Study On The Functional Protection Of Melatonin On Leydig Cells In Mice Fed With High-Fat Diet

Background Obesity caused by high-fat diet has adverse effects on male hormone production and fertility.It may lead to a series of pathological changes in the body Obesity can cause dysfunction of hypothalamic-pituitary-gonadal axis and chronic inflammation of tissues.Changes in the testicular micro environment can lead to dysfunction of testicular tissue and cells,contributing to hypogonadism in men,and finally resulting in male infertility.Studies have shown that melatonin has a variety of physiological functions,such as regulating mood,body weight,sleep,immunity,anti-oxidation and circadian rhythm,but its effects on male reproduction need to be further investigatedObjective To investigate the damage of high-fat diet on the testosterone synthesis ability of mouse Leydig cells,and the mechanism by which melatonin exerts a protective effect by inhibiting oxidative stress and endoplasmic reticulum stress in testicular tissue,and further investigate the mechanism of melatonin on stressed TM3 cells,so as to clarify its protective effect on the function of mouse testicular Leydig cellsMethods Twenty four male C57BL/6J mice,7 weeks old,were purchased from Laboratory Animal Centre,Anhui Medical University and divided into three groups feeding with high-fat diet as HFD group,or high-fat diet with additional melatonin intragastric administration as HFD+MT group,or a normal chow for negative control as CTRL group for 12 weeks.Body weight,serum lipid and hormone were measured after 12 weeks.Tissue samples were taken for subsequent experiments.The morphological change of testis was observed by HE and IHC staining.The protein expressions of OS,ER stress and testosterone synthesis were detected by western blotting and qRT-PCR.TM3 cells were cultured and divided into CTRL group,H2O2 group and MT+H2O2 group.OS of TM3 cells were induced by H2O2,cell viability was measured using CCK8 method,and ROS were detected by flow cytometry.Changes of protein expressions of testosterone synthesis,OS,ER stress and mitochondrial disfunction were detected by western blotting.Results1.Effects of melatonin on body weight,serum lipids and hormones in three groups of mice:the body weight of the HFD group and the weight of the epididymal adipose tissue were significantly higher than those of the CTRL group,while the HFD+MT group decreased compared with the HFD group(P<0.05).There was no significant difference in testicular tissue weighing between the three groups.The serum level of TG in the HFD group increased compared with the CTRL group,but melatonin treatment has no significant effect on serum lipids.Melatonin supplementation inhibited the decrease of serum T and LH level compared with HFD group(P<0.05).2.Morphological observation of testicular tissue in three groups of mice:The results of HE staining showed that the testicular tissue structure of the CTRL group mice was intact,and the spermatogonial cells were in a circular arrangement and neatly arranged;Leydig cells were prominent and polygonal,with clear cell boundaries and an abundant of cytoplasm.Compared with the CTRL group,the Leydig cells in the HFD group became smaller,with irregular morphology and less cytoplasm;the spermatogenic cells became polygonal and were disorderly arrangement.Compared with the HFD group,the HFD+MT group showed a recovery in cell morphology and arrangement,which was close to the normal morphology.The results of immunohistochemistry showed that 3?-HSD,the brown-yellow positive staining,was expressed in the cytoplasm of testicular Leydig cells.The positive expression of the HFD group was significantly weaker than that of the CTRL group(P<0.05),and enhanced in the HFD+MT group compared with the HFD group(P<0.01)3.Protective effect of melatonin on testicular steroidogenesis in mice fed a high-fat diet:western blotting showed that the protein expression levels of StAR and P450scc in the HFD group were s ignificantly lower than those in the CTRL group,and improved compared with that of HFD group after melatonin treatement(P<0.05).The results of RT-PCR showed that the mRNA expressions of Star,P450scc and Hsdl7b was down-regulated in the HFD group compared with the CTRL group,and up-regulated when melatonin supplemented(P<0.05)4.Melatonin inhibited OS and ER stress in the testicular tissues of mice fed on high-fat diet:as shown in Western blottingting,the expressions of SIRT1 and SOD2 were significantly lower in the HFD group than in the CTRL group,which in the melatonin-treated group were up-regulated compared to the HFD group(P<0.05).The protein expression of GRP78 in the HFD group was significantly higher than that in the CTRL group,which in the melatonin-treated group was lower than the HFD group(P<0.05).As shown in qRT-PCR,the mRNA expression of Sod2 and Gpx4 were lower in the HFD group than in the CTRL group,and increased after treated with melatonin The mRNA expression of Grp78 and Chop in the HFD group was higher than that that in the control group,which was significantly decreased after treated with melatonin(P<0.05)5.The protective effect of melatonin on H2O2-induced functional damage of TM3 cells:to further explore the mechanism of melatonin on injured Leydig cells,TM3 cells were cultured in vitro and OS was induced by H2O2 to detect the protective effect of melatonin on its function.The results of CCK8 showed that the cell viability decreased in the H2O2 group and increased in the MT+H2O2 group(P<0.05).The results of flow cytometry showed that ROS level were increased in the H2O2 group compared with CTRL group,while melatonin intervention significantly reduced ROS level in the MT+H2O2 group compared with H2O2 group(P<0.05).The results of western blotting showed that the protein/enzyme expressions of testosterone synthesis related StAR in the H2O2 group were down-regulated compared with the HFD group,and up-regulated in the MT+H2O2 group compared with the HFD group(P<0.05).The OS related protein expressions of SIRTI,SOD2 and GPx4 in the H2O2 group were down-regulated compared with the CTRL group,and up-regulated in MT+H2O2 group compared with H2O2 group(P<0.05).The expression of GRP78 in the H2O2 group was higher than that in the CTRL group,and decreased after melatonin treatment(P<0.05).There was no significant trend showed in CHOP protein expression(P>0.05).The mitochondrial function related targets COXIV and CytC decreased in the H2O2 group compared with CTRL group,and increased in MT+H2O2 group compared with H2O2 group(P<0.05)Conclusion Melatonin could improve the changes in the testicular structure of mice induced by high-fat diet,protecting the function of Leydig cells,and have therapeutic value for hypogonadism in men caused by high-fat diet in clinical practice.