| Background and purpose:Chemotherapy-induced premature ovarian failure(CIPOF)refers to an endocrine disease in which women have reduced estrogen levels before the age of 40 due to the chemotherapy drugs,accompanied by various degrees of perimenopausal symptoms.The main clinical manifestations are menstrual disorders,night sweats,hot flashes,irritability,and infertility.At the same time,the incidence of estrogen reduction-related diseases such as osteoporosis and cardiovascular disease is increased,which seriously affects the quality of life of female patients after chemotherapy.There is no effective treatment plan for CIPOF,and the common clinical symptomatic treatment of hormone replacement(HRT)can not effectively reverse the ovarian function in patients with POF,and the long-term use will increase the incidence of breast cancer,thrombosis and other diseases.Commonly used clinical hormone replacement therapy(HRT)cannot effectively reverse the ovarian function of POF patients,and long-term use will increase the incidence of breast cancer,thrombosis and other diseases.At present,the pathogenesis of ovarian failure caused by chemotherapy drugs has not yet been elucidated,and further exploration of CIPOF pathogenesis is needed to look for safe and effective ovarian protective agents.The gut microbiota is closely involved in many physiological and pathological processes such as human immune regulation,nutrient absorption,and metabolism,and plays an important role in maintaining the health of the body.With the introduction of the concept of "estrogen-gut microbial axis",the gut microbiota has been confirmed to be directly related to various gynecological endocrine diseases such as polycystic ovary syndrome.Gut microbiota plays an important role in the process of drug metabolism and absorption.It can bind to a variety of enzymes in the host and affect the efficacy and side effects of platinum and other chemotherapeutic drugs.Relevant studies have reported that the gut microbiota can affect the host’s responsiveness to chemotherapeutic drugs,which can promote the efficacy of chemotherapeutic drugs,reduce the effect of anticancer drugs,and neutralize the toxicity of chemotherapeutic drugs.Therefore,from the perspective of gut microbiota,exploring the side effects of ovarian failure caused by chemotherapeutic drugs and looking for protective agents to improve ovarian function impairment after chemotherapy will be an important "breakthrough" for current clinical treatment.This study intends to gut microbiota as the starting point,from population cohort,multiomics analysis,in vitro and external experiments and other different levels of CIPOF intestinal flora host interaction mechanism,identify key metabolites and their bacteria,clarify the molecular mechanism of granule apoptosis involved in CIPOF,in order to open up new direction for CIPOF pathogenesis and open up new ideas for CIPOF.Content and methods:1.The microbiota combined with the metabolome analyzed the CIPOF microbiota host interaction mode to construct the characteristic intestinal flora and metabolic profiles of CIPOF patients.(1)Stool and peripheral blood samples of 30 CIPOF patients and healthy women(Control)were collected;metagenomic shotgun sequencing(WGS)was used to obtain CIPOF gut microbiota and functional spectrum.(2)The CIPOF mouse model was constructed using Cis-platinum,the intestinal metabolism profile of CIPOF mice was obtained by liquid-phase tandem mass spectrometry(LC-MS/MS),and the intestinal bacteria profile was obtained by 16 S rRNA gene sequencing.(3)Correlation analysis of microbiota and metabolome data,analysis of the interaction between CIPOF flora and host,and identification of key bacterial species and metabolites.2.Combined antibiotics and fecal bacteria transplantation experiment(FMT)to explore the causal relationship between gut microbiota imbalance and CIPOF.(1)After using antibiotics to eliminate the basic gut microbiota of mice,a CIPOF model was constructed by Cis-platinum to evaluate the symptoms of mouse POF,including: ELISA method to detect serum estradiol(E2)and follicle stimulating hormone(FSH)levels,HE staining and follicle count Detect ovarian tissue structure and follicular development at various levels,observe the structure of ovarian antral follicles with transmission electron microscope,and Tunel detects ovarian granulosa cell apoptosis.(2)The CIPOF gut microbiota was reconstructed in the mouse intestine by FMT,and a CIPOF model was constructed to evaluate the symptoms of POF in mice to explore the causal relationship between the gut microbiota and CIPOF.3.To evaluate the protective effect of the differential metabolite2,4-dicarboxybenzoic acid(2,4-DHBA)producing bacteria Lactobacillus reuteri(L.reuteri)and Lactobacillus brevis(L.brevis)on CIPOF.(1)QPCR was used to detect the abundance of L.reuteri and L.brevis in the feces of CIPOF mice.(2)Targeted mass spectrometry was used to detect the ability of L.reuteri and L.brevis to produce 2,4-DHBA.(3)Gavage C57BL/6 mice with L.reuteri and L.brevis to construct a CIPOF model,the ovarian protective effects of L.reuteri and L.brevis were evaluated from the aspects of mouse body weight,ovarian weight,ovarian tissue structure,follicular development at various levels,follicle count,and ovarian granulosa cell apoptosis.4.Explore the molecular mechanism of 2,4-DHBA regulating granular cell apoptosis and participating in the pathogenesis of CIPOF.(1)C57BL/6 mice were pretreated with 2,4-DHBA to construct a CIPOF model to verify the efficacy and safety of 2,4-DHBA.(2)After 2,4-DHBA pretreated ovarian granulosa cell line(KGN)cells,Cis-platinum was used to stimulate the cells,and the cell proliferation and apoptosis were detected by the CCK-8 method and Annexin V-FITC/PI apoptosis kit.(3)Transcriptome sequencing was used to screen the differential genes of granulosa cells in CIPOF mice treated with 2,4-DHBA,and the expression levels of differential genes in the ovaries of CIPOF mice were detected by QPCR and WB.(4)The KGN cell line was transfected with a plasmid overexpressing the target gene or knocked out the target gene si RNA to detect the effect of 2,4-DHBA on Cis-platinum-induced KGN cell apoptosis.Result:1.Compared with the Control group,the alpha diversity of CIPOF gut microbiota was significantly reduced,and the beta diversity was significantly different;the abundance of conditional pathogenic bacteria such as Oscillospira increased,and the abundance of beneficial bacteria such as Lactobacillus reduced.2.After Cis-platinum stimulation,the distribution of intestinal metabolites in mice changed significantly.Differential metabolites are mainly involved in the taurine(Taurine)/ phenylalanine(Phenylalanine)metabolic pathway,with downregulation of 50 metabolites represented by 2,4-DHBA,and upregulation of 94 metabolites represented by γ-Glutamylglutamine.3.Correlation analysis of microbiome and metabolome data shows that Lactobacillus and other microorganisms are positively correlated with the content of2,4-DHBA,which is a related group of 2,4-DHBA-producing bacteria.4.Combining antibiotics to eliminate the gut microbiota of CIPOF mice can improve mouse body weight and ovarian weight,serum E2 and FSH levels,increase the number of primordial follicles,and relieve the structural damage of ovarian antral follicles and the apoptosis of granulosa cells.5.Compared with CIPOF mice fed with normal mouse fecal bacteria liquid,transplantation of CIPOF mouse fecal bacteria liquid increased body weight and ovarian weight,serum E2 and FSH levels of CIPOF mice,increased the number of atretic follicles,and aggravated the structural damage of ovarian antral follicles And the apoptosis of granular cells.6.L.reuteri and L.brevis can improve the weight loss of ovaries in CIPOF mice,increase the number of primordial follicles,and relieve the structural damage of ovarian antral follicles and the apoptosis of granulosa cells.7.Supplementation of the differential metabolite 2,4-DHBA can improve body weight and ovarian weight loss in CIPOF mice,increase the number of primordial follicles,reduce the number of atresia follicles,and alleviate the structural damage of ovarian antral follicles and the apoptosis of granulosa cells.8.2,4-DHBA can ameliorate the decrease in proliferation and apoptosis of KGN cells induced by Cis-platinum.9.After 2,4-DHBA pretreatment,the expression level of SOX7 gene in the ovarian tissue of CIPOF mice decreased.10.Knockdown of SOX7 gene can improve the apoptosis of KGN cells caused by Cis-platinum,and 2,4-DHBA can significantly improve the apoptosis of KGN cells caused by SOX7 overexpression.Conclution:1.The composition and function of the gut microbiota of CIPOF hosts were significantly disturbed,with probiotics such as L.reuteri and L.brevis as well as the flora metabolites 2,4-DHBA content decreased.2.The transplantation of CIPOF feces can aggravate the ovarian toxicity of Cis-platinum,and the gut microbiota participates in the pathogenesis of CIPOF.3.L.reuteri and L.brevis can improve host CIPOF symptoms by producing2,4-DHBA.4.2,4-DHBA can improve the pathogenesis of CIPOF by down-regulating SOX7 to inhibit ovarian granulosa cell apoptosis. |
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