| Background: As life expectancy increases and the number of older adults increases and age-related diseases are of interest to many researchers.More and more people pay attention to delaying aging and preventing the occurrence and development of age-related diseases.Reproductive aging is related to the health level and quality of life of the middle-aged and elderly population and is one of the hot topics in the aging research field.Male fertility,including serum testosterone levels,declines with aging.After 35 to 40,testosterone begins to decline at a gradual rate of 1 to 3 percent per year,eventually leading to gonads in older men as they live longer.Mitochondrial dysfunction and autophagy deficiency are considered to be critical pathophysiological mechanisms of male hypogonadism and decreased testosterone synthesis in Leydig cells.Testosterone synthesis factors mainly include Leydig cells’ ability to synthesize testosterone,changes in the extracellular environment,hypothalamic-pituitary regulation axis,and so on.Leydig cells are the primary source of androgen in the male body,and testosterone synthesis is mainly completed in mitochondria and endoplasmic reticulum.Testosterone synthesis is accompanied by ROS production,and testicular tissue is prone to oxidative stress(OS).Mitochondria are the primary source of ROS,and mitochondrial dysfunction leads to OS.ROS is an upstream regulator of autophagy,and OS induces autophagy under certain conditions.Compared with other tissues in vivo,Leydig cells had a higher level of basal autophagy,and the autophagy activity decreased with aging,which affected the ability of testosterone synthesis.Autophagy deficiency may lead to the increase of ROS level and OS in senescent testicular tissue.Modulation of autophagy affects testosterone synthesis and spermatogenesis.Resveratrol is a polyphenolic compound that has antioxidant properties and is found in a variety of foods,particularly grapes and red wine.RSV has a wide range of pharmacological effects,such as anti-aging,anti-cancer,anti-inflammatory,and anti-oxidation.The broad anti-aging effects of RSV are considered to be the most attractive.The molecular mechanisms of RSV are complex and can target multiple pathways simultaneously.In terms of male reproduction,many studies have shown that RSV has protective effects,such as increased sperm production,improved sperm motility,and antioxidant defense,reduced lipid peroxidation,and enhanced sperm mitochondrial activity and DNA integrity.Some studies have shown that RSV protects mesenchymal cells from environmental stimuli such as hydrogen peroxide,benzopyrene,and nicotine.Because there has been no study on the dysfunction of testosterone synthesis in Leydig cells related to RSV senescence delay.This study was aimed to investigate the roles of autophagy and mitochondrial biosynthesis in the decreased testosterone synthesis function of Leydig cells induced by aging,as well as the influence of RSV on the protective effect of anti-reproductive aging and the molecular mechanism.Objective:To study the effect of aging on the testosterone synthesis ability of mouse Leydig cells,and the mechanism of resveratrol protecting the decreased testosterone synthesis function of aging mice by regulating the interaction between testicular autophagy and mitochondrial biosynthesis.Methods:(1)In vivo experiment,45 2-month-old(Young)male C57 BL/6J mice were fed with or without resveratrol for 18 months,and divided into an Aged group(Aged)and Aged group plus resveratrol(Aged+RSV).Serum testosterone(T)was measured by radioimmunoassay.The expressions of STAR,3β-HSD,and Beclin-1,Atg5,Atg7 and LC3 were detected by Western Blot.The expression of 3β-HSD in the stroma of testis was detected by immunohistochemical staining.(2)In vitro experiment,D-galactose was used to induce senescence of TM3 cells,and CCK-8 was used to detect cell viability.The expression of age-related protein P21 was detected by Western Blot.The number of senescent cells was detected by β-galactosidase staining,and the concentration of D-galactose-induced senescent TM3 cells was determined to be 10g/L for 48 h.After induced by D-gal(10g/L)for 48 h and RSV(10u M)for 24 h,the expression levels of T biosynthesis-related proteins,autophagy-related proteins,mitochondrial biosynthesis-related proteins and antioxidant enzyme related proteins were detected by Western Blot.Mito SOX fluorescence staining was used to detect the level of mitochondrial reactive oxygen species.Mitotracker Green fluorescent probe was used to detect mitochondrial structure and morphology.After Atg7 si RNA knockdown,D-galactose(10g/L)induction for 48 h and RSV(10u M)induction for 24 h,the expression levels of T biosynthesis-related proteins,autophagy-related proteins,mitochondrial biosynthesis-related proteins and antioxidant enzyme related proteins in TM3 cells were detected by Western Blot.Mito SOX fluorescence staining was used to detect the level of mitochondrial reactive oxygen species.Mitotracker Green fluorescent probe was used to detect mitochondrial structure and morphology.Result:(1)RSV delayed the decrease of testosterone synthesis in testicular stromal cells of elderly mice: RSV consumption significantly increased the decrease of serum testosterone T,testosterone synthesis related protein STAR and 3β-HSD in elderly mice,suggesting that RSV could alleviate the age-dependent decrease of testosterone level in mice.(2)RSV inhibited testis autophagy deficiency and protected mitochondrial dysfunction in elderly mice: The protein expression levels of autophagy-related proteins and mitochondrial function-related proteins in mice fed RSV for a long time were higher than those in the elderly group,indicating that RSV could inhibit the trend of autophagy and mitochondrial function decline with age.(3)D-gal induced senescence of TM3 cells and the protective effect of RSV: TM3 cells were treated with D-gal 10g/L for 48 h.The senescence induced by TM3 cells was demonstrated to be successful by β-galactoside staining,CCK-8 detection of cell viability,and P21 detection of senescence-related protein marker.The expression of autophagy-related proteins,mitochondrial function-related proteins,and antioxidant enzyme-related proteins in TM3 cells induced by RSV 10 u M for 24 h was detected by WB.Mito Tracker red fluorescence was used to detect mitochondrial reactive oxygen species and Mito Tracker green fluorescence was used to label mitochondrial morphology.The results showed that RSV effectively inhibited the decrease of autophagy,mitochondrial dysfunction and oxidative stress in D-gal induced senescence TM3 cells.(4)Atg7 knockdown affected autophagy and mitochondrial function in D-gal induced senescent TM3 cells: Atg7 knockdown resulted in autophagy deficiency in senescent TM3 cells.WB detected autophagy-related proteins and mitochondrial function-related proteins and found that the expression of the senescent hi protein was significantly reduced after Atg7 knockdown.These results suggest that autophagy deficiency may further affect senescent mitochondrial dysfunction.(5)The effect of autophagy deficiency on testis stromal cells of aging mice and the protective effect of RSV on the function of testosterone synthesis: After Atg7 knockdown,the expression of testosterone biosynthesis-related proteins St AR and 3β-HSD in D-gal induced aging TM3 cells were detected and significantly decreased,indicating that the testosterone synthesis ability of aging TM3 cells was impaired by autophagy deficiency;TM3 cells were treated with RSV,and the expression of T synthesis protein was detected.It was found that RSV did not increase the expression of T synthesis protein,indicating that autophagy deficiency seriously weakened the protective effect of RSV on steroidogenesis.Conclusion:(1)Autophagy and mitochondrial dysfunction are closely related to decreased testosterone synthesis in Leydig cells caused by aging.Resveratrol can inhibit autophagy and mitochondrial dysfunction in Leydig cells,producing the anti-aging protective effect.(2)Autophagy deficiency caused by ATG7 knockdown leads to mitochondrial biosynthesis disorder in TM3 cells,which affects testosterone synthesis and the retarding effect of RSV on senile Leydig cells,further demonstrating the role of autophagy mechanism in RSV’s retarding effect on reproductive aging. |
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