Effects Of Bacillus Subtilis Cell Wall On The Expression Of β-defensin-1 In Sheep Rumen Epithelial Cells And Its Signaling Pathway

Defensins are cationic Peptides rich in disulfide bonds.They are widely distrib uted in animals and Play a role in killing bacteria,fungi,viruses and other microor ganisms.They are part of the innate immune system.The effect of Bacillus subtilis cell wall on the expression of Sheepβ-defensin-1（SBD-1）in Ovine Ruminal Epith elial Cells（ORECs）cultured in vitro and its possible signal transduction pathway w ere studied.In order to investigate whether the cell wall of Bacillus subtilis can affect the expression of ORECs SBD-1,Bacillus subtilis was crushed by repeated freezing an d thawing combined with ultrasonic crushing and its cell wall was collected success fully.The cell walls of Bacillus subtilis at different concentrations(0,25,50,100,200,400μg·m L^-1)were co-cultured with ORECs for 8 h.Fluorescence quantitative PCR（RT-q PCR）and enzyme-linked immunosorbent assay（ELISA）were used to det ect the optimal stimulation concentration of SBD-1 expression induced by Bacillus s ubtilis cell wall.Then ORECs were stimulated at different time（0,2,4,8,12,24h）with the optimal stimulation concentration to find out the optimal stimulation ti me of SBD-1 expression induced by Bacillus subtilis cell wall.The results showed that the cell wall of Bacillus subtilis was successfully Prepared,and the optimal sti mulation concentration was 50μg·m L^-1,and the optimal stimulation time was 12 h.The optimal stimulation conditions for SBD-1 expression induced by Bacillus s ubtilis cell wall were selected to stimulate ORECs to conduct a Preliminary study o n the signaling Pathway.The m RNA and protein expressions of pathway factors（T LR-2,My D88,NF-κB,p38,JNK,ERK1/2）before and after stimulation were detect ed by RT-q PCR and Western Blot.NF-κB,p38,JNK and ERK1/2 were blocked by pathway inhibitors and their effects on SBD-1 induction were detected.The results show:The m RNA and protein expressions of TLR-2,My D88,NF-κB,p38,JNK a nd ERK1/2 increased after ORECs stimulation under optimal conditions,and the m RNA expression of SDD-1 decreased after each signaling pathway was blocked by pathway inhibitors.In summary,TLR-2,My D88,NF-κB and MAPKs were involved in the expression of SBD-1 induced by Bacillus subtilis cell wall.