Study On β-defensin-1 Expression And Signaling Pathways Induced By Inactivated Bacillus Subtilis In Rumen Epithelial Cells Of Sheep

Defensins are amphiphilic cationic peptides with a broad spectrum of antimicrobial activity that are normally produced by the body.They are widely distributed in a variety of biological species and an important part of innate immunity.Sheepβ-defensin-1 is expressed throughout the gastrointestinal tract of sheep,and plays an important role in maintaining gastrointestinal homeostasis and regulating gastrointestinal immunity.This lab early studies showed that Bacillus Subtilis can induce the the expression of SBD-1 in Ovine ruminal epithelial cells,in order to further understand bacillus subtilis ORECs SBD-1 the expression of effective components,this experiment with inactivated after processing of bacillus subtilis induced ORECs,to detect the expression of the SBD-1,and preliminarily explores the activate signaling pathways.The purpose of this study was to investigate the effect of inactivated Bacillus subtilis on the expression of sheepβ-defesin-1（SBD-1）in ORECs sheep by using rumen epithelial cells cultured in vitro as an experimental model.Firstly,ORECs were induced by different concentrations of inactivated Bacillus subtilis(10⁷,10⁸,10⁹,10¹⁰,10¹¹ cfu/m L)for 8 h.The changes of the expression level of ORECs SBD-1 at m RNA and protein levels were detected by q PCR and ELISA methods,and the optimal stimulation concentration was determined to be the bacterial liquid concentration with the highest expression of ORECs SBD-1 induced by inactivated Bacillus subtilis.The expression of SBD-1 in ORECs was detected by q PCR and ELISA for different time（2,4,8,12 h）induced by the inactivated Bacillus subtilis at this concentration,so as to select the best time to induce the expression of SBD-1.The results showed that 10⁸～10¹¹ cfu/m L of inactivated Bacillus subtilis could significantly promote the expression of SBD-1 in ORECs,and the highest expression of SBD-1 m RNA was observed when the concentration of inactivated Bacillus subtilis was 10¹⁰ cfu/m L for 8 h of ORECs induction,and the difference was very significant compared with the control group（P<0.01）,The expression of SBD-1 protein reached the maximum after 12 h induction of ORECs by10¹⁰ cfu/m L Bacillus subtilis inactivation,and the difference was extremely significant compared with the control group（P<0.01）.The experimental group with the best effect on inducing SBD-1 expression of ORECs induced by inactivated Bacillus subtilis was selected to conduct a preliminary study on the signaling pathway inducing SBD-1 expression by q PCR method.The results showed that m RNA expression levels of TLR-2,My D88,p38,JNK,and ERK1/2 were significantly up-regulated in the experimental group compared with the unstimulated group.After blocking ORECs with specific inhibitors of JNK,ERK1/2 and p38 signaling pathways,the induction test was conducted with inactivated Bacillus subtilis to detect the expression of SBD-1m RNA.The results showed that the m RNA expression of SBD-1 in ORECs was significantly decreased after blocking treatment.According to the above experiments,it can be concluded that TLR-2,My D88 and MAPKs are involved in the inactivation of Bacillus subtilis to induce the expression of ORECs SBD-1.