Endometrial Microbiota In Chronic Endometritis And The Regulatory Mechanism Of Lactobacillus On Inflammatory Injury Of Endometrial Cells

Objective This study aims to investigate and compare the abundance,diversity,community and species differences of the endometrial microbiota between infertile patients with and without chronic endometritis（CE）,explore the regulation effect of Lactobacillus iners（ATCC55195）and Lactobacillus acidophilus（ATCC4356）on the induction of cytokine（IL-8,TNF-α and IL-1β）of human endometrial epithelial cells by lipopolysaccharide（LPS）,and preliminarily explore its molecular mechanism.Method Part I: 16 Sr RNA gene sequencing and bioinformatics analysis were applied to analyze the endometrial microbiota of infertile patients with and without CE,so as to explore the abundance,diversity,community and species differences of the two groups.Part II: The model of the LPS-induced inflammation of human endometrial epithelial cells was established,and the blank control group and LPS group were set.The level of cytokine（IL-8,TNF-α and IL-1β）in cell culture medium were determined by ELISA;the expression of cytokine（IL-8,TNF-α and IL-1β m RNA）and genes（NF-κB and IKBα）related to NF-κB pathway were determined by q RT-PCR;the protein expression of IKBα and NF-κB was measured by Western bolt.Part III: The model of how inert Lactobacillus iners and Lactobacillus acidophilus act on human endometrial epithelial cells with LPS respectively was constructed.According to the adding order of LPS and Lactobacillus,they were divided into LPS + inert Lactobacillus iners group（LPS+ATCC55195）,LPS + Lactobacillus acidophilus group（LPS+ATCC4356）,Inert Lactobacillus iners + LPS group（ATCC55195+LPS）and Lactobacillus acidophilus + LPS group（ATCC4356+LPS）.The levels of cytokine（IL-8,TNF-α and IL-1β）in cell culture medium were determined by ELISA;the expression of cytokine（IL-8,TNF-α and IL-1β m RNA）and genes（NF-κB and IKBα）related to NF-κB pathway were determined by q RT-PCR;the protein expression of IKBα and NF-κB was measured by Western bolt;the nuclear translocation of NF-κB was detected by immunofluorescence.Result 1.There were microbial communities in the endometrium of infertile patients with and without CE.The dominant species at the phylum level was Proteobacteria;the dominant species at the genus level was Ralstonia.There was no obvious difference in abundance or diversity between the two groups,and no statistically significant community differences were found between the two groups.2.No difference between the species at the level of endometrial microbiota was identified between infertile patients with and without chronic endometritis;at the level of class,the species showing differences were Erysipelotrichia and TA18;at the level of order,the species showing differences were Lactobacillales,Obscuribacterales and Erysipelotrichales;at the level of family,there were 14 species showing differences,such as Lactobacillaceae,SM2D12,Coxiellaceae,etc;at the level of genus,there were 22 species showing differences,including Lactobacillus,Muricoccus,Cloacibacterium,etc;at the level of species,there were 22 species showing differences,including Lactobacillus iners,bacterium_enrichment_culture_clone_DSR₅4,Dialister_propionicifaciens,etc.Lactobacillales,Lactobacillaceae,Lactobacillus and Lactobacillus iners are all of the highest abundance among the different species at the same level,and it was found that their abundance was significantly reduced in patients with chronic endometritis.3.LPS can promote the release of cytokine IL-8,TNF-α and IL-1β,upregulat the expression of cytokine（IL-8,TNF-α and IL-1β m RNA）and genes（NF-κB and IKBα）related to NF-κB pathway,and promote the phosphorylation of IKBα and the nuclear translocation of the transcription factor NF-κB.4.Pretreatment and post-inflammatory inert with Lactobacillus iners（ATCC55195）,and Lactobacillus acidophilus（ATCC4356）can inhibit the release of the lipopolysaccharide（LPS）-induced cytokine（TNF-α and IL-1β）of human endometrial epithelial cells.While the inhibitory effect on cytokine（IL-8）only plays a role in the inflammatory state.5.Pretreatment and post-inflammatory inert with Lactobacillus iners（ATCC55195）,and Lactobacillus acidophilus（ATCC4356）can downregulate the expression of cytokine（IL-8,TNF-α and IL-1β m RNA）and genes（NF-κB and IKBα）related to NF-κB pathway,and suppress the phosphorylation of IKBα and the nuclear translocation of the transcription factor NF-κB.Conclusion 1.There were microbial communities in the endometrium of infertile patients with and without chronic endometritis.No difference was identified in community abundance or diversity between the two groups,and the difference in community between the two groups was not statistically significant.2.There were species differences at the levels of class,order,family,genus and species in the endometrium between infertile patients with and without chronic endometritis.Lactobacillales,Lactobacillaceae,Lactobacillus and Lactobacillus iners are all of the highest abundance among the different species at the same level,and it was found that their abundance was significantly reduced in patients with chronic endometritis.3.A certain abundance of Lactobacillus in endometrium of infertile patients may have a protective effect on chronic endometritis.4.Pretreatment and post-inflammatory inert with Lactobacillus iners（ATCC55195）,and Lactobacillus acidophilus（ATCC4356）can inhibit the release of the lipopolysaccharide（LPS）-induced cytokine（TNF-α and IL-1β）of human endometrial epithelial cells.While the inhibitory effect on cytokine（IL-8）only plays a role in the inflammatory state.This inhibitory mechanism involves NF-κB signal pathway.However,different strains of Lactobacillus or different action time lead to different regulation effects.The application of Lactobacillus iners and Lactobacillus acidophilus has certain clinical significance for preventing the occurrence of endometritis or alleviating the inflammation.