摘要
为探究Toll样受体2/4(TLR2/4)、核苷酸结合寡聚结构域样受体1/2(NOD1/2)在产肠毒素大肠杆菌(ETEC)诱导的猪肠道上皮细胞(IPEC-J2)炎症反应中的作用,本研究以不同MOI(0.1、0.02、0.01)的ETEC感染IPEC-J2,2 h后,采用MTT法检测IPEC-J2的细胞活力,以筛选ETEC感染IPEC-J2的最适MOI。结果显示,经MOI 0.01的ETEC感染后IPEC-J2的细胞活力极显著低于对照组(P<0.001)。因此,采用最适MOI 0.01的ETEC感染IPEC-J2,分别培养不同时间后,采用荧光定量PCR(qPCR)检测ETEC感染IPEC-J2中的TLR2/4、NOD1/2及促炎性细胞因子IL-6、IL-8、肿瘤细胞坏死因子(TNF-α)mRNA的转录水平;利用TLR2/4混合抑制剂AMG9810及NOD1/2混合抑制剂NOD-IN-1分别与IPEC-J2共作用6 h,再经ETEC感染2 h后经qPCR检测TLR2/4、NOD1/2及下游促炎性细胞因子IL-6、IL-8、TNF-α m RNA的转录水平。q PCR结果显示,与对照组相比,ETEC感染的IPEC-J2中TLR2 m RNA转录水平(感染后30 min及120 min)及TLR4 m RNA转录水平(感染后105 min及120 min)均极显著升高(P<0.01、P<0.001),其余时间段与对照组无显著差异;IPEC-J2 NOD1 mRNA转录水平(感染60 min及以后)、NOD2 mRNA转录水平(感染后30 min~120 min)均极显著高于对照组(P<0.01、P<0.001),且IL-6、IL-8(感染后30 min除外)、TNF-α mRNA的转录水平随感染时间延长均极显著升高(P<0.01)。与不加抑制剂的感染组相比,经两种受体抑制剂作用后再感染ETEC的IPEC-J2中TLR2/4、NOD1/2 mRNA的转录水平均显著(P<0.05)或极显著(P<0.01)下降;且其中的IL-6、IL-8、TNF-α mRNA的转录水平均极显著降低(P<0.01)。上述结果首次表明,ETEC感染可通过激活IPEC-J2受体TLR2/4、NOD1/2进而诱导下游促炎性细胞因子IL-6、IL-8、TNF-α mRNA转录水平的升高。本研究为阐释ETEC诱导猪肠道炎症反应的致病机制提供了参考依据。
In order to explore the role of Toll like receptor 2/4 (TLR2/4) and nucleotide-binding oligomerization domain-like receptor 1/2(NOD1/2) in the inflammatory response of porcine intestinal epithelial cells (IPEC-J2) induced by enterotoxigenic Escherichia coli (ETEC),IPEC-J2 were infected with ETEC at different MOI (0.1,0.02,0.01).Two hours later,the cell viability of IPEC-J2 was detected by MTT method to screen the optimal MOI for IPEC-J2 infected with ETEC.The results showed that the cell viability of IPEC-J2 infected with ETEC at MOI 0.01was significantly lower than that of the control group (P<0.001).Therefore,IPEC-J2 was infected with ETEC at the most suitable MOI 0.01.After incubation for different times,the mRNA levels of TLR2/4,NOD1/2 and pro-inflammatory cytokines IL-6,IL-8 and TNF-α in IPEC-J2 infected with ETEC were detected by RT-PCR;IPEC-J2 cells were treated respectively with TLR2/4 mixed inhibitor AMG9810 and NOD1/2 mixed inhibitor NOD-IN-1 for 6 hours,and then infected with ETEC for 2 hours.The mRNA levels of TLR2/4,NOD1/2 and pro-inflammatory cytokines IL-6,IL-8 and TNF-α were detected by RT-PCR.RT-PCR results showed that compared with the control group,the mRNA levels of TLR2(30 minutes and 120 minutes after infection) and TLR4 (105 minutes and 120 minutes after infection) were significantly higher (P<0.01,P<0.001) in ETEC-infected IPEC-J2,and there was no significant difference in the other time periods in contrast to the control group;the IPEC-J2NOD1 mRNA level (60 minutes after infection) and NOD2 mRNA level (30 minutes to 120 minutes after infection) were significantly higher than those in the control group (P<0.01,P<0.001).The transcription levels of IL-6,IL-8 (except 30 minutes after infection) and TNF-α significantly increased with the extension of infection time (P<0.01).Compared with the infection group without inhibitors,the transcription levels of TLR2/4and NOD1/2 in IPEC-J2 treated with two receptor inhibitors significantly or extremely significantly decreased (P<0.05,P<0.01);The transcription levels of IL-6,IL-8 and TNF-α significantly decreased (P<0.01).These results show for the first time that ETEC infection can induce the transcription levels of proinflammatory cytokines IL-6,IL-8 and TNF-α increase by activating IPEC-J2 receptors TLR2/4 and NOD1/2,which provides a reference basis for explaining the pathogenic mechanism of ETEC induced intestinal inflammation in pigs.
作者
李雪滢
韩阳春
黄静
李霞
曾聪
肖乐
刘峻源
曾雯玉
彭远义
王自力
LI Xue-ying;HAN Yang-chun;HUANG Jing;LI Xia;ZENG Cong;XIAO Le;LIU Jun-yuan;ZENG Wen-yu;PENG Yuan-yi;WANG Zi-li(College of Veterinary Medicine,Southwest University,Chongqing 400715,China;College of Animal Science,Zhejiang University,Hangzhou 310030,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2022年第7期750-756,共7页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金项目(31872507)。
