Mechanism Of PCOS Induced By The Gut Dysbiosis-mediated Blockage Of NAD~+ Synthesis

Background: Polycystic ovary syndrome（PCOS）is one of the most common reproductive and endocrine disorders in women of childbearing age.The global prevalence of PCOS among women of childbearing age is 8%¹3%,and it is increasing year by year.It often manifests as menstrual disorders,obesity,hirsutism and other symptoms,and in severe cases,infertility and even secondary cardiovascular diseases,which have an adverse impact on the physical and mental health and quality of life of patients.So far,domestic and international scholars generally believe that PCOS is a disease caused by a variety of factors,and its etiology and pathogenesis usually involve genetics,insulin resistance,endocrine hormone inflammation and gut microbiota,but its specific mechanism has not been clarified.In recent years,with the maturity of gene sequencing technology and metabolomics,the study of gut microbiota in PCOS patients has attracted extensive attention.Studies have shown that there are significant differences in the gut microbiota of patients with PCOS and healthy people.It has also been demonstrated through animal experiments that the use of DHEA to induce PCOS-like symptoms in rats is accompanied by changes in the gut microbiota of rats.These studies suggest a potential link between gut microbiota and PCOS,but the specific mechanism is unclear.Objectives: PCOS can seriously damage the physical health and reproductive function of women of childbearing age,and its clinical treatment effect is poor due to its complex etiology and many influencing factors.Therefore,exploring the pathogenesis of PCOS may provide new therapeutic targets for PCOS and enhance the clinical efficacy of PCOS.Therefore,this study took the gut microbiota as the starting point,and aimed to explore the potential relationship between the disorder of gut microbiota and the occurrence and development of PCOS.Finally,speculate on the mechanism of how it worksMethods: 1.Healthy participants and PCOS patients were recruited through clinical trials,divided into Control group and PCOS group.Collect their age,height,weight,and other clinical statistics.Blood samples were collected to detect the levels of sex hormones and metabolic indexes,and fecal samples were collected for 16 S rDNA sequencing and metabolomic analysis of gut microbiota,to explore the structural differences and metabolic differences of gut microbiota between PCOS patients and healthy individuals.Then to identify differential metabolites that may have an impact on PCOS symptoms.2.After clarifying the differences in gut microbiota between PCOS patients and healthy participants,animal experiments were conducted to revalidate them.First,the mice were randomly divided into three groups,Control group,H-FMT group and PFMT group.The mice in the FMT group were treated with compound antibiotics to make them in a pseudo-sterile state,and then the feces of PCOS patients and healthy participants were transplanted to the mice,and the fecal microbial transplantation（FMT）model was established.And then the gut microbiota structure and metabolomics were detected.These four metabolites are all related to tryptophan（Trp）metabolism,and Trp metabolism affects the synthesis of NAD⁺ in humans,so the above results suggest that 3-HAA may affect PCOS by affecting NAD⁺ synthesis.3.After identifying the differential metabolites which affect PCOS,we used DHEA-induced PCOS-like mice models for animal experiments.The relationship between differential metabolites and PCOS was verified by supplementing the differential metabolites and their related upstream and downstream substances.and its mechanism of action was speculated.Results:1.First,we found that compared with healthy women,the biodiversity of the intestinal microbiota of PCOS patients was significantly reduced,and the structure of the microbiota was changed: Bifidobacteria and Actinomycetes were enriched in the fecal samples of PCOS patients,while the relative abundance of Bacteroides,UCG₀02,Alistipes,and Roseburia was significantly reduced.Metabolomic analysis of gut microbiota showed that a total of 706 metabolites were changed between the two groups,and the levels of 423 metabolites in the PCOS group decreased and 283 metabolites increased compared with the healthy group.These metabolites contain 3-hydroxyanthranilic acid（3-HAA）,an important precursor to the synthetic kynurenine pathway（KP）of NAD⁺ in humans.2.Secondly,we established FMT mice models by transplanting feces from healthy subjects and PCOS patients to mice,and successfully induced PCOS-like symptoms of abnormal glucose metabolism,interrupted estrous cycle,disordered sex hormone levels,and histopathological changes in the ovaries in the P-FMT group mice.The 16 S rDNA sequencing of the intestinal microbiota was then performed on Control,H-FMT,and PFMT mice.Then,biodiversity analysis and microbiota identification were carried out to verify that FMT could change the intestinal microbiota β biodiversity and microbiota composition structure of mice.Then,metabolomics analysis confirmed that the microbial metabolic function of the gut microbiota in the H-FMT group and the P-FMT group was changed.A total of 513 metabolites were identified as having their levels changed,of which 278 metabolites had increased and 235 metabolites had decreased in the P-FMT group.The results were then compared to those of humans,and it was determined that 37 metabolites showed similar changes,with 12 metabolites having elevated levels and 25 metabolites decreasing.Four of the differential metabolites shared by these humans and mice showed the same changing trend,with 3-HAA and 5-dehydroquinic acid（5-DQA）levels significantly decreasing in the PCOS group and PFMT species,while 5-hydroxytryptamine（5-HT）and N’-formyl-L-Kynurenine（FK）levels were increasing.Finally,compared with the control group and the H-FMT group,the metabolite 3-HAA in the P-FMT group was significantly reduced,which was consistent with the results of fecal metabolomics analysis in PCOS patients,suggesting that 3-HAA may be closely related to the occurrence and development of PCOS.3.Finally,we used DHEA to induce PCOS-like symptoms in mice and establish the PCOS-like mice models.The alleviating effect of DHEA-induced PCOS-like symptoms in mice was then verified by supplementing mice with 3-HAA.Subsequently,we administered NMN（a key precursor in the NAD⁺ synthetic salvage pathway）or FK866（an inhibitor of rate-limiting enzymes in the NAD⁺ synthetic rescue pathway）to demonstrate that promoting NAD⁺ synthesis improved PCOS symptoms in mice,while inhibiting NAD⁺ synthesis aggravated PCOS symptoms.NAD⁺ has been shown to be strongly associated with ferroptosis.Therefore,we treated mice with Fer-1（an inhibitor of ferroptosis）and found that 3-HAA and Fer-1 had comparable relief effects on DHEA-induced PCOS-like symptoms in mice,and the combination of the two did not significantly increase the remission.Finally,we speculate that the mechanism of PCOS may be that gut microbiota can reducing the levels of 3-HAA,which leads to the blockage of NAD⁺ synthesis in the body,and then activate the ferroptosis,ultimately lead to the occurrence of PCOS.Conclusions: The one of the mechanisms of PCOS may be the gut microbiota decrease levels of 3-HAA,which blocks the kynurenine pathway,one of the NAD⁺ synthesis pathways,and then activates cell ferroptosis,which may promote the occurrence and development of PCOS.