H3 relaxin alleviated vascular injury  in rats with type 1 diabetes by inhibiting endoplasmic reticulum stress and inflammation

H3 relaxin alleviated vascular injury in rats with type 1 diabetes by inhibiting endoplasmic reticulum stress and inflammation | 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 Research Article H3 relaxin alleviated vascular injury in rats with type 1 diabetes by inhibiting endoplasmic reticulum stress and inflammation Xiaohui Zhang, Kelaier Yang, Jinyu Chi, Wenjia Chen, Xiao Ma, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-435725/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 Human recombinant relaxin-3 (H3 relaxin ),a small molecule peptide hormone, ameliorated myocardial injury after myocardial infarction or isoprenaline injection by inhibiting apoptosis and fibrosis. However, whether H3 relaxin protects vascular function in rats with type 1 diabetes and its mechanism are unknown. In type 1 diabetes rats model induced by streptozotocin (STZ), rats were subcutaneously injected H3 relaxin (2 µg/kg/d or 0.2 µg/kg/d) for 2 weeks. At 4 or 8 weeks after STZ injection, we detected the expression of fibrosis (type I and III collagen), ERS (endoplasmic reticulum stress) and NLRP3 inflammasome activation in the aortas and inflammation markers in the plasma from rats with diabetes. Compared with the diabetic rats, H3 relaxin treatment exhibited markedly decreased plasma oxidative stress markers (TNF-a and MDA) levels. The protein expression levels of type I and III collagen in the aortas were increased in rats with diabetes, inhibited by H3 relaxin. H3 relaxin treatment inhibited ERS (GRP78 and CHOP) and NLRP3 inflammasome activation in the aortas of diabetic rats. These results suggest that H3 relaxin inhibited fibrosis, ERS and inflammation activation in the aortas of type 1 diabetic rats. Molecular Biology H3 relaxin type 1 diabetes NLRP3 inflammasome ERS fibrosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Arterial stiffness is an early stage of arterial dysfunction in patients with diabetes, which pathophysiologic mechanism is that high glucose induces impaired endothelial function. Endothelial dysfunction aggravates media thickness and fibrosis, in turn worsen arterial stiffness[ 1 ]. Arterial Fibrosis is an active process that collagen, fibronectin and adhesion proteins accumulate, adhesion molecules and integrins increase, and the extracellular matrix (ECM) remodel, which are involved in the arterial complications of diabetes[ 2 ]. Another pathogenic mechanism of arterial dysfunction in diabetes is that high glucose mediated vascular inflammation[ 3 , 4 ]. ROS mediated oxidative stress is considered to be responsible for the progression and development of diabetic arterial dysfunction[ 5 , 6 ]. Our previous studies suggest that the ROS mediated NLRP3 inflammasome activation plays a key role in the progression of diabetic cardiomyopathy[ 7 , 8 ]. The NLRP3 inflammasome, a complex, comprised of the apoptosis-associated speck-like protein (ASC), NLRP3 and pro-caspase-1[ 9 ]. Once NLRP3 inflammasome was activated, then pro-caspase-1 transformed into cleaved caspase-1, which can activate IL-1β and IL-18 involved in arterial injury in diabetes. In addition, ROS induced by high glucose promotes ERS, aggravated inflammation and insulin resistance in diabetic vascular dysfunction. ERS may be involved in the mechanism of diabetic nephropathy, for example, ERS marker CCAAT/enhancer-binding protein homologous protein (CHOP) knockout ameliorated diabetic nephropathy in streptozotocin-induced diabetes mice[ 10 ]. In the retina of rats with streptozotocin-induced diabetes, ERS resulted in increased inflammation and vascular permeability[ 11 ]. Previous investigations suggested that acute ERS inhibition in aortic rings protected mice from diabetes-induced endothelial dysfunction[ 12 ]. Relaxin-3, an ancestral peptide of the human relaxin subclass of the insulin superfamily, was predominantly expressed in the central nervous system and regulated feeding, arousal, memory, learning and central responses to physiological stressors[ 13 – 16 ]. Recent data indicated that H3 relaxin alleviated ischaemic injury by reducing hypoxia-induced production of ROS[ 17 ]. H3 relaxin inhibited myocardial injury induced by isoproterenol or in cardiac-restricted transgenic overexpression of β2-AR mice[ 18 , 19 ]. Our recent study reported that H3 relaxin prevented cardiac injury and fibrosis by regulating the activation of NLRP3 inflammasome in diabetic cardiomyopathy[ 7 , 8 ]. However, whether H3 relaxin protected the vascular complications of diabetes from inhibiting the activation of ERS and the NLRP3 inflammasome was unknown. In this study, we sought to define whether H3 relaxin attenuates the vascular complications of experimental diabetes. Moreover, we explored the mechanism that H3 relaxin inhibited vascular injury. 2. Materials And Methods 2.1. Animals and reagents Male SD rats (Second Affiliated Hospital of Harbin Medical University, Harbin, China; weight, 200 ~ 250g) experiments were conducted at First Affiliated Hospital of Harbin Medical University, and the present study was approved by the Laboratory Animal Management and Ethics Review Committee of First Affiliated Hospital of Harbin Medical University. STZ was purchased from Sigma (St. Louis, MO, USA), and H3 relaxin was obtained from Phoenix Pharmaceuticals (Belmont, CA, USA). 2.2 Experimental diabetic rats model and groups Diabetes was prepared in SD rats by intraperitoneal injection of 65 mg/kg STZ once as described previously[ 7 ]. Rats were randomly divided into 4 groups: control group, DM group, A group and B group. A group: at 2 or 6 weeks after STZ administration, the rats were injected with subcutaneous 2 µg/kg/d H3 relaxin for 2 weeks. B group: at 2 or 6 weeks after STZ administration, the rats were injected with subcutaneous 0.2 µg/kg/d H3 relaxin for 2 weeks. 2.3 Hematoxylin and eosin (HE) staining and Masson staining At 8 weeks after STZ injection, the aortas were dissected from the rats. HE-stained and Masson trichrome-stained sections were photographed using a light microscope (Olympus). The areas of fibrosis in 4 randomly selected visual fields were measured with a threshold function under 40× magnification. 2.4 Western blotting The aorta proteins extract (40 µg) from four groups were separated and transferred to PVDF membrane. Membranes were incubated in antibodies, including anti-β-actin (ZSGB-BIO, BeiJing, China), anti-GRP78(Abcam, Cambridge, UK), anti-CHOP (Abcam, Cambridge, UK), anti-NLRP3(Bioss, Beijing, China), anti-IL-1β (Novus Biologicals, CO, USA), anti-IL-18(Novus Biologicals, CO, USA), anti-I-collagen (Bioss, Beijing, China) and anti-III-collagen(Bioss, Beijing, China). Then PVDF membranes were incubated in horseradish peroxidase–linked secondary antibody for 1 hour. Bands were quantified with NIH Image Software. 2.5 Measurements of plasma MDA and TNF-a The blood of SD rats was centrifugated into obtaining plasma at 1000g for 10 min, then MDA was detected using ELISAs (Nanjing Jiancheng) and TNF-a was measured as described previously[ 7 ]. 2.6 Relaxin and receptor expressions quantified by real-time PCR Aorta tissues in control and DM group rats were collected at 4 and 8 weeks after STZ administration. Total RNA was isolated, reverse transcribed and quantified according to the manufacturer’s instructions (TaKaRa, Dalian, China) as described previously[ 20 ]. 2.7 Statistical analyses Experiments were carried out in 8 rats per group in triplicate, and data were expressed as the means ± SE by GraphPad Prism 7.0 software. Data were analysed by one-way ANOVA, followed by a Newman-Keuls multiple comparison test. P < 0.05 was considered significant. 3. Results 3.1 H3 relaxin ameliorated aorta injury in diabetic rats Histological analysis of the vascular structure was performed by HE staining of the aorta specimens at 8 weeks after STZ injection. We observed that disordered elastic fibres in the aortas of rats with diabetes compared with those of controls by HE staining; however, H3 relaxin treatment restored the vascular structure (Fig. 1 a). Masson staining showed that vascular smooth muscle cells were stained red and interstitial collagen was blue. The collagen network of adjacent cells is intact in control rats. Extracellular matrix deposition in vascular wall increased significantly in diabetic group. More, the collagen network around the cell breaks and arrange disorders in diabetic rats, and the content of collagen in H3 relaxin treatment group are less than diabetes group and elastic fibres arrange more regular than diabetes group (Fig. 1 b,c,d). Compared with the controls, the contents of MDA at 8 weeks in the DM group were markedly increased, whereas were significantly decreased after H3 relaxin administration. Compared with the controls, the levels of TNF-a at 4 weeks in the DM group were markedly increased, wherea were significantly decreased after H3 relaxin adiminstration (Fig. 2 ). 3.2 H3 relaxin improved vascular fibrosis in the aortas of rats with diabetes Extracellular matrix contains structural proteins, such as Type I and III collagen, adhesion molecules, integrins. The expression of types I and III collagen were increased in the aortas of STZ-treated diabetic rats, whereas were inhibited by H3 relaxin treatment in diabetic rats (Fig. 3 ). 3.3 H3 relaxin inhibited ERS in the aortas of rats with diabetes To explore whether ERS participated in H3 relaxin protection against vascular injury in rats with diabetes, the levels of ERS markers, including GRP78 and CHOP, were evaluated by western blotting. We found that compared with control, the expression of GRP78 and CHOP were significantly higher in the DM group, however, were lower after H3 relaxin treatment in diabetic rats (Fig. 4 ). 3.4 H3 relaxin inhibited NLRP3 inflammasome activation Previous investigations have reported that the activation of NLRP3 inflammasome was involved in the pathophysiologic mechanism in complications of diabetes. In our work, the expression levels of NLRP3, IL-1β and IL-18 proteins were increased in DM group, decreased by H3 relaxin administration (Fig. 5 ). 3.5 Relaxin-1/3 and their receptor expressions in aortas In the aortas of diabetic rats, the mRNA expression of Relaxin-1 significantly was upregulated at 4w after STZ injection (Fig. 6 a); however, the mRNA expression of Relaxin-3 was upregulated at 4w and 8w after STZ injection (Fig. 6 b). In addition, the mRNA expression of relaxin family peptide receptor 1 ( RXFP1) was significantly upregulated at 8w after STZ injection (Fig. 6 c); RXFP3 mRNA expression was also upregulated at 4w and 8w after STZ injection (Fig. 6 d). 4. Discussion The aims of this study were to identify whether H3 relaxin protected against vascular injury induced by diabetes in vivo and potential mechanism. Our results demonstrated that vascular fibrosis, NLRP3 inflammasome activation and ERS were involved in the vascular complications of diabetes and that H3 relaxin improved vascular injury by inhibiting fibrosis, ERS and NLRP3 inflammasome activation. In higher primates, relaxin family peptides contains seven members, which are relaxin-1, 2, 3 and insulin-like peptides (INSL) 3, 4, 5, 6. However, in rats, there are six members in relaxin family peptides, including relaxin-1 (similar to human relaxin-2), relaxin-3 and INSL-3, 4, 5, 6[ 13 ]. The relaxin family peptides receptors contains RXFP1, 2, 3, 4, natural ligand of human relaxin-2 (rats relaxin-1) is RXFP1 which plays protective effects in the cardiovascular disease. Recent studies reported that relaxin-3 can inhibit myocardial injury by binding to RXFP1 in rats with myocardial infarction[ 19 ], although RXFP3, a natural receptor of relaxin-3, is mainly located in the brain. Later, study found that in rat atrial and ventricular cells, there existed relaxin-3 mRNA which expression was up-regulated in the myocardium after isoproterenol administration[ 18 ]. Our recent study reported that H3 relaxin improved cardiac injury by regulating the activation of NLRP3 inflammasome in diabetic cardiomyopathy[ 7 , 8 ]. This study focus on whether H3 relaxin inhibited diabetic vascular injury, and we choose the dose of 0.2 or 2ug/kg/day relaxin-3 treatment for diabetic rats as zhang et al. described previously[ 18 ]. Interestingly, we found both doses of H3 relaxin were effective to a similar degree, and we will choose the very low dose H3 relaxin in future experiment. This study limitations contained that we did not test plasma relaxin-1 and relaxin-3 expression, so we can not clarify the issue that why both high and low dose H3 relaxin had similar protective effects in vascular injury in type 1 diabetes rats. We will aviod this question in future. We found that the body weight increased and the glucose level decreased after H3 relaxin injection in diabetic rats as reported in our previous study[ 7 ], and which maybe a mechanism of that H3 relaxin inhibited vascular fibrosis, need to be verificated. Unfortunately, we did not detected the level of insulin, we will avoid this issue in future. In addition, we found that Relaxin-1 , 3 and RXFP1, 3 mRNA were significantly up-regulated after STZ in the aortas of diabetic rats according to real-time PCR[ 20 ]. The reason in increased expression of RXFP1 and RXFP3 in aortas of diabetic rats may be due to increased expression of endogous relaxin-1/3, however, these results clarified that endogenous relaxin-1/3 and receptors participated in the mechanism of vascular injury in diabetic rats. The mechanism of vascular complications of diabetes contains endothelial dysfunction, oxidative stress and arterial remodelling. Oxidative stress is a determining mechanism in vascular complication of diabetes. Increased plasma TNF-a and IL-6 are associated with vascular dysfunction in patients with type 2 diabetes[ 21 ]. NF- κ B activity increased, TNF-a and intercellular adhesion molecule (ICAM) upregulated in vascular tissues of type 2 diabetic rats[ 22 ]. Aljwaid H et al. found that compared with control, MDA levels were higher in patients with diabetes, which was related with higher levels of oxidized ascorbate. These studies clarfied that oxidative stress was involved in the mechanism of vascular dysfunction in diabetes[ 23 ]. Recent data indicate that H3 relaxin protects against ischaemic injury by reducing the hypoxia-induced production of ROS. In our study, we found that plasma MDA and TNF-a levels were increased in rats with diabetes, and H3 relaxin inhibited vascular dysfunction by regulating MDA and TNF-a levels in rats with diabetes. The expression of ERS markers were increased in kidney from patients with diabetes, were involved in the pathogenesis of diabetic nephropathy, however, CHOP knockout improved kidney injury in diabetic mice[ 10 ]. In addition, ER stress activated inflammatory factors and mediated increased vascular permeability in retina of diabetes[ 11 ]. In a recent study, we found that H3 relaxin inhibited the ERS mediated apoptosis of myocardial cells induced by high glucose[ 24 ]. In our study, we found that compared with the controls, the expression of CHOP and GRP78 increased in the aortas of diabetic rats, which were inhibited by H3 relaxin administration, indicated that H3 relaxin inhibited ERS-induced vascular injury. High glucose-induced NLRP3 inflammasome activation prompted vascular complications in diabetes. In vivo, NLRP3 inflammasome was activated in the aortas of rats after a high glucose diet, and inhibited by rutin administration[ 25 ]. Aberrantly, in atherosclerotic pig aortas, the expression of NLPR3, ASC and IL-1β were increased, along with NF-κB activation and endothelial dysfunction. In patients with coronary heart disease, increased NLRP3 expression in aortas was positively associated the severity of coronary artery disease[ 27 ]. In addition, our findings are consistent; compared with the controls, the expression and activation of the NLRP3 inflammasome were increased in the aortas of rats with diabetes, and were inhibited by H3 relaxin. In conclusion, H3 relaxin is a potential candidate for treating diabetes-associated vascular complications by inhibiting fibrosis, the activation of ERS and NLRP3 inflammasome. Declarations Author contributions: Xiaohui Zhang and Kelaier Yang: conception, design and analysis of data, performed the data analyses, and wrote the manuscript. Xiao Ma and Xinhua Yin: contributed to the conception of the study. Jinyu Chi and Wenjia Chen: contributed signifcantly to analysis and manuscript preparation. Compliance with ethical standards Ethical approval ： This study was conducted after obtaining First Affiliated Hospital of Harbin Medical Univwersity’s ethical committee approval. Acknowledgement This work was supported by the Natural Science Foundation of China (No. 81500288), Pastdoctoral Scientific Research Developmental Fund of Heilongjiang Province (LBH-Q18080,LBH-Q19133), The Innovation of Foundation of the First Affiliated Hospital of Harbin Medical University(2019M08), Natural Science Foundation of Heilongjiang Province (LH2020H034), and The Outstanding Youth Foundation of the First Affiliated Hospital of Harbin Medical University(HYD2020JQ0005). Conflict of interest statement On behalf of all authors, the corresponding author states that there are no conflicts of interest. Data statement Study data are available. References Savoia C, Burger D, Nishigaki N et al (2011)Angiotensin II and the vascular phenotype in hypertension , Expert Rev Mol Med 13 : e11 Wynn TA, Ramalingam TR ( 2013 ) Mechanisms of fibrosis: therapeutic translation for fibrotic disease, Nat Med 2012;18:1028–1040. Chen K, Zhang J, Zhang W, et al. 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( 2020 ) Selective deficiency in endothelial PTP1B protects from diabetes and endoplasmic reticulum stress-associated endothelial dysfunction via preventing endothelial cell apoptosis.Biomed Pharmacother 127:110200 Bathgate RA, Samuel CS , Burazin TC et al (2002)Human relaxin gene 3 (H3) and the equivalent mouse relaxin (M3) gene. Novel members of therelaxin peptide family . J Biol Chem 2 : 1148-57 Ma S , Olucha-Bordonau FE , Hossain MA, et al. (2009 ) Modulation of hippocampal theta oscillations and spatial memory by relaxin-3 neurons of the nucleus incertus . Learn Mem 16 : 730 – 42 McGowan BM, Stanley SA, Smith KL, et al. ( 2005 ) Central relaxin-3 administration causes hyperphagia in male Wistar rats. Endocrinology 146:3295 –3295 300 McGowan BM, Stanley SA, Smith KL, et al. ( 2006 ) Effects of acute and chronic relaxin-3 on food intake and energy expenditure in rats. Regul Pept 136:72 –72 7 Willcox JM, Summerlee AJ, ( 2014 ) Relaxin protects astrocytes from hypoxia in vitro. PLoS One 9:e90864 Zhang J , Qi YF , Geng B, et al. (2005 ) Effect of relaxin on myocardial ischemia injury induced by isoproterenol . Peptides 26 : 1632-9 Hossain MA , Man BC , Zhao C, et al. ( 2011 ) H3 relaxin demonstrates antifibrotic properties via the RXFP1 receptor . Biochemistry 50 : 1368-75 Zhang X, Pan L, Yang K, et al. ( 2017 ) Alterations of relaxin and its receptor system components in experimental diabetic cardiomyopathy rats. Cell Tissue Res 370:297–304 Natali A, Toschi E, Baldeweg S, et al. ( 2006 ) Clustering of insulin resistance with vascular dysfunction and low-grade inflammation in type 2 diabetes. Diabetes 55: 1133–1140 Bitar MS, Ayed AK, Abdel-Halim SM et al. ( 2010 ) Inflammation and apoptosis in aortic tissues of aged type II diabetes: amelioration with α-lipoic acid through phosphatidylinositol 3-kinase/Akt-dependent mechanism. Life Sci 86:844–853 Aljwaid H, White DL, Collard KJ, et al. ( 2015 ) Non-transferrin-bound iron is associated with biomarkers of oxidative stress, inflammation and endothelial dysfunction in type 2 diabetes. J Diabetes Complications 29:943–949 Zhang X, Ma X, Zhao M, et al. ( 2015 ) H2 and H3 relaxin inhibit high glucose-induced apoptosis in neonatal rat ventricular myocytes. Biochimie 108:59–67 Wang W, Wu QH, Sui Y, et al. ( 2017 ) Rutin protects endothelial dysfunction by disturbing Nox4 and ROS-sensitive NLRP3 inflammasome. Biomed Pharmacother 86:32–40 Li Y, Xu S, Jiang B, et al. ( 2013 ) Activation of sterol regulatory element binding protein and NLRP3 inflammasome in atherosclerotic lesion development in diabetic pigs. PLoS One 8:e67532 Zheng F, Xing S, Gong Z, et al. ( 2013)NLRP3 inflammasomes show high expression in aorta of patients with atherosclerosis. 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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-435725","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":22551342,"identity":"20333fea-e61e-4566-90f6-3a5d01d378ad","order_by":0,"name":"Xiaohui Zhang","email":"","orcid":"","institution":"First Affiliated Hospital of Harbin Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaohui","middleName":"","lastName":"Zhang","suffix":""},{"id":22551343,"identity":"832f179a-0daf-4f48-9838-eab8735e6d1e","order_by":1,"name":"Kelaier Yang","email":"","orcid":"","institution":"First Affiliated Hospital of Harbin Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kelaier","middleName":"","lastName":"Yang","suffix":""},{"id":22551344,"identity":"5ed7489c-c47d-4bc4-a0b4-d313919fde3e","order_by":2,"name":"Jinyu Chi","email":"","orcid":"","institution":"First Affiliated Hospital of Harbin Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jinyu","middleName":"","lastName":"Chi","suffix":""},{"id":22551345,"identity":"9ad42754-681f-429f-afaf-22aedceb570a","order_by":3,"name":"Wenjia Chen","email":"","orcid":"","institution":"First Affiliated Hospital of Harbin Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wenjia","middleName":"","lastName":"Chen","suffix":""},{"id":22551346,"identity":"4e510175-856c-4990-b1e1-c743a19e3c08","order_by":4,"name":"Xiao Ma","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYJCCAx8qauSANBvROhgPzjhzzBimRYIYLcyHeduYExuI1qI7I4HhMM8ZtvQNx5ufPWDcYVNHUIvZmQMMB+dUyORuOHPM3IDxTBphW8yONzAceHOGLXfDjQQzCca2w0RoOQwMMaBf0g3uP/8G1PKfOFsOArUkGNzgAdlygAgtIL8AA9lw5pmcMonEtmTJBoJabiQwfwBGpTzf8ePbJD622fETtIWBgf8DmFI4ACQSiFCPAPIEHTQKRsEoGAUjFgAAR6pCslYKOZ8AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-3460-4892","institution":"Second Affiliated Hospital of Harbin Medical University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"","lastName":"Ma","suffix":""},{"id":22551347,"identity":"e40d5f82-ac63-4a08-8393-5a32673dbad9","order_by":5,"name":"Xinhua Yin","email":"","orcid":"","institution":"First Affiliated Hospital of Harbin Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xinhua","middleName":"","lastName":"Yin","suffix":""}],"badges":[],"createdAt":"2021-04-17 23:13:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-435725/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-435725/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":8341487,"identity":"d6f566cc-28ec-41da-b660-581bd4929d17","added_by":"auto","created_at":"2021-04-22 17:18:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":143224,"visible":true,"origin":"","legend":"HE and Masson staining in the aortas of rats with diabetes. \n(a) HE staining in the aortas of rats with diabetes at 8 weeks after STZ injection(×200). (b) Masson staining in the aortas of rats with diabetes at 8 weeks after STZ injection(×200). (c) Masson staining in the aortas of rats with diabetes at 8 weeks after STZ injection(×400). (d) Quantification of fibrosis area analysed by Masson staining. \n","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-435725/v1/8418a27b0e5cb87dc2f83fe2.png"},{"id":8341411,"identity":"1a4bca07-4fa5-410e-b9c7-90b6622565b9","added_by":"auto","created_at":"2021-04-22 17:15:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":12657,"visible":true,"origin":"","legend":"H3 relaxin decreased plasma MDA and TNF-a expression in diabetic rats\n (a) Protein expression of MDA at 4 and 8 weeks in the plasma of diabetic rats. (b) Protein expression of TNF-a at 4 and 8 weeks in the plasma of diabetic rats. *P \u003c 0.05 vs. control, **P \u003c 0.01 vs. control, #P \u003c 0.05 vs. DM, ##P \u003c 0.01 vs. DM.\n","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-435725/v1/5f49f1b7548be5f06050a8ad.png"},{"id":8341410,"identity":"6a885590-7897-4e89-a7e3-7b34a13c879f","added_by":"auto","created_at":"2021-04-22 17:15:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":43339,"visible":true,"origin":"","legend":"Effect of H3 relaxin treatment on vascular fibrosis in the aortas of diabetic rats\n(a) The protein expression of fibrosis markers was analysed by western blotting. (b) The protein levels of collagen III were normalized to β-actin (collagen III/β-actin). (c) The protein levels of collagen I were normalized to β-actin (collagen I/β-actin). *P \u003c 0.05 vs. control, **P \u003c 0.01 vs. control, #P \u003c 0.05 vs. DM, ##P \u003c 0.01 vs. DM.\n","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-435725/v1/a0cc0bcca7052f7dd3f8d6e4.png"},{"id":8341020,"identity":"410718c4-0480-4d92-9aea-9882826e67f9","added_by":"auto","created_at":"2021-04-22 17:12:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":25271,"visible":true,"origin":"","legend":"Effect of H3 relaxin treatment on ERS in the aortas of diabetic rats\n(a) GRP78 and CHOP protein expression levels were analysed by western blotting. (b) The protein levels of GRP78 were normalized to β-actin (GRP78/β-actin). (c) The protein levels of CHOP were normalized to β-actin (CHOP/β-actin). *P \u003c 0.05 vs. control, **P \u003c 0.01 vs. control, #P \u003c 0.05 vs. DM, ##P \u003c 0.01 vs. DM.\n","description":"","filename":"OnlineFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-435725/v1/b46a223822c34e5e423f8eeb.png"},{"id":8341024,"identity":"f3b6068d-c97f-4363-8d2e-b0864d38e686","added_by":"auto","created_at":"2021-04-22 17:12:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":54088,"visible":true,"origin":"","legend":"H3 relaxin inhibited NLRP3 inflammasome activation \n(a) Protein expression of NLRP3 inflammasome markers (NLRP3, IL-1β and IL-18) at 4 and 8 weeks in the aortas of diabetic rats. (b) The protein levels of NLRP3 were normalized to β-actin (NLRP3/β-actin). (c) The protein levels of IL-1β were normalized to β-actin (IL-1β/β-actin). (d) The protein levels of IL-18 were normalized to β-actin (IL-18/β-actin). *P \u003c 0.05 vs. control, **P \u003c 0.01 vs. control, #P \u003c 0.05 vs. DM, ##P \u003c 0.01 vs. DM.\n","description":"","filename":"OnlineFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-435725/v1/51780df1e08c48b08bb0fa16.png"},{"id":8341413,"identity":"cb2b5a9c-745e-4ccc-87a7-bc88a537b07e","added_by":"auto","created_at":"2021-04-22 17:15:47","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":14967,"visible":true,"origin":"","legend":"Relaxin-1, relaxin-3 and their receptors RXFP1 and RXFP3 levels in aortas of experimental diabetic rats. \nReal-time PCR detected the expression of relaxin-1 (a), relaxin-3 (b), RXFP1 (c) and RXFP3 mRNA (d) at 4 w and 8 w in the aortas of experimental diabetic rats. The results are relative to the β-actin level. *P \u003c 0.05 vs. control, **P \u003c 0.01 vs. control.\n","description":"","filename":"OnlineFigure6.png","url":"https://assets-eu.researchsquare.com/files/rs-435725/v1/18afee44faed3ec8b03e3f4e.png"},{"id":13688030,"identity":"5072bc4b-8a20-4532-a54b-75708e9a1cc9","added_by":"auto","created_at":"2021-09-17 12:23:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":748642,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-435725/v1/d923d81b-06ea-4946-a344-3f1cdf281534.pdf"}],"financialInterests":"","formattedTitle":"H3 relaxin alleviated vascular injury in rats with type 1 diabetes by inhibiting endoplasmic reticulum stress and inflammation","fulltext":[{"header":"1. Introduction","content":" \u003cp\u003eArterial stiffness is an early stage of arterial dysfunction in patients with diabetes, which pathophysiologic mechanism is that high glucose induces impaired endothelial function. Endothelial dysfunction aggravates media thickness and fibrosis, in turn worsen arterial stiffness[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Arterial Fibrosis is an active process that collagen, fibronectin and adhesion proteins accumulate, adhesion molecules and integrins increase, and the extracellular matrix (ECM) remodel, which are involved in the arterial complications of diabetes[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Another pathogenic mechanism of arterial dysfunction in diabetes is that high glucose mediated vascular inflammation[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. ROS mediated oxidative stress is considered to be responsible for the progression and development of diabetic arterial dysfunction[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Our previous studies suggest that the ROS mediated NLRP3 inflammasome activation plays a key role in the progression of diabetic cardiomyopathy[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The NLRP3 inflammasome, a complex, comprised of the apoptosis-associated speck-like protein (ASC), NLRP3 and pro-caspase-1[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Once NLRP3 inflammasome was activated, then pro-caspase-1 transformed into cleaved caspase-1, which can activate IL-1β and IL-18 involved in arterial injury in diabetes. In addition, ROS induced by high glucose promotes ERS, aggravated inflammation and insulin resistance in diabetic vascular dysfunction. ERS may be involved in the mechanism of diabetic nephropathy, for example, ERS marker CCAAT/enhancer-binding protein homologous protein (CHOP) knockout ameliorated diabetic nephropathy in streptozotocin-induced diabetes mice[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In the retina of rats with streptozotocin-induced diabetes, ERS resulted in increased inflammation and vascular permeability[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Previous investigations suggested that acute ERS inhibition in aortic rings protected mice from diabetes-induced endothelial dysfunction[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRelaxin-3, an ancestral peptide of the human relaxin subclass of the insulin superfamily, was predominantly expressed in the central nervous system and regulated feeding, arousal, memory, learning and central responses to physiological stressors[\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Recent data indicated that H3 relaxin alleviated ischaemic injury by reducing hypoxia-induced production of ROS[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. H3 relaxin inhibited myocardial injury induced by isoproterenol or in cardiac-restricted transgenic overexpression of β2-AR mice[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Our recent study reported that H3 relaxin prevented cardiac injury and fibrosis by regulating the activation of NLRP3 inflammasome in diabetic cardiomyopathy[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, whether H3 relaxin protected the vascular complications of diabetes from inhibiting the activation of ERS and the NLRP3 inflammasome was unknown. In this study, we sought to define whether H3 relaxin attenuates the vascular complications of experimental diabetes. Moreover, we explored the mechanism that H3 relaxin inhibited vascular injury.\u003c/p\u003e "},{"header":"2. Materials And Methods","content":" \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Animals and reagents\u003c/h2\u003e \u003cp\u003e Male SD rats (Second Affiliated Hospital of Harbin Medical University, Harbin, China; weight, 200\u0026thinsp;~\u0026thinsp;250g) experiments were conducted at First Affiliated Hospital of Harbin Medical University, and the present study was approved by the Laboratory Animal Management and Ethics Review Committee of First Affiliated Hospital of Harbin Medical University. STZ was purchased from Sigma (St. Louis, MO, USA), and H3 relaxin was obtained from Phoenix Pharmaceuticals (Belmont, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Experimental diabetic rats model and groups\u003c/h2\u003e \u003cp\u003eDiabetes was prepared in SD rats by intraperitoneal injection of 65 mg/kg STZ once as described previously[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Rats were randomly divided into 4 groups: control group, DM group, A group and B group. A group: at 2 or 6 weeks after STZ administration, the rats were injected with subcutaneous 2 \u0026micro;g/kg/d H3 relaxin for 2 weeks. B group: at 2 or 6 weeks after STZ administration, the rats were injected with subcutaneous 0.2 \u0026micro;g/kg/d H3 relaxin for 2 weeks.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Hematoxylin and eosin (HE) staining and Masson staining\u003c/h2\u003e \u003cp\u003eAt 8 weeks after STZ injection, the aortas were dissected from the rats. HE-stained and Masson trichrome-stained sections were photographed using a light microscope (Olympus). The areas of fibrosis in 4 randomly selected visual fields were measured with a threshold function under 40\u0026times; magnification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Western blotting\u003c/h2\u003e \u003cp\u003eThe aorta proteins extract (40 \u0026micro;g) from four groups were separated and transferred to PVDF membrane. Membranes were incubated in antibodies, including anti-β-actin (ZSGB-BIO, BeiJing, China), anti-GRP78(Abcam, Cambridge, UK), anti-CHOP (Abcam, Cambridge, UK), anti-NLRP3(Bioss, Beijing, China), anti-IL-1β (Novus Biologicals, CO, USA), anti-IL-18(Novus Biologicals, CO, USA), anti-I-collagen (Bioss, Beijing, China) and anti-III-collagen(Bioss, Beijing, China). Then PVDF membranes were incubated in horseradish peroxidase\u0026ndash;linked secondary antibody for 1 hour. Bands were quantified with NIH Image Software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Measurements of plasma MDA and TNF-a\u003c/h2\u003e \u003cp\u003eThe blood of SD rats was centrifugated into obtaining plasma at 1000g for 10 min, then MDA was detected using ELISAs (Nanjing Jiancheng) and TNF-a was measured as described previously[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Relaxin and receptor expressions quantified by real-time PCR\u003c/h2\u003e \u003cp\u003eAorta tissues in control and DM group rats were collected at 4 and 8 weeks after STZ administration. Total RNA was isolated, reverse transcribed and quantified according to the manufacturer\u0026rsquo;s instructions (TaKaRa, Dalian, China) as described previously[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Statistical analyses\u003c/h2\u003e \u003cp\u003eExperiments were carried out in 8 rats per group in triplicate, and data were expressed as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;SE by GraphPad Prism 7.0 software. Data were analysed by one-way ANOVA, followed by a Newman-Keuls multiple comparison test. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e \u003c/div\u003e "},{"header":"3. Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003ch2\u003e3.1 H3 relaxin ameliorated aorta injury in diabetic rats\u003c/h2\u003e\n\u003cp\u003eHistological analysis of the vascular structure was performed by HE staining of the aorta specimens at 8 weeks after STZ injection. We observed that disordered elastic fibres in the aortas of rats with diabetes compared with those of controls by HE staining; however, H3 relaxin treatment restored the vascular structure (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea). Masson staining showed that vascular smooth muscle cells were stained red and interstitial collagen was blue. The collagen network of adjacent cells is intact in control rats. Extracellular matrix deposition in vascular wall increased significantly in diabetic group. More, the collagen network around the cell breaks and arrange disorders in diabetic rats, and the content of collagen in H3 relaxin treatment group are less than diabetes group and elastic fibres arrange more regular than diabetes group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb,c,d).\u003c/p\u003e\n\u003cp\u003eCompared with the controls, the contents of MDA at 8 weeks in the DM group were markedly increased, whereas were significantly decreased after H3 relaxin administration. Compared with the controls, the levels of TNF-a at 4 weeks in the DM group were markedly increased, wherea were significantly decreased after H3 relaxin adiminstration (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003e\u003cstrong\u003e3.2 H3 relaxin improved vascular fibrosis in the aortas of rats with diabetes\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eExtracellular matrix contains structural proteins, such as Type I and III collagen, adhesion molecules, integrins. The expression of types I and III collagen were increased in the aortas of STZ-treated diabetic rats, whereas were inhibited by H3 relaxin treatment in diabetic rats (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n\u003ch2\u003e\u003cstrong\u003e3.3 H3 relaxin inhibited ERS in the aortas of rats with diabetes\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eTo explore whether ERS participated in H3 relaxin protection against vascular injury in rats with diabetes, the levels of ERS markers, including GRP78 and CHOP, were evaluated by western blotting. We found that compared with control, the expression of GRP78 and CHOP were significantly higher in the DM group, however, were lower after H3 relaxin treatment in diabetic rats (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003ch2\u003e3.4 H3 relaxin inhibited NLRP3 inflammasome activation\u003c/h2\u003e\n\u003cp\u003ePrevious investigations have reported that the activation of NLRP3 inflammasome was involved in the pathophysiologic mechanism in complications of diabetes. In our work, the expression levels of NLRP3, IL-1\u0026beta; and IL-18 proteins were increased in DM group, decreased by H3 relaxin administration (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003e3.5 Relaxin-1/3 and their receptor expressions in aortas\u003c/h2\u003e\n\u003cp\u003eIn the aortas of diabetic rats, the mRNA expression of \u003cem\u003eRelaxin-1\u003c/em\u003e significantly was upregulated at 4w after STZ injection (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea); however, the mRNA expression of \u003cem\u003eRelaxin-3\u003c/em\u003e was upregulated at 4w and 8w after STZ injection (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eb). In addition, the mRNA expression of relaxin family peptide receptor 1 (\u003cem\u003eRXFP1) was\u003c/em\u003e significantly upregulated at 8w after STZ injection (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ec); \u003cem\u003eRXFP3\u003c/em\u003e mRNA expression was also upregulated at 4w and 8w after STZ injection (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ed).\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe aims of this study were to identify whether H3 relaxin protected against vascular injury induced by diabetes in vivo and potential mechanism. Our results demonstrated that vascular fibrosis, NLRP3 inflammasome activation and ERS were involved in the vascular complications of diabetes and that H3 relaxin improved vascular injury by inhibiting fibrosis, ERS and NLRP3 inflammasome activation.\u003c/p\u003e\n\u003cp\u003eIn higher primates, relaxin family peptides contains seven members, which are relaxin-1, 2, 3 and insulin-like peptides (INSL) 3, 4, 5, 6. However, in rats, there are six members in relaxin family peptides, including relaxin-1 (similar to human relaxin-2), relaxin-3 and INSL-3, 4, 5, 6[\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e]. The relaxin family peptides receptors contains RXFP1, 2, 3, 4, natural ligand of human relaxin-2 (rats relaxin-1) is RXFP1 which plays protective effects in the cardiovascular disease. Recent studies reported that relaxin-3 can inhibit myocardial injury by binding to RXFP1 in rats with myocardial infarction[\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e], although RXFP3, a natural receptor of relaxin-3, is mainly located in the brain. Later, study found that in rat atrial and ventricular cells, there existed relaxin-3 mRNA which expression was up-regulated in the myocardium after isoproterenol administration[\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]. Our recent study reported that H3 relaxin improved cardiac injury by regulating the activation of NLRP3 inflammasome in diabetic cardiomyopathy[\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e]. This study focus on whether H3 relaxin inhibited diabetic vascular injury, and we choose the dose of 0.2 or 2ug/kg/day relaxin-3 treatment for diabetic rats as zhang et al. described previously[\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]. Interestingly, we found both doses of H3 relaxin were effective to a similar degree, and we will choose the very low dose H3 relaxin in future experiment. This study limitations contained that we did not test plasma relaxin-1 and relaxin-3 expression, so we can not clarify the issue that why both high and low dose H3 relaxin had similar protective effects in vascular injury in type 1 diabetes rats. We will aviod this question in future.\u003c/p\u003e\n\u003cp\u003eWe found that the body weight increased and the glucose level decreased after H3 relaxin injection in diabetic rats as reported in our previous study[\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e], and which maybe a mechanism of that H3 relaxin inhibited vascular fibrosis, need to be verificated. Unfortunately, we did not detected the level of insulin, we will avoid this issue in future. In addition, we found that \u003cem\u003eRelaxin-1\u003c/em\u003e, \u003cem\u003e3\u003c/em\u003e and \u003cem\u003eRXFP1, 3\u003c/em\u003e mRNA were significantly up-regulated after STZ in the aortas of diabetic rats according to real-time PCR[\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e]. The reason in increased expression of \u003cem\u003eRXFP1 and RXFP3\u003c/em\u003e in aortas of diabetic rats may be due to increased expression of endogous relaxin-1/3, however, these results clarified that endogenous relaxin-1/3 and receptors participated in the mechanism of vascular injury in diabetic rats.\u003c/p\u003e\n\u003cp\u003eThe mechanism of vascular complications of diabetes contains endothelial dysfunction, oxidative stress and arterial remodelling. Oxidative stress is a determining mechanism in vascular complication of diabetes. Increased plasma TNF-a and IL-6 are associated with vascular dysfunction in patients with type 2 diabetes[\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e]. NF-\u003cem\u003e\u0026kappa;\u003c/em\u003eB activity increased, TNF-a and intercellular adhesion molecule (ICAM) upregulated in vascular tissues of type 2 diabetic rats[\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e]. Aljwaid H et al. found that compared with control, MDA levels were higher in patients with diabetes, which was related with higher levels of oxidized ascorbate. These studies clarfied that oxidative stress was involved in the mechanism of vascular dysfunction in diabetes[\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. Recent data indicate that H3 relaxin protects against ischaemic injury by reducing the hypoxia-induced production of ROS. In our study, we found that plasma MDA and TNF-a levels were increased in rats with diabetes, and H3 relaxin inhibited vascular dysfunction by regulating MDA and TNF-a levels in rats with diabetes.\u003c/p\u003e\n\u003cp\u003eThe expression of ERS markers were increased in kidney from patients with diabetes, were involved in the pathogenesis of diabetic nephropathy, however, CHOP knockout improved kidney injury in diabetic mice[\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e]. In addition, ER stress activated inflammatory factors and mediated increased vascular permeability in retina of diabetes[\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e]. In a recent study, we found that H3 relaxin inhibited the ERS mediated apoptosis of myocardial cells induced by high glucose[\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]. In our study, we found that compared with the controls, the expression of CHOP and GRP78 increased in the aortas of diabetic rats, which were inhibited by H3 relaxin administration, indicated that H3 relaxin inhibited ERS-induced vascular injury.\u003c/p\u003e\n\u003cp\u003eHigh glucose-induced NLRP3 inflammasome activation prompted vascular complications in diabetes. In vivo, NLRP3 inflammasome was activated in the aortas of rats after a high glucose diet, and inhibited by rutin administration[\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e]. Aberrantly, in atherosclerotic pig aortas, the expression of NLPR3, ASC and IL-1\u0026beta; were increased, along with NF-\u0026kappa;B activation and endothelial dysfunction. In patients with coronary heart disease, increased NLRP3 expression in aortas was positively associated the severity of coronary artery disease[\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]. In addition, our findings are consistent; compared with the controls, the expression and activation of the NLRP3 inflammasome were increased in the aortas of rats with diabetes, and were inhibited by H3 relaxin.\u003c/p\u003e\n\u003cp\u003eIn conclusion, H3 relaxin is a potential candidate for treating diabetes-associated vascular complications by inhibiting fibrosis, the activation of ERS and NLRP3 inflammasome.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e Xiaohui Zhang and Kelaier Yang: conception, design and analysis of data, performed the data analyses, and wrote the manuscript. Xiao Ma and Xinhua Yin: contributed to the conception of the study. Jinyu Chi and Wenjia Chen: contributed signifcantly to analysis and manuscript preparation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with ethical standards \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003eThis study was conducted after obtaining First Affiliated Hospital of Harbin Medical Univwersity\u0026rsquo;s ethical committee approval.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Natural Science Foundation of China (No. 81500288), Pastdoctoral Scientific Research Developmental Fund of Heilongjiang Province (LBH-Q18080,LBH-Q19133), The Innovation of Foundation of the First Affiliated Hospital of Harbin Medical University(2019M08), Natural Science Foundation of Heilongjiang Province (LH2020H034), and The Outstanding Youth Foundation of the First Affiliated Hospital of Harbin Medical University(HYD2020JQ0005).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest statement \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOn behalf of all authors, the corresponding author states that there are no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStudy data are available.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSavoia C, Burger D, Nishigaki N et al \u003cem\u003e(2011)Angiotensin II and the vascular phenotype in hypertension\u003c/em\u003e, \u003cem\u003eExpert Rev Mol Med 13\u003c/em\u003e: \u003cem\u003ee11\u003c/em\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWynn TA, Ramalingam TR \u003cem\u003e(\u003c/em\u003e2013\u003cem\u003e) Mechanisms of fibrosis: therapeutic translation for fibrotic disease, Nat Med 2012;18:1028\u0026ndash;1040.\u003c/em\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen K, Zhang J, Zhang W, \u003cem\u003eet al. 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Heart Lung Circ 22:746 \u0026ndash;746 50\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":false,"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":"H3 relaxin, type 1 diabetes, NLRP3 inflammasome, ERS, fibrosis","lastPublishedDoi":"10.21203/rs.3.rs-435725/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-435725/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHuman recombinant relaxin-3 (H3 relaxin ),a small molecule peptide hormone, ameliorated myocardial injury after myocardial infarction or isoprenaline injection by inhibiting apoptosis and fibrosis. However, whether H3 relaxin protects vascular function in rats with type 1 diabetes and its mechanism are unknown. In type 1 diabetes rats model induced by streptozotocin (STZ), rats were subcutaneously injected H3 relaxin (2 \u0026micro;g/kg/d or 0.2 \u0026micro;g/kg/d) for 2 weeks. At 4 or 8 weeks after STZ injection, we detected the expression of fibrosis (type I and III collagen), ERS (endoplasmic reticulum stress) and NLRP3 inflammasome activation in the aortas and inflammation markers in the plasma from rats with diabetes. Compared with the diabetic rats, H3 relaxin treatment exhibited markedly decreased plasma oxidative stress markers (TNF-a and MDA) levels. The protein expression levels of type I and III collagen in the aortas were increased in rats with diabetes, inhibited by H3 relaxin. H3 relaxin treatment inhibited ERS (GRP78 and CHOP) and NLRP3 inflammasome activation in the aortas of diabetic rats. These results suggest that H3 relaxin inhibited fibrosis, ERS and inflammation activation in the aortas of type 1 diabetic rats.\u003c/p\u003e","manuscriptTitle":"H3 relaxin alleviated vascular injury in rats with type 1 diabetes by inhibiting endoplasmic reticulum stress and inflammation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-04-22 17:12:45","doi":"10.21203/rs.3.rs-435725/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":"872779c5-f267-4b4b-8225-39ef4ffda7fe","owner":[],"postedDate":"April 22nd, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":3837185,"name":"Molecular Biology"}],"tags":[],"updatedAt":"2021-04-22T17:15:47+00:00","versionOfRecord":[],"versionCreatedAt":"2021-04-22 17:12:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-435725","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-435725","identity":"rs-435725","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}