Melatonin Ameliorates Diabetic Hyperglycemia-induced Impairment Of Leydig Cell Steroidogenic Function Through Activation Of SIRT1 Pathway

Background Diabetes mellitus（DM）is a common metabolic disease characterized by hyperglycemia.Clinical studies show that diabetes can damage male reproductive function,but the mechanism of diabetes damaging male gonad function is not completely clear.Melatonin（MT）,as an essential antioxidant targeting mitochondrion,also plays a regulatory role in autophagy and mitochondrial autophagy,and its protective role in metabolic damage caused by diabetes,obesity,aging,etc.,has gradually attracted attention.Sirtuin 1（SIRT1）is an NAD+-dependent deacetylase,which plays an essential role in many metabolic and physiological processes,and the part of MT in diabetes-related reproductive damage deserves further study.Objective To explore the mechanism of SIRT1 pathway regulating mitochondrial biosynthesis and mitochondrial autophagy in the damage of testosterone synthesis caused by diabetes and the protective effect of MT.Method Twenty male SD rats were randomly divided into the control group,high glucose model group,MT treatment group,and insulin treatment group.Intraperitoneal injection of streptozotocin（STZ）was used to establish the model of type I diabetes.After successful modeling,MT treatment and insulin treatment were used as controls.Morphological changes were observed in the rat testis tissue sections.The total protein was extracted and analyzed by Western blotting for SIRT1 pathway protein,testosterone synthesis-related protein/enzyme,mitochondrial function-related protein,and autophagy-related protein.In vitro experiment,a mouse TM3 Leydig cell line was divided into a control group,a high glucose group,and an MT treatment group.The Sirt1 gene was knocked down by si RNA to study the effect of SIRT1 on MT protection against high glucose damage.To analyze the mechanism of MT up-regulating SIRT1 under high-sugar background,the high-sugar group was treated with antioxidant（NAC）in advance to study the relationship between the effect of MT on SIRT1 level,mitochondrial autophagy,and mitochondrial synthesis and ROS level.The DCFH-DA probe detected the changes of ROS in TM3 cells.Mito SOX noticed the production level of m ROS;MDC staining was used to detect the formation of autophagy bodies.Immunofluorescence technique mitochondrial autophagy level;JC-1 fluorescent probe was used to detect mitochondrial membrane potential.Mito Tracker labeled the number and morphology of mitochondria;Western blot was used to detect the expression levels of testosterone synthesis-related proteins/enzymes,autophagy and mitochondrial autophagy related proteins,mitochondrial biosynthesis-related proteins,and SIRT1/AMPK in TM3 cells of each group.Results 1.MT alleviates the adverse effects of high glucose on testosterone synthesis:In the tall glucose model of rat testis and TM3 cells in the DM group,the expression levels of rate-limiting enzymes related to testosterone synthesis（St AR,3β-HSD,and P450scc）were significantly lower than those in a control group,greatly improved the MT treatment group.2.Effects of MT on AMPK/SIRT1 pathway and mitochondrial function: The expression levels of SIRT1,AMPK,p-AMPK,mitochondrial synthesis,and its function-related proteins（mt TFA,NRF1,ATPB,and COXIV）in rat testis and TM3 cells treated with high glucose in DM group were all lower than those in a control group,and increased after MT treatment.SIRT1 and PGC-1α in TM3 cells cytoplasm were the same as the above trends.In addition,the observation of an electron microscope found that the swelling and degeneration of mitochondria were evident in the testis of DM rats,which was improved in the MT treatment group.JC-1 fluorescent probe was used to detect the mitochondrial membrane potential（MMP）of TM3 cells,Mito Tracker was used to mark the morphology of mitochondria,and Mito SOX was used to detect the mitochondrial reactive oxygen species（m ROS）.The above results showed that MMP in TM3 cells decreased,the number of damaged mitochondria increased,and the production of m ROS increased under high glucose treatment.MT treatment can significantly increase MMP and reduce the level of m ROS.It has been proved that MT may promote mitochondrial biosynthesis and protect mitochondrial synthesis function by activating the SIRT1/PGC-1α/NRF1 pathway under high glucose background.3.Effects of MT on autophagy and mitophagy: by detecting the expression of autophagy-related proteins（Beclin-1,Atg7,and LC3）in rat testis and TM3 cells and marking autophagy bodies in TM3 cells by MDC,it was found that the expression of autophagy-related proteins in DM rats and TM3 cells treated with high glucose decreased,autophagy bodies decreased,and autophagy level recovered significantly after mt treatment.It is suggested that MT therapy may improve the function damage of Leydig cells induced by hyperglycemia by activating the AMPK/SIRT1 pathway and autophagy-related gene expression.The immunofluorescence co-localization of BNIP3 L,mitochondrial marker protein COXIV,and autophagy marker protein LC3 was detected to evaluate the level of mitochondrial autophagy in TM3 cells.The results showed that under high glucose,the mitochondrial autophagy activity decreased,and MT could up-regulate the mitochondrial autophagy activity related to BNIP3 L.4.Effect of knocking down Sirt1 on MT’s resistance to high glucose damage:Knocking down SIRT1 in TM3 cells showed that the impact of MT on increasing the expression level of testosterone synthesis rate-limiting enzyme in a high glucose environment,activating autophagy and mitochondrial autophagy activity,and protecting mitochondrial synthesis and morphological recovery were all inhibited.5.Up-regulation of SIRT1 by MT is related to ROS level: after TM3 cells were treated with high glucose,ROS level was significantly increased,and antioxidant enzyme GPX4 was down-regulated.The addition of MT has an anti-oxidative stress effect.Added NAC pretreatment was to neutralize ROS before MT treatment to study further the mechanism of MT activating SIRT1 under the background of high glucose.The results showed that SIRT1,AMPK,LC3,and mt TFA were significantly lower than those without NAC pretreatment,which indicated that neutralizing ROS could block the stimulation of MT to SIRT1/AMPK pathway in TM3 cells and promote autophagy and mitochondrial biosynthesis.Conclusion 1.DM hyperglycemia leads to the decrease of testosterone synthesis function,accompanied by autophagy defect,mitochondrial dysfunction,and oxidative stress,which is improved after MT treatment.2.MT treatment can promote mitochondrial biosynthesis and regulate autophagy/mitochondrial autophagy levels by activating the AMPK/SIRT1 pathway,improving mitochondrial dysfunction caused by high glucose,and protecting testosterone synthesis.3.The activation of the SIRT1 pathway by MT under high glucose background is related to ROS levels and oxidative stress.