热应激麻鸡感染大肠杆菌后空肠TLR4-TBK1信号通路免疫因子变化研究

Research on changes of immune factors in the jejunum TLR4-TBK1 signaling pathway after heat-stressed Ma chickens infected with Escherichia coli

为了探索热应激麻鸡感染大肠杆菌后对空肠中TLR4-TBK1信号通路及相关免疫因子的影响,试验将24只雄性麻鸡随机均分为3组,即空白组、大肠杆菌组(E.coli组)和热应激+大肠杆菌组(HS+E.coli组)。HS+E.coli组麻鸡于41℃热应激12 h后恢复7 d,随后E.coli组和HS+E.coli组麻鸡均灌胃给予1×109 cfu/mL大肠杆菌O157:H71 mL,空白组麻鸡给予相同剂量的无菌生理盐水。于感染大肠杆菌后第4天处死麻鸡,取一段空肠进行病理组织学检查,并通过Western-blot法测定β干扰素(IFN-β)、γ干扰素(IFN-γ)、高迁移率族蛋白B1(HMGB1)、干扰素调节因子3(IRF3)、磷酸化干扰素调节因子3(p-IRF3)、热休克蛋白70(HSP70)、Toll样受体4(TLR4)和TANK结合激酶1(TBK1)等相关蛋白的表达情况。结果表明:E.coli组与空白组相比,大肠杆菌感染降低了空肠中绒毛高度并增加了隐窝深度,使空肠中IFN-β、IFN-γ、HMGB1、p-IRF3、TLR4蛋白水平极显著升高(P<0.01),IRF3(P<0.01)、HSP70(P<0.05)、TBK1(P<0.01)蛋白表达水平降低。而HS+E.coli组与E.coli组相比,热应激降低了空肠的隐窝深度,空肠中IFN-β(P<0.01)、IFN-γ(P<0.01)、p-IRF3(P<0.01)、TBK1(P<0.05)和TLR4(P<0.01)蛋白表达水平降低。说明热应激可以通过下调TLR4-TBK1信号通路相关免疫因子表达抑制大肠杆菌感染麻鸡空肠的免疫反应。

In order to explore the effect of heat stress on the TLR4-TBK1 signaling pathway and related immune factors in the jejunum of Ma chicken infected with Escherichia coli,in the experiment,24 male Ma chickens were randomly divided into three groups,namely blank group,Escherichia coli group(E.coli group)and heat stress+Escherichia coli group(HS+E.coli group).Ma chickens in the HS+E.coli group recovered for seven days after heat stress at 41℃for 12 hours,and subsequently,both E.coli group and HS+E.coli group were given 1 mL E.coli O157:H7 of 1×109 cfu/mL by gavage;the blank group was given the same dose of sterile saline.On the 4 th day after E.coli infection,the Ma chickens were killed,and a section of jejunum was taken for histopathological examination.Western-blot method was used to determine the expression of related proteins such asβ-interferon(IFN-β),γ-interferon(IFN-γ),high mobility group protein B1(HMGB1),interferon regulatory factor 3(IRF3),phosphorylated interferon regulation factor 3(p-IRF3),heat shock protein 70(HSP70),Toll-like receptor 4(TLR4)and TANK binding kinase 1(TBK1).The results showed that compared with the control group,Escherichia coli infection decreased villus height and increased crypt depth in jejunum in E.coli group,and the protein levels of IFN-β,IFN-γ,HMGB1,p-IRF3 and TLR4 in jejunum were extremely significantly increased(P<0.01),while the protein levels of IRF3(P<0.01),HSP70(P<0.05)and TBK1(P<0.01)were extremely significantly decreased.Compared with E.coli group,heat stress decreased crypt depth of jejunum in HS+E.coli group,and decreased protein expression levels of IFN-β(P<0.01),IFN-γ(P<0.01),p-IRF3(P<0.01),TBK1(P<0.05)and TLR4(P<0.01)in jejunum.The results suggested that heat stress could inhibit the intestinal immune response through down-regulating TLR4-TBK1 signaling pathway in Ma chicken infected with Escherichia coli.