Study Effects Of S.cerevisiae And Inactivate S.c On The Expression Of ?-defensin-1 In Ruminal Explants Of Sheep

Defensin is a broad-spectrum anti-microbial,anti-tumor and immune-modulating cationic peptide widely distributed in many biological groups.It is considered as a substitute for antibiotics due to its unique biological mechanism.At present,there are two kinds of ?-defensins identified in sheep,namely ?-defensin-1,-2(sheep ?-defensin-1,-2,SBD-1,SBD-2).SBD-2 is only expressed in the tongue and ileum,while SBD-1 is expressed in both the respiratory and digestive tracts.Studies have confirmed that probiotic Saccharomyces cerevisiae(S.c)can promote the expression of SBD-1 in rumen epithelial cells of sheep,and the studies on the expression of induction of SBD-1 in rumen explants of sheep have not been reported.The rumen explants of sheep retain a certain degree of histological features,and can be used as an inducing object to simulate the rumen environment of sheep.The aim of this study was to investigate the effects of probiotic S.c and inactivate S.c on the expression of SBD-1 in rumen explants of sheep,and to preliminary explore the signaling pathways that may mediate this process.The explants were cultured according to the rumen explant culture program of the sheep that has been established by the research team.Co-cultured S.c and inactivated S.c with explants for 24h at different concentrations(104,105,106,107,108,109 CFU·mL-1),the expression of SBD-1 mRNA and protein in rumen explants were detected by qPCR and ELISA to determine the maximum concentration of SBD-1 expression induced by S.c and inactivated S.c.Then,stimulated the rumen explants with this concentration of S.c and inactivated S.c for different periods of time(2,4,8,12,16,20,24h),SBD-1 mRNA expression and protein changes in rumen explants were also detected by qPCR and ELISA,so as to screen out the best time for the induction of SBD-1 expression by live S.c and inactivated S.c.The results showed that probiotic S.c and inactivated S.c could promote the expression of SBD-1 in rumen explants of sheep,and the expression of SBD-1 in the rumen explants of sheep reached maximum after induction for 16h at the concentration of 107 CFU·mL-1 live S.c,and 108 CFU·mL-1 inactivated S.c.qPCR was used to detect the relative expression of mRNAs(TLR-2,MyD88,NF-?B,p38,ERK1/2,JNK)in the signaling pathway of rumen explants stimulated by inactivate S.c to determine whether NF-?B and MAPK are involved in signal transduction.Three specific inhibitors SB202190,PD98059 and SP600125(respectively inhibition of p38,ERK1/2 and JNK pathways)of MAPK and PDTC pathway of NF-?B pathway-specific inhibitors were used to pretreat the rumen explants of sheep,and stimulated explants with the optimal induction conditions of inactivated S.c,and used qPCR to detect the expression of SBD-1 mRNA in explants to determine the participation of the two pathways.The relative expression levels of various mRNAs(TLR-2,MyD88,NF-?B,p38,ERK1/2 and JNK)in the signaling pathway were increased in different degrees after the induction of explants by inactivate S.c.Pretreat sheep rumen explants with inhibitors(PDTC,SB202190,PD98059 and SP600125),the relative expression of SBD-1 mRNA in rumen explants of sheep was decreased after induction of explants by inactivated S.c,and the relative expression of SBD-1 mRNA in explants decreased most after inhibition of p38-MAPK pathway.Therefore,both NF-?B and MAPK signaling pathways are involved in the signal transduction during induction,and the p38-MAPK may be the main pathway.