Role Of Luman/CREB3 Mediated UPR In Inflammatory Response Of Goat Endometrial Epithelial Cells

Endometritis is an important obstetric disease which cause the low efficiency of dairy goats.Escherichia coli is one of the main pathogens causing endometritis.As a pathogenic factor,E.coli LPS plays a key role in the survival and infection of the body.Endoplasmic reticulum stress and UPR plays a key role in endocarditis.Luman,as a transcription factor binding to the endoplasmic reticulum membrane,also plays an important regulatory role,but the specific regulatory mechanism remains unclear.Therefore,we constructed Luman lentivirus-interfering goat endometrial epithelial cell line and explored the regulation mechanism of Luman and UPR in the expression of related inflammatory factors under LPS stimulation.Further clarify the regulation of endoplasmic reticulum stress and UPR in innate immunity which can provide a theoretical basis for the treatment of endometritis.The experimental results are as follows:(1)Construction of goat Luman lentivirus interference vector,qPCR,Western Blot and immunofluorescence detection interference efficiency,which can up to 70%.CCK8 kit and BeyoClickTM EdU-555 kit detect cell proliferation,founded that the cell proliferation was significantly increased.The cell cycle was detected by flow cytometry.The expression of Cyclin A,Cyclin B and Cyclin D was detected by qPCR.It was found that the G1 phase was significantly decreased and the expression of Cyclin D was significantly increased.(2)Immunofluorescence localization indicated revealed that Luman was mainly localized in the cytoplasm and also expressed in the nucleus in gEECs.(3)LPS stimulated gEECs,ELISA detected the expression of IL-1?,IL-6,TNF-?,qPCR detected IL-1?,IL-6,TLR4,CD14,PTGS1,PTGS2 and PTGES,founded they were significantly increased.The expression of endoplasmic reticulum stress-related proteins GRP78,phosphoIRE1 and XBP1 were significantly increased.(4)LPS stimulated gEECs shRNA-negative group and shluman group,Western Blot detected UPR-related protein expression,founded that Luman interference group cells showed higher sensitivity to LPS.Compared with the shRNA-negative group,GRP78,phosphoIRE1,XBP1 increased significantly in shluman group.The expression of IL-1?,IL-6 and TNF-? was detected by ELISA,which were also significantly increased in shLuman group.IL-1?,IL-6 and TLR4 CD14,PTGS1,PTGS2 and PTGES were detected at mRNA level,they also showed significant changes.The expression of IRE1 and related inflammatory factors were significantly decreased in the shLuman group after treated with 4-PBA.In summary,this study investigated the role of Luman-mediated UPR in the inflammatory response of goat endometrial epithelial cells by constructing a goat Luman lentivirus interfering cell line.It was demonstrated that Luman and UPR are involved in the cellular inflammatory response induced by LPS,and may mainly affect the secretion of related inflammatory factors through the IRE1-XBP1 pathway.This study extends our understanding of the mechanisms by which Luman and UPR regulate innate immunity,and provides new clues for studying the biological functions of Luman and UPR.