Study On β-defensin-1 Expression And Signaling Pathways Induced By S.cerevisiae Cultures And Extracts In Ovine Ruminal Epithelial Cells

Defensins,multifunctional cationic amphiphilic antimicrobial peptides,are produced by epithelial cells and immune cells.They have a wide range of antibacterial,antifungal and antiviral activities and can maintain the physiological health of mucosal epithelium.Sheep β-defensin-1(sBD-1)is a defensin expressed mainly in the trachea and the entire digestive tract of sheep.Previous studies have clearly shown that probiotics such as Saccharomyces cerevisiae can induce the expression of Ovine ruminal epithelial cells(ORECs)and Ovine ruminal explants(OREs)sBD-1.However,the effects of S.cerevisiae cultures and extracts on the expression of ORECs sBD-1 have not been reported.In this study,ORECs cultured in vitro were used as test subjects,and the MTT method was used to detect 0-400 μg/mL concentrations of S.cerevisiae cultures and extracts that had no toxic effect on ORECs.First,after inducing ORECs for different time periods(0,6,12,18,24,30 h)with 200 μg/mL S.cerevisiae cultures and extracts respectively,the relative expression of sBD-1 mRNA was detected by qPCR to determine the time of the S.cerevisiae cultures and extracts induced ORECs sBD-1 mRNA highest expression;Then,after inducing ORECs with different concentration gradients(0.100,200,300,400 μg/mL)S.cerevisiae cultures and extracts respectively,qPCR is used to detect sBD-1 mRNA expression.Finally,treat the ORECs with the optimal stimulus concentration and induction time of the S.cerevisiae cultures and extracts to compare the effects of the two inducing the expression of ORECs sBD-1 mRNA.The results showed that both S.cerevisiae cultures and extracts induced ORECs sBD-1 mRNA expression.ORECs sBD-1 mRNA expression was highest when the concentration of S.cerevisiae cultures were 400 μg/mL and the concentration of S.cerevisiae extracts were 200 μg/mL and ORECs were induced for 6 h,respectively.And the induction effect of S.cerevisiae extracts is better than that of S.cerevisiae cultures.S.cerevisiae extracts with better induction effect was selected,and the signal pathway of sBD-1 expression induced by S.cerevisiae extracts was studied by qPCR technology.The results showed that the relative expression levels of TLR-2,MyD88,and MAPKs(p38,JNK,and ERK1/2)mRNA in ORECs stimulated by S.cerevisiae extracts were all up-regulated compared to unstimulated,while the relative expression levels of NF-κB mRNA were not.Then the p38,JNK and ERK1/2 pathways were blocked respectively,and their effects on sBD-1 mRNA expression induced by S.cerevisiae extracts were detected.The results showed that the relative expression of sBD-1 mRNA was significantly reduced.In summary,this study shows that TLR-2,MyD88,and MAPKs are involved in regulating the expression of ORECs sBD-1 gene induced by S.cerevisiae extracts.