E.coli O78和LAB对牦牛肠道上皮细胞MAPK/Zonulin通路的影响

Effects of Ecoli O78 and LAB on MAPK/Zonulin Pathway of Intestinal Epithelial Cells in Yaks

为探究牦牛致病性大肠杆菌(E.coli O78)和乳酸杆菌(LAB)对牦牛肠道上皮屏障MAPK/Zonulin通路的影响作用,采用牦牛上皮细胞作为实验模型,Transwell构建单层上皮细胞屏障,设立阴性对照组(Control组)、阳性对照组(Model组,加入1×10^(5)CFU E.coli O78)、低剂量组乳酸杆菌组(LLAB组,加入1×10^(5)CFU E.coli O78和1×10^(5)CFU LAB)、高剂量乳酸杆菌组(HLAB组,加入加入1×10^(5)CFU E.coli O78和1×10^(7)CFU LAB),采用RT-qPCR和Western-blot技术测定各组P38、ERK、JNK、Zonulin的基因和蛋白表达水平。结果:与Control组相比,Model组的P38、ERK、JNK、Zonulin的基因表达水平极显著上升(P<0.01),而其蛋白表达水平呈现显著上升的趋势(P<0.05);与Model组相比,LLAB组P38的基因表达水平极显著下降(P<0.01),ERK、JNK、Zonulin的基因表达水平表现显著下降的趋势(P<0.05),P38、ERK、Zonulin的蛋白表达水平则呈现极显著下降的趋势(P<0.01),JNK的蛋白表达水平表现为显著下降(P<0.05);与Model组相比,HALB组P38、ERK、JNK的基因的表达水平表现为显著下降(P<0.05),Zonulin的基因表达水平表现为极显著下降(P<0.01),P38、ERK、JNK、Zonulin的蛋白表达水平呈现极显著下降的趋势(P<0.01)。结论:E.coli O78可通过上调MAPK通路关键基因P38、ERK、JNK的基因表达水平,同时上调Zonulin蛋白使肠道屏障受损;LAB可以减轻E.coli O78引起的肠道屏障受损,这一作用是通过下调MAPK通路关键基因P38、ERK、JNK的基因表达水平,同时下调Zonulin蛋白表达水平来实现的。

To investigate the effects of E.coli O78 and LAB on the MAPK/Zonulin pathway of the intestinal epithelial barrier in yak,an experimental model using yak epithelial cells was established,and the single-layer epithelial cell barrier was constructed using a Transwell.A control group(Control group),a model group(Model group,with 1×10^(5)CFU E.coli O78 added),a low-dose group(LLAB group,with 1×10^(5)CFU E.coli O78 and 1×10^(5)CFU LAB added),and a high-dose group(HLAB group,with 1×10^(5)CFU E.coli O78 and 1×10^(7)CFU LAB added)were set up.The gene and protein expression levels of P38,ERK,JNK,and Zonulin in each group were measured using RT-qPCR and Western-blot techniques.The results showed that compared with the Control group,the gene expression levels of P38,ERK,JNK,and Zonulin in the Model group were significantly increased(P<0.01),while their protein expression levels showed a significant upward trend(P<0.05);compared with the Model group,the gene expression levels of P38 in the LLAB group were significantly decreased(P<0.01),while the gene expression levels of ERK,JNK,and Zonulin showed a significant downward trend(P<0.05),and the protein expression levels of P38,ERK,and Zonulin showed an extremely significant downward trend(P<0.01),while the protein expression level of JNK showed a significant downward trend(P<0.05);compared with the Model group,the gene expression levels of P38,ERK,and JNK in the HLAB group were significantly decreased(P<0.05),while the gene expression level of Zonulin was significantly decreased(P<0.01),and the protein expression levels of P38,ERK,JNK,and Zonulin showed an extremely significant downward trend(P<0.01).Conclusion:E.coli O78 can damage the intestinal barrier by upregulating the key gene P38,ERKand JNK in the MAPK pathway,as well as the upregulation of zonulin protein;LAB can alleviate intestinal barrier damage caused by E.coli O78 by downregulating the key gene P38,ERK and JNK in the MAPK pathway,while downregulating the expression level of zonulin protein.