Based On High-throughput Sequencing And Regulation Of Nf-κb Signal Pathway Research On The Effects Of Vaginal Microbiota On The Occurrence,development And Mechanism Of Endometriosis

Endometriosis,referred to as EMS for short,is a disease characterized by estrogen dependence,chronic inflammation,infertility,abdominal pain and other symptoms.Endometrial tissue appears outside the uterus(histology),affecting about10% of childbearing age women.The definite diagnosis of endometriosis mainly depends on the pathological examination after surgical resection of the ectopic focus.At present,there are many theories involved in its pathogenesis,and there is no single theory to explain various types of endometriosis.In the proposed pathogenesis of endometriosis,the theory of immunity and inflammation is widely accepted.The activation of neutrophils and macrophages leads to the release of pro-inflammatory cytokines such as interleukin(IL)-6 and the angiogenesis of endometriosis,which leads to the growth and survival of endometriosis.Most of the proteins involved in the formation of the pro-inflammatory environment of endometriosis are mediated by nuclear factor kappa B(NF-κ B).It has been shown to promote cell proliferation in endometriosis through toll like receptor myeloid differentiation factor 88(TLR-My D88)signal pathway.In this process,lipopolysaccharides(LPS)from bacteria bind to receptors(TLRs)on endometriosis cells and through phosphorylation and ubiquitination of I κB(NF-κB inhibitory protein)activate classical NF-κ B signal pathway,so that p65/p50 dimer(previously identified by I κ B inactivation)is phosphorylated to p-p65/p50(activation)and subsequently binds to DNA.This may lead to the up regulation of a series of target genes,and releasr IL-6,IL-1 β and macrophage migration inhibitory factor(MIF).In recent years,the development of high-throughput sequencing technology and microbiome research have attracted more and more attention.There are many reports on the impact of intestinal microbiota on EMS patients,but there are few researches on the impact of vaginal microbiota on EMS patients.Vagina is a lumen with more than 300 kinds of bacterial colonization(NAT commun.in february 2020).A variety of bacteria are closely related to gynecological diseases,such as bacterial vaginosis(BV)and sexually transmitted diseases(STD).Researches have shown that the progression of these diseases may be alleviated by restoring normal vaginal microbiota.Based on the previous literature reports that the occurrence of many diseases is closely related to microbial imbalance,it is speculated that the occurrence of endometriosis may also be related to vaginal microbiota disorder.To this end,we first used high-throughput sequencing technology to analyze the diversity of vaginal microbiota between healthy women of childbearing age and patients with endometriosis and its relationship with the occurrence and development of endometriosis;Then the EMS mouse model was established,and the intervention research was carried out at the animal level.Using molecular biology technology,we further explored the influence of vaginal microbiota on the occurrence,development and mechanism of endometriosis based on the regulation of NF-κB signal pathway,and provided new thinking for further revealing the occurrence and development mechanism of endometriosis and broadening the treatment ideas of endometriosis.Part I Research on the relationship between vaginal microbiota diversity,the occurrence and development of endometriosis Objective:We intended to analyze the diversity of vaginal microbiota in patients with endometriosis and healthy women of childbearing age and its relationship with the occurrence and development of endometriosis.Methods:1.After the approval of the Ethics Committee(registration number of the clinical trial registration center: chictr20000039078),16 patients with endometriosis who visited the Second Affiliated Hospital of Nanchang University from September2020 to March 2021 were randomly selected as the endometriosis group(all confirmed by postoperative pathology)and 18 healthy women of childbearing age without known related diseases as the healthy women of childbearing age group.They were included in the study after obtaining informed consent.The endometriosis group and the healthy women of childbearing age group adopted the unified inclusion and exclusion criteria.The basic clinical information(age,BMI,number of abortions,etc.)and blood test results(AMH,CA125,NLR representing inflammation,etc.)of the subjects were recorded.Inclusion criteria of endometriosis group: 1.Age 18-50 years old;2.patients clinically diagnosed as endometriosis according to symptoms,signs and auxiliary examinations;3.No sexual life,vaginal flushing and vaginal medication within 3days,no cervical treatment within 7 days,and no antibiotics within 30 days;4.there is no abnormality in the vaginal secretion test,and the white blood cell count and neutrophil ratio in the blood routine test are within the normal range.Inclusion criteria for healthy women of childbearing age: 1.Age 18-50 years old;Menstrual cycle(28 ± 7 days);2.health examination population;3.No sexual life,vaginal flushing and vaginal medication within 3 days;No cervical treatment within 7 days;No antibiotics were used within 30 days;4.no abnormality found in vaginal secretions;The white blood cell count and neutrophil ratio in blood routine test were within the normal range.2.Sampling: the sampling method of vaginal secretion samples is as follows:for outpatient or inpatient,take the lithotomy position,support the vaginal wall with a sterile vaginal dilator,and use a disposable sterile swab to take samples in the vagina.Immediately after collection(transportation time shall not exceed 30 min),put it into-80℃ refrigerator for freezing storage.3.High throughput sequencing and analysis: 16 patients with endometriosis and18 healthy women of childbearing age collected 1 vaginal secretion sample from each individual for high-throughput sequencing analysis of vaginal microbiota.Results:1.According to the r-ASRM(American Society of reproductive medicine Revised)score,9 patients in the endometriosis group were stage 3-4,and 7 patients were stage 1-2.2.Comparison of general clinical indexes between the two groups: there was no significant difference in age and body mass index between endometriosis group and healthy women of childbearing age group(P > 0.05);The number of abortions in endometriosis group was 0.88 ± 0.96,and that in healthy women of childbearing age group was 0.11 ± 0.32.The number of abortions in endometriosis group was higher than that in healthy women of childbearing age group(P < 0.05);The AMH of endometriosis group was 1.13 ± 1,and that of healthy women of childbearing age group was 4.07 ± 1.09.The AMH of endometriosis group was lower than that of healthy women of childbearing age group(P < 0.05);CA125 was 67.33 ± 28.63 in endometriosis group and 14.43 ± 4.49 in healthy women of childbearing age group.CA125 in endometriosis group was higher than that in healthy women of childbearing age group(P < 0.05);The NLR of endometriosis group was 2.26 ± 0.8,and that of healthy women of childbearing age group was 1.82 ± 0.38.The NLR of endometriosis group was higher than that of healthy women of childbearing age group(P < 0.05).3.Comparison of vaginal microbiota diversity between the two groups:(1)There was no significant difference between Shannon index(estimated total species)and Chao1 index(community diversity)of two groups α diversity(P > 0.05).According to distance matrix and PCo A analysis,the β diversity of vaginal microorganisms in endometriosis group was different from that of healthy women of childbearing age(21.8% vs 12.6%,unweighted Uni Frac distance method).The two groups of vaginal bacterial communities were composed of 154 genera belonging to28 phyla.(2)At the phylum and genus levels,the vaginal microbiota composition of endometriosis group was different from that of healthy women of childbearing age:the abundance comparison of dominant genera shows that actinomycetes(especially harmful gardnerella)were significantly enriched in endometriosis patients(from0.23% to 21.59%),while Firmicutes(especially beneficial Lactobacillus)were decreased in endometriosis patients(from 98.25% to 68.79%).In order to further determine which bacterial taxa were different between the endometriosis group and the control group,we conducted a LDA effect size(lefse)analysis(LDA threshold3.5)and identified the genera with significant differences,including alloscardovia,cloacibacterium,gardnerella,etc.4.The correlation analysis between vaginal microbiota and high-throughput sequencing in endometriosis group showed that the relative abundance of vaginal Lactobacillus was negatively correlated with blood neutrophils(r=-0.5039,P < 0.05);Megasphaera and shuttleworthia were positively correlated with blood CA125(r=0.5455,P < 0.05)and NLR(r=0.6035,P < 0.05).Conclusions:1.Compared with the vaginal microbiota of healthy women of childbearing age,there is no significant difference in the vaginal microbiota of patients with endometriosis in α diversity,while β Diversity varies.2.The relative abundance of beneficial bacteria(Lactobacillus,etc.)decreased and that of harmful bacteria(Gardnerella,etc.)increased in patients with endometriosis;The decrease of Lactobacillus relative abundance in vaginal microbiota was positively correlated with the level of blood neutrophils.Megasphaera and shuttleworthia were positively correlated with blood CA125 and NLR.Part II Effects of vaginal microbiota on the progression of endometriosis in mice Objective:We want to research the mechanism of the effects of vaginal microbiota on the progression of endometriosis in mice.Methods:1.The endometriosis model of mice was established by intraperitoneal injection of endometrial segments: the donor mice were intraperitoneally injected with estrogen for 1-2 personality cycles,anesthetized with 2% isoflurane and sacrificed after cervical spondylectomy;The endometrium was cut into pieces with a diameter of about 1mm and injected intraperitoneally into the recipient mice at a ratio of 1:2;After 3 weeks,the ectopic foci were confirmed by ultrasound to be colonized and grown in the abdomen and pelvis of the recipient mice.2.Experimental group: the mice were divided into healthy control group(Group C)and endometriosis model group(group M).The vagina of mice only used absorbable gelatin sponge water once every 3 days for 3 weeks);Mixed antibiotic group(ABX group,containing 0.5g/L vancomycin,1g/L neomycin,1g/L metronidazole and 1g/L ampicillin solution,the absorbable gelatin sponge soaked in the mixed antibiotic was placed in the vagina of model mice every 3 days for 3weeks);Parthenolide(NF-κ B signal pathway blocker,10mg/kg intraperitoneal injection of model mice,4 times a week for 3 weeks).3.Histological examination: high resolution small animal ultrasound was used to observe the changes of abdominal and pelvic ectopic foci once a week.After 3weeks of treatment,the mice were anesthetized with 2% isoflurane and decapitated,and the lesions were dissected and observed.He staining and immunohistochemistry(Ki-67 and Iba-1)were used for histological examination of endometrium and ectopic lesions.4.Molecular biological detection: detection of inflammatory cytokines(IL-1β,IL-6 and TNF-α)in mouse peritoneal fluid by q-PCR.Detection of key proteins of NF-κB signal pathway(TLR4,My D88,p65,p-p65)in mouse ectopic endometrium changes in by WB.Results:1.Three weeks after the intervention of mouse endometriosis modeling,compared with the model group(23.74 ± 3.70 mm of ectopic focus),the mice treated with ABX(20.33 ± 3.45 mm of ectopic focus)and parthenolide(16.63 ± 2.00 mm of ectopic focus)had less endometriosis(P < 0.05),and compared with the ABX group,the parthenolide group had less endometriosis(P < 0.05).2.He staining showed that the ectopic lesions in the model group had typical endometriosis like structures,including thick epithelial layers and gland regions,while the ectopic lesions in the ABX(mixed antibiotic vnma)group and the small white chrysanthemum lactone group had thinner epithelial regions and fewer glands;Compared with the model group,the lesion matrix area of ABX mice and parthenolide mice was smaller.Compared with the model group,the proliferation marker Ki-67 positive epithelial cells and macrophage marker Iba-1 decreased in ABX mice and parthenolide mice(especially parthenolide mice).3.Heterotopic focus NF-κB pathway related protein and peritoneal fluid inflammatory factor levels: compared with ectopic lesions in the model group,the expression of NF-κb pathway related proteins in the ABX group decreased,while parthenolide also inhibited p65/p-p65.Similarly,compared with the model group,IL-1β,TNF-α and IL-6 in the peritoneal fluid of ABX mice and parthenolide mice concentration decreased.Conclusion:After intraperitoneal injection of parthenolide and vaginal use of mixed antibiotics(VNMA),The expression of NF-κB pathway related proteins decreased,the level of inflammatory factors in mouse peritoneal fluid decreased,and the histological proliferation level and volume of heterotopic lesions decreased.Context Conclusion: compared with the vaginal microbiota of healthy women of childbearing age,the diversity of vaginal microbiota in patients with endometriosis was changed(β Diversity).There is a decrease in the relative abundance of beneficial bacteria(Lactobacillus,etc.)and an increase in the relative abundance of harmful bacteria(Gardnerella,etc.)in the vaginal microbiota.The vaginal use of mixed antibiotic solution(VNMA)in mice could block NF-κB pathway and inhibited the progression of endometriosis,but the inhibitory effect was weaker than that of parthenolide.