酿酒酵母菌诱导绵羊瘤胃上皮细胞SBD-1表达的信号通路初步研究

摘要

文章旨在探索核因子-κB (NF-κB)和丝裂原活化蛋白激酶(MAPKs)通路是否介导益生性酿酒酵母菌(Saccharomyces cerevisiae)诱导绵羊瘤胃上皮细胞(RECs)β-防御素-1(SBD-1)基因的转录.首先建立绵羊RECs培养体系作为体外试验模型,选用诱导SBD-1转录最高的菌液浓度和诱导培养时间进行信号通路初步研究,采用实时荧光定量逆转录PCR (RT-qPCR)对已建立的诱导SBD-1转录模型中的细胞膜受体——Toll样受体2(TLR2)、信号衔接蛋白——髓样分化因子(MyD88)以及NF-κB和MAPKs通路中的相关因子基因转录变化进行检测;然后选用NF-κB和MAPKs通路中的4种特异性抑制剂(即NF-κB通路特异性抑制剂PDTC、P38通路特异性抑制剂SB202190、ERK 1/2通路特异性抑制剂PD98059、JNK通路特异性抑制剂SP600125)通过单独或相互组合处理细胞后再进行诱导培养,同时采用RT-qPCR的方法检测用抑制剂处理绵羊RECs后SBD-1 mRNA的转录水平.结果表明:酿酒酵母菌刺激RECs后,NF-κB和MAPKs通路中各因子NF-κB、P38、JNK、ERK1/2、细胞膜受体TLR2与信号衔接蛋白MyD88的mRNA水平与未刺激组相比均有所升高,且呈显著性差异(P＜0.01或P＜0.05);通过单独或组合添加抑制剂后再诱导,均发现特异性抑制剂PDTC、SB202190、SP600125、PD98059可极显著抑制酿酒酵母菌对RECs SBD-1的上调作用(P＜0.01),且P38通路特异性抑制剂SB202190的抑制效果最明显.结果提示,酿酒酵母菌诱导绵羊RECs SBD-1的转录可能与TLR2-MyD88-NF-κB/MAPKs通路有关,但以TLR2-MyD88-MAPKs中的TLR2-MyD88 P38通路为主要的信号通路.%In order to explore the regulation mechanism of probiotic Saccharomyces cerevisiae inducing Sheep Beta-Defensin-1 (SBD-1) gene expression in sheep rumen epithelial cells (RECs),we studied the NF-κB and MAPKs signal pathways.Firstly,the RECs of sheep were cultured successfully as in vitro experimental model,and then selected the concentration and incubation time of S.cerevisiae,inducing SBD-1 expression highest,to preliminarily study the subsequent signaling pathways.Real-time fluorescence quantitative PCR (RT-qPCR) was conducted to determine mRNA expressive variation of the toll like receptor 2 (TLR2),myeloid differentiation factor88 (MyD88) and related factors of Nuclear factor-kappa B (NF-κB) and Mitogen-activated protein kinases (MAPKs) pathways in the established model to induce SBD-1.And then four specific inhibitors of NF-κB and MAPKs pathways were chosen,which were PDTC (NF-κB signaling pathway inhibitor),SB202190 (P38 signaling pathway inhibitor),PD98059 (ERK1/2 signaling pathway inhibitor) and SP600125 (JNK signaling pathway inhibitor).Four kinds of inhibitors were used to treat cells by single or combined with each other and then further induced culture,while using RT-qPCR methods for detecting expression level of RECs SBD-1 mRNA after inhibitor treatment.The results showed that the cells stimulated by S.cerevisiae resulted in the transcription of NF-κB,P38,JNK,ERK1/2,TLR2 and MyD88 were significantly increased (P＜0.05).And PDTC,SB202190,SP600125 and PD98059 significantly inhibited (P＜0.01)the SBD-1 transcription of stimulated cells that previously treated with the inhibitor or inhibitors,and the SB202190 had the best inhibitory effect.The results suggest that the pathways of S.cerevisiae inducing SBD-1 transcription may be related to TLR2-MyD88-NF-κB/MAPKs,but the TLR2-MyD88-P38 of TLR2-MyD88-MAPKs may be as main signaling pathway.