| Objectives:In this study,the mechanism of apoptosis mediated by restriction of niacinamide adenine dinucleotide(NAD~+)synthesis in granulosa cells(GCs)of polycystic ovary syndrome(PCOS)was investigated,which provides a potential therapeutic targets for improving follicular dysplasia of PCOS.Methods:1.Analysis of differential expression profiles of m RNA in ovarian GCs from PCOS patients:transcriptome data of human ovarian GCs were downloaded from the GEO database and bioinformatics analysis of differentially expressed genes was performed.2.The PCOS rat model was induced by letrozole combined with high-fat diet.The model was identified by weight monitoring,vaginal smear monitoring of estrous cycle,serum testosterone(T)detection by radioimmunoassay and ovarian morphology observation by H&E staining.3.ELISA and Western Blot(WB)were used to detect the contents of NAD~+and lactate in ovary of PCOS rats,as well as the acetylation level of total ovarian protein.4.Analysis of changes in the expression of apoptosis-related factors(BAX,BCL-2 and CASPASE-3),NAD~+synthesis key enzyme(nicotinamide phosphoribosyltransferase,NAMPT),NAD~+-dependent deacetylase(Sirtuin 2,SIRT2)and lactate synthesis key enzyme(lactate dehydrogenase A,LDHA)in the ovaries of PCOS rats:(1)m RNA and protein expression of BAX,BCL-2,CASPASE-3,NAMPT,SIRT2 and LDHA were detected in rat ovaries of each group by q RT-PCR and WB;(2)Expression localization of BAX,BCL-2,CASPASE-3,NAMPT,SIRT2 and LDHA in rat ovaries was detected by immunohistochemistry;(3)Pearson correlation analysis was used to evaluate the correlation between NAD~+,lactate and apoptosis-related factors.5.FK866 was added to inhibit the expression of NAMPT in KGN cells:(1)Cell viability was measured by CCK-8;(2)The apoptosis rate and apoptosis-related factors were detected by flow cytometry,q RT-PCR and WB;(3)The expressions of NAMPT,SIRT2 and LDHA were detected by q RT-PCR and WB;(4)The content of NAD~+in cells was detected by ELISA;(5)The content of lactate in cell medium was detected by ELISA.6.Overexpression of LDHA in 293T cells was supplemented with FK866 to inhibit the expression of NAMPT:(1)The acetylation of LDHA was detected by co-precipitation(Co-IP)technique;(2)The content of lactate in cell medium was detected by ELISA.7.AGK2 was added to inhibit SIRT2expression in KGN cells:(1)The expressions of SIRT2 and LDHA were detected by q RT-PCR and WB;(2)The content of lactate in cell medium was detected by ELISA.Results:1.The transcriptome data of ovarian GCs from PCOS patients and control women showed that 207 genes were differentially expressed in GCs,including 143 up-regulated genes and 64 down-regulated genes.GO and KEGG enrichment analysis showed that glycolysis and niacin-niacinamide metabolic pathways were significantly enriched,and heat map cluster analysis showed that the expressions of LDHA and SIRT2 were significantly down-regulated in PCOS group.2.Compared with the control group,the body weight(374.12±11.99 vs270.13±8.67,P<0.01)of the PCOS group was significantly increased,the estrous cycle was stagnated in the interphase,the serum T level(4.85±1.48 vs 0.25±0.20,P<0.01)was significantly increased,the number of cystic follicles(11.33±2.42 vs2.50±1.05,P<0.01)in the ovary was significantly increased and the number of corpus luteum(3.50±1.05 vs 9.67±1.97,P<0.01)was significantly decreased.3.The contents of NAD~+(0.44±0.04 vs 0.75±0.01,P<0.01)and lactate(0.39±0.01vs 0.50±0.03,P<0.05)in ovarian tissue of PCOS group were significantly decreased,while the acetylation of total protein was significantly enhanced.4.The expressions of BAX and CASPASE-3 in ovarian granulosa cells of PCOS group were significantly up-regulated(P<0.05),while the expressions of BCL-2,NAMPT,SIRT2 and LDHA were significantly down-regulated(P<0.05);By Pearson correlation analysis,the NAD~+level in ovarian tissue of PCOS rats was correlated with Bax(r=-0.98,P<0.01),Caspase-3(r=-0.99,P<0.01)was negatively correlated with Bcl-2(r=0.99,P<0.01),and NAD~+was positively correlated with lactate level(r=0.93,P<0.01)showed a significant positive correlation.5.After silencing of NAMPT,KGN cell viability decreased,apoptosis rate increased,expression of BAX and CASPASE-3 was upregulated,and expression of BCL-2,NAMPT,SIRT2 and LDHA was downregulated.6.Silencing of LDHA on the basis of overexpression of NAMPT,acetylation of LDHA was enhanced and lactate(6.94±0.20 vs 8.55±0.55,P<0.05)was decreased,7.After SIRT2 silencing,the expression of SIRT2 and LDHA was downregulated in KGN cells,and lactate(7.08±0.04 vs 7.46±0.18,P<0.05)were decreased.Conclusion:The contents of NAD~+and lacate in ovary of PCOS rats were significantly decreased,and the apoptosis of GCs was significantly increased.Restriction of NAD~+synthesis can promote the apoptosis of GCs,increase the acetylation level of LDHA and inhibit its expression,block the glycolysis process and reduce the lactate level,thus inhibiting the energy supply required for follicle development. |
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