| Objective: As a complicated etiology with high genetic heterogeneity,Premature ovarian insufficiency(POI)is a hot topic in female endocrinology research,which seriously affects the fertility of women of childbearing age.At present,there is no effective treatment for POI.In this study,the effect of NMN on ovarian follicle development in rats with cyclophosphamide induced POI was investigated,in order to better provide a potential therapeutic strategy for the clinic and provide new ideas for the study of targeted drugs for the regulation of POI.Methods: POI rat model was established by the classical method of intraperitoneal injection of cyclophosphamide(CTX).Fifty-nine eight-week-old female SD rats were given adaptive feeding for 6 days and randomly divided into two groups,including 15 in the Control group and 44 in the model group.Model group was intraperitoneally injected with CTX for 2 weeks,and control group was intraperitoneally injected with equal volume of normal saline.The POI group was randomly divided into treatment group(CTX-POI-NMN)and model group(CTX-POI)after the establishment of estrous cycle and serum hormone identification model.Part I: The changes of estrous cycle in each group during modeling and treatment were monitored by vaginal smear.The abdominal aorta blood of anesthetized rats was collected to detect the levels of sex hormones such as follicle-stimulating hormone(FSH)and estrogen(E2)in the serum of the rats.The ovaries of rats were removed,and the morphology of ovary tissue was observed and the number of follicles at all levels was counted by hematoxylin-Eosin(HE)staining.The remaining 3 females in each group were cooped up with a male for 3 days after conception,and the male mice were removed.The difference in litter number was calculated 25 days later.quantitative Real-Time PCR(q RT-PCR)and Immunohistochemistry(IHC)were used to determine m RNA and antigen expression levels of Bax and Bcl-2related factors in rat ovarian tissue regulation.Part II: quantitative Real-Time PCR(q RT-PCR)and Immunohistochemistry;IHC)were used to determine the m RNA and antigen expression levels of niacinamide phosphoribose transferase(NAMPT),silencing information regulatory protein 4(SIRT4)and human FOXO3(FOXO3).Liquid chromatography-mass spectrometryand high performance liquid chromatography-tandem mass spectrometry were used to determine the effect of ovarian energy metabolites.Transcriptome analysis was used to analyze the differential expression of genes and their molecular signaling pathways.Results:(1)By estrous cycle,serum sex hormones,ovarian morphology,number of follicles,fertility and differential expression of apoptotic factors in ovarian tissue,it was found that estrous cycle disorder and FSH of POI rats were higher than that of normal group(5.21±0.24 vs 1.61±0.91,P<0.01).E2 significantly decreased(83.46±11.36 vs 133.90±10.68,P<0.01),and the number of atresia follicles increased(110.00±28.601 vs 36.50±9.387,P<0.01).The number of primal follicles decreased significantly(8.83±0.983 vs 14.17±5.076,P<0.05).The number of other follicles decreased(primary: 37.67±6.772 vs47.67±13.866,P>0.05;Secondary follicles: 41.00±7.266 vs 44.83±8.400,P>0.05),the litter number of POI rats was significantly lower than that of normal group(2.00±3.464 vs 15.33±2.082,P<0.01).Moreover,the content of pro-apoptotic gene(Bax)was significantly increased while the expression of anti-apoptotic gene(Bcl-2)was significantly decreased,indicating that cyclophosphamide induced POI in rats was successful.After NMN treatment,the expression of estrous cycle,serum sex hormones,fertility and apoptosis factors in ovarian tissue were not different from the control group.Compared with the model group,the number of atretic follicles was significantly decreased(110.00±28.60 vs 26.17±15.54,P<0.01).The number of primary follicles increased significantly(8.83±0.98 vs 13.17±4.62,P<0.01),while the number of primary and secondary follicles increased(37.67±6.77 vs 42.00±9.10,P>0.05;41.00±7.266 vs 44.83±8.400,P>0.05),suggesting that NMN treatment can improve follicular recruitment development in POI rats.(2)Through targeted metabolomics,it was concluded that NMN supplementation could increase the level of NAD+ and ATP in vivo.Transcriptome screening and analysis of the differential gene expression in the ovaries of rats in each group showed that the molecular pathway was NAMpt-SIRT4-FOXO3 predicted by biogenic analysis.The expression of NAMPT,SIRT4 and FOXO3 in ovarian tissues was detected by QRT-PCR and IHC,and the expression of NAMPT in CTX-POI group was significantly decreased(P<0.05),while the content of SIIRT4 was significantly increased(P>0.05)compared with the Control group.The content of FOXO3 was significantly increased(P<0.01),the expression of NAMPT was significantly increased after NMN treatment(P<0.05),and the contents of SIIRT4 and FOXO3 were significantly decreased(P<0.01).It was further verified that NMN may regulate follicular development,protect ovarian reserve and improve fertility in POI rats through the signaling pathway of NAMpt-SIRT4-FOXO3.Conclusions:NMN can significantly improve the endocrine function of POI rats,inhibit the apoptosis of granulosa cells,reduce the formation of atresia follicles and promote the development of follicles at all levels,thus alleviating and treating POI rats with POI symptoms of primary ovarian dysfunction.The mechanism may be that NMN significantly increases the level of OVARIAN NAD+ through the NAMPT-FOXO3-SIRT4 signaling pathway,thereby increasing the level of ovarian ATP,inhibiting the apoptosis of follicular granulosa cells,maintaining the energy required for follicular development,and promoting follicular development.The NAMpt-SIRT4-FOXO3 signaling pathway is expected to provide a novel therapeutic strategy and drug target for human intervention to control primal follicular overactivation and improve follicular loss and fertility in POI patients. |
PDF Full Text Request