Biochanin A has anti-inflammatory effects on diet-induced obesity and ovariectomy in mice

Biochanin A has anti-inflammatory effects on diet-induced obesity and ovariectomy in mice | 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 Biochanin A has anti-inflammatory effects on diet-induced obesity and ovariectomy in mice Jéssica Maria Dantas Araújo Aragão, Luana Heimfarth, Wemerson de Santana Neres, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5188359/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 Biochanin A (BCA) is a phytoestrogen widely studied for its ability to alleviate menopausal symptoms and treat metabolic diseases in the presence or absence of estrogen. This study was conducted to investigate the effect of BCA in ovariectomized (OVX) mice subjected to a high-fat diet (HFD). To this end, female C57BL6 mice were randomly divided into 5 groups: SHAM (sham-operated) with HFD, OVX with a standard diet (SD) or HFD, and two other OVX groups with HFD treated with BCA (2 mg/kg or 6 mg/kg, i.p.) during the last 30 days. The experiment lasted 15 weeks, after which it was observed that the OVX HFD animals presented a compromised metabolic profile compared to the SHAM HFD or OVX SD animals. When evaluating the BCA-treated groups in comparison to the OVX HFD group, it was demonstrated that there was less inflammation in the AT due to the reduction in crown-like structures (CLS) and the increase in the adipocyte area. This effect was complemented by an increase in the levels of the cytokines IL-5 and IL-10 and an increase in the expression of Mrc1, a marker of M2 macrophages, and Pparγ, a key regulator of tissue metabolism. Furthermore, in the liver, BCA reduced the degree of hepatic steatosis and the expression of Nos2. We concluded that BCA exerted an anti-inflammatory response in the liver, particularly in the AT, indicating a resolution profile despite not altering the animals' metabolic profile. This study demonstrated, for the first time, the anti-inflammatory effect of BCA on tissues affected by lipotoxicity caused by high-fat diet consumption, exacerbated by ovariectomy. Physiology phytoestrogens high-fat diet inflammation female castration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Obesity is the fifth most common cause of death worldwide (Camacho and Ruppel 2017 ). The risk of metabolic alterations associated with obesity increases in women during the onset of menopause, predisposing them to cardiovascular, hepatic, and diabetic diseases (Davis et al. 2015 ). Bilateral ovariectomy is an animal model that mimics the metabolic changes experienced during menopause (Medina-Contreras et al. 2020 ). Like humans, ovariectomized mice and rats develop insulin resistance, visceral adiposity, and impaired muscular glucose metabolism, key risk factors for metabolic syndrome (Campbell & Febbraio 2002 ; Ko & Kim 2020 ). Obesity results in a proinflammatory state in various tissues, starting in metabolically active cells such as adipocytes, hepatocytes, or myocytes and activating inflammation and immune response signaling pathways. Adipocyte hypertrophy/hyperplasia leads to hypoxia, tissue necrosis, and consequently, recruitment of immune cells that release proinflammatory cytokines (Longo et al. 2019 ). Additionally, estrogen plays an anti-inflammatory role in ovariectomized mice by increasing the expression of markers of proresolutive macrophages (Pepe et al. 2017 ). In vitro models support these findings; in coculture of adipocytes with macrophages, estrogen reduces the expression of proinflammatory cytokines under metabolic stress conditions (or after TNF-α stimulation) (Eaton & Sethi 2019 ). A reduction in endogenous estrogen has deleterious effects on the inflammatory response in these individuals. Therefore, to address the development of these pathologies, hormone replacement therapy (HRT) with estrogens has several beneficial effects (Lovre et al. 2016 ). However, a series of contraindications, such as increased risk of cardiovascular events and breast cancer, have been reported (Fait 2019 ) (Bolton 2016 ; Chen 2011 ; Rossouw et al. 2002 ). In light of this evidence, there is growing interest in safer molecules such as phytoestrogens (Moreira et al. 2014 ) to reduce the use or minimize the adverse effects of estrogen-based HRT. Among these phytoestrogens, biochanin A (BCA) is an isoflavone isolated from the leaves and stems of Trifolium lugens L . and is widely used to alleviate postmenopausal-related problems (Vlaisavljevic et al. 2014 ; Yu et al. 2019 ). BCA has been shown to have antioxidant, anticancer, anti-inflammatory, antidiabetic, hepatoprotective, and other biological activities and has been extensively investigated (Felix et al. 2022 ). Among its effects on metabolism, BCA promoted a beige phenotype, increasing mitochondrial biogenesis regulation in mouse adipocytes (3T3-L1) (Choi et al. 2019 ; Pagel-Langenickel et al. 2008 ). Additionally, in rats with obesity-induced cardiomyopathy, adiponectin inhibited the exacerbated production of free radicals and proinflammatory cytokines and reduced NF-κB activation in the heart (Rani et al. 2021 ). BCA is a potent molecule that exerts biological effects through multiple signal transduction pathways involved in cell differentiation, inflammation, and metabolism (Felix et al. 2022 ). However, studies focused on obesity have mainly examined the effect of this isoflavone on metabolism. Therefore, this study was conducted to evaluate whether BCA has an effect on inflammation in obesity-targeted tissues that develop after ovariectomy. 2. Materials and Methods 2.1. Animals Thirty-five C57BL/6 healthy nonpregnant female mice obtained from the Central Animal House of the Federal University of Sergipe (UFS) were used. The animals, aged 17–20 weeks, were maintained on a shelf with a controlled temperature (23 ± 2°C), an air exhaust system, and a 12/12 h light/dark cycle, and food and water were provided ad libitum . The animals were kept in polypropylene cages with dimensions of 29 × 18 × 16 cm in groups of 2 to 3 per cage. The animals were divided into groups based on the average weight of each cage. The experimental protocols were approved by the Animal Research Ethics Committee of the Federal University of Sergipe under registration numbers 57/16 and 14/18. 2.2. Ovariectomy The mice underwent bilateral ovariectomy (OVX) surgery. The removal of the ovaries was performed through a dorsal incision after trichotomy and asepsis of the region, with the animals under anesthesia with ketamine (100 mg/kg) and xylazine (10 mg/kg). Some mice underwent sham surgery, in which the ovaries were exposed and immediately returned to the anatomical region. After that, the animals were kept in cages for a period of 15 days before starting the induction of obesity. 2.3. Obesity induction and treatment A high-fat diet (HFD-26% carbohydrate, 59% lipids and 15% protein, with lard as a lipid source) was obtained from the company PragSoluções Biociências©. The standard diet (SD) consisted of 62% carbohydrates, 13% lipids, and 25% proteins. Both diets contained a mix of vitamins and minerals according to the AIN-93 recommendation. The standard diet (SD) was provided to the OVX SD group, while the HFD was provided to the SHAM HFD, OVX HFD, and BCA 2 mg/kg or 6 mg/kg (i.p.) groups. After 9 full weeks (at the 10th week), BCA treatment was administered daily for 5 weeks. BCA was diluted in saline and 0.5% dimethyl sulfoxide (DMSO). Another group received 100 µL of vehicle (saline or 0.5% DMSO) via i.p. injection during the same period. Body weight was monitored twice a week. After this period, the animals were euthanized with an overdose of ketamine (100 mg/kg) and xylazine (10 mg/kg) for the collection of blood, adipose tissue, liver, spleen, kidney, gastrocnemius muscle, heart, and uterus. 2.4. Intraperitoneal glucose tolerance test Mice were fasted for 5 hours to perform the glucose tolerance test. Three days before eutanasia, 1.5 mg/g D-glucose was administered to the animals, and glucose was measured via a glucometer (Bioland G500, Controller-SC®, Brazil) in a drop of blood obtained from the tail vein at 0, 15, 30, 60 and 120 min postinjection. 2.5. Biochemical parameters Blood was collected from the retro-orbital plexus after a 10-hour fast. The blood was centrifuged at 10,000 rpm and 10°C for 10 minutes to separate the serum, which was subsequently stored at -20°C for later analysis. The concentrations of glucose and total cholesterol were investigated using commercial kits following the manufacturer's instructions (Labtest®). 2.6. Histology The tissues were weighed, and samples of perigonadal AT from the same anatomical region were collected from all the animals. The tissues were stored in 4% paraformaldehyde, where they spent a minimum of 6 h and a maximum of 24 h at 5°C. Liver samples were kept in 10% buffered formalin at 25°C for at least 24 h for fixation. The tissues were immersed in graded alcohol and xylene and embedded in paraffin. Sections measuring approximately 5 µm were stained with hematoxylin-eosin (H&E), observed with an optical microscope and analyzed by a blinded evaluator. 2.6.1. Adipose tissue (AT) analysis The images captured from the perigonadal AT were used to determine the number of crown-like structures (CLSs) and adipocyte areas. Using ImageJ (RRID:SCR_003070) software, adipocytes were assessed by drawing ellipses around the cell membranes. The software scale was adjusted to a 100 µm scale at 400x magnification. In each analyzed image, 5 to 10 adipocytes were circumscribed, totaling at least 100 adipocytes per mouse. At 400x magnification, a covert evaluator counted the CLSs along the entire length of the tissue, and the result was normalized to 50 fields. 2.6.2. Liver analysis To determine the percentage of hepatic steatosis in liver samples, four histological sections of each specimen were analyzed. In each section, ten histological fields 0.06 mm 2 in length (magnification 400x) were selected and photomicrographed (with a Leica DFC340FX digital camera coupled to an Olympus CX31 optical microscope). The fields were selected by systematic randomization (for each selected field, two neglected fields followed). The determination of the percentage of liver tissue section area undergoing steatotic vacuolization was performed using the image analysis software ImageJ (RRID:SCR_003070). The program was previously calibrated, and once the measurement scale (%) was defined, the measurements were obtained through manual grayscale thresholding. The total section area under analysis (0.06 mm 2 ) was considered 100%, and the percentage of area identified by the thresholding procedure was subtracted from this value to obtain the percentage of steatosis area in each histological field (400x). All morphometric measurements were carried out by a hidden evaluator and are expressed as a percentage of liver tissue section. 2.7. Triglyceride content in the liver Total liver fat was quantified according to the principles of the method proposed by Folch et al. ( 1957 ), with slight modifications. Briefly, 1 g of liver tissue was homogenized with 1.5 ml of chloroform:methanol (2:1). The mixture was then filtered through fine filter paper, and for every 1 ml of filtrate, 200 µl of saline solution was added. The tubes were gently shaken by inversion three times. The homogenate was subsequently centrifuged for 10 minutes at 3000 rpm to separate the phases. A known volume of the lower phase (chloroform phase) was transferred to a previously weighed container, taken to an oven (60°C) for evaporation and resuspended in 1 ml of isopropanol. An aliquot was used for analysis of total triglycerides (Labtest, Brazil). Finally, the results were normalized to the weight of the tissue used in the analysis. 2.8. Enzyme-linked immunosorbent assay (ELISA) The proinflammatory cytokines IL-6 (Invitrogen™) and the anti-inflammatory cytokines IL-10 and IL-5 (R&D Systems©) were quantified in the perigonadal AT, liver and muscle using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's protocol. The results were subsequently normalized to the total protein concentration. 2.9. Quantitative real-time PCR Total RNA was extracted from tissues using TRIzol (Thermo Fisher Scientific, USA). RNA quality and concentrations were measured by spectrophotometer using the 260/280 nm ratio. RNA (1 µg) was reverse transcribed to cDNA using the TaqMan™ High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, USA) following the manufacturer's instructions. Relative quantification by real-time PCR was performed using TaqManTM Fast Advanced Master Mix (Thermo Fisher Scientific, USA) in a QuantiStudio™ 5 Real-Time PCR System thermocycler (Thermo Fisher Scientific, USA) under the following conditions: 2 min at 50°C, 10 min at 95°C, 40 cycles of 15 s at 95°C and 1 min at 60°C. Amplification data were analyzed using QuantiStudio™ design & analysis software v1.5.2. The following genes were evaluated via TaqMan assays: Mrc1 (Mm01329359_m1), Arg1 (Mm00475988_m1), Nos2 (Mm00440502_m1), and Pparγ (Mm00440940_m1). Gapdh (Mm99999915_g1; Thermo Fisher Scientific, USA) was used as a housekeeping gene. Relative quantities were determined using the 2 −ΔΔCt method. 2.10. Statistical analysis The results are expressed as the mean ± standard error of the mean (SEM) or median and interquartile range for scores of histological parameters. Statistical analysis of the data was performed using the GraphPad Prism 8 program (RRID:SCR_002798). The Shapiro‒Wilk normality test was applied to the groups to assess whether the data followed a normal or parametric distribution. After confirming the normality of the data, one-way or two-way ANOVA was performed, followed by Bonferroni's or Kruskal‒Wallis’s post hoc test for multiple comparisons. Values of p < 0.05 were considered significant. 3. Results 3.1. BCA improves inflammation in adipose tissue without altering metabolism The absence of estrogen caused by ovariectomy leads to metabolic dysfunctions in several tissues. First, an increase in weight was observed in the SHAM HFD group compared with the OVX, BCA 2 mg/kg and BCA 6 mg/kg groups beginning in the 3rd week of consumption of the high-fat diet (Fig. 1). A). Regarding diet consumption, the OVX HFD group showed an increase in weight starting from the 5th week compared to that of the OVX SD group (p = 0.0297). Although treatment with BCA did not reduce weight, at the 15th week, there was no significant difference in weight between the BCA 6 mg/kg group (39.8 ± 2.2 g) and the SHAM group (31.2 ± 2.3 g), unlike in the OVX groups (42.4 ± 2.8 g) and the BCA 2 mg/kg group (41.6 ± 2.1 g) (Fig. 1). A). Weight gain was consistent with the adiposity index, in which the high-fat diet increased weight gain in the OVX SD group compared to the OVX HFD group (4.4 ± 0.6 vs. 18.3 ± 0.8; p < 0.0001), and ovariectomy added to this effect in the SHAM HFD group compared to the OVX HFD group (7.8 ± 0.5 vs. 18.3 ± 0.8; p < 0.0001). Treatment did not change this parameter compared to that in the OVX HFD group (Fig. 1.B). Histology analysis of adipose tissue revealed that obesity induced by HFD increased the adipocyte area in the OVX SD vs. OVX HFD groups (p < 0.0001) and that ovariectomy enhanced this effect in the SHAM HFD vs. OVX HFD groups (p < 0.0001). Unlike what was observed in the weight analysis, treatment with a dose of BCA at 6 mg/kg, but not at 2 mg/kg, promoted a reduction in adipocyte area (p < 0.0001 and p = 0.2033) (Fig. 1. C). The crown-like structure (CLS) count is a widely used parameter for demonstrating inflammation in adipose tissue (Fig. 1). E) (Cinti et al. 2005 ). We observed that the CLS count in the tissue increased with HFD consumption in OVX SD vs OVX HFD animals (10.4 ± 3.1 vs 28.7 ± 3.8; p = 0.0004) and that this parameter also increased after surgery in SHAM HFD vs OVX HFD animals (18.9 ± 2.5 vs 28.7 ± 3.8; p = 0.0483). After treatment with 2 mg/kg or 6 mg/kg BCA, the WBC count was lower than that in the OVX HFD group (10.4 ± 1, 11.6 ± 1.3 vs 28.7 ± 3.8; p = 0.0001, p = 0.0001) (Fig. 1D). The glucose tolerance test demonstrated that animals in the OVX SD group had lower glucose levels at 30 and 60 min than did those in the OVX HFD group (p = 0.0170, p = 0.0313) (Fig. 2. A). The scientific literature demonstrates that ovariectomy changes the blood glucose levels of animals (Louet et al. 2004 ), and this can be seen in our data, in which the OVX HFD, BCA 2 mg/kg and BCA 6 mg/kg groups had a greater AUC in the test than did the SHAM HFD (29125 ± 854, 27078 ± 559, 28270 ± 923 vs 24209 ± 1342). However, neither the BCA 2 mg/kg nor the BCA 6 mg/kg treatment changed the glucose concentration (Fig. 2. B). Fasting blood glucose and serum cholesterol levels were measured. As expected, compared with HFD consumption, HFD consumption increased fasting blood glucose and cholesterol levels in the OVX HFD animals (264 ± 22.2 vs 160 ± 25.7; p = 0.0255 and 148.4 ± 9.6 vs 85.3 ± 12; p = 0.0061). When comparing the SHAM HFD and OVX HFD groups, we observed that only fasting blood glucose was greater in the ovariectomized group (132 ± 18 vs 264 ± 22.2; p = 0.011). Treatment with BCA did not alter the blood glucose or cholesterol levels of the animals at either of the doses used (Fig. 2. C and D). To assess the effect of HFD consumption and OVX on tissue weight (liver, muscle, heart, spleen, kidney, and uterus), the index was calculated using tibia length. Only the heart indices were greater in the OVX HFD animals than in the SHAM HFD (p = 0.0487) and OVX SD (p = 0.0321) animals. The groups treated with either dose of BCA showed no change in this index compared to the OVX HFD or SHAM HFD groups. The uterus volume was lower in the ovariectomized animals, OVX HFD, 2 mg/kg BCA, and 6 mg/kg BCA groups than in the SHAM HFD group, as expected (p < 0.0001, p < 0.0001, and p < 0.0001, respectively) (Table 1). This demonstrated the effectiveness of the ovariectomy 3.2. BCA treatment improves steatosis in the liver When assessing liver damage, large intracytoplasmic fat vacuoles accumulated, promoting cell ballooning. To a lesser extent, areas of microvesicular steatosis were evident and characterized by the formation of multiple intracytoplasmic lipid vacuoles. Mononuclear inflammatory infiltrates were observed in all the groups that consumed a HFD; however, these infiltrates were considered mild and had an irregular distribution (Fig. 3. A, C, D, E). The percentage of patients with hepatic steatosis was calculated only for the groups that received a HFD. The OVX group had a greater percentage of hepatic steatosis than the SHAM HFD group (88 ± 2.4% vs 75 ± 1%; p = 0.0095) (Fig. 3. F), and both doses of BCA reduced hepatic steatosis compared to that in the OVX HFD group (32 ± 6.4% and 33.4 ± 7%, p < 0.0001 and p < 0.0001). The quantification of TAG in the liver was performed for all groups (Fig. 3. G). As expected, the OVX SD group had a lower TAG content in the liver than the OVX HFD group (149.4 ± 13.4 vs 876.4 ± 201.7; p = 0.0027), and ovariectomy also promoted an increase in this content in the OVX HFD vs. SHAM HFD animals (876.4 ± 201.7 vs 233.7 ± 28.2; p = 0.0091). Finally, compared with those in the OVX HFD group, the animals in the BCA 6 mg/kg group, but not those in the 2 mg/kg dose group, had lower TAG levels (876.4 ± 201.7 vs 362.3 ± 28.9; p = 0.0178) (Fig. 3. G). 3.3. Effect of BCA on Anti-inflammatory activity in adipose tissue (AT) The inflammatory profile of the tissues was investigated by measuring the concentrations of proinflammatory cytokines (IL-6) and anti-inflammatory cytokines (IL-5 and IL-10) in tissues affected by lipotoxicity, such as AT (adipose tissue), liver, and muscle (Fig. 4). The concentration of the cytokine IL-6 was not altered in the AT or liver in the OVX HFD, BCA 2 mg/kg, or 6 mg/kg groups (Fig. 4. A and D). However, in the muscle, treatment with 2 mg/kg BCA reduced the IL-6 concentration compared to that in the OVX HFD group (0.019 ± 0.016 vs 0.6 ± 0.1; p = 0.0342) (Fig. 4. G). Regarding the anti-inflammatory cytokines, an increase in the IL-5 concentration was observed in the ATs of animals treated only with 2 mg/kg BCA (311.4 ± 58.6 vs 100.7 ± 16; p = 0.0023) and in the IL-10 concentration at both BCA doses (199 ± 18.4, 167.4 ± 10.7 vs 81.7 ± 9.3; p < 0.0001, p = 0.0004) compared to those in the OVX HFD group (Fig. 4. B, C). Thus, the expression of genes linked to macrophage polarization and PPARγ was evaluated. We found that, in AT, there was no change in the expression of the Nos2 mRNA (which represents type M1 macrophages) (Fig. 5. A). However, in the BCA 6 mg/kg group, there was greater expression of Mrc1 mRNA (M2 macrophages) than in the OVX HFD group (12.5 ± 1.6 vs 4.7 ± 1.8, p = 0.0320) (Fig. 5. B). Furthermore, there was greater expression of Pparγ mRNA in the 6 mg/kg BCA group than in the OVX HFD group (11.5 ± 0.4 vs 3.1 ± 1.2, p = 0.0338) (Fig. 5. C) In the liver, BCA at 2 mg/kg or 6 mg/kg reduced the expression of the genes associated with the M1 macrophage profile ( Nos2 ), but not M2 ( Arg1 ) or Pparγ , compared to that in the OVX HFD group (0.32 ± 0.09, 0.4 ± 0.1 vs. 2.65 ± 0.5; p > 0.0001, p > 0.0001) (Fig. 5. E-F). 4. Discussion The anti-inflammatory effect of biochanin A (BCA) has been described in various in vitro and in vivo models (Anuranjana et al. 2023 ; Feng and Lai 2023 ; Felix et al. 2020 ). Dietary consumption of BCA has previously been investigated in cell lines, mice, and male rats, and the anti-obesity effect of boric acid has been shown to increase (Rathinasamy et al. 2020 ; Choi et al. 2019 ; Su et al. 2013 ). The dataset found in the literature on the anti-inflammatory effect of BCA demonstrates that this isoflavone acts on various targets to prevent/reduce the inflammatory process, such as through the inhibition of NF-κB and MAPK via positive regulation of PPAR-γ (Felix et al. 2022 ; Sarfraz et al. 2020 ). Nevertheless, no study has investigated the anti-inflammatory effect of this phytoestrogen on menopause in the context of obesity. Therefore, the present study was the first to evaluate the anti-inflammatory effect of BCA after establishing obesity in OVX animals. As a phytoestrogen, BCA has a structure similar to that of estrogen. Therefore, these effects may be dependent on interactions with estrogen receptor beta (ER-β) or GPR30 (Felix et al. 2021 ; Escande et al. 2006 ). Ovariectomy is a widely used surgery in the scientific literature that depletes endogenous estrogen in animals (oophorectomy in humans), mimicking the physiological condition of menopause. The hypoestrogenic effect of ovariectomy leads to increased visceral fat, inflammation, oxidative stress, and metabolic complications such as dyslipidemia, hepatic steatosis, and predisposition to cardiovascular diseases (Medina-Contreras et al. 2020 ; Davis et al. 2015 ). This was clearly demonstrated in our study, with an increase in weight and adiposity and a worsened glycemic profile in the OVX HFD animals. During the development of obesity, the expansion of adipose tissue (AT) culminates in a web of inflammatory and metabolic events that challenge proposed therapies. In addition, in the menopausal phase, estrogen deficiency makes the environment even less favorable for treatment. In the early days after the consumption of a HFD, prior to adipocyte hypertrophy, there is an increase in the number of neutrophils that release enzymes and degrade IRS-1, inducing insulin resistance (IR) (Delgado-Rizo et al. 2017 ; Elgazar-Carmon et al. 2008 ). As weeks progress, highly inflammatory macrophages influx into ATs, forming CLSs and releasing chemokines and cytokines that recruit these leukocytes (Kosteli et al. 2010 ). We did not observe metabolic changes in the animals (weight, blood glucose, or cholesterol); however, a reduction in inflammation markers in the adipose tissue (AT) was identified. CLSs are a pathological hallmark of obesity, and the elimination of adipocyte residues is suggested to be an important function of macrophages in obese individuals. The frequency of adipocyte death is positively correlated with increased adipocyte size in obese mice (Cinti et al. 2005 ). Consistent with these findings, OVX animals had increased CLS counts, and BCA showed anti-inflammatory potential by reducing CLS. Subsequently, we demonstrated that BCA reduces steatosis and TAG accumulation in the liver. The antisteatotic effect of BCA was previously investigated by Park et al. ( 2016 ), who demonstrated that, in male mice fed BCA, phytoestrogen promoted the recovery of metabolites linked to lipogenesis and beta-oxidation and suppressed the expression of enzymes linked to glucose metabolism. Recently, Fan et al. ( 2021 ) showed that NAFLD progression was prevented in rats fed a BCA diet, and this effect may be linked to the reduction in PPARγ expression. Unfortunately, we did not find differences in the expression of this receptor in the liver. However, it is possible to associate this result with the fact that the isoform primarily expressed in the liver is PPARα, and the expression of PPARγ in female mice may differ from that in male mice in terms of the levels of the PPARγ1 and PPARγ2 isoforms (Berthier et al. 2021 ). The anti-inflammatory effect of BCA in the liver was also observed. We found low expression of the Nos2 gene, which encodes the enzyme inducible nitric oxide synthase (iNOS), considered a hallmark of inflammation, which is activated during obesity (Qian et al. 2019 ). The enzyme is expressed in Kupffer cells (Zhang & Lang 2023 ) and in nonalcoholic fatty liver disease (NAFLD). This enzyme has been strongly associated with insulin resistance due to impaired autophagy mechanisms in hepatocytes during diet-induced obesity (Qian et al. 2019 ) and in Kupffer cells and is linked to the development of NAFLD (Jin et al. 2023 ). Although we do not have enough data to confirm this phenomenon, these findings indicate that the protective effect of BCA on the liver against steatosis may be linked to a reduction in the expression of the enzyme. AT is governed by molecules and immune cells that dictate the inflammatory profile. Eosinophils are important cytokine-producing leukocytes with a Th2 profile that include cytokines such as IL-5 and IL-10 and support metabolic homeostasis in AT during a healthy state (Castoldi et al. 2016 ; Qiu et al. 2014 ; Molofsky et al. 2013 ). Lee et al. ( 2018 ) observed that these cells are important for improving the tissue metabolic profile in eosinophil-deficient mice and are inversely associated with adiposity and IR (Wu et al. 2011 ). Another group reported that the return of these cells, induced by IL-5, to a physiological state did not restore glucose tolerance in obese animals (Lee et al. 2018 ). Thus, the effect found in the present study corroborates these findings, as the increase in IL-5 in the AT of obese ovariectomized animals treated with BCA did not improve glucose intolerance. Another important aspect of AT inflammation during obesity is the accumulation of macrophages. The flow of saturated fatty acids to ATs during HFD consumption is an important regulator of inflammation, monocyte recruitment, and polarization to M1 (Coenen et al. 2007 ; Lumeng et al. 2007 ; Weisberg et al. 2003 ). The role of IL-10 in inducing M2 macrophages has been well described (Castoldi et al. 2016 ). This cytokine is secreted by macrophages and regulatory T lymphocytes and exerts anti-inflammatory effects, increasing sensitivity to insulin, opposing TNF-α in inducing IR in 3T3L adipocytes (Lumeng et al. 2007 ). Under healthy conditions, adipose tissue (AT) produces IL-13 along with PPARγ and PPARβ/δ, which promote the activation of M2 macrophages and suppress the activation of M1 macrophages via the production of adiponectin and IL-10 (Olefsky & Glass 2010 ). Thus, a likely shift in the macrophage profile toward M2 was observed in the ATs of animals treated with BCA, represented by an increase in the anti-inflammatory cytokines IL-5 and IL-10 and the gene expression of Mrc1 (mannose receptor), which are linked to the M2 macrophage phenotype (Zhou et al. 2018 ). These findings reinforce the anti-inflammatory effect of BCA on the AT of obese mice. Although we did not explore the mechanism of action of BCA in depth, we propose that these results in AT are linked to the PPARγ receptor, which is highly expressed in tissue. This study has limitations regarding the route of administration, but it demonstrates this important finding in a menopause obesity model. We showed here that the effect of BCA was not directly related to the antihyperlipidemic and antihyperglycemic effects already demonstrated in the literature but rather to its anti-inflammatory effect. Thus, the mechanisms of action involving the activation of the PPARγ receptor, NFκB, and MAPK to promote the observed results should be further investigated. Declarations Ethics approval This study was performed in accordance with the principles of the Declaration of Helsinki. Approval was granted by the Animal Research Ethics Committee of the Federal University of Sergipe under registration numbers 57/16 and 14/18. Funding This work was supported by the National Council for Scientific and Technological Development (CNPQ) and the Higher Education Personnel Improvement Coordination (CAPES). EAC is a beneficiary of the CNPq scientific productivity grant (CNPq: 315369/2021-3). Availability of data and materials The authors declare that the data supporting the findings of this study are available within the paper. Should any raw data files be needed in another format, they are available from the corresponding author upon reasonable request. Competing Interests The authors declare that there are no competing interests. Author contributions The authors contributed to this study as follows: Jéssica M. D. Araújo: conceptualization, methodology, formal analysis and investigation, writing - original draft preparation review and editing; Luana Heimfarth: methodology, formal analysis and investigation, writing - review and editing; Wemerson S. Neres: methodology, formal analysis and investigation, writing - review and editing; Franciel B. Félix: formal analysis and investigation, writing - review and editing; Patrícia R. Santos: methodology, formal analysis and investigation, writing - review and editing; Fabíula F. Abreu: formal analysis and investigation, writing - review and editing; Luana M. Cercato: formal analysis and investigation, writing - review and editing; Ana C. S. Nascimento: formal analysis and investigation, writing - review and editing; Alan B. S. Vasconcelos: formal analysis and investigation, writing - review and editing; Rosilene C. Soares: writing - review and editing, resources; Ricardo Albuquerque: methodology, formal analysis and investigation, writing - review and editing; Greice I. Heidend: methodology, formal analysis and investigation All the authors reviewed and approved the final version of the manuscript. The authors declare that all the data were generated in house and that no paper mill was used. References Anuranjana PV, Beegum F, K P D, George KT, Viswanatha GL, Nayak PG, Kanwal A, Kishore A, Shenoy RR, Nandakumar K (2023) Mechanisms Behind the Pharmacological Application of Biochanin-A: A review. F1000Res 12:107. https://doi.org/10.12688/f1000research.126059.3 Berthier A, Johanns M, Zummo FP, Lefebvre P, Staels B (2021) PPARs in liver physiology. Biochim Biophys Acta Mol Basis Dis 1867(5):166097. https://doi.org/10.1016/j.bbadis.2021.166097 Bolton JL (2016) Menopausal Hormone Therapy, Age, and Chronic Diseases: Perspectives on Statistical Trends. Chem Res Toxicol 29:1583–1590. https://doi.org/10.1021/acs.chemrestox.6b00272 Camacho S, Ruppel A (2017) Is the calorie concept a real solution to the obesity epidemic? Glob Health Action 1289650. https://doi.org/10.1080/16549716.2017.1289650 Campbell SE, Febbraio MA (2002) Effect of the ovarian hormones on GLUT4 expression and contraction-stimulated glucose uptake. Am J Physiol Endocrinol Metab 282:E1139–E1146. https://doi.org/10.1152/ajpendo.00184.2001 Castoldi A, Naffah de Souza C, Câmara NO, Moraes-Vieira PM (2016) The Macrophage Switch in Obesity Development. Front Immunol 6:637. https://doi.org/10.3389/fimmu.2015.00637 Chen WY (2011) Postmenopausal hormone therapy and breast cancer risk: current status and unanswered questions. Endocrinol Metab Clin North Am 40. https://doi.org/10.1016/j.ecl.2011.05.006 . 509 – 18 Choi EM, Suh KS, Park SY, Chin SO, Rhee SY, Chon S (2019) Biochanin A prevents 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced adipocyte dysfunction in cultured 3T3-L1 cells. J Environ Sci Health Tox Hazard Subst Environ Eng 54:865–873. https://doi.org/10.1080/10934529.2019.1603746 Cinti S, Mitchell G, Barbatelli G, Murano I, Ceresi E, Faloia E, Wang S, Fortier M, Greenberg AS, Obin MS (2005) Adipocyte death defines macrophage localization and function in adipose tissue of obese mice and humans. J Lipid Res 46:2347–2355. https://doi.org/10.1194/jlr.M500294-JLR200 Coenen KR, Gruen ML, Chait A, Hasty AH (2007) Diet-induced increases in adiposity, but not plasma lipids, promote macrophage infiltration into white adipose tissue. Diabetes 3:64–73. https://doi.org/10.2337/db06-1375 Davis SR, Lambrinoudaki I, Lumsden M, Mishra GD, Pal L, Rees M, Santoro N, Simoncini T (2015) Menopause. Nat Rev Dis Primers 15004. https://doi.org/10.1038/nrdp.2015.4 Delgado-Rizo V, Martínez-Guzmán MA, Iñiguez-Gutierrez L, García-Orozco A, Alvarado-Navarro A, Fafutis-Morris M (2017) Neutrophil Extracellular Traps and Its Implications in Inflammation: An Overview. Front Immunol 8:81. https://doi.org/10.3389/fimmu.2017.00081 Eaton SA, Sethi JK (2019) Immunometabolic Links between Estrogen, Adipose Tissue and Female Reproductive Metabolism. Biology (Basel) 8:8. https://doi.org/10.3390/biology8010008 Elgazar-Carmon V, Rudich A, Hadad N, Levy R (2008) Neutrophils transiently infiltrate intra-abdominal fat early in the course of high-fat feeding. J Lipid Res 9:1894–1903. https://doi.org/10.1194/jlr.M800132-JLR200 Escande A, Pillon A, Servant N, Cravedi JP, Larrea F, Muhn P, Nicolas JC, Cavaillès V, Balaguer P (2006) Evaluation of ligand selectivity using reporter cell lines stably expressing estrogen receptor alpha or beta. Biochem Pharmacol 10:1459–1469. https://doi.org/10.1016/j.bcp.2006.02.002 Fait T (2019) Menopause hormone therapy: latest developments and clinical practice. Drugs Context 8:212551. https://doi.org/10.7573/dic.212551 Fan Y, Yan LT, Yao Z, Xiong GY (2021) Biochanin A Regulates Cholesterol Metabolism Further Delays the Progression of Nonalcoholic Fatty Liver Disease. Diabetes Metab Syndr Obes 14:3161–3172. https://doi.org/10.2147/DMSO.S315471 Felix FB, Araújo JMD, de Souza EV, Pinho V, Camargo EA, Corrêa CB, Grespan R (2020) Biochanin A attenuates zymosan-induced arthritis in mice similarly to 17-β estradiol: an alternative to hormone replacement therapy? Inflamm Res 12:1245–1256. https://doi.org/10.1007/s00011-020-01403-4 Felix FB, Vago JP, Beltrami VA, Araújo JMD, Grespan R, Teixeira MM, Pinho V (2022) Biochanin A as a modulator of the inflammatory response: An updated overview and therapeutic potential. Pharmacol Res 180. https://doi.org/10.1016/j.phrs.2022.106246 Felix FB, Vago JP, Fernandes DO, Martins DG, Moreira IZ, Gonçalves WA, Costa WC, Araújo JMD, Queiroz-Junior CM, Campolina-Silva GH, Soriani FM, Sousa LP, Grespan R, Teixeira MM, Pinho V (2021) Biochanin A Regulates Key Steps of Inflammation Resolution in a Model of Antigen-Induced Arthritis via GPR30/PKA-Dependent Mechanism. Front Pharmacol 12:662308. https://doi.org/10.3389/fphar.2021.662308 Feng ZJ, Lai WF (2023) Chemical and Biological Properties of Biochanin A and Its Pharmaceutical Applications. Pharmaceutics 4:1105. https://doi.org/10.3390/pharmaceutics15041105 Folch J, Lees M, Sloane Stanley GH (1957) A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 226:497–509. https://doi.org/10.1016/S0021-9258(18)64849-5 Jin G, Yao X, Liu D, Zhang J, Zhang X, Yang Y, Bi Y, Zhang H, Dong G, Tang H, Cheng S, Hong F, Si M (2023) Inducible nitric oxide synthase accelerates nonalcoholic fatty liver disease progression by regulating macrophage autophagy. Immun Inflamm Dis 12:e1114. https://doi.org/10.1002/iid3.1114 Ko SH, Kim HS (2020) Menopause-Associated Lipid Metabolic Disorders and Foods Beneficial for Postmenopausal Women. Nutrients 12:202. https://doi.org/10.3390/nu12010202 Kosteli A, Sugaru E, Haemmerle G, Martin JF, Lei J, Zechner R, Ferrante AW Jr (2010) Weight loss and lipolysis promote a dynamic immune response in murine adipose tissue. J Clin Invest 10:3466–3479. https://doi.org/10.1172/JCI42845 Lee EH, Itan M, Jang J, Gu HJ, Rozenberg P, Mingler MK, Wen T, Yoon J, Park SY, Roh JY, Choi CS, Park WJ, Munitz A, Jung Y (2018) Eosinophils support adipocyte maturation and promote glucose tolerance in obesity. Sci Rep 1:9894. https://doi.org/10.1038/s41598-018-28371-4 Longo M, Zatterale F, Naderi J, Parrillo L, Formisano P, Raciti GA, Beguinot F, Miele C (2019) Adipose Tissue Dysfunction as Determinant of Obesity-Associated Metabolic Complications. Int J Mol Sci 20:2358. https://doi.org/10.3390/ijms20092358 Louet JF, LeMay C, Mauvais-Jarvis F (2004) Antidiabetic actions of estrogen: insight from human and genetic mouse models. Curr Atheroscler Rep 3:180–185. https://doi.org/10.1007/s11883-004-0030-9 Lovre D, Lindsey SH, Mauvais-Jarvis F (2016) Effect of menopausal hormone therapy on components of the metabolic syndrome. Ther Adv Cardiovasc Dis 11:33–43. https://doi.org/10.1177/1753944716649358 Lumeng CN, Deyoung SM, Bodzin JL, Saltiel AR (2007) Increased inflammatory properties of adipose tissue macrophages recruited during diet-induced obesity. Diabetes 1:16–23. https://doi.org/10.2337/db06-1076 Medina-Contreras J, Villalobos-Molina R, Zarain-Herzberg A, Balderas-Villalobos J (2020) Ovariectomized rodents as a menopausal metabolic syndrome model. A minireview. Mol Cell Biochem 475:261–276. https://doi.org/10.1007/s11010-020-03879-4 Molofsky AB, Nussbaum JC, Liang HE, Van Dyken SJ, Cheng LE, Mohapatra A, Chawla A, Locksley RM (2013) Innate lymphoid type 2 cells sustain visceral adipose tissue eosinophils and alternatively activated macrophages. J Exp Med 3:535–549. https://doi.org/10.1084/jem.20121964 Moreira AC, Silva AM, Santos MS, Sardão VA (2014) Phytoestrogens as alternative hormone replacement therapy in menopause: What is real, what is unknown. J Steroid Biochem Mol Biol 143:61–71. https://doi.org/10.1016/j.jsbmb.2014.01.016 Olefsky JM, Glass CK (2010) Macrophages, inflammation, and insulin resistance. Annu Rev Physiol 72:219–246. https://doi.org/10.1146/annurev-physiol-021909-135846 Pagel-Langenickel I, Bao J, Joseph JJ, Schwartz DR, Mantell BS, Xu X, Raghavachari N, Sack MN (2008) PGC-1alpha integrates insulin signaling, mitochondrial regulation, and bioenergetic function in skeletal muscle. J Biol Chem 283:22464–22472. https://doi.org/10.1074/jbc.M800842200 Park HS, Hur HJ, Kim SH, Park SJ, Hong MJ, Sung MJ, Kwon DY, Kim MS (2016) Biochanin A improves hepatic steatosis and insulin resistance by regulating the hepatic lipid and glucose metabolic pathways in diet-induced obese mice. Mol Nutr Food Res 9:1944–1955. https://doi.org/10.1002/mnfr.201500689 Pepe G, Braga D, Renzi TA, Villa A, Bolego C, D'Avila F, Barlassina C, Maggi A, Locati M, Vegeto E (2017) Self-renewal and phenotypic conversion are the main physiological responses of macrophages to the endogenous estrogen surge. Sci Rep 20:44270. https://doi.org/10.1038/srep44270 Qian Q, Zhang Z, Li M, Savage K, Cheng D, Rauckhorst AJ, Ankrum JA, Taylor EB, Ding WX, Xiao Y, Cao HJ, Yang L (2019) Hepatic Lysosomal iNOS Activity Impairs Autophagy in Obesity. Cell Mol Gastroenterol Hepatol 1:95–110. https://doi.org/10.1016/j.jcmgh.2019.03.005 Qiu Y, Nguyen KD, Odegaard JI, Cui X, Tian X, Locksley RM, Palmiter RD, Chawla A (2014) Eosinophils and type 2 cytokine signaling in macrophages orchestrate development of functional beige fat. Cell 6:1292–1308. https://doi.org/10.1016/j.cell.2014.03.066 Rani AJI, Uddandrao VVS, Vadivukkarasi GSGSPCSSPTPPS S (2021) Biochanin A attenuates obesity cardiomyopathy in rats by inhibiting oxidative stress and inflammation through the Nrf-2 pathway. Arch Physiol Biochem 1–16. https://doi.org/10.1080/13813455.2021.1874017 Rathinasamy AJIR, Uddandrao VVS, Raveendran N, Sasikumar V (2020) Antiobesity Effect of Biochanin-A: Effect on Trace Element Metabolism in High Fat Diet-Induced Obesity in Rats. Cardiovasc Hematol Agents Med Chem 1:21–30. https://doi.org/10.2174/1871524920666200207101920 Rossouw JE, Anderson GL, Prentice RL, LaCroix AZ, Kooperberg C, Stefanick ML, Jackson RD, Beresford SA, Howard BV, Johnson KC, Kotchen JM, Ockene J (2002) Writing Group for the Women's Health Initiative Investigators. Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results From the Women's Health Initiative randomized controlled trial. JAMA 288:321–333. 10.1001/jama.288.3.321 Sarfraz A, Javeed M, Shah MA, Hussain G, Shafiq N, Sarfraz I, Riaz A, Sadiqa A, Zara R, Zafar S, Kanwal L, Sarker SD, Rasul A (2020) Biochanin A: A novel bioactive multifunctional compound from nature. Sci Total Environ 722:137907. https://doi.org/10.1016/j.scitotenv.2020.137907 Su SJ, Yeh YT, Su SH, Chang KL, Shyu HW, Chen KM, Yeh H (2013) Biochanin a promotes osteogenic but inhibits adipogenic differentiation: evidence with primary adipose-derived stem cells. Evid Based Complement Alternat Med 846039. https://doi.org/10.1155/2013/846039 Vlaisavljevic S, Kaurinovic B, Popovic M, Djurendic-Brenesel M, Vasiljevic B, Cvetkovic D, Vasiljevic S (2014) Trifolium pratense L. as a potential natural antioxidante. Molecules 19:713–725. https://doi.org/10.3390/molecules19010713 Weisberg SP, McCann D, Desai M, Rosenbaum M, Leibel RL, Ferrante AW Jr (2003) Obesity is associated with macrophage accumulation in adipose tissue. J Clin Invest 12:1796–1808. https://doi.org/10.1172/JCI19246 Wu D, Molofsky AB, Liang HE, Ricardo-Gonzalez RR, Jouihan HA, Bando JK, Chawla A, Locksley RM (2011) Eosinophils sustain adipose alternatively activated macrophages associated with glucose homeostasis. Science 6026:243–247. 10.1126/science.1201475 Yu C, Zhang P, Lou L, Wang Y (2019) Perspectives Regarding the Role of Biochanin A in Humans. Front Pharmacol 10:793. https://doi.org/10.3389/fphar.2019.00793 Zhang W, Lang R (2023) Macrophage metabolism in nonalcoholic fatty liver disease. Front Immunol 14:1257596. https://doi.org/10.3389/fimmu.2023.1257596 Zhou Y, Do DC, Ishmael FT, Squadrito ML, Tang HM, Tang HL, Hsu MH, Qiu L, Li C, Zhang Y, Becker KG, Wan M, Huang SK, Gao P (2018) Mannose receptor modulates macrophage polarization and allergic inflammation through miR-511-3p. J Allergy Clin Immunol 1:350–364. https://doi.org/10.1016/j.jaci.2017.04.049 Additional Declarations The authors declare no competing interests. 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. 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(A) Weight evolution over 15 weeks. (B) The adiposityindex was normalized to thetibia length. (C) Area of 100 adipocytes per animal. (D) Crown-like structurecount (CLS) on ATsnormalized to 50 fields. (E) Representative image of CLSs in perigonadal ATs stained with H\u0026amp;E and evaluated at 400x magnification. The results are expressed as the mean ± SEM or in a violin plot with median titers and arange of 5–95%, as evaluated by one-way or two-way ANOVA followed by Bonferroni’s posttestor Dunn’s posttest. *p\u0026gt; 0.05, ***p\u0026gt;0.0001 vs the OVX HFD group; #p\u0026gt; 0.05 vs the SHAM HFD group.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-5188359/v1/b405077b4ba44ad533d58bbb.png"},{"id":65859289,"identity":"6d9b6cfe-188a-47dc-ae96-820ef99bb7a1","added_by":"auto","created_at":"2024-10-03 15:47:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":469847,"visible":true,"origin":"","legend":"\u003cp\u003eAssessment of the metabolic profiles of the animals. (A) A tolerance test was performed by fasting the animals for 5 hours. Blood glucose was measured at 0, 15, 30, 60 and 120 min after the administration of D-glucose (1.5 mg/g of weight). (B) The area under the test curve (AUC) was calculated with the GraphPad Prism program. The biochemical parameters (C) fasting glucose and (D) cholesterol were also measured after the animals had fasted for 10 hours. The results are expressed as the mean ± SEM and were analyzed by one-way or two-way ANOVA followed by Bonferroni post hoc correction. *p\u0026gt; 0.05 vs OVX HFD or #p\u0026gt; 0.05 vs SHAM HFD.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-5188359/v1/8b0c49f54ab3785c7fb7df11.png"},{"id":65859288,"identity":"54e2202a-c5b4-4565-ad36-333eba3fb3a3","added_by":"auto","created_at":"2024-10-03 15:47:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1437264,"visible":true,"origin":"","legend":"\u003cp\u003eEffectof BCA treatment on histopathological changes in the liver. The livers were stained with H\u0026amp;E, and images were taken at 400x magnification (60 µm scale bar). (A–E) Representative images of each group showing large intracellular fat vacuoles promoting cellular ballooning and peripheral nuclear displacement (macrovesicular), more prominent in the centrolobular regions, in the animals that received the HFD (A, C, D, E). Mononuclear inflammatory infiltrates werepresent in all four groups with steatosis, but they wereconsidered mild and hadirregular focal distributions. (F) Percentage of liver steatosis in the groups that received the HFD. (G) The triglyceride concentration wasnormalized to thetotal tissue weight (mg). The results are expressed as the mean ± SEM or in violin plots with median titers and a range of 5–95%, as evaluated by one-way or two-way ANOVA followed by Bonferroni’s posttestor Dunn’s posttest. *p\u0026gt; 0.05, **p\u0026gt; 0.001, ***p\u0026gt;0.0001 vs OVX HFD group or #p\u0026gt; 0.05 vs SHAM HFD.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-5188359/v1/692d0a75a666f5cf31ae1103.png"},{"id":65858957,"identity":"70df9b2d-cf35-4a3b-87a9-682ee729920c","added_by":"auto","created_at":"2024-10-03 15:39:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":589146,"visible":true,"origin":"","legend":"\u003cp\u003eProfile of cytokines in adipose tissue and liver. Proinflammatory cytokines(IL-6) and anti-inflammatory cytokines (IL-5 and IL-10) were evaluated in the perigonadalAT (A, B, C), liver (D, E, F) and muscle (G, H, I) tissues. The results areexpressed in violin plots with median titers and a range of 5–95%. One-way ANOVA followed by Bonferroni or Dunn’s post hoc test was used. *p\u0026gt; 0.05, **p\u0026gt; 0.001, ***p\u0026gt;0.0001 vs OVX HFD.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-5188359/v1/aa29eabaf242e2e4aa2fa2f7.png"},{"id":65858958,"identity":"4e5a4b76-cd47-408c-ad05-86d37c1415a1","added_by":"auto","created_at":"2024-10-03 15:39:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":390060,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-5188359/v1/f72006da50231b6536b6573a.png"},{"id":65859868,"identity":"4885f315-fe77-457a-a316-31e5229f24af","added_by":"auto","created_at":"2024-10-03 15:55:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3842230,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5188359/v1/ad7c2c86-a720-4fce-87b8-f8309a368bc6.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eBiochanin A has anti-inflammatory effects on diet-induced obesity and ovariectomy in mice\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eObesity is the fifth most common cause of death worldwide (Camacho and Ruppel \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The risk of metabolic alterations associated with obesity increases in women during the onset of menopause, predisposing them to cardiovascular, hepatic, and diabetic diseases (Davis et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBilateral ovariectomy is an animal model that mimics the metabolic changes experienced during menopause (Medina-Contreras et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Like humans, ovariectomized mice and rats develop insulin resistance, visceral adiposity, and impaired muscular glucose metabolism, key risk factors for metabolic syndrome (Campbell \u0026amp; Febbraio \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Ko \u0026amp; Kim \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eObesity results in a proinflammatory state in various tissues, starting in metabolically active cells such as adipocytes, hepatocytes, or myocytes and activating inflammation and immune response signaling pathways. Adipocyte hypertrophy/hyperplasia leads to hypoxia, tissue necrosis, and consequently, recruitment of immune cells that release proinflammatory cytokines (Longo et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Additionally, estrogen plays an anti-inflammatory role in ovariectomized mice by increasing the expression of markers of proresolutive macrophages (Pepe et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). \u003cem\u003eIn vitro\u003c/em\u003e models support these findings; in coculture of adipocytes with macrophages, estrogen reduces the expression of proinflammatory cytokines under metabolic stress conditions (or after TNF-α stimulation) (Eaton \u0026amp; Sethi \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA reduction in endogenous estrogen has deleterious effects on the inflammatory response in these individuals. Therefore, to address the development of these pathologies, hormone replacement therapy (HRT) with estrogens has several beneficial effects (Lovre et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). However, a series of contraindications, such as increased risk of cardiovascular events and breast cancer, have been reported (Fait \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) (Bolton \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Chen \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Rossouw et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). In light of this evidence, there is growing interest in safer molecules such as phytoestrogens (Moreira et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) to reduce the use or minimize the adverse effects of estrogen-based HRT.\u003c/p\u003e \u003cp\u003eAmong these phytoestrogens, biochanin A (BCA) is an isoflavone isolated from the leaves and stems of \u003cem\u003eTrifolium lugens L\u003c/em\u003e. and is widely used to alleviate postmenopausal-related problems (Vlaisavljevic et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Yu et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). BCA has been shown to have antioxidant, anticancer, anti-inflammatory, antidiabetic, hepatoprotective, and other biological activities and has been extensively investigated (Felix et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAmong its effects on metabolism, BCA promoted a beige phenotype, increasing mitochondrial biogenesis regulation in mouse adipocytes (3T3-L1) (Choi et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Pagel-Langenickel et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Additionally, in rats with obesity-induced cardiomyopathy, adiponectin inhibited the exacerbated production of free radicals and proinflammatory cytokines and reduced NF-κB activation in the heart (Rani et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBCA is a potent molecule that exerts biological effects through multiple signal transduction pathways involved in cell differentiation, inflammation, and metabolism (Felix et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, studies focused on obesity have mainly examined the effect of this isoflavone on metabolism. Therefore, this study was conducted to evaluate whether BCA has an effect on inflammation in obesity-targeted tissues that develop after ovariectomy.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Animals\u003c/h2\u003e \u003cp\u003eThirty-five C57BL/6 healthy nonpregnant female mice obtained from the Central Animal House of the Federal University of Sergipe (UFS) were used. The animals, aged 17\u0026ndash;20 weeks, were maintained on a shelf with a controlled temperature (23\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C), an air exhaust system, and a 12/12 h light/dark cycle, and food and water were provided \u003cem\u003ead libitum\u003c/em\u003e. The animals were kept in polypropylene cages with dimensions of 29 \u0026times; 18 \u0026times; 16 cm in groups of 2 to 3 per cage. The animals were divided into groups based on the average weight of each cage. The experimental protocols were approved by the Animal Research Ethics Committee of the Federal University of Sergipe under registration numbers 57/16 and 14/18.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Ovariectomy\u003c/h2\u003e \u003cp\u003eThe mice underwent bilateral ovariectomy (OVX) surgery. The removal of the ovaries was performed through a dorsal incision after trichotomy and asepsis of the region, with the animals under anesthesia with ketamine (100 mg/kg) and xylazine (10 mg/kg). Some mice underwent sham surgery, in which the ovaries were exposed and immediately returned to the anatomical region. After that, the animals were kept in cages for a period of 15 days before starting the induction of obesity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Obesity induction and treatment\u003c/h2\u003e \u003cp\u003eA high-fat diet (HFD-26% carbohydrate, 59% lipids and 15% protein, with lard as a lipid source) was obtained from the company PragSolu\u0026ccedil;\u0026otilde;es Bioci\u0026ecirc;ncias\u0026copy;. The standard diet (SD) consisted of 62% carbohydrates, 13% lipids, and 25% proteins. Both diets contained a mix of vitamins and minerals according to the AIN-93 recommendation. The standard diet (SD) was provided to the OVX SD group, while the HFD was provided to the SHAM HFD, OVX HFD, and BCA 2 mg/kg or 6 mg/kg (i.p.) groups. After 9 full weeks (at the 10th week), BCA treatment was administered daily for 5 weeks. BCA was diluted in saline and 0.5% dimethyl sulfoxide (DMSO). Another group received 100 \u0026micro;L of vehicle (saline or 0.5% DMSO) via i.p. injection during the same period. Body weight was monitored twice a week. After this period, the animals were euthanized with an overdose of ketamine (100 mg/kg) and xylazine (10 mg/kg) for the collection of blood, adipose tissue, liver, spleen, kidney, gastrocnemius muscle, heart, and uterus.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Intraperitoneal glucose tolerance test\u003c/h2\u003e \u003cp\u003eMice were fasted for 5 hours to perform the glucose tolerance test. Three days before eutanasia, 1.5 mg/g D-glucose was administered to the animals, and glucose was measured via a glucometer (Bioland G500, Controller-SC\u0026reg;, Brazil) in a drop of blood obtained from the tail vein at 0, 15, 30, 60 and 120 min postinjection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Biochemical parameters\u003c/h2\u003e \u003cp\u003eBlood was collected from the retro-orbital plexus after a 10-hour fast. The blood was centrifuged at 10,000 rpm and 10\u0026deg;C for 10 minutes to separate the serum, which was subsequently stored at -20\u0026deg;C for later analysis. The concentrations of glucose and total cholesterol were investigated using commercial kits following the manufacturer's instructions (Labtest\u0026reg;).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Histology\u003c/h2\u003e \u003cp\u003eThe tissues were weighed, and samples of perigonadal AT from the same anatomical region were collected from all the animals. The tissues were stored in 4% paraformaldehyde, where they spent a minimum of 6 h and a maximum of 24 h at 5\u0026deg;C. Liver samples were kept in 10% buffered formalin at 25\u0026deg;C for at least 24 h for fixation. The tissues were immersed in graded alcohol and xylene and embedded in paraffin. Sections measuring approximately 5 \u0026micro;m were stained with hematoxylin-eosin (H\u0026amp;E), observed with an optical microscope and analyzed by a blinded evaluator.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.6.1. Adipose tissue (AT) analysis\u003c/h2\u003e \u003cp\u003eThe images captured from the perigonadal AT were used to determine the number of crown-like structures (CLSs) and adipocyte areas. Using ImageJ (RRID:SCR_003070) software, adipocytes were assessed by drawing ellipses around the cell membranes. The software scale was adjusted to a 100 \u0026micro;m scale at 400x magnification. In each analyzed image, 5 to 10 adipocytes were circumscribed, totaling at least 100 adipocytes per mouse.\u003c/p\u003e \u003cp\u003eAt 400x magnification, a covert evaluator counted the CLSs along the entire length of the tissue, and the result was normalized to 50 fields.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.6.2. Liver analysis\u003c/h2\u003e \u003cp\u003eTo determine the percentage of hepatic steatosis in liver samples, four histological sections of each specimen were analyzed. In each section, ten histological fields 0.06 mm\u003csup\u003e2\u003c/sup\u003e in length (magnification 400x) were selected and photomicrographed (with a Leica DFC340FX digital camera coupled to an Olympus CX31 optical microscope). The fields were selected by systematic randomization (for each selected field, two neglected fields followed). The determination of the percentage of liver tissue section area undergoing steatotic vacuolization was performed using the image analysis software ImageJ (RRID:SCR_003070). The program was previously calibrated, and once the measurement scale (%) was defined, the measurements were obtained through manual grayscale thresholding. The total section area under analysis (0.06 mm\u003csup\u003e2\u003c/sup\u003e) was considered 100%, and the percentage of area identified by the thresholding procedure was subtracted from this value to obtain the percentage of steatosis area in each histological field (400x). All morphometric measurements were carried out by a hidden evaluator and are expressed as a percentage of liver tissue section.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e2.7. Triglyceride content in the liver\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eTotal liver fat was quantified according to the principles of the method proposed by Folch et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1957\u003c/span\u003e), with slight modifications. Briefly, 1 g of liver tissue was homogenized with 1.5 ml of chloroform:methanol (2:1). The mixture was then filtered through fine filter paper, and for every 1 ml of filtrate, 200 \u0026micro;l of saline solution was added. The tubes were gently shaken by inversion three times. The homogenate was subsequently centrifuged for 10 minutes at 3000 rpm to separate the phases. A known volume of the lower phase (chloroform phase) was transferred to a previously weighed container, taken to an oven (60\u0026deg;C) for evaporation and resuspended in 1 ml of isopropanol. An aliquot was used for analysis of total triglycerides (Labtest, Brazil). Finally, the results were normalized to the weight of the tissue used in the analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Enzyme-linked immunosorbent assay (ELISA)\u003c/h2\u003e \u003cp\u003eThe proinflammatory cytokines IL-6 (Invitrogen\u0026trade;) and the anti-inflammatory cytokines IL-10 and IL-5 (R\u0026amp;D Systems\u0026copy;) were quantified in the perigonadal AT, liver and muscle using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's protocol. The results were subsequently normalized to the total protein concentration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Quantitative real-time PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from tissues using TRIzol (Thermo Fisher Scientific, USA). RNA quality and concentrations were measured by spectrophotometer using the 260/280 nm ratio. RNA (1 \u0026micro;g) was reverse transcribed to cDNA using the TaqMan\u0026trade; High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, USA) following the manufacturer's instructions. Relative quantification by real-time PCR was performed using TaqManTM Fast Advanced Master Mix (Thermo Fisher Scientific, USA) in a QuantiStudio\u0026trade; 5 Real-Time PCR System thermocycler (Thermo Fisher Scientific, USA) under the following conditions: 2 min at 50\u0026deg;C, 10 min at 95\u0026deg;C, 40 cycles of 15 s at 95\u0026deg;C and 1 min at 60\u0026deg;C. Amplification data were analyzed using QuantiStudio\u0026trade; design \u0026amp; analysis software v1.5.2. The following genes were evaluated via TaqMan assays: Mrc1 (Mm01329359_m1), Arg1 (Mm00475988_m1), Nos2 (Mm00440502_m1), and Pparγ (Mm00440940_m1). Gapdh (Mm99999915_g1; Thermo Fisher Scientific, USA) was used as a housekeeping gene. Relative quantities were determined using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Statistical analysis\u003c/h2\u003e \u003cp\u003eThe results are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM) or median and interquartile range for scores of histological parameters. Statistical analysis of the data was performed using the GraphPad Prism 8 program (RRID:SCR_002798). The Shapiro‒Wilk normality test was applied to the groups to assess whether the data followed a normal or parametric distribution. After confirming the normality of the data, \u003cem\u003eone-way\u003c/em\u003e or \u003cem\u003etwo-way\u003c/em\u003e ANOVA was performed, followed by Bonferroni's or Kruskal‒Wallis\u0026rsquo;s post hoc test for multiple comparisons. Values of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.1. BCA improves inflammation in adipose tissue without altering metabolism\u003c/h2\u003e \u003cp\u003eThe absence of estrogen caused by ovariectomy leads to metabolic dysfunctions in several tissues. First, an increase in weight was observed in the SHAM HFD group compared with the OVX, BCA 2 mg/kg and BCA 6 mg/kg groups beginning in the 3rd week of consumption of the high-fat diet (Fig.\u0026nbsp;1). A). Regarding diet consumption, the OVX HFD group showed an increase in weight starting from the 5th week compared to that of the OVX SD group (p\u0026thinsp;=\u0026thinsp;0.0297).\u003c/p\u003e \u003cp\u003eAlthough treatment with BCA did not reduce weight, at the 15th week, there was no significant difference in weight between the BCA 6 mg/kg group (39.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2 g) and the SHAM group (31.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3 g), unlike in the OVX groups (42.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8 g) and the BCA 2 mg/kg group (41.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1 g) (Fig.\u0026nbsp;1). A).\u003c/p\u003e \u003cp\u003eWeight gain was consistent with the adiposity index, in which the high-fat diet increased weight gain in the OVX SD group compared to the OVX HFD group (4.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 vs. 18.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), and ovariectomy added to this effect in the SHAM HFD group compared to the OVX HFD group (7.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 vs. 18.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Treatment did not change this parameter compared to that in the OVX HFD group (Fig.\u0026nbsp;1.B).\u003c/p\u003e \u003cp\u003eHistology analysis of adipose tissue revealed that obesity induced by HFD increased the adipocyte area in the OVX SD vs. OVX HFD groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and that ovariectomy enhanced this effect in the SHAM HFD vs. OVX HFD groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Unlike what was observed in the weight analysis, treatment with a dose of BCA at 6 mg/kg, but not at 2 mg/kg, promoted a reduction in adipocyte area (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 and p\u0026thinsp;=\u0026thinsp;0.2033) (Fig.\u0026nbsp;1. C).\u003c/p\u003e \u003cp\u003eThe crown-like structure (CLS) count is a widely used parameter for demonstrating inflammation in adipose tissue (Fig.\u0026nbsp;1). E) (Cinti et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). We observed that the CLS count in the tissue increased with HFD consumption in OVX SD vs OVX HFD animals (10.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1 vs 28.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8; p\u0026thinsp;=\u0026thinsp;0.0004) and that this parameter also increased after surgery in SHAM HFD vs OVX HFD animals (18.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5 vs 28.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8; p\u0026thinsp;=\u0026thinsp;0.0483). After treatment with 2 mg/kg or 6 mg/kg BCA, the WBC count was lower than that in the OVX HFD group (10.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1, 11.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 vs 28.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8; p\u0026thinsp;=\u0026thinsp;0.0001, p\u0026thinsp;=\u0026thinsp;0.0001) (Fig.\u0026nbsp;1D).\u003c/p\u003e \u003cp\u003eThe glucose tolerance test demonstrated that animals in the OVX SD group had lower glucose levels at 30 and 60 min than did those in the OVX HFD group (p\u0026thinsp;=\u0026thinsp;0.0170, p\u0026thinsp;=\u0026thinsp;0.0313) (Fig.\u0026nbsp;2. A).\u003c/p\u003e \u003cp\u003eThe scientific literature demonstrates that ovariectomy changes the blood glucose levels of animals (Louet et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), and this can be seen in our data, in which the OVX HFD, BCA 2 mg/kg and BCA 6 mg/kg groups had a greater AUC in the test than did the SHAM HFD (29125\u0026thinsp;\u0026plusmn;\u0026thinsp;854, 27078\u0026thinsp;\u0026plusmn;\u0026thinsp;559, 28270\u0026thinsp;\u0026plusmn;\u0026thinsp;923 vs 24209\u0026thinsp;\u0026plusmn;\u0026thinsp;1342). However, neither the BCA 2 mg/kg nor the BCA 6 mg/kg treatment changed the glucose concentration (Fig.\u0026nbsp;2. B).\u003c/p\u003e \u003cp\u003eFasting blood glucose and serum cholesterol levels were measured. As expected, compared with HFD consumption, HFD consumption increased fasting blood glucose and cholesterol levels in the OVX HFD animals (264\u0026thinsp;\u0026plusmn;\u0026thinsp;22.2 vs 160\u0026thinsp;\u0026plusmn;\u0026thinsp;25.7; p\u0026thinsp;=\u0026thinsp;0.0255 and 148.4\u0026thinsp;\u0026plusmn;\u0026thinsp;9.6 vs 85.3\u0026thinsp;\u0026plusmn;\u0026thinsp;12; p\u0026thinsp;=\u0026thinsp;0.0061). When comparing the SHAM HFD and OVX HFD groups, we observed that only fasting blood glucose was greater in the ovariectomized group (132\u0026thinsp;\u0026plusmn;\u0026thinsp;18 vs 264\u0026thinsp;\u0026plusmn;\u0026thinsp;22.2; p\u0026thinsp;=\u0026thinsp;0.011). Treatment with BCA did not alter the blood glucose or cholesterol levels of the animals at either of the doses used (Fig.\u0026nbsp;2. C and D).\u003c/p\u003e \u003cp\u003eTo assess the effect of HFD consumption and OVX on tissue weight (liver, muscle, heart, spleen, kidney, and uterus), the index was calculated using tibia length. Only the heart indices were greater in the OVX HFD animals than in the SHAM HFD (p\u0026thinsp;=\u0026thinsp;0.0487) and OVX SD (p\u0026thinsp;=\u0026thinsp;0.0321) animals. The groups treated with either dose of BCA showed no change in this index compared to the OVX HFD or SHAM HFD groups.\u003c/p\u003e \u003cp\u003eThe uterus volume was lower in the ovariectomized animals, OVX HFD, 2 mg/kg BCA, and 6 mg/kg BCA groups than in the SHAM HFD group, as expected (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, and p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, respectively) (Table\u0026nbsp;1). This demonstrated the effectiveness of the ovariectomy\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.2. BCA treatment improves steatosis in the liver\u003c/h2\u003e \u003cp\u003eWhen assessing liver damage, large intracytoplasmic fat vacuoles accumulated, promoting cell ballooning. To a lesser extent, areas of microvesicular steatosis were evident and characterized by the formation of multiple intracytoplasmic lipid vacuoles. Mononuclear inflammatory infiltrates were observed in all the groups that consumed a HFD; however, these infiltrates were considered mild and had an irregular distribution (Fig.\u0026nbsp;3. A, C, D, E).\u003c/p\u003e \u003cp\u003eThe percentage of patients with hepatic steatosis was calculated only for the groups that received a HFD. The OVX group had a greater percentage of hepatic steatosis than the SHAM HFD group (88\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4% vs 75\u0026thinsp;\u0026plusmn;\u0026thinsp;1%; p\u0026thinsp;=\u0026thinsp;0.0095) (Fig.\u0026nbsp;3. F), and both doses of BCA reduced hepatic steatosis compared to that in the OVX HFD group (32\u0026thinsp;\u0026plusmn;\u0026thinsp;6.4% and 33.4\u0026thinsp;\u0026plusmn;\u0026thinsp;7%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003cp\u003eThe quantification of TAG in the liver was performed for all groups (Fig.\u0026nbsp;3. G). As expected, the OVX SD group had a lower TAG content in the liver than the OVX HFD group (149.4\u0026thinsp;\u0026plusmn;\u0026thinsp;13.4 vs 876.4\u0026thinsp;\u0026plusmn;\u0026thinsp;201.7; p\u0026thinsp;=\u0026thinsp;0.0027), and ovariectomy also promoted an increase in this content in the OVX HFD vs. SHAM HFD animals (876.4\u0026thinsp;\u0026plusmn;\u0026thinsp;201.7 vs 233.7\u0026thinsp;\u0026plusmn;\u0026thinsp;28.2; p\u0026thinsp;=\u0026thinsp;0.0091). Finally, compared with those in the OVX HFD group, the animals in the BCA 6 mg/kg group, but not those in the 2 mg/kg dose group, had lower TAG levels (876.4\u0026thinsp;\u0026plusmn;\u0026thinsp;201.7 vs 362.3\u0026thinsp;\u0026plusmn;\u0026thinsp;28.9; p\u0026thinsp;=\u0026thinsp;0.0178) (Fig.\u0026nbsp;3. G).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Effect of BCA on Anti-inflammatory activity in adipose tissue (AT)\u003c/h2\u003e \u003cp\u003eThe inflammatory profile of the tissues was investigated by measuring the concentrations of proinflammatory cytokines (IL-6) and anti-inflammatory cytokines (IL-5 and IL-10) in tissues affected by lipotoxicity, such as AT (adipose tissue), liver, and muscle (Fig.\u0026nbsp;4). The concentration of the cytokine IL-6 was not altered in the AT or liver in the OVX HFD, BCA 2 mg/kg, or 6 mg/kg groups (Fig.\u0026nbsp;4. A and D). However, in the muscle, treatment with 2 mg/kg BCA reduced the IL-6 concentration compared to that in the OVX HFD group (0.019\u0026thinsp;\u0026plusmn;\u0026thinsp;0.016 vs 0.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1; p\u0026thinsp;=\u0026thinsp;0.0342) (Fig.\u0026nbsp;4. G). Regarding the anti-inflammatory cytokines, an increase in the IL-5 concentration was observed in the ATs of animals treated only with 2 mg/kg BCA (311.4\u0026thinsp;\u0026plusmn;\u0026thinsp;58.6 vs 100.7\u0026thinsp;\u0026plusmn;\u0026thinsp;16; p\u0026thinsp;=\u0026thinsp;0.0023) and in the IL-10 concentration at both BCA doses (199\u0026thinsp;\u0026plusmn;\u0026thinsp;18.4, 167.4\u0026thinsp;\u0026plusmn;\u0026thinsp;10.7 vs 81.7\u0026thinsp;\u0026plusmn;\u0026thinsp;9.3; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, p\u0026thinsp;=\u0026thinsp;0.0004) compared to those in the OVX HFD group (Fig.\u0026nbsp;4. B, C).\u003c/p\u003e \u003cp\u003eThus, the expression of genes linked to macrophage polarization and PPARγ was evaluated.\u003c/p\u003e \u003cp\u003eWe found that, in AT, there was no change in the expression of the \u003cem\u003eNos2\u003c/em\u003e mRNA (which represents type M1 macrophages) (Fig.\u0026nbsp;5. A). However, in the BCA 6 mg/kg group, there was greater expression of \u003cem\u003eMrc1\u003c/em\u003e mRNA (M2 macrophages) than in the OVX HFD group (12.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6 vs 4.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8, p\u0026thinsp;=\u0026thinsp;0.0320) (Fig.\u0026nbsp;5. B). Furthermore, there was greater expression of \u003cem\u003ePparγ\u003c/em\u003e mRNA in the 6 mg/kg BCA group than in the OVX HFD group (11.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 vs 3.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2, p\u0026thinsp;=\u0026thinsp;0.0338) (Fig.\u0026nbsp;5. C)\u003c/p\u003e \u003cp\u003eIn the liver, BCA at 2 mg/kg or 6 mg/kg reduced the expression of the genes associated with the M1 macrophage profile (\u003cem\u003eNos2\u003c/em\u003e), but not M2 (\u003cem\u003eArg1\u003c/em\u003e) or \u003cem\u003ePparγ\u003c/em\u003e, compared to that in the OVX HFD group (0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09, 0.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 vs. 2.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5; p\u0026thinsp;\u0026gt;\u0026thinsp;0.0001, p\u0026thinsp;\u0026gt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;5. E-F).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe anti-inflammatory effect of biochanin A (BCA) has been described in various \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e models (Anuranjana et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Feng and Lai \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Felix et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Dietary consumption of BCA has previously been investigated in cell lines, mice, and male rats, and the anti-obesity effect of boric acid has been shown to increase (Rathinasamy et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Choi et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Su et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe dataset found in the literature on the anti-inflammatory effect of BCA demonstrates that this isoflavone acts on various targets to prevent/reduce the inflammatory process, such as through the inhibition of NF-κB and MAPK via positive regulation of PPAR-γ (Felix et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Sarfraz et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Nevertheless, no study has investigated the anti-inflammatory effect of this phytoestrogen on menopause in the context of obesity. Therefore, the present study was the first to evaluate the anti-inflammatory effect of BCA after establishing obesity in OVX animals.\u003c/p\u003e \u003cp\u003eAs a phytoestrogen, BCA has a structure similar to that of estrogen. Therefore, these effects may be dependent on interactions with estrogen receptor beta (ER-β) or GPR30 (Felix et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Escande et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Ovariectomy is a widely used surgery in the scientific literature that depletes endogenous estrogen in animals (oophorectomy in humans), mimicking the physiological condition of menopause. The hypoestrogenic effect of ovariectomy leads to increased visceral fat, inflammation, oxidative stress, and metabolic complications such as dyslipidemia, hepatic steatosis, and predisposition to cardiovascular diseases (Medina-Contreras et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Davis et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). This was clearly demonstrated in our study, with an increase in weight and adiposity and a worsened glycemic profile in the OVX HFD animals.\u003c/p\u003e \u003cp\u003eDuring the development of obesity, the expansion of adipose tissue (AT) culminates in a web of inflammatory and metabolic events that challenge proposed therapies. In addition, in the menopausal phase, estrogen deficiency makes the environment even less favorable for treatment.\u003c/p\u003e \u003cp\u003eIn the early days after the consumption of a HFD, prior to adipocyte hypertrophy, there is an increase in the number of neutrophils that release enzymes and degrade IRS-1, inducing insulin resistance (IR) (Delgado-Rizo et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Elgazar-Carmon et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). As weeks progress, highly inflammatory macrophages influx into ATs, forming CLSs and releasing chemokines and cytokines that recruit these leukocytes (Kosteli et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). We did not observe metabolic changes in the animals (weight, blood glucose, or cholesterol); however, a reduction in inflammation markers in the adipose tissue (AT) was identified.\u003c/p\u003e \u003cp\u003eCLSs are a pathological hallmark of obesity, and the elimination of adipocyte residues is suggested to be an important function of macrophages in obese individuals. The frequency of adipocyte death is positively correlated with increased adipocyte size in obese mice (Cinti et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Consistent with these findings, OVX animals had increased CLS counts, and BCA showed anti-inflammatory potential by reducing CLS.\u003c/p\u003e \u003cp\u003eSubsequently, we demonstrated that BCA reduces steatosis and TAG accumulation in the liver. The antisteatotic effect of BCA was previously investigated by Park et al. (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), who demonstrated that, in male mice fed BCA, phytoestrogen promoted the recovery of metabolites linked to lipogenesis and beta-oxidation and suppressed the expression of enzymes linked to glucose metabolism. Recently, Fan et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) showed that NAFLD progression was prevented in rats fed a BCA diet, and this effect may be linked to the reduction in PPARγ expression. Unfortunately, we did not find differences in the expression of this receptor in the liver. However, it is possible to associate this result with the fact that the isoform primarily expressed in the liver is PPARα, and the expression of PPARγ in female mice may differ from that in male mice in terms of the levels of the PPARγ1 and PPARγ2 isoforms (Berthier et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe anti-inflammatory effect of BCA in the liver was also observed. We found low expression of the Nos2 gene, which encodes the enzyme inducible nitric oxide synthase (iNOS), considered a hallmark of inflammation, which is activated during obesity (Qian et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The enzyme is expressed in Kupffer cells (Zhang \u0026amp; Lang \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and in nonalcoholic fatty liver disease (NAFLD). This enzyme has been strongly associated with insulin resistance due to impaired autophagy mechanisms in hepatocytes during diet-induced obesity (Qian et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and in Kupffer cells and is linked to the development of NAFLD (Jin et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Although we do not have enough data to confirm this phenomenon, these findings indicate that the protective effect of BCA on the liver against steatosis may be linked to a reduction in the expression of the enzyme.\u003c/p\u003e \u003cp\u003eAT is governed by molecules and immune cells that dictate the inflammatory profile. Eosinophils are important cytokine-producing leukocytes with a Th2 profile that include cytokines such as IL-5 and IL-10 and support metabolic homeostasis in AT during a healthy state (Castoldi et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Qiu et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Molofsky et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLee et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) observed that these cells are important for improving the tissue metabolic profile in eosinophil-deficient mice and are inversely associated with adiposity and IR (Wu et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Another group reported that the return of these cells, induced by IL-5, to a physiological state did not restore glucose tolerance in obese animals (Lee et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Thus, the effect found in the present study corroborates these findings, as the increase in IL-5 in the AT of obese ovariectomized animals treated with BCA did not improve glucose intolerance.\u003c/p\u003e \u003cp\u003eAnother important aspect of AT inflammation during obesity is the accumulation of macrophages. The flow of saturated fatty acids to ATs during HFD consumption is an important regulator of inflammation, monocyte recruitment, and polarization to M1 (Coenen et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Lumeng et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Weisberg et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe role of IL-10 in inducing M2 macrophages has been well described (Castoldi et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). This cytokine is secreted by macrophages and regulatory T lymphocytes and exerts anti-inflammatory effects, increasing sensitivity to insulin, opposing TNF-α in inducing IR in 3T3L adipocytes (Lumeng et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eUnder healthy conditions, adipose tissue (AT) produces IL-13 along with PPARγ and PPARβ/δ, which promote the activation of M2 macrophages and suppress the activation of M1 macrophages via the production of adiponectin and IL-10 (Olefsky \u0026amp; Glass \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Thus, a likely shift in the macrophage profile toward M2 was observed in the ATs of animals treated with BCA, represented by an increase in the anti-inflammatory cytokines IL-5 and IL-10 and the gene expression of Mrc1 (mannose receptor), which are linked to the M2 macrophage phenotype (Zhou et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). These findings reinforce the anti-inflammatory effect of BCA on the AT of obese mice. Although we did not explore the mechanism of action of BCA in depth, we propose that these results in AT are linked to the PPARγ receptor, which is highly expressed in tissue.\u003c/p\u003e \u003cp\u003eThis study has limitations regarding the route of administration, but it demonstrates this important finding in a menopause obesity model. We showed here that the effect of BCA was not directly related to the antihyperlipidemic and antihyperglycemic effects already demonstrated in the literature but rather to its anti-inflammatory effect. Thus, the mechanisms of action involving the activation of the PPARγ receptor, NFκB, and MAPK to promote the observed results should be further investigated.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was performed in\u0026nbsp;accordance\u0026nbsp;with the principles of the Declaration of Helsinki. Approval was granted by the Animal Research Ethics Committee of the Federal University of Sergipe under registration numbers 57/16 and 14/18.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by\u0026nbsp;the\u0026nbsp;National Council for Scientific and Technological Development (CNPQ) and the Higher Education Personnel Improvement Coordination (CAPES). EAC is a beneficiary of\u0026nbsp;the\u0026nbsp;CNPq scientific productivity grant (CNPq: 315369/2021-3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the data supporting the findings of this study are available within the paper. Should any raw data files be needed in another format,\u0026nbsp;they are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors contributed to this study as follows: \u003cstrong\u003eJ\u0026eacute;ssica M. D. Ara\u0026uacute;jo:\u003c/strong\u003e conceptualization, methodology, formal analysis and investigation, writing - original draft preparation review and editing; \u003cstrong\u003eLuana Heimfarth:\u003c/strong\u003e methodology, formal analysis and investigation, writing - review and editing; \u003cstrong\u003eWemerson S. Neres:\u0026nbsp;\u003c/strong\u003emethodology, formal analysis and investigation, writing - review and editing; \u003cstrong\u003eFranciel B. F\u0026eacute;lix:\u003c/strong\u003e formal analysis and investigation, writing - review and editing; \u003cstrong\u003ePatr\u0026iacute;cia R. Santos:\u003c/strong\u003e methodology, formal analysis and investigation, writing - review and editing; \u003cstrong\u003eFab\u0026iacute;ula F. Abreu:\u0026nbsp;\u003c/strong\u003eformal analysis and investigation, writing - review and editing; \u003cstrong\u003eLuana M. Cercato:\u003c/strong\u003e formal analysis and investigation, writing - review and editing; \u003cstrong\u003eAna C. S. Nascimento:\u003c/strong\u003e formal analysis and investigation, writing - review and editing; \u003cstrong\u003eAlan B. S. Vasconcelos:\u003c/strong\u003e formal analysis and investigation, writing - review and editing; \u003cstrong\u003eRosilene C. Soares:\u003c/strong\u003e writing - review and editing, resources; \u003cstrong\u003eRicardo Albuquerque:\u003c/strong\u003e methodology,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eformal analysis and investigation, writing - review and editing;\u0026nbsp;\u003cstrong\u003eGreice I. Heidend:\u003c/strong\u003e methodology, formal analysis and investigation\u003c/p\u003e\n\u003cp\u003eAll the authors reviewed and approved the final version of the manuscript. The authors declare that all the data were generated in house and that no paper mill was used.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAnuranjana PV, Beegum F, K P D, George KT, Viswanatha GL, Nayak PG, Kanwal A, Kishore A, Shenoy RR, Nandakumar K (2023) Mechanisms Behind the Pharmacological Application of Biochanin-A: A review. F1000Res 12:107. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.12688/f1000research.126059.3\u003c/span\u003e\u003cspan address=\"10.12688/f1000research.126059.3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBerthier A, Johanns M, Zummo FP, Lefebvre P, Staels B (2021) PPARs in liver physiology. Biochim Biophys Acta Mol Basis Dis 1867(5):166097. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bbadis.2021.166097\u003c/span\u003e\u003cspan address=\"10.1016/j.bbadis.2021.166097\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBolton JL (2016) Menopausal Hormone Therapy, Age, and Chronic Diseases: Perspectives on Statistical Trends. Chem Res Toxicol 29:1583\u0026ndash;1590. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.chemrestox.6b00272\u003c/span\u003e\u003cspan address=\"10.1021/acs.chemrestox.6b00272\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCamacho S, Ruppel A (2017) Is the calorie concept a real solution to the obesity epidemic? Glob Health Action 1289650. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/16549716.2017.1289650\u003c/span\u003e\u003cspan address=\"10.1080/16549716.2017.1289650\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCampbell SE, Febbraio MA (2002) Effect of the ovarian hormones on GLUT4 expression and contraction-stimulated glucose uptake. Am J Physiol Endocrinol Metab 282:E1139\u0026ndash;E1146. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1152/ajpendo.00184.2001\u003c/span\u003e\u003cspan address=\"10.1152/ajpendo.00184.2001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCastoldi A, Naffah de Souza C, C\u0026acirc;mara NO, Moraes-Vieira PM (2016) The Macrophage Switch in Obesity Development. Front Immunol 6:637. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fimmu.2015.00637\u003c/span\u003e\u003cspan address=\"10.3389/fimmu.2015.00637\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen WY (2011) Postmenopausal hormone therapy and breast cancer risk: current status and unanswered questions. Endocrinol Metab Clin North Am 40. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ecl.2011.05.006\u003c/span\u003e\u003cspan address=\"10.1016/j.ecl.2011.05.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. 509\u0026thinsp;\u0026ndash;\u0026thinsp;18\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChoi EM, Suh KS, Park SY, Chin SO, Rhee SY, Chon S (2019) Biochanin A prevents 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced adipocyte dysfunction in cultured 3T3-L1 cells. J Environ Sci Health Tox Hazard Subst Environ Eng 54:865\u0026ndash;873. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/10934529.2019.1603746\u003c/span\u003e\u003cspan address=\"10.1080/10934529.2019.1603746\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCinti S, Mitchell G, Barbatelli G, Murano I, Ceresi E, Faloia E, Wang S, Fortier M, Greenberg AS, Obin MS (2005) Adipocyte death defines macrophage localization and function in adipose tissue of obese mice and humans. J Lipid Res 46:2347\u0026ndash;2355. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1194/jlr.M500294-JLR200\u003c/span\u003e\u003cspan address=\"10.1194/jlr.M500294-JLR200\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoenen KR, Gruen ML, Chait A, Hasty AH (2007) Diet-induced increases in adiposity, but not plasma lipids, promote macrophage infiltration into white adipose tissue. Diabetes 3:64\u0026ndash;73. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2337/db06-1375\u003c/span\u003e\u003cspan address=\"10.2337/db06-1375\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDavis SR, Lambrinoudaki I, Lumsden M, Mishra GD, Pal L, Rees M, Santoro N, Simoncini T (2015) Menopause. Nat Rev Dis Primers 15004. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nrdp.2015.4\u003c/span\u003e\u003cspan address=\"10.1038/nrdp.2015.4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDelgado-Rizo V, Mart\u0026iacute;nez-Guzm\u0026aacute;n MA, I\u0026ntilde;iguez-Gutierrez L, Garc\u0026iacute;a-Orozco A, Alvarado-Navarro A, Fafutis-Morris M (2017) Neutrophil Extracellular Traps and Its Implications in Inflammation: An Overview. Front Immunol 8:81. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fimmu.2017.00081\u003c/span\u003e\u003cspan address=\"10.3389/fimmu.2017.00081\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEaton SA, Sethi JK (2019) Immunometabolic Links between Estrogen, Adipose Tissue and Female Reproductive Metabolism. Biology (Basel) 8:8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/biology8010008\u003c/span\u003e\u003cspan address=\"10.3390/biology8010008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElgazar-Carmon V, Rudich A, Hadad N, Levy R (2008) Neutrophils transiently infiltrate intra-abdominal fat early in the course of high-fat feeding. J Lipid Res 9:1894\u0026ndash;1903. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1194/jlr.M800132-JLR200\u003c/span\u003e\u003cspan address=\"10.1194/jlr.M800132-JLR200\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEscande A, Pillon A, Servant N, Cravedi JP, Larrea F, Muhn P, Nicolas JC, Cavaill\u0026egrave;s V, Balaguer P (2006) Evaluation of ligand selectivity using reporter cell lines stably expressing estrogen receptor alpha or beta. Biochem Pharmacol 10:1459\u0026ndash;1469. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bcp.2006.02.002\u003c/span\u003e\u003cspan address=\"10.1016/j.bcp.2006.02.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFait T (2019) Menopause hormone therapy: latest developments and clinical practice. Drugs Context 8:212551. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.7573/dic.212551\u003c/span\u003e\u003cspan address=\"10.7573/dic.212551\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFan Y, Yan LT, Yao Z, Xiong GY (2021) Biochanin A Regulates Cholesterol Metabolism Further Delays the Progression of Nonalcoholic Fatty Liver Disease. Diabetes Metab Syndr Obes 14:3161\u0026ndash;3172. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2147/DMSO.S315471\u003c/span\u003e\u003cspan address=\"10.2147/DMSO.S315471\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFelix FB, Ara\u0026uacute;jo JMD, de Souza EV, Pinho V, Camargo EA, Corr\u0026ecirc;a CB, Grespan R (2020) Biochanin A attenuates zymosan-induced arthritis in mice similarly to 17-β estradiol: an alternative to hormone replacement therapy? Inflamm Res 12:1245\u0026ndash;1256. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00011-020-01403-4\u003c/span\u003e\u003cspan address=\"10.1007/s00011-020-01403-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFelix FB, Vago JP, Beltrami VA, Ara\u0026uacute;jo JMD, Grespan R, Teixeira MM, Pinho V (2022) Biochanin A as a modulator of the inflammatory response: An updated overview and therapeutic potential. Pharmacol Res 180. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.phrs.2022.106246\u003c/span\u003e\u003cspan address=\"10.1016/j.phrs.2022.106246\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFelix FB, Vago JP, Fernandes DO, Martins DG, Moreira IZ, Gon\u0026ccedil;alves WA, Costa WC, Ara\u0026uacute;jo JMD, Queiroz-Junior CM, Campolina-Silva GH, Soriani FM, Sousa LP, Grespan R, Teixeira MM, Pinho V (2021) Biochanin A Regulates Key Steps of Inflammation Resolution in a Model of Antigen-Induced Arthritis via GPR30/PKA-Dependent Mechanism. Front Pharmacol 12:662308. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fphar.2021.662308\u003c/span\u003e\u003cspan address=\"10.3389/fphar.2021.662308\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeng ZJ, Lai WF (2023) Chemical and Biological Properties of Biochanin A and Its Pharmaceutical Applications. Pharmaceutics 4:1105. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/pharmaceutics15041105\u003c/span\u003e\u003cspan address=\"10.3390/pharmaceutics15041105\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFolch J, Lees M, Sloane Stanley GH (1957) A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 226:497\u0026ndash;509. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0021-9258(18)64849-5\u003c/span\u003e\u003cspan address=\"10.1016/S0021-9258(18)64849-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJin G, Yao X, Liu D, Zhang J, Zhang X, Yang Y, Bi Y, Zhang H, Dong G, Tang H, Cheng S, Hong F, Si M (2023) Inducible nitric oxide synthase accelerates nonalcoholic fatty liver disease progression by regulating macrophage autophagy. Immun Inflamm Dis 12:e1114. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/iid3.1114\u003c/span\u003e\u003cspan address=\"10.1002/iid3.1114\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKo SH, Kim HS (2020) Menopause-Associated Lipid Metabolic Disorders and Foods Beneficial for Postmenopausal Women. Nutrients 12:202. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/nu12010202\u003c/span\u003e\u003cspan address=\"10.3390/nu12010202\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKosteli A, Sugaru E, Haemmerle G, Martin JF, Lei J, Zechner R, Ferrante AW Jr (2010) Weight loss and lipolysis promote a dynamic immune response in murine adipose tissue. J Clin Invest 10:3466\u0026ndash;3479. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1172/JCI42845\u003c/span\u003e\u003cspan address=\"10.1172/JCI42845\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee EH, Itan M, Jang J, Gu HJ, Rozenberg P, Mingler MK, Wen T, Yoon J, Park SY, Roh JY, Choi CS, Park WJ, Munitz A, Jung Y (2018) Eosinophils support adipocyte maturation and promote glucose tolerance in obesity. Sci Rep 1:9894. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-018-28371-4\u003c/span\u003e\u003cspan address=\"10.1038/s41598-018-28371-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLongo M, Zatterale F, Naderi J, Parrillo L, Formisano P, Raciti GA, Beguinot F, Miele C (2019) Adipose Tissue Dysfunction as Determinant of Obesity-Associated Metabolic Complications. Int J Mol Sci 20:2358. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijms20092358\u003c/span\u003e\u003cspan address=\"10.3390/ijms20092358\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLouet JF, LeMay C, Mauvais-Jarvis F (2004) Antidiabetic actions of estrogen: insight from human and genetic mouse models. Curr Atheroscler Rep 3:180\u0026ndash;185. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11883-004-0030-9\u003c/span\u003e\u003cspan address=\"10.1007/s11883-004-0030-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLovre D, Lindsey SH, Mauvais-Jarvis F (2016) Effect of menopausal hormone therapy on components of the metabolic syndrome. Ther Adv Cardiovasc Dis 11:33\u0026ndash;43. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1177/1753944716649358\u003c/span\u003e\u003cspan address=\"10.1177/1753944716649358\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLumeng CN, Deyoung SM, Bodzin JL, Saltiel AR (2007) Increased inflammatory properties of adipose tissue macrophages recruited during diet-induced obesity. Diabetes 1:16\u0026ndash;23. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2337/db06-1076\u003c/span\u003e\u003cspan address=\"10.2337/db06-1076\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMedina-Contreras J, Villalobos-Molina R, Zarain-Herzberg A, Balderas-Villalobos J (2020) Ovariectomized rodents as a menopausal metabolic syndrome model. A minireview. Mol Cell Biochem 475:261\u0026ndash;276. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11010-020-03879-4\u003c/span\u003e\u003cspan address=\"10.1007/s11010-020-03879-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMolofsky AB, Nussbaum JC, Liang HE, Van Dyken SJ, Cheng LE, Mohapatra A, Chawla A, Locksley RM (2013) Innate lymphoid type 2 cells sustain visceral adipose tissue eosinophils and alternatively activated macrophages. J Exp Med 3:535\u0026ndash;549. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1084/jem.20121964\u003c/span\u003e\u003cspan address=\"10.1084/jem.20121964\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoreira AC, Silva AM, Santos MS, Sard\u0026atilde;o VA (2014) Phytoestrogens as alternative hormone replacement therapy in menopause: What is real, what is unknown. J Steroid Biochem Mol Biol 143:61\u0026ndash;71. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jsbmb.2014.01.016\u003c/span\u003e\u003cspan address=\"10.1016/j.jsbmb.2014.01.016\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOlefsky JM, Glass CK (2010) Macrophages, inflammation, and insulin resistance. Annu Rev Physiol 72:219\u0026ndash;246. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1146/annurev-physiol-021909-135846\u003c/span\u003e\u003cspan address=\"10.1146/annurev-physiol-021909-135846\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePagel-Langenickel I, Bao J, Joseph JJ, Schwartz DR, Mantell BS, Xu X, Raghavachari N, Sack MN (2008) PGC-1alpha integrates insulin signaling, mitochondrial regulation, and bioenergetic function in skeletal muscle. J Biol Chem 283:22464\u0026ndash;22472. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1074/jbc.M800842200\u003c/span\u003e\u003cspan address=\"10.1074/jbc.M800842200\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePark HS, Hur HJ, Kim SH, Park SJ, Hong MJ, Sung MJ, Kwon DY, Kim MS (2016) Biochanin A improves hepatic steatosis and insulin resistance by regulating the hepatic lipid and glucose metabolic pathways in diet-induced obese mice. Mol Nutr Food Res 9:1944\u0026ndash;1955. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/mnfr.201500689\u003c/span\u003e\u003cspan address=\"10.1002/mnfr.201500689\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePepe G, Braga D, Renzi TA, Villa A, Bolego C, D'Avila F, Barlassina C, Maggi A, Locati M, Vegeto E (2017) Self-renewal and phenotypic conversion are the main physiological responses of macrophages to the endogenous estrogen surge. Sci Rep 20:44270. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/srep44270\u003c/span\u003e\u003cspan address=\"10.1038/srep44270\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQian Q, Zhang Z, Li M, Savage K, Cheng D, Rauckhorst AJ, Ankrum JA, Taylor EB, Ding WX, Xiao Y, Cao HJ, Yang L (2019) Hepatic Lysosomal iNOS Activity Impairs Autophagy in Obesity. Cell Mol Gastroenterol Hepatol 1:95\u0026ndash;110. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jcmgh.2019.03.005\u003c/span\u003e\u003cspan address=\"10.1016/j.jcmgh.2019.03.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQiu Y, Nguyen KD, Odegaard JI, Cui X, Tian X, Locksley RM, Palmiter RD, Chawla A (2014) Eosinophils and type 2 cytokine signaling in macrophages orchestrate development of functional beige fat. Cell 6:1292\u0026ndash;1308. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cell.2014.03.066\u003c/span\u003e\u003cspan address=\"10.1016/j.cell.2014.03.066\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRani AJI, Uddandrao VVS, Vadivukkarasi GSGSPCSSPTPPS S (2021) Biochanin A attenuates obesity cardiomyopathy in rats by inhibiting oxidative stress and inflammation through the Nrf-2 pathway. Arch Physiol Biochem 1\u0026ndash;16. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/13813455.2021.1874017\u003c/span\u003e\u003cspan address=\"10.1080/13813455.2021.1874017\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRathinasamy AJIR, Uddandrao VVS, Raveendran N, Sasikumar V (2020) Antiobesity Effect of Biochanin-A: Effect on Trace Element Metabolism in High Fat Diet-Induced Obesity in Rats. Cardiovasc Hematol Agents Med Chem 1:21\u0026ndash;30. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2174/1871524920666200207101920\u003c/span\u003e\u003cspan address=\"10.2174/1871524920666200207101920\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRossouw JE, Anderson GL, Prentice RL, LaCroix AZ, Kooperberg C, Stefanick ML, Jackson RD, Beresford SA, Howard BV, Johnson KC, Kotchen JM, Ockene J (2002) Writing Group for the Women's Health Initiative Investigators. Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results From the Women's Health Initiative randomized controlled trial. JAMA 288:321\u0026ndash;333. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1001/jama.288.3.321\u003c/span\u003e\u003cspan address=\"10.1001/jama.288.3.321\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSarfraz A, Javeed M, Shah MA, Hussain G, Shafiq N, Sarfraz I, Riaz A, Sadiqa A, Zara R, Zafar S, Kanwal L, Sarker SD, Rasul A (2020) Biochanin A: A novel bioactive multifunctional compound from nature. Sci Total Environ 722:137907. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.scitotenv.2020.137907\u003c/span\u003e\u003cspan address=\"10.1016/j.scitotenv.2020.137907\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSu SJ, Yeh YT, Su SH, Chang KL, Shyu HW, Chen KM, Yeh H (2013) Biochanin a promotes osteogenic but inhibits adipogenic differentiation: evidence with primary adipose-derived stem cells. Evid Based Complement Alternat Med 846039. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1155/2013/846039\u003c/span\u003e\u003cspan address=\"10.1155/2013/846039\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVlaisavljevic S, Kaurinovic B, Popovic M, Djurendic-Brenesel M, Vasiljevic B, Cvetkovic D, Vasiljevic S (2014) \u003cem\u003eTrifolium pratense\u003c/em\u003e L. as a potential natural antioxidante. Molecules 19:713\u0026ndash;725. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/molecules19010713\u003c/span\u003e\u003cspan address=\"10.3390/molecules19010713\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeisberg SP, McCann D, Desai M, Rosenbaum M, Leibel RL, Ferrante AW Jr (2003) Obesity is associated with macrophage accumulation in adipose tissue. J Clin Invest 12:1796\u0026ndash;1808. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1172/JCI19246\u003c/span\u003e\u003cspan address=\"10.1172/JCI19246\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu D, Molofsky AB, Liang HE, Ricardo-Gonzalez RR, Jouihan HA, Bando JK, Chawla A, Locksley RM (2011) Eosinophils sustain adipose alternatively activated macrophages associated with glucose homeostasis. Science 6026:243\u0026ndash;247. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1126/science.1201475\u003c/span\u003e\u003cspan address=\"10.1126/science.1201475\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu C, Zhang P, Lou L, Wang Y (2019) Perspectives Regarding the Role of Biochanin A in Humans. Front Pharmacol 10:793. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fphar.2019.00793\u003c/span\u003e\u003cspan address=\"10.3389/fphar.2019.00793\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang W, Lang R (2023) Macrophage metabolism in nonalcoholic fatty liver disease. Front Immunol 14:1257596. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fimmu.2023.1257596\u003c/span\u003e\u003cspan address=\"10.3389/fimmu.2023.1257596\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou Y, Do DC, Ishmael FT, Squadrito ML, Tang HM, Tang HL, Hsu MH, Qiu L, Li C, Zhang Y, Becker KG, Wan M, Huang SK, Gao P (2018) Mannose receptor modulates macrophage polarization and allergic inflammation through miR-511-3p. J Allergy Clin Immunol 1:350\u0026ndash;364. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jaci.2017.04.049\u003c/span\u003e\u003cspan address=\"10.1016/j.jaci.2017.04.049\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"a73f55c5-d0c3-4ebf-b0db-a5c04815027e","identifier":"10.13039/501100003593","name":"Conselho Nacional de Desenvolvimento Científico e Tecnológico","awardNumber":"315369/2021-3","order_by":0}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Federal University of Sergipe","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":"phytoestrogens, high-fat diet, inflammation, female castration","lastPublishedDoi":"10.21203/rs.3.rs-5188359/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5188359/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBiochanin A (BCA) is a phytoestrogen widely studied for its ability to alleviate menopausal symptoms and treat metabolic diseases in the presence or absence of estrogen. This study was conducted to investigate the effect of BCA in ovariectomized (OVX) mice subjected to a high-fat diet (HFD). To this end, female C57BL6 mice were randomly divided into 5 groups: SHAM (sham-operated) with HFD, OVX with a standard diet (SD) or HFD, and two other OVX groups with HFD treated with BCA (2 mg/kg or 6 mg/kg, i.p.) during the last 30 days. The experiment lasted 15 weeks, after which it was observed that the OVX HFD animals presented a compromised metabolic profile compared to the SHAM HFD or OVX SD animals. When evaluating the BCA-treated groups in comparison to the OVX HFD group, it was demonstrated that there was less inflammation in the AT due to the reduction in crown-like structures (CLS) and the increase in the adipocyte area. This effect was complemented by an increase in the levels of the cytokines IL-5 and IL-10 and an increase in the expression of Mrc1, a marker of M2 macrophages, and Pparγ, a key regulator of tissue metabolism. Furthermore, in the liver, BCA reduced the degree of hepatic steatosis and the expression of Nos2. We concluded that BCA exerted an anti-inflammatory response in the liver, particularly in the AT, indicating a resolution profile despite not altering the animals' metabolic profile. This study demonstrated, for the first time, the anti-inflammatory effect of BCA on tissues affected by lipotoxicity caused by high-fat diet consumption, exacerbated by ovariectomy.\u003c/p\u003e","manuscriptTitle":"Biochanin A has anti-inflammatory effects on diet-induced obesity and ovariectomy in mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-03 15:39:08","doi":"10.21203/rs.3.rs-5188359/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":"ea5ebf9b-87ba-422f-8799-d4d12b074861","owner":[],"postedDate":"October 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":38429722,"name":"Physiology"}],"tags":[],"updatedAt":"2024-10-03T15:39:08+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-03 15:39:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5188359","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5188359","identity":"rs-5188359","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}