Improving mitochondrial function and alleviating oxidative stress in aged women with ovarian insufficiency:the role of melatonin through the SIRT3/SOD2 pathway
Objective To investigate the potential effects of melatonin on improving mitochondrial function and reducing oxidative stress in granulosa cells in aged women with ovarian insufficiency in vitro,as well as explore the underlying mechanisms.Methods Granulosa cells were extracted from waste follicular fluid obtained from patients undergoing assisted reproductive technology at the Reproductive Medicine Center of the Second Affiliated Hospital of Zhengzhou University from March to June 2022.According to the age of the patients,they were divided into two groups:the aged group(age≥38 years old,6 cases)and the young control group(age<35 years old,6 cases).The mitochondrial ultrastructure of the granulosa cells was examined using transmission electron microscopy.Intracellular ATP levels were measured using an ATP detection kit,while mitochondrial DNA(mtDNA)copy number was assessed using real-time fluorescence quantitative PCR.Mitochondrial membrane potential was evaluated using the JC-1 fluorescent probe,reactive oxygen species(ROS)content was measured using the MitoSOX™ Red mitochondrial oxide indicator,and protein expressions of SIRT3 and SOD2 were determined using Western blotting.According to the random number table method,samples from the aged group were randomly allocated to either the melatonin treatment group or blank control group(5 cases in each group)to assess the impact of in vitro melatonin treatment on the aforementioned mitochondrial parameters.SIRT3 in granular cells was down-regulated by transfection of siRNA,and the above indexes were detected before and after melatonin addition and compared with the negative control group.Results In comparison to the young group,the aged group exhibited distinct differences in the ultrastructure of granulosa cell mitochondria.Specifically,the mitochondrial structure appeared unclear,with sparse and irregularly arranged ridges.Furthermore,significant reductions were observed in ATP levels(P=0.012),mtDNA copy number(P=0.005),and mitochondrial membrane potential(P=0.009)in the aged group,while ROS content was increased(P=0.003).Additionally,the levels of SIRT3 and SOD2 were significantly decreased(P<0.001 and P=0.002,respectively).These differences were statistically significant.Following in vitro melatonin culture,improvements were observed in the mitochondrial ultrastructure,as well as increases in ATP levels(P<0.001),mtDNA copy number(P=0.038),and mitochondrial membrane potential(P=0.002).Correspondingly,SIRT3 and SOD2 levels increased(P= 0.011 and P=0.031,respectively),while ROS content decreased(P<0.001).These changes were statistically significant.After siRNA transfection,the expression of SIRT3 in the granulosa cells was significantly down-regulated(P<0.001).After melatonin treatment,the ATP levels(P<0.001),the mtDNA copy number(P=0.001),and the mitochondrial membrane potential(P<0.001)were all lower than those in the negative control group without SIRT3 downregulation,and the ROS content was higher than that in the negative control group(P<0.001),with statistical differences.Similarly,the effects of melatonin on reducing ROS were also significantly diminished.Conclusion In vitro melatonin culture has the potential to enhance mitochondrial function and alleviate oxidative stress in granulosa cells from aged women with ovarian insufficiency.Furthermore,in addition to its direct antioxidative properties,melatonin may regulate the levels of SIRT3 and SOD2 to reduce ROS.
MelatoninGranulosa cellsAgingMitochondriaOxidative stressReactive oxygen species
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