| Long-term high-concentrate feeding can easily lead to ruminal subacute acidosis(SARA)in cows,which is manifested by abnormally elevated peripheral blood lipopolysaccharide(LPS)levels,and high levels of LPS can cause systemic inflammatory reactions,especially causing various Abnormal epithelial-mesenchymal transition(EMT)occurs in the epithelial cells of tissues and organs.In the early stage,the peripheral blood of about 400 high-yielding dairy cows in the farm was tested,and it was found that the LPS level was positively correlated with the feed intake and lactation volume,and the peak value of LPS(0.11-0.15 μg/L)happened just in the embryo implantation window period.It can be inferred that high levels of LPS will cause unnecessary epithelial-mesenchymal transition in the endometrial epithelium,resulting in failure of embryo implantation and a reduction in the reproductive rate of dairy cows.This may be the reason for the short service life of dairy cows in my country and the high active culling rate.one.In this study,cow endometrial epithelial cells(bEECS)were treated with LPS for 48 h to simulate the endometrial epithelial environment under high LPS levels.By analyzing the changes of the key pathway TGFβ1/SMAD3 and the main regulator ZEB1 that regulate the occurrence of EMT,three EMT marker genes(E-cadherin,N-cadherin,Vimentin)were detected,combined with cell migration rate and cell morphology changes,the specific mechanism of LPS-induced EMT in bEECs was analyzed,and the research results are as follows:1.LPS treated bEECs and EMT occurred.10 ug/m L LPS treatment can significantly promote the proliferation of bEECs,and increase the expression levels of TGFβ1/SMAD3 and ZEB1;Western Blot analysis found that LPS treatment decreased E-cadherin and increased N-cadherin(N-cadherin)The expression levels of Vimentin and vimentin were consistent with the results of immunofluorescence detection.In addition,LPS treatment increased the migration rate of bEECs cells and induced the transformation of cell morphology to elongated spindle,showing the morphological characteristics of mesenchymal cells.It shows that LPS activates TGFβ1/SMAD3 pathway,and up-regulates ZEB1 to induce EMT in bEECs.2.miR-200 b inhibits EMT in bEECs by targeting ZEB1.After treatment of bEECs with LPS and overexpression of ZEB1 with the activator butyl benzyl phthalate(BBP),compared with the control group without BBP activation,the expression level of E-cadherin was significantly decreased,while the expression levels of N-cadherin and Vimentin significantly increased,further indicating that ZEB1 directly induces EMT in bEECs.At the same time,it was found that LPS treatment could significantly reduce the expression level of Bta-miR-200b(miR-200b)m RNA in bEECs,and the dual-luciferase reporter system showed that miR-200 b could target ZEB1 3’UTR.Treatment of bEECs with miR-200 b mimic can inhibit the occurrence of EMT.It was shown that miR-200 b inhibited EMT by binding to ZEB1-3’UTR to reduce the expression of ZEB1.3.Transcriptome sequencing was used to analyze the differential expression of genes related to inflammation and embryo implantation.Further verification of inflammation and embryo implantation-related gene changes,the results found that after LPS induction,a total of 177 genes showed significant differences in expression,among which inflammationrelated genes CXCL8,CXCL5,CXCL3 and SAA3 were significantly up-regulated,embryo implantation-related genes Bo LA,BOLA-DQA2 was significantly upregulated,while IGF2 and ITGA5 were significantly downregulated.Using KEGG enrichment analysis,it was found that the differential genes were mainly in inflammation-related pathways such as TNF pathway,NF-kappa B pathway,IL-17 pathway,NOD-like receptor pathway and Toll-like receptor pathway.It shows that high level of LPS will indeed cause inflammatory reaction of endometrial epithelial cells,which is not conducive to embryo implantation.In summary,LPS upregulates the expression of ZEB1 by activating TGFβ1/SMAD3,and induces EMT in bovine endometrial epithelial cells,while miR-200 b can inhibit the occurrence of EMT by targeting ZEB1,indicating that high levels of LPS can induce cow endometrial epithelial cells to undergo EMT.Abnormal occurrence of EMT in epithelial cells,thereby inhibiting embryo implantation. |
