Study On The Anti-inflammatory Mechanism Of Rabbit Endometrial Epithelial Cells α7nAChR Mediated MAPK Signaling Pathway

Postpartum female animals undergo changes of the uterus in anatomical structure,physiology,and immune function.What’s more,due to the relative shortage of mineral ions and the negative energy balance,the female animals are prone to ascending infection of reproductive tract bacteria after delivery,which eventually lead to endometritis.The inflammation plays an important role in the body’s metabolism,defense and tissue regeneration,however,an excessive inflammation can result in tissue damage.Bacterial infection is one of the main causes of endometritis.The body mainly recognizes bacterial components through pattern recognition receptors which can activate intracellular signaling pathways,induce a large amount of secretion of chemokines and cytokines,and finally promote inflammation in the body.The serine-threonine protein kinase family formed by mitogen-activated protein kinase(MAPK)can respond to a variety of biological processes such as extracellular and intracellular stimuli,cell-mediated growth and immunity and inflammation.Short-term stimulation of LPS,the main virulence factor of Gram-negative bacteria,which can promote the phosphorylation of MAPK family ERKs,JNKs and P38 MAPKs,meanwhile,it can also activate the MAPK signaling pathway to induce the release of inflammatory cytokines.In this study,it was found that the phosphorylation levels of MAPK and NF-κB were inhibited after 12 hours of LPS stimulation by culturing rabbit endometrial epithelial cells in vitro.This result is contrary to that of short-term stimulation,therfore,there may be a potential cellular self-regulation mechanism which possibly be related to the "cholinergic anti-inflammatory pathway".On one hand,the research used different concentrations of LPS,α7nAChR inhibitors and agonists to treat cells,meanwhile,the western blot was also used to measure the expression of key proteins in the MAPK signaling pathway.Further use fluorescence quantitative method and ELISA method to detect the secretion of inflammatory cytokines in rabbit endometrial epithelial cells,moreover,the influence of key proteins in the NF-κB signaling pathway were detected by Western blot.The whole reaserch procedure above aimed to explore the possible mechanism of cell self-regulation by exploring the role of α7 nicotinic acetylcholine receptor(α7nAChR)mediates and regulates the MAPK signaling pathway in rabbit endometrial epithelial cells.The results of the experiment showed that the phosphorylation levels of ERK,JNK and P38 MAPK in rabbit endometrial epithelial cells stimulated by 100 ng/m L LPS for 12 hours were significantly lower than those in the control group.Moreover,The α7nAChR inhibitor methyl taurophylline(MLA)could significantly increase the phosphorylation levels of ERK,JNK,and P38 MAPK.On the other hand,and the phosphorylation levels of ERK,JNK,and P38 MAPK were not significantly different from those treated with MLA alone after co-treatment of cells with the α7nAChR agonists PNU282987 and nicotine treated with LPS respectively;what’s more,under the combined effect of MLA,LPS,the α7nAChR agonists PNU282987 and nicotine,the phosphorylation levels of ERK,JNK and P38 MAPK are significantly inhibited.The preliminary results of the study indicate that the α7 nicotinic acetylcholine receptor(α7nAChR)mediates and regulates the MAPK signaling pathway which is a potential mechanism for cells to avoid inflammatory overreaction and self-regulation.