Study On The Expression And Signaling Pathway Of β-defensin-1 Induced By Bacillus Subtilis Culture In Sheep Rumen Epithelial Cells

Defensins are a class of cationic antibacterial active peptides that are widely found in animals and plants in recent years.They have extensive antimicrobial and immunomodulatory effects and are an important part of the body’s natural immune system.Sheepβ-defesin-1 is expressed in the trachea and digestive tract of sheep.In this experiment,the culture supernatant of Bacillus subtilis was used as a stimulus to investigate its effect on the expression of SBD-1 in sheep ruminal epithelial cells,and to conduct a preliminary study on the main signaling pathway activated by it.In this experiment,ORECs were first cultured in vitro,and then the culture supernatants corresponding to Bacillus subtilis at different concentrations(10⁷,10⁸,10⁹,10¹⁰,10¹¹cfu/m L)stimulated ORECs for 8 h,and then fluorescence quantitative PCR（q PCR）was used to stimulate the ORECs for 8 h.The optimal stimulating concentration of Bacillus subtilis culture supernatant to induce SBD-1 expression was screened with enzyme-linked immunosorbent assay（ELISA）,and the culture supernatant corresponding to this concentration of Bacillus subtilis was used to stimulate ORECs at different times（2,4,8,12,24 h）,and q PCR and ELISA were used to select the optimal stimulation time for Bacillus subtilis culture supernatant to induce SBD-1 expression.The results showed that when the concentration of Bacillus subtilis was 10¹¹cfu/m L,the corresponding culture supernatant induced ORECs for 24 h,the m RNA and protein expression of SBD-1 reached the highest level.The optimal conditions of Bacillus subtilis culture supernatant to stimulate the expression of SBD-1 in ORECs were selected,and the signal pathway of inducing SBD-1expression was preliminarily studied by q PCR method.q PCR technology was used to detect the m RNA expression changes of pathway factors（TLR-2,My D88,NF-κB,JNK,ERK1/2,p38）before and after stimulation,and then specific inhibitors of each signaling pathway were used to block NF-κB JNK,ERK1/2 and p38,the changes of SBD-1expression were detected.The results showed that compared with the unstimulated group,the relative m RNA expressions of TLR-2,My D88,NF-κB,JNK,ERK1/2 and p38 were all increased.After using specific inhibitors of each signaling pathway,SBD-1 The relative expression of m RNA decreased.In conclusion,TLR-2-My D88-NF-κB and TLR-2-My D88-MAPKs were involved in the process of Bacillus subtilis culture supernatant stimulating the expression of SBD-1 in ORECs.