Autophagy Involved In Resveratrol Protective Effect On Steroidogenesis In Mice Fed A High-fat Diet

Background Excessive consumption of fatty foods in modern society has led to an increasing number of obese people.Excessive dietary fat will increase the lipid content of adipose and non-fat tissues,and cause chronic inflammation of tissues through lipotoxicity,resulting in cell functional disordor and death.Mitochondrial dysfunction and oxidative stress may be the main pathogenesis of delayed hypogonadism caused by factors such as obesity.Because the rate-limiting step of testosterone synthesis is completed in mitochondria and testicular mesenchymal cells have a high basal autophagy activity,studies have shown that the therapeutic effect of resveratrol(RSV)on metabolic diseases is related to its role in regulating autophagy,but autophagy The role of mechanism in the high-fat diet(HFD)leading to reduced testosterone synthesis and the protective effect of RSV is urgently studied,to further clarify the therapeutic value of resveratrol in male reproduction.Objective To study the adverse effects of obesity-related metabolic diseases caused by high-fat diets on testosterone synthesis and the role of autophagy in the protection mechanism of resveratrol,and to regulate the degree of autophagy of TM3 cells in vitro to further study autophagy The effect on the ability of testosterone synthesis and the protective mechanism of resveratrol,to clarify the protective mechanism of resveratrol on Leydig cell function in testes of high-fat diet mice.Methods Sixty 8-week-old male C57 BL / 6J male rats were randomly divided into four groups: a high-fat diet(HFD group)or a high-fat diet supplemented with resveratrol(HFD + RSV group,400 mg/kg/day),the control group used basic diet without adding RSV(Ctrl group)or adding the same amount of RSV(Ctrl + RSV group).All the diets were maintained for 20 weeks,and the modeling was completed.Bodyweight,blood lipids,hormones,and tissue samples were measured.HE staining and immunohistochemistry(IHC)was used to observe testicular morphological changes.Western blot was used to detect testosterone synthesis,oxidative stress(OS),autophagy and mitochondrial function-related indicators.Besides,TM3 cells were treated with HCG、 RSV、 RAPA or 3-MA and cultured.OS was induced by H 2 O 2.Cell viability was measured by the CCK8 method,mitochondrial membrane potential(MMP)was detected by flow cytometry,and inverted fluorescence was detected.The number of autophagosomes was observed under a microscope.C ells were collected and tested for changes in testosterone synthesis,oxidative stress,autophagy,and mitochondrial function-related proteins by Western blot.Result1.The protective effect of RSV on testosterone synthesis in high-fat diet mice: the addition of RSV can significantly reduce the weight gain and testosterone levels of mice caused by HFD.Tests of St AR and 3β-HSD proteins in testicular tissue and in vitro isolation of Leydig cells to stimulate testosterone synthesis experiments showed that RSV improves the testosterone synthesis loss caused by HFD.2.RSV reduces OS and mitochondrial dysfunction in testicular tissues of high-fat diet mice: RSV inhibits HFD-induced down-regulation of expression levels of mitochondrial function-related proteins mt TFA,Cox-4 and oxidative stress-related proteins/enzymes SOD2 and GPX4 Reduced antioxidant capacity.3.RSV inhibits testicular tissue autophagy in high-fat diet mice: observation ofautophagosomes and changes in LC 3II / I,Beclin 1,and ATG7 expressions indicate that RSV inhibits testicular tissue autophagy disorders caused by HFD.TM3 cell line was treated with RAPA and 3MA.The results showed that autophagy activity was closely related to the expression level of St AR in TM3 cells.4.The role of autophagy in protecting TM3 cells from RSV oxidative damage:treatment of TM3 cells with H2O2 induces oxidative stress,RAPA or 3-MA pretreatment to study the effect of changes in autophagy level on RSV antioxidant protection.Compared with the RSV group alone,the expression of LC3Ⅱ / Ⅰ and St AR protein in the 3-MA + RSV group was significantly down-regulated,mitochondrial dysfunction,and 3-MA inhibited autophagy,which weakened RSV protection;RAPA + RSV group also had significant testosterone synthesis.Decrease,reduce cell viability.Conclusion The autophagy mechanism plays a key role in the protective function of RSV on Leydig cell testosterone synthesis in high-fat diet mice,and the regulation of autophagy affects RSV’s reduction of mitochondrial dysfunction and oxidative stress.RSV has important significance in the treatment of hypogonadism caused by obesity-related metabolic diseases.