Study On The Mechanism Of Modified Guishen Pill To Ameliorate Polycystic Ovary Syndrome

Background: Polycystic ovary syndrome(PCOS)is characterized by persistent anovulation,hyperandrogenemia,and polycystic ovarian changes.It is the most common reproductive endocrine disorder in women of childbearing age,with high incidence,heterogeneity,lifelong and refractory characteristics,and is also the main cause of female infertility.At present,the western medicine treatment drugs for PCOS mainly include ovulation stimulants,insulin sensitizers and androgen lowering drugs.However,it is limited to symptomatic treatment,poor long-term efficacy,and prone to OHSS,premature ovarian failure,severe gastrointestinal adverse reactions and other side effects.Therefore,we need to actively seek safe and effective treatment strategies from Chinese medicine.Modified Guishen pill(MGP)is derived from the classic prescription Guishen pill in Jingyue Quanshu by Zhang Jingyue,which has been widely used in the clinical treatment of PCOS with remarkable curative effect.However,due to the complex components of modified Guishen pill,the molecular mechanism of its treatment of PCOS remains to be explored.Therefore,this study explored the effects of modified Guishen pill on PCOS from the perspectives of gut microbiota,network pharmacology and metabolomics,clarified the mechanism of drug action,and searched for the most likely targeting molecules.Objective:(1)The gut microbiota of rats was detected by 16 S r DNA high-throughput gene sequencing technology to explore the effect of modified Guishen pill intervention on the gut microbiota of letrozoleinduced PCOS model rats and to extract the most likely targeted flora category.Moreover,to explore the possible therapeutic effect and mechanism of MGP on PCOS combined with the quantitative detection results of genes and proteins.(2)A comprehensive strategy integrating metabolomics and network pharmacology was used to reveal the mechanism of modified Guishen pill for the treatment of PCOS from the perspectives of metabolism and gene,and to find the most likely target molecule.Methods:(1)Six-week-old specific-pathogen free(SPF)female Sprague-Dawley rats were treated with letrozole for 21 d to establish the PCOS model.(2)The rats were randomly divided into six groups: 1)blank,2)model,3)diane-35,4)high-dose MGP(H.MGP),5)medium-dose MGP(M.MGP),and 6)low-dose MGP(L.MGP).(3)HE,ELISA and Gram staining were used to detect the therapeutic effect of MGP on letrozole-induced PCOS rats.(4)ELISA,IHC,RT-q PCR and WB were used to detect the antiinflammatory effect of MGP on letrozole-induced PCOS rats and its related mechanisms.(5)16S r DNA sequencing of fecal samples was used to evaluate the effect of MGP on the composition and relative abundance of gut microbiota in PCOS rats,so as to find the most likely targeted gut microbiota.(6)Plasma metabolomics was used to authenticate differential metabolites though UHPLC-QE-MS and enriched related metabolic pathways using the Metabo Analyst platform.(7)Using network pharmacology to explore the endogenous targets of MGP in the treatment of PCOS.The combined network of metabolomics and network pharmacology was established based on cytoscape to comprehensively analyze and predict the hub targets of MGP treatment for PCOS.(8)ELISA,IHC,RT-QPCR and WB were used to verify the role of core targets in MGP treatment of PCOS and related mechanisms though animal experiments.Results:(1)MGP effectively restored estrous cycle disorder and reduced the number of cystic follicles and promoted the formation of granulosa cells and corpus lutea in PCOS rats.(2)After treatment with MGP,the plasma levels of FSH,E2 were significantly raised while LH,T and LH/FSH ratios were reduced.Moreover,the MGP-treated rats had lower IL-1β,TNF-α,IL-6 and CRP,and higher IL-10 levels in plasma and ovaries than PCOS model rats.Meanwhile,MGP markedly reversed the increase in immune infiltration areas of IL-1β,IL-6,TNF-α,CD68,and the decrease in IL-10 immune infiltration areas in ovaries of PCOS rats.Furthermore,at the protein level,MGP treatment resulted in decreased levels of CD68,p-p65,p-IκBα,p-ERK,p-JNK,p-p38 but increased IκBα expression;at the gene level,MGP significantly increased the expressions of IL-10,CD163,CD206 and CD200 R,and decreased the levels of CD68,CD86,IL-1β,IL-6,TNF-α,CRP,p65,ERK,JNK and p38 in letrozole-induced PCOS rats.(3)16S r DNA sequencing of the gut microbiota indicated that MGP regulated gut microbiota structure and restored gut microbiota dysbiosis.More importantly,MGP significantly increased the relative abundance of bifidobacteria at all the order,family,and genus levels.(4)Correlation analysis showed that gut microbiota genus were significantly correlated with the host phenotype of letrozole-induced PCOS rats.(5)Fifty-four potential differential metabolites were involved in the treatment of PCOS by MGP.These potential differential metabolites are mainly involved in the metabolism of amino acids and fatty acids and may be the main biomarkers of MGP in the treatment of PCOS.(6)The hub genes derived from network pharmacology were consistent with the metabolomic analysis results to varying degrees.(7)The comprehensive analysis identified that a key new target for e NOS,five key metabolites: ornithine,citrulline,l-glutamic acid,acetylornithine and hydroxyproline and one related pathway: arginine and proline metabolism.(8)Animal experiments have verified the expression and localization of e NOS in the ovaries and further noted that MGP could significantly improve insulin resistance,reduce apoptosis and oxidative stress in letrozole-induced PCOS rats,which might be related to the up-regulation of e NOS expression.Conclusions:(1)MGP can effectively improve ovarian function in letrozole-induced PCOS rats,and that it has an anti-inflammatory effect in PCOS rats,which may be associated with the reduction in macrophage infiltration and blocking of the NF-κB and MAPK pathways.Meanwhile,MGP can regulate gut microbiota homeostasis and remodel the gut microbiota structure,especially significantly increase the relative abundance of bifidobacteria in letrozole-induced PCOS rats.These results have provided solid evidence for MGP to treat PCOS.(2)The novel potential target of e NOS and arginine and proline metabolism pathway are key links in MGP treatment of PCOS through a comprehensive strategy integrating metabolomics and network pharmacology.And animal experiments have demonstrated that MGP can improve insulin resistance and reduce apoptosis and oxidative stress in letrozole-induced PCOS rats by up-regulating the expression of e NOS.Overall,this study provides a prototype to clarify the potential pharmacological mechanism of the traditional Chinese medicine compound.