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-1mRNA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-1expression 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 differentiationfactor88（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（P38signaling pathway inhibitor）,PD98059（ERK1/2signaling 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-1mRNA 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 MyD88were significantly increased（P〈0.05）.And PDTC,SB202190,SP600125 and PD98059significantly inhibited（P〈0.01）the SBD-1transcription 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.cerevisiaeinducing SBD-1transcription may be related to TLR2-MyD88-NF-κB/MAPKs,but the TLR2-MyD88-P38 of TLR2-MyD88-MAPKs may be as main signaling pathway.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2017年第3期561-569,共9页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家自然科学基金(31160491
31560682)
关键词
β-防御素-1
瘤胃上皮细胞
酿酒酵母菌
抑制剂
信号通路
实时荧光定量PCR
β-defensin-1
ruminal epithelial cells
Saccharomyces cerevisiae
inhibitors
signaling pathways
real-time fluorescence quantitative PCR
