerts Anticancer Effects in Human Triple-Negative Breast Cancer Cells Triggering Autophagy-Dependent Apoptotic Cell Death. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e \u003cb\u003e24\u003c/b\u003e (7), 6764. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://doi:10.3390/ijms24076764\u003c/span\u003e\u003cspan address=\"http://doi:10.3390/ijms24076764\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCorvino, A. et al. Pills of Multi-Target H\u003csub\u003e2\u003c/sub\u003eS Donating Molecules for Complex Diseases. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e \u003cb\u003e25\u003c/b\u003e (13), 7014. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://doi:10.3390/ijms25137014\u003c/span\u003e\u003cspan address=\"http://doi:10.3390/ijms25137014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, L. et al. Characterization of a novel, water-soluble hydrogen sulfide\u0026ndash;releasing molecule (GYY4137) new insights into the biology of hydrogen sulfide. \u003cem\u003eCirculation\u003c/em\u003e \u003cb\u003e117\u003c/b\u003e (18), 2351\u0026ndash;2360. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1161/CIRCULATIONAHA.107.753467\u003c/span\u003e\u003cspan address=\"https://doi:10.1161/CIRCULATIONAHA.107.753467\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, Z. et al. The hydrogen sulfide donor, GYY 4137, exhibits anti-atherosclerotic activity in high fat fed apolipoprotein E\u0026ndash;/\u0026ndash; mice. \u003cem\u003eBr. J. Pharmacol.\u003c/em\u003e \u003cb\u003e169\u003c/b\u003e (8), 1795\u0026ndash;1809. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1111/bph.12246\u003c/span\u003e\u003cspan address=\"https://doi:10.1111/bph.12246\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeng, G. et al. Hydrogen sulfide donor GYY4137 protects against myocardial fibrosis. Oxid Med Cell Longev 2015:691070. (2015). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1155/2015/691070\u003c/span\u003e\u003cspan address=\"https://doi:10.1155/2015/691070\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePechanova, O., Bernatova, I., Pelouch, V. \u0026amp; Simko, F. Protein remodelling of the heart in NO-deficient hypertension: the effect of captopril. \u003cem\u003eJ. Mol. Cell. Cardiol.\u003c/em\u003e \u003cb\u003e29\u003c/b\u003e (12), 3365\u0026ndash;3374. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1006/jmcc.1997.0566\u003c/span\u003e\u003cspan address=\"https://doi:10.1006/jmcc.1997.0566\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1997).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReddy, G. K. \u0026amp; Enwemeka, C. S. A simplified method for the analysis of hydroxyproline in biological tissues. Clin Biochem 29(3):225-9. (1996). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://doi:10.1016/0009-9120(96)00003-6\u003c/span\u003e\u003cspan address=\"http://doi:10.1016/0009-9120(96)00003-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. PMID: 8740508.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGasparova, Z. et al. High-fructose intake-induced dyslipidemia and oxidative stress accompanied by hippocampal dysfunctions in hypertensive but not hypertriacylglycerolemic rats. \u003cem\u003eGen. Physiol. Biophys.\u003c/em\u003e \u003cb\u003e42\u003c/b\u003e, 25\u0026ndash;36. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://doi:10.4149/gpb_2022053\u003c/span\u003e\u003cspan address=\"http://doi:10.4149/gpb_2022053\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKitts, D. D., Yuan, Y. V. \u0026amp; Godin, D. V. Plasma and lipoprotein lipid composition and hepatic antioxidant status in spontaneously hypertensive (SHR) and normotensive (WKY) rats. \u003cem\u003eCan. J. Physiol. Pharmacol.\u003c/em\u003e \u003cb\u003e76\u003c/b\u003e (2), 202\u0026ndash;209 (1998).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYuan, Y. \u0026amp; Kitts, D. D. Dietary fat source and cholesterol interactions alter plasma lipids and tissue susceptibility to oxidation in spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats. \u003cem\u003eMol. Cell. Biochem.\u003c/em\u003e \u003cb\u003e232\u003c/b\u003e (1\u0026ndash;2), 33\u0026ndash;47. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1023/a:1014837131439\u003c/span\u003e\u003cspan address=\"https://doi:10.1023/a:1014837131439\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2002).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHojna, S. et al. Hepatoprotective and cardioprotective effects of empagliflozin in spontaneously hypertensive rats fed a high-fat diet. \u003cem\u003eBiomed. Pharm.\u003c/em\u003e \u003cb\u003e174\u003c/b\u003e, 116520. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biopha.2024.116520\u003c/span\u003e\u003cspan address=\"10.1016/j.biopha.2024.116520\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKurbel, S. Arterial Hypertension Due to Fructose Ingestion: Model Based on Intermittent Osmotic Fluid Trapping in the Small Bowel. \u003cem\u003eTheor. Biol. Med. Model.\u003c/em\u003e \u003cb\u003e7\u003c/b\u003e, 27. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://doi:10.1186/1742-4682-7-27\u003c/span\u003e\u003cspan address=\"http://doi:10.1186/1742-4682-7-27\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShiou, Y. L., Huang, I. C., Lin, H. T. \u0026amp; Lee, H. C. High fat diet aggravates atrial and ventricular remodeling of hypertensive heart disease in aging rats. \u003cem\u003eJ. Formos. Med. Assoc.\u003c/em\u003e \u003cb\u003e117\u003c/b\u003e (7), 621\u0026ndash;631. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1016/j.jfma.2017.08.008\u003c/span\u003e\u003cspan address=\"https://doi:10.1016/j.jfma.2017.08.008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGabbiani, G. The myofibroblast in wound healing and fibrocontractive diseases. \u003cem\u003eJ. Pathol.\u003c/em\u003e \u003cb\u003e200\u003c/b\u003e (4), 500\u0026ndash;503. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://doi:10.1002/path.1427\u003c/span\u003e\u003cspan address=\"http://doi:10.1002/path.1427\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2003).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFrangogiannis, N. G. Transforming growth factor-β in myocardial disease. \u003cem\u003eNat. Rev. Cardiol.\u003c/em\u003e \u003cb\u003e19\u003c/b\u003e (7), 435\u0026ndash;455. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://doi:10.1038/s41569-021-00646-w\u003c/span\u003e\u003cspan address=\"http://doi:10.1038/s41569-021-00646-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePřeček, J., Hutyra, M., Kov\u0026aacute;čik, F., Ors\u0026aacute;g, J. \u0026amp; T\u0026aacute;borsk\u0026yacute;, M. Biomarkers of renal function in prognostic stratification of patients with acute coronary syndrome. \u003cem\u003eCor et Vasa\u003c/em\u003e. \u003cb\u003e60\u003c/b\u003e (2), e148\u0026ndash;154. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1016/j.crvasa.2017.05.018\u003c/span\u003e\u003cspan address=\"https://doi:10.1016/j.crvasa.2017.05.018\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBosse, J. D. et al. A low-carbohydrate/high-fat diet reduces blood pressure in spontaneously hypertensive rats without deleterious changes in insulin resistance. \u003cem\u003eAm. J. Physiol. Heart Circ. Physiol.\u003c/em\u003e \u003cb\u003e304\u003c/b\u003e (12), H1733\u0026ndash;H1742. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1152/ajpheart.00631.2012\u003c/span\u003e\u003cspan address=\"https://doi:10.1152/ajpheart.00631.2012\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaddad, Y. \u0026amp; Couture, R. Interplay between the kinin B1 receptor and inducible nitric oxide synthase in insulin resistance. \u003cem\u003eBr. J. Pharmacol.\u003c/em\u003e \u003cb\u003e173\u003c/b\u003e (12), 1988\u0026ndash;2000. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/bph.13491\u003c/span\u003e\u003cspan address=\"10.1111/bph.13491\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2016). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://\u003c/span\u003e\u003cspan address=\"https://\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang, N. et al. High-fat diet up-regulates caveolin-1 expression in aorta of diet-induced obese but not in diet-resistant rats. \u003cem\u003eCardiovasc. Res.\u003c/em\u003e \u003cb\u003e76\u003c/b\u003e (1), 167\u0026ndash;174. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1016/j.cardiores.2007.05.028\u003c/span\u003e\u003cspan address=\"https://doi:10.1016/j.cardiores.2007.05.028\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eF\u0026eacute;l\u0026eacute;tou, M. Endothelium-dependent hyperpolarization and endothelial dysfunction. \u003cem\u003eJ. Cardiovasc. Pharmacol.\u003c/em\u003e \u003cb\u003e67\u003c/b\u003e (5), 373\u0026ndash;387. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1097/FJC.0000000000000346\u003c/span\u003e\u003cspan address=\"https://doi:10.1097/FJC.0000000000000346\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJebelovszki, E. et al. High-fat diet-induced obesity leads to increased NO sensitivity of rat coronary arterioles: role of soluble guanylate cyclase activation. \u003cem\u003eAm. J. Physiol. Heart Circ. Physiol.\u003c/em\u003e \u003cb\u003e294\u003c/b\u003e (6), H2558\u0026ndash;H2564. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1152/ajpheart.01198.2007\u003c/span\u003e\u003cspan address=\"https://doi:10.1152/ajpheart.01198.2007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang, G. et al. H\u003csub\u003e2\u003c/sub\u003eS is an endothelium-derived hyperpolarizing factor. \u003cem\u003eAntiox Red Sig\u003c/em\u003e. \u003cb\u003e19\u003c/b\u003e (14), 1634\u0026ndash;1646. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1089/ars.2012.4805\u003c/span\u003e\u003cspan address=\"https://doi:10.1089/ars.2012.4805\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCacanyiova, S. et al. The vasoactive role of perivascular adipose tissue and the sulfide signaling pathway in a nonobese model of metabolic syndrome. \u003cem\u003eBiomolecules\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e (1), 108. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.3390/biom11010108\u003c/span\u003e\u003cspan address=\"https://doi:10.3390/biom11010108\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, W. et al. Inhibition of ROS and inflammation by an imidazopyridine derivative X22 attenuate high fat diet-induced arterial injuries. \u003cem\u003eVascul Pharmacol.\u003c/em\u003e \u003cb\u003e72\u003c/b\u003e, 153\u0026ndash;162. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1016/j.vph.2015.05.006\u003c/span\u003e\u003cspan address=\"https://doi:10.1016/j.vph.2015.05.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePanchal, S. K. et al. High-carbohydrate, high-fat diet\u0026ndash;induced metabolic syndrome and cardiovascular remodeling in rats. \u003cem\u003eJ. Cardiovasc. Pharmacol.\u003c/em\u003e \u003cb\u003e57\u003c/b\u003e (5), 611\u0026ndash;624. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1097/FJC.0b013e31821b1379\u003c/span\u003e\u003cspan address=\"https://doi:10.1097/FJC.0b013e31821b1379\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBerenyiova, A., Drobna, M., Cebova, M., Kristek, F. \u0026amp; Cacanyiova, S. Changes in the vasoactive effects of nitric oxide, hydrogen sulfide and the structure of the rat thoracic aorta: the role of age and essential hypertension. \u003cem\u003eJ. Physiol. Pharmacol.\u003c/em\u003e \u003cb\u003e69\u003c/b\u003e (4). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.26402/jpp.2018.4.05\u003c/span\u003e\u003cspan address=\"https://doi:10.26402/jpp.2018.4.05\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNeves, K. B. et al. Chemerin reduces vascular nitric oxide/cGMP signalling in rat aorta: a link to vascular dysfunction in obesity? \u003cem\u003eClin. Sci.\u003c/em\u003e \u003cb\u003e127\u003c/b\u003e (2), 111\u0026ndash;122. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://doi:10.1042/CS20130286\u003c/span\u003e\u003cspan address=\"http://doi:10.1042/CS20130286\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSzpakowicz, A. et al. Serum Chemerin Concentration Is Associated with Proinflammatory Status in Chronic Coronary Syndrome Biomolecules \u003cb\u003e11\u003c/b\u003e: 1149. (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/biom11081149\u003c/span\u003e\u003cspan address=\"10.3390/biom11081149\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEichelmann, F. et al. Chemerin as a Biomarker Linking Inflammation and Cardiovascular Diseases. \u003cem\u003eJ. Am. Coll. Cardiol.\u003c/em\u003e \u003cb\u003e73\u003c/b\u003e (3), 378\u0026ndash;379. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jacc.2018.10.058\u003c/span\u003e\u003cspan address=\"10.1016/j.jacc.2018.10.058\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTrovato, F. M., Castrogiovanni, P., Szychlinska, M. A., Purrello, F. \u0026amp; Musumeci, G. Impact of Western and Mediterranean diets and vitamin D on muscle fibers of sedentary rats. \u003cem\u003eNutrients\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e (2), 231. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.3390/nu10020231\u003c/span\u003e\u003cspan address=\"https://doi:10.3390/nu10020231\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCasili, G. et al. Inhibition of the 3\u0026ndash;mercaptopyruvate sulfurtransferase\u0026mdash;hydrogen sulfide system promotes cellular lipid accumulation. \u003cem\u003eGeroScience\u003c/em\u003e \u003cb\u003e44\u003c/b\u003e, 2271\u0026ndash;2289. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11357-022-00600-9\u003c/span\u003e\u003cspan address=\"10.1007/s11357-022-00600-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao, S. et al. Hydrogen sulfide alleviates liver injury via S-sulfhydrated-Keap1/Nrf2/LRP1 pathway. \u003cem\u003eHepatol\u003c/em\u003e \u003cb\u003e73\u003c/b\u003e (1), 282\u0026ndash;302. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://doi:10.1002/hep.31247\u003c/span\u003e\u003cspan address=\"http://doi:10.1002/hep.31247\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGeng, B. et al. Increase or decrease hydrogen sulfide exert opposite lipolysis, but reduce global insulin resistance in high fatty diet induced obese mice. \u003cem\u003ePLoS One\u003c/em\u003e. \u003cb\u003e8\u003c/b\u003e (9), e73892. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1371/journal.pone.0073892\u003c/span\u003e\u003cspan address=\"https://doi:10.1371/journal.pone.0073892\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQabazard, B., Yousif, M., Mousa, A. \u0026amp; Phillips, O. A. GYY4137 attenuates functional impairment of corpus cavernosum and reduces fibrosis in rats with STZ-induced diabetes by inhibiting the TGF-β1/Smad/CTGF pathway. \u003cem\u003eBiomed. Pharm.\u003c/em\u003e \u003cb\u003e138\u003c/b\u003e, 111486. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biopha.2021.111486\u003c/span\u003e\u003cspan address=\"10.1016/j.biopha.2021.111486\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlshahwan, H. et al. Hydrogen sulfide donor GYY4137 attenuates vascular complications in mesenteric bed of streptozotocin-induced diabetic rats. \u003cem\u003eEur. J. Pharmacol.\u003c/em\u003e \u003cb\u003e933\u003c/b\u003e, 175265. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://doi:10.1016/j.ejphar.2022.175265\u003c/span\u003e\u003cspan address=\"http://doi:10.1016/j.ejphar.2022.175265\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022). Epub 2022 Sep 12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCebova, M. \u0026amp; Kristek, F. Age-dependent ultrastructural changes of coronary artery in spontaneously hypertensive rats. \u003cem\u003eGen. Physiol. Biophys.\u003c/em\u003e \u003cb\u003e30\u003c/b\u003e (4), 364\u0026ndash;372. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4149/gpb_2011_04_364\u003c/span\u003e\u003cspan address=\"10.4149/gpb_2011_04_364\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2011). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://\u003c/span\u003e\u003cspan address=\"http://\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSimko, F. et al. Sacubitril/Valsartan and Ivabradine Attenuate Left Ventricular Remodelling and Dysfunction in Spontaneously Hypertensive Rats: Different Interactions with the Renin\u0026ndash;Angiotensin\u0026ndash;Aldosterone System. \u003cem\u003eBiomedicines\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e (8), 1844. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.3390/biomedicines10081844\u003c/span\u003e\u003cspan address=\"https://doi:10.3390/biomedicines10081844\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNag, S., Patel, S., Mani, S. \u0026amp; Hussain, T. Role of angiotensin type 2 receptor in improving lipid metabolism and preventing adiposity. \u003cem\u003eMol. Cell. Biochem.\u003c/em\u003e \u003cb\u003e461\u003c/b\u003e (1\u0026ndash;2), 195\u0026ndash;204. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s11010-019-03602-y\u003c/span\u003e\u003cspan address=\"10.1007/s11010-019-03602-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://\u003c/span\u003e\u003cspan address=\"https://\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLaggner, H. et al. The novel gaseous vasorelaxant hydrogen sulfide inhibits angiotensin-converting enzyme activity of endothelial cells. \u003cem\u003eJ. Hypertens.\u003c/em\u003e \u003cb\u003e25\u003c/b\u003e (10), 2100\u0026ndash;2104. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1097/HJH.0b013e32829b8fd0\u003c/span\u003e\u003cspan address=\"https://doi:10.1097/HJH.0b013e32829b8fd0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDrobna, M. et al. Captopril Partially Decreases the Effect of H\u003csub\u003e2\u003c/sub\u003eS on Rat Blood Pressure and Inhibits H\u003csub\u003e2\u003c/sub\u003eS-Induced Nitric Oxide Release From S-Nitrosoglutathione. \u003cem\u003ePhysiol. Res.\u003c/em\u003e \u003cb\u003e64\u003c/b\u003e (4), 479\u0026ndash;486. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.33549/physiolres.932772\u003c/span\u003e\u003cspan address=\"https://doi:10.33549/physiolres.932772\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAltaany, Z., Ju, Y., Yang, G. \u0026amp; Wang, R. The coordination of S-sulfhydration, S-nitrosylation, and phosphorylation of endothelial nitric oxide synthase by hydrogen sulfide. \u003cem\u003eSci. Signal.\u003c/em\u003e \u003cb\u003e7\u003c/b\u003e (342), ra87. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1126/scisignal.2005478\u003c/span\u003e\u003cspan address=\"https://doi:10.1126/scisignal.2005478\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrumbach, I. M., Chen, W., Mertens, S. A. \u0026amp; Harrison, D. G. A negative feedback mechanism involving nitric oxide and nuclear factor kappa-B modulates endothelial nitric oxide synthase transcription. \u003cem\u003eJ. Mol. Cell. Cardiol.\u003c/em\u003e \u003cb\u003e39\u003c/b\u003e (4), 595\u0026ndash;603. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1016/j.yjmcc.2005.06.012\u003c/span\u003e\u003cspan address=\"https://doi:10.1016/j.yjmcc.2005.06.012\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSoares, A. G., Catelli de Carvalho, M. H. \u0026amp; Akamine, E. Obesity Induces Artery-Specific Alterations: Evaluation of Vascular Function and Inflammatory and Smooth Muscle Phenotypic Markers. \u003cem\u003eBioMed. Res. Int. 17: Article ID\u003c/em\u003e. \u003cb\u003e5038602\u003c/b\u003e, 10pages. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1155/2017/5038602\u003c/span\u003e\u003cspan address=\"10.1155/2017/5038602\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, X., Ma, Z. \u0026amp; Zhu, Y. Z. Regional Heterogeneity of Perivascular Adipose Tissue: Morphology, Origin, and Secretome. Front Pharmacol 12: Article 697720. (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://doi:10.3389/fphar.2021.697720\u003c/span\u003e\u003cspan address=\"http://doi:10.3389/fphar.2021.697720\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhatia, M. \u0026amp; Gaddam, R. R. Hydrogen sulfide in inflammation: a novel mediator and therapeutic target. \u003cem\u003eARS\u003c/em\u003e \u003cb\u003e34\u003c/b\u003e (17), 1368\u0026ndash;1377. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1089/ars.2020.8211\u003c/span\u003e\u003cspan address=\"https://doi:10.1089/ars.2020.8211\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, L. et al. The complex effects of the slow‐releasing hydrogen sulfide donor GYY 4137 in a model of acute joint inflammation and in human cartilage cells. \u003cem\u003eJ. Cell. Mol. Med.\u003c/em\u003e \u003cb\u003e17\u003c/b\u003e (3), 365\u0026ndash;376. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1111/jcmm.12016\u003c/span\u003e\u003cspan address=\"https://doi:10.1111/jcmm.12016\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCui, W., Chen, J., Yu, F., Liu, W. \u0026amp; He, M. GYY4137 protected the integrity of the blood-brain barrier via activation of the Nrf2/ARE pathway in mice with sepsis. \u003cem\u003eFASEB J.\u003c/em\u003e \u003cb\u003e35\u003c/b\u003e (9), e21902. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1096/fj.202100074R\u003c/span\u003e\u003cspan address=\"https://doi:10.1096/fj.202100074R\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"hydrogen sulfide, GYY-4137, high-fat diet, spontaneously hypertensive rat, vascular","lastPublishedDoi":"10.21203/rs.3.rs-6030564/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6030564/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMetabolic syndrome is a growing global health burden, resulting in an urgent need for new therapeutic strategies. We evaluated the effects of 3 weeks of treatment with slow-releasing H\u003csub\u003e2\u003c/sub\u003eS donor GYY-4137 on adiposity, systolic blood pressure (sBP), plasma biochemical indices, vascular function and nitric oxide (NO) and hydrogen sulfide (H\u003csub\u003e2\u003c/sub\u003eS) pathways in the isolated thoracic aortas (TAs) and mesenteric arteries (MAs) from spontaneously hypertensive rats (SHRs) fed a high-fat diet (HFD) for 8 weeks. Although the HFD increased TAs relaxation, it induced cardiac remodeling, decreased adrenergic contraction, reduced NO participation in vasoactive responses, decreased NO-synthase (NOS) activity, altered the expression of the endothelial NOS (reduced), inducible NOS, and tumor necrosis factor alpha (TNFα) (both increased) proteins and increased adiposity and plasma chemerin levels. Treatment with GYY-4137 reduced sBP, improved relaxation of the MA, partially restored the contractility of the TA, generally restored NO signaling, and decreased the expression of the inducible NOS and TNFα proteins and plasma chemerin. Thus, a slow H\u003csub\u003e2\u003c/sub\u003eS-releasing donor could partially ameliorate metabolic changes induced by increased fat intake during essential hypertension and trigger beneficial vasoactive effects associated with the restoration of NO signaling and suppression of inflammation.\u003c/p\u003e","manuscriptTitle":"The Effect of High Fat Diet and H2S Donor GYY-4137 on Vascular Function in Spontaneously Hypertensive Rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-17 20:50:55","doi":"10.21203/rs.3.rs-6030564/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f514658f-995f-4371-b59f-5fc65b1e3d54","owner":[],"postedDate":"April 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":47220904,"name":"Biological sciences/Physiology"},{"id":47220905,"name":"Health sciences/Diseases"},{"id":47220906,"name":"Health sciences/Pathogenesis"}],"tags":[],"updatedAt":"2025-04-21T08:08:35+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-17 20:50:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6030564","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6030564","identity":"rs-6030564","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}