The Role Of Transcription Factor Nrf2 In LPS-induced Injury Of Bovine Endometrial Epithelial Cells

Endometritis is a common and multiple disease in dairy cows,which significantly affects milk yield and reproductive performance.Endometrial inflammation with endometrial tissue damage and affect the normal physiological function of the uterus.As a result,the pregnancy rate of dairy cows is reduced,the calving interval is prolonged,and even be eliminated.However,the precise regulatory mechanism is still not entirely clear due to the complicated causes of endometritis in dairy cows,involving numerous regulatory elements.Under the action of various of stress factors,endoplasmic reticulum stress and oxidative stress will occur in cells,in which endoplasmic reticulum stress plays a role in a variety of inflammatory diseases.Studies have shown that nuclear factor E2 related factor 2（Nuclearfactorerythroid2-relatedfactor2,Nrf2）,as a key factor of cellular oxidative stress,plays an important regulator role in a variety of inflammatory responses.However,it’s unclear the mechanism of how Nrf2 participates in the endoplasmic reticulum stress signal pathway and affects the inflammatory response.In view of this,this study took bovine endometrial epithelial cells（BEECs）as the research object,induced BEECs inflammation model in vitro by LPS,and explored the effect of Nrf2 in LPS-induced damage to bovine endometrial epithelial cells using immunofluorescence,RT-qPCR,Western blot,RNA interference,flow cytometry and other experimental methods.The following are the main results:（1）The results of immunohistochemistry showed that Nrf2 was mainly expressed in the luminal and glandular epithelium of the endometrium,and the expression of Nrf2 in the endometritis group was significantly higher than that in the healthy group（P<0.05）.In the model of BEECs inflammation induced by LPS,the expression of mRNA and protein of Nrf2increased significantly（P<0.01,P<0.0001）.At the same time,endoplasmic reticulum stress was activated and,the expression of GRP78,ATF6,PERK and ATF4 and phosphorylation of e IF2αwere significantly increased（P<0.05,P<0.01）.（2）Inhibition of Nrf2 expression could attenuate inflammatory response and activation of PERK pathway induced by LPS in BEECs.Knockdown of Nrf2 expression in endometrial epithelial cells by siNrf2 transfection reduced the proliferation of endometrial epithelial cells and the LPS-induced inflammatory response of BEECs.The expression of inflammatory factors TNF-α,IL-6,IL-8 and NF-κB（p65）decreased significantly（P<0.05,P<0.0001）;the outflow of Ca²⁺from endoplasmic reticulum decreased,and the expression of PERK pathway related genes GRP78,PERK,ATF4 and CHOP and the phosphorylation level of e IF2αwere inhibited（P<0.05,P<0.01）.（3）Activation of Nrf2 expression enhanced the inflammatory response of BEECs induced by LPS and the activation of PERK pathway.After activating Nrf2 expression by TBHQ treatment,the expression of BEECs inflammatory cytokines induced by LPS increased,such as TNF-α,IL-6,IL-8 and NF-κB（p65）expressions were significantly increased（P<0.05,P<0.001）.The Ca²⁺efflux from endoplasmic reticulum and the expression levels of PERK pathway related genes were increased（P<0.05）.In summary,the present study identified that Nrf2,as a nuclear transcriptional regulatory factor,regulates the expression of inflammatory factors in BEECs through PERK pathway in the unfolded protein response in bovine endometritis,which reveals the role of Nrf2 in LPS-induced inflammatory response in BEECs.This study provides experimental basis for further study on the pathogenesis of endometritis in dairy cows.