Mogroside V Improves Follicular Development And Ovulation In PCOS Rats Through Regulating Glycolysis

Objective: In this study,we induced a PCOS rat model by oral administration of letrozole combined with a high-fat diet and then treated with mogroside V(MV)to evaluate the protective effects of MV on endocrine and follicle development in PCOS rats,and to clarify its mechanism in PCOS rats.Methods: SPF-level SD female rats(5 weeks of age)were fed adaptively for 1 week.45 female SD rats were randomly divided into 3groups: the control group(CON group,n=15),the PCOS model group(PCOS group,n=15)and the PCOS + MV group(POCS-MV group,n=15).The CON group fed with ordinary diet and oral administration solvent(0.1%sodium carboxymethyl cellulose)for 30 days,both PCOS group rats and PCOS-MV group rats were all given high-fat diet and oral administration of letrozole(1 mg/kg d)for 30 days.From the 21 st day of modeling,vaginal secretion smears were collected for 10 consecutive days.After successful modeling,he control group rats were fed with ordinary diet,the PCOS group continued to be fed with high-fat diet,and the pcos-mv group rats were fed with ordinary diet and oral administration of MV(600mg/kg/day)for 30 days.Vaginal smears were collected for 10 consecutive days from the 21 st day of MV administration.After the last day of the treatment,the rats were properly anesthetized,blood was collected and the reproductive hormones were measured by radioimmunoassay;the ovaries were excised.The development of ovarian follicles was observed by hematoxylin eosin staining(HE);The fertility of rats was measured by mating with male rats.Metabolomics was employed to test the concentration of ovarian energy metabolites in each group;Transcriptome sequencing was used to analyze the gene expression level,the differentially expressed genes of glycolysis pathway were enriched and screened by bioinformatic analysis.The expressions of lactate dehydrogenase A(Ldha),hexokinase 2(Hk2)and M2 pyruvate kinase(Pkm2)in ovaries were detected by real-time fluorescence quantitative PCR(q RT-PCR),Western blotting and immunohistochemistry.KGN cells were cultured in vitro and administrated with androgen combined with different concentrations of MV(0,25,50,100 nmol /L).The cell viability was measured by CCK-8assay and the concentration of lactic acid and pyruvate were measured by ELISA.QRT-PCR and Western blotting were applied to detect the expression of LDHA,HK2 and PKM2.Results: MV administration not only significantly reduced the body weight and ovary weight,but also attenuated the disrupted estrous cycle and decreased the level of testosterone.MV restored the follicular development,especially by increasing the number of corpus luteum and the thickness of the granular layer in young-adult PCOS rats.Moreover,metabolomics showed that MV markedly increased the levels of DGlucose 6-phosphate,lactate and GTP,while decreased the level of pyruvate.Bioinformatic analysis revealed that MV recovered multiple metabolism-related processes including gluconeogenesis,glycolysis and glucose metabolic process.Further real-time quantitative PCR analysis showed that MV upregulated the expression of lactate dehydrogenase A(Ldha),hexokinase 2(Hk2)and pyruvate kinase M2(Pkm2).Western blotting and immunohistochemistry analysis showed that MV restored the expression of lactate dehydrogenase A(Ldha),hexokinase 2(Hk2)and pyruvate kinase M2(Pkm2).Cultured KGN cells showed that high concentration of testosterone could reduce the cell viability,inhibit the production of lactate and pyruvate.The key rate limiting enzyme genes of glycolysis genes(ldh A,h K2 and pkm2)were significantly down regulated.However MV can reverse the effect of testosterone,increase KGN cell viability,promote the production of lactate and pyruvate.and up-regulatd the expression of LDHA,HK2 and PKM2.(P < 0.05).Conclusions: Collectively,these findings indicated that MV could effectively improve the ovarian microenvironment by upregulating the expression of LDHA,HK2 and PKM2 in granulosa cells and enhancing lactate and energy production,which may contribute to follicle development and ovulation of young-adult PCOS rats.