Effect Of Bacillus Subtilis Peptidoglycan On The Expression Of SBD-1 In Sheep Rumen Epithelial Cells And Preliminary Study Of Signaling Pathway

With the implementation of the“Anti-antibiotic” policy in feed additives,it has become the focus of researchers to look for additives that can substitute for antibiotics.Defensins have been widely studied as potential substitutes for antibiotics..The purpose of this study was to investigate the effect of peptidoglycan derived from Bacillus subtilis cell wall on the expression of Sheep β-defensin-1(SBD-1)in Ovine ruminal epithelial cells(ORECs)and the signaling pathway activated by the peptidoglycan.Firstly,ORECs were cultured by trypsin continuous digestion method.The cell growth curve,immunofluorescence and PCR were used to detect whether the cultured cells were ORECs or not.Secondly,the optimal stimulation conditions were obtained by determining the expression levels of SBD-1 m RNA and protein at different time(2,4,6,8,10,12 h)and concentration(0,10,20,30,40,50 μg/m L)of ORECs.Then,the m RNA and protein expressions of ORECs signaling pathway factors(TLR-2,MYD88,P38,NF-ΚB,ERK1/2,JNK)were detected under the optimal stimulation conditions.Finally,the m RNA and protein expression levels of ORECs SBD-1 stimulated by peptidoglycan after treatment with signaling pathwayspecific inhibitors,respectively,in vitro culture of ORECs were examined.The results showed that:(1)the cultured cells were in accordance with the growth trend of ORECs.Compared with the control group,the cells showed positive staining by immunofluorescence assay and oval or polygon shape.PCR results showed that the cells expressed e-cadherin and Alkaline phosphatase genes,which confirmed that the cultured cells were ORECs.(2)q PCR and ELISAresults showed that the expression of SBD-1 could be induced by the peptidoglycan of Bacillus subtilis.(3)q PCR and WB results showed that the expression of TLR-2 receptor and My D88 could activate NF-κB and MAPK(ERK1/2 and JNK)signal pathway.(4)The results of PCR and ELISA showed that the expression of SBD-1 in ORECs treated with the specific inhibitor decreased to some extent,which indicated that NF-κB and MAPK(ERK1/2 and JNK)signal pathway co-mediated the expression of SBD-1.The peptidoglycan of Bacillus subtilis can induce the expression of SBD-1 in ORECs cultured in vitro,and changes with the concentration of peptidoglycan and induction time.The expression of ORECs SBD-1 induced by Bacillus subtilis peptidoglycan is regulated by NF-κB and MAPK(ERK1/2 and JNK),and NF-κB is the most important.This study revealed the molecular mechanism of the expression of ORECs SBD-1 induced by Bacillus subtilis peptidoglycan in vitro.At the same time,it provides a theoretical basis for the safe,effective and reasonable development of Bacillus subtilis peptidoglycan as a feed additive.