The Mechanism Of NMN Activating SIRT2-mediated Glycolysis Pathway To Improve Follicular Development In PCOS Rats

Objective: Clarify the effect of NMN intervention to improve the follicular development of PCOS rats and explain the mechanism of NMN activating the SIRT2-mediated glycolysis pathway to improve the follicular development of PCOS rats.Methods: The rat model of PCOS was induced by letrozole combined with high-fat diet.After NMN administration,detecting serum hormone content,estrous cycle and ovarian morphology in order to analyze the effect of NMN on the follicular development of PCOS rats.Analysing and screening glycolysis metabolites and rate-limiting enzyme genes by targeting energy metabonomics and transcriptome sequencing.The expression of glycolysis rate-limiting enzyme and SIRT2 was verifyed via immunohistochemistry and western blot.Analyze the changes in glycolysis rate of granular cells by adding testosterone,NMN and SIRT2 specific inhibitor AGK2 to KGN cells.Results: 1.The body weight,serum testosterone,insulin concentration and insulin resistance index of PCOS rats increase significantly compared with the control group.NMN administration can decrease the body weight,serum testosterone,insulin concentration and insulin resistance index.2.The estrous cycle of PCOS rats is disordered and the ovarian weight increases.NMN administration can recover the estrus cycle and downregulate the ovarian weight.HE staining of the ovary showed that PCOS rats had more cystic dilated follicles and atresia follicles,thinner granular cell layer and less corpus luteum.The follicular development of rats was improved with the corpus luteum and granulosa cells increased after NMN administration.3.Targeted energy metabonomics showed disorder ovarian energy metabolism with increased pyruvate concentration and decreased lactic acid concentration in PCOS rats.NMN administration improved ovarian energy metabolism and glycolytic pathway of PCOS rats.4.Heat map analysis showed that the glycolysis rate-limiting enzyme-related genes HK2,PKM2,and LDHA were significantly down-regulated in PCOS and reversed after NMN administration,as well as in QRT-PCR,immunohistochemistry,and western blot.5.Transcriptomics analysis showed disorder gene expression of acetylation and deacetylation in ovarian tissue of PCOS rats.The expression of SIRT2 was significantly reduced in PCOS,increased after NMN administration,as well as in QRT-PCR,immunohistochemistry,and western blot.6.Cultured granulosa cells in vitro,testosterone can promote the production of pyruvate and inhibit the production of lactic acid,while reducing the m RNA and protein levels of HK2,PKM2,LDHA and SIRT2,which is reversed after NMN administration.After adding the SIRT2 inhibitor AGK2,the improvement effect of NMN disappears,suggesting NMN works through activating SIRT2.Conclusions: NMN activates the expression of SIRT2 in granulosa cells and enhances its glycolysis rate to improve follicular development in PCOS rats.