原儿茶酸对肠毒素大肠杆菌K88诱导的猪小肠上皮细胞程序性坏死和Toll样受体4信号通路的影响

Effects of Protocatechuic Acid on Necroptosis and Toll Like Receptor 4 Signaling Pathway in Porcine Small Intestinal Epithelial Cells Induced by Enterotoxigenic Escherichia coli K88

本试验旨在研究原儿茶酸(PCA)对肠毒素大肠杆菌K88(ETEC K88)诱导的猪小肠上皮细胞(IPEC-1细胞)程序性坏死和Toll样受体4(TLR4)信号通路的影响。试验采用2×2因子设计,分为4个组:对照组、PCA组(40μmol/L PCA作用24 h)、ETEC K88组(50倍细胞数ETEC K88作用2 h)和PCA+ETEC K88(40μmol/L PCA作用24 h+50倍细胞数ETEC K88作用2 h)组,每组3个重复。结果显示:1)与对照组相比,ETEC K88组细胞活力显著降低(P<0.05),细胞上清液乳酸脱氢酶(LDH)活性显著升高(P<0.05);与ETEC K88组相比,PCA+ETEC K88组细胞活力显著升高(P<0.05),细胞上清液LDH活性显著降低(P<0.05)。2)与对照组相比,ETEC K88刺激导致细胞形态损伤,超微结构破坏,表现为细胞膜破裂,细胞核皱缩,染色质外溢,线粒体出现肿胀并且空泡化;ETEC K88组细胞坏死率显著升高(P<0.05)。与ETEC K88组相比,添加PCA能够保护细胞形态,PCA+ETEC K88组细胞坏死率显著降低(P<0.05)。3)与对照组相比,ETEC K88组肿瘤坏死因子-α(TNF-α)、TLR4、脂多糖结合蛋白(LBP)、髓样分化蛋白2(MD2)、白细胞介素受体相关激酶1(IRAK1)、肿瘤坏死因子受体相关因子6(TRAF6)和髓样分化因子88(MyD 88)的mRNA相对表达显著升高(P<0.05);与ETEC K88组相比,PCA+ETEC K88组TNF-α、TLR4、LBP、MD2、IRAK1、TRAF6和MyD88的mRNA相对表达量显著降低(P<0.05)。4)与对照组相比,ETEC K88组肿瘤坏死因子受体1(TNFR1)、Fas相关死亡结构域(FADD)、混合系列蛋白激酶样结构域(MLKL)、高迁移率蛋白1(HMGB1)、动力相关蛋白1(Drp1)和磷酸甘油酸变位酶5(PGAM5)的mRNA相对表达量显著升高(P<0.05);与ETEC K88组相比,PCA+ETEC K88组TNFR1、FADD、HMGB1、Drp1和PGAM5的mRNA相对表达量显著降低(P<0.05)。由此可见,PCA可能通过抑制细胞程序性坏死和TLR4信号通路的激活,缓解ETEC K88诱导的IPEC-1细胞的炎症反应和损伤。

This experiment was conducted to study the effects of protocatechuic acid(PCA)on necroptosis and Toll like receptor 4(TLR4)signaling pathway in porcine small intestinal epithelial cells(IPEC-1 cells)induced by enterotoxigenic Escherichia coli K88(ETEC K88).The experiment used a 2×2 factor design,divided into four groups:control group,PCA group(40μmol/L PCA treated 24 h),ETEC K88 group(50 times the number of cells ETEC K88 treated 2 h)and PCA+ETEC K88 group(40μmol/L PCA treated 24 h+50 times the number of cells ETEC K88 treated 2 h),each group contained 3 replicates.The results showed as follows:1)compared with the control group,the cell viability of ETEC K88 group was significantly decreased(P<0.05),and the cell supernatant lactate dehydrogenase(LDH)activity was significantly increased(P<0.05);compared with the ETEC K88 group,the cell viability of PCA+ETEC K88 group was significantly increased(P<0.05),and the cell supernatant LDH activity was significantly decreased(P<0.05).2)Compared with the control group,ETEC K88 treatment resulted in morphological damage and ultrastructural destruction of cells,such as cell membrane rupture,nuclear shrinkage,chromatin overflow,swelling and vacuolation of mitochondria,leading to an increase in cell necrosis rate;the cell necrosis rate of ETEC K88 group was significantly increased(P<0.05).Compared with the ETEC K88 group,adding PCA can protect cell morphology;the cell necrosis rate of PCA+ETEC K88 group was significantly decreased(P<0.05).3)Compared with the control group,the mRNA relative expression levels of tumor necrosis factor-α(TNF-α),TLR4,lipopolysaccharide binding protein(LBP),myeloid differentiation protein 2(MD2),interleukin receptor-related kinase 1(IRAK1),tumor necrosis factor receptor-related factor 6(TRAF6)and myeloid differentiation factor 88(MyD88)of ETEC K88 group were significantly increased(P<0.05);compared with the ETEC K88 group,the mRNA relative expression levels of TNF-α,TLR4,LBP,MD2,IRAK1,TRAF6 and MyD88 of PCA+ETEC K88 group were significantly decreased(P<0.05).4)Compared with the control group,the mRNA relative expression levels of tumor necrosis factor receptor 1(TNFR1),Fas associated death domain(FADD),mixed lineage kinase like domain protein(MLKL),high mobility protein 1(HMGB1),dynamin related protein 1(Drp1)and phosphoglycerate mutase 5(PGAM5)of ETEC K88 group were significantly increased(P<0.05);compared with the ETEC K88 group,the mRNA relative expression levels of TNFR1,FADD,HMGB1,Drp1 and PGAM5 of PCA+ETEC K88 group were significantly decreased(P<0.05).In conclusion,PCA may alleviate the inflammatory reaction and injury of IPEC-1 cells induced by ETEC K88 by inhibiting activation of necroptosis and TLR4 signaling pathway.