| Bovine endometritis is one of the main causes of infertility in dairy cows,which brings serious economic losses to the dairy industry.In order to further investigate the pathogenesis of bovine endometritis,clinical cases with endometritis in dairy cows were evaluated clinically and histologically.Meanwhile,ncRNAs(lncRNAs and microRNAs)in endometritis tissues of healthy and diseased dairy cows were detected by high-throughput sequencing technology.The significantly differently expressed lncRNAs and microRNAs between the two groups were screened.Bioinformatics analysis was conducted on the differentially expressed lncRNAs and microRNAs to explore their regulatory roles in the occurrence and development of endometritis in dairy cows.Based on the comprehensive analysis of the sequencing results,we selected bta-miR-21-5p as the object of further study.To explore how BTA-miR-21-5p regulates the inflammatory response process of bovine endometrial epithelial cells in dairy cows,bovine endometrial epithelial cells(BEECs)induced by LPS were used as an inflammatory model in vitro to observe the changes in the expression of related molecules by overexpression and inhibition of the expression of BTA-miR-21-5p in BEECs.This study provided a new theoretical basis for the pathogenesis of endometritis in dairy cows from the perspective of ncRNA.The research contents of this paper include:(1)Clinical and histological evaluation of endometritis in dairy cowsIn this study,dairy cows within 28 to 35 days postpartum were selected for diagnosis of endometritis by examining the characteristics of vaginal mucus.Meanwhile,dairy cows with other diseases and recent medication records were excluded during the diagnosis process.Vaginal mucus was collected,and the neutrophils in the mucus were counted after rapid diff staining.Endometrial tissue samples were collected by endometrial biopsy apparatus for histopathological evaluation.The expression of inflammatory factors(IL-1?,IL-6,TNF-?)in endometrial tissues was detected by RT-qPCR method to determine the inflammatory state of endometrial tissues.The results shouwed that:1)34 cows of 144 postpartum cows were diagnosed with endometritis,with an incidence of23.6%;2)The proportion of neutrophils in the mucus of healthy cows was 13.76% ± 0.91,and that of neutrophils in the vaginal mucus of endometritis cows was 27% ± 0.86.3)Histopathological examination showed that the endometrium of dairy cows in the healthy group was intact,and the endometrium epithelial cells were arranged neatly and covered the stromal layer completely.The endometrial epithelium of cows with endometritis was destroyed and endometrial epithelial cells were necrotic.Neutrophils and lymphocytes were infiltrated in the epidermis.Stromal layer edema,bleeding,infiltration of lymphocytes and plasma cells;4)The expression levels of inflammatory cytokines IL-1?,IL-6 and TNF-? in endometritis of cows were significantly increased.(2)Screening and analysis of long non-coding RNA(lncRNA)associated with endometritis in dairy cowsStudies have found that lncRNAs can participate in important life processes such as ontogenesis,substance metabolism and cell differentiation of the body,and are closely related to the occurrence of a variety of diseases.However,the role of long non-coding RNA in endometritis in dairy cows has not been reported.This sectionBased on the clinical evaluation,histomathological evaluation and molecular biological evaluation of dairy cow endometritis in the previous chapter,dairy cows were screened out and divided into healthy group(Control,CN,n=2)and endometritis group(E,n=4).The changes of lncRNA expression profiles in endometrial tissues of cows in two groups were studied.Total RNA was extracted from endometrial tissues in the N group and the E group,and the transcripts in the samples of the two groups were detected by high-throughput sequencing platform,and the coding ability of the transcripts was analyzed.According to their coding ability,the transcripts were divided into lncRNA and mRNA.According to the identified expressions of lncRNAs and mRNAs in the samples of the two groups,the lncRNAs and mRNAs with significant differences in expression between the two groups were screened.Through GO enrichment analysis and KEGG signaling pathway analysis,the potential functions of differential lncRNA and mRNA in the process of endometritis in dairy cows were studied.The results showed that a total of6201 differentially expressed lncRNAs were detected,among which 696 were significantly differentially expressed.A total of 21,184 differentially expressed mRNAs were detected,among which 2357 mRNAs were significantly differentially expressed.Among all the significantly differentially expressed lncRNAs and mRNAs,we focused on the known lncRNAs and mRNAs.A total of 239 known lncRNAs were differentially expressed in endometrial tissue samples of group E,of which 109 were up-regulated and 130 were down-regulated.A total of 1,900 known mRNAs were significantly differentially expressed in endometrial tissue samples from group E,among which 1,123 mRNAs were up-regulated and 777 mRNAs were down-regulated.The biological processes involved in the differentially expressed mRNAs in the uterine tissues of group E were mainly related to stimulation response,organism immunity,reproductive process,cell killing,tight junction,etc.The main signaling pathways involved include MAPK signaling pathway,Toll-like receptor mediated signaling pathway,phagocytosis,complement system and so on.The target genes of differentially expressed lncRNAs are mainly involved in biological processes such as stimulation response,immune regulation,reproductive process and extracellular matrix.The main signaling pathways involved in regulation were Staphylococcus aureus infection,c AMP signaling pathway,innate immune deficiency,amino acid metabolism,etc.The above results indicate that endometritis has a significant influence on the expression profile of lncRNA in endometrium tissue of cows,and lncRNA can be involved in regulating local innate immune defense function of endometrium tissue.3)Screening and analysis of microRNA related to endometritis in dairy cowsMultiple studies have shown that MicroRNA is extensively involved in the regulation of a variety of diseases in mammals,including cow endometritis.In this chapter,we carried out high-throughput sequencing on the two groups of samples screened in the previous chapter(group CN and group E),identified the microRNAs in the endometrial tissues of the group CN and group E,focused on the microRNAs differentially expressed in the endometrial tissues of disaffected cattle,and carried out GO enrichment analysis and KEGG signaling pathway analysis.To explore the biological processes and signaling pathways involved.The results of this part showed that a total of 235 microRNAs were differentially expressed between the CN group and the E group,among which 189 were up-regulated and 46 were down-regulated.Among all the microRNAs that were significantly differentially expressed,we focused on the microRNAs that were highly expressed in endometrial tissues.The expression is high in uterine tissue,Moreover,the significantly up-regulated microRNAs included bta-miR-204,bta-miR-145,bta-miR-222,bta-miR-200 c,bta-miR-10174-3p,bta-miR-423-5p,bta-miR-21-5p,and bta-miR-3596;The microRNAs with high expression levels and significantly down-regulated in uterine tissues were bta-miR-196 a,bta-miR-378,bta-miR-24-3p,bta-miR-3604,bta-miR-1,bta-miR-26 a,and bta-miR-23b-3p.GO and KEGG analysis of the differentially expressed microRNAs revealed that they are mainly involved in cell damage,regulation of immune function,stimulation response,reproductive process,extracellular matrix regulation,cell connectivity,etc.,and are also involved in mediating the regulation of innate immunity,affecting bacterial infection,and participating in the expression of metabolic genes and inflammatory genes.The above results indicate that microRNAs may be involved in the occurrence and development of endometritis through multiple pathways.(4)bta-miR-21-5p promoted LPS-induced inflammatory response of bovine endometrial epithelial cells through MyD88-dependent signaling pathwayBased on the sequencing results in the previous studies,we selected bta-miR-21-5p as the object of further study to study its influence on the occurrence and development of endometritis,as well as the related molecular mechanisms involved in the regulation of the disease.In order to reveal the process of regulating endometrial inflammation by bta-miR-21-5p,cow endometrial epithelial cells were isolated and cultured by tissue digestion method in this part of the study,and the inflammatory model was established by stimulating cells with 3?g/mL LPS.The expression of BTA-miR-21-5p in cells was interfered by small interfering RNA,and the gene expression of related inflammatory cytokines(IL-1?,IL-6,TNF-?)was detected by RT-qPCR.The expression of key molecules in the MyD88 signaling pathway was detected by Western Blotting and immunofluorescence staining,and the molecular mechanism of the effect of bta-miR-21-5p on the inflammatory response of BEECs was preliminarily explored.1)The results showed that the overexpression of Bta-miR-21-5p promoted the inflammatory response of cells,and significantly increased the gene expression of inflammatory cytokines(IL-1?,IL-6,TNF-?).However,inhibiting the expression of bta-miR-21-5p can improve the inflammatory response induced by LPS in endometrial epithelial cells,and the gene expression of inflammatory cytokines in cells is significantly reduced.2)Overexpression of Bta-miR-21-5p significantly increased the expression level of TLR4 protein,the expression level of IRAK4 protein,a key molecule dependent on the MyD88 signaling pathway,and the phosphorylation level of transcription factor NF-?B in BEECs;When the expression of BTA-miR-21-5p was inhibited,the opposite result was obtained.In addition,interference with the expression of BTA-miR-21-5p in BEECs had no significant effect on the expression of TRIF protein,a key molecule independent of the MyD88 signaling pathway.These results suggest that Bta-miR-21-5p may be a key signaling molecule promoting the development of endometritis in dairy cows,and can play a pro-inflammatory role through the MyD88-dependent signaling pathway,which may be a key target molecule for the diagnosis and treatment of endometritis in dairy cows.(5)bta-miR-21-5p can promote LPS-induced inflammatory injury of endometrial epithelial cells in dairy cows10?g/mL LPS was used to stimulate BEECs as an in vitro inflammatory injury model.RT-qPCR was used to detect the expression levels of apoptosis related genes Bcl-2 and Bax,and Western Blotting was used.The expression of key molecules in the p38MAKP/JNK pathway was detected.The results showed that the overexpression of Bta-miR-21-5p increased the expression of Bax,while the decrease of Bcl-2 expression and the inhibition of Bta-miR-21-5p resulted in the opposite result.These results suggested that BTA-miR-21-5p could induce cell injury by promoting apoptosis.In addition,bta-miR-21-5p also increased the level of p38 phosphorylation and the expression of JNK.These results suggested that bta-miR-21-5p may promote the injury of endometrial epithelial cells in dairy cows through the p38MAKP/JNK pathway. |
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