电针调节AKT/NF-κB信号通路炎症反应修复受损肠黏膜屏障的机制研究

Mechanism Study of Electro-Acupuncture Repairing Damaged Intestinal Mucosal Barrier by Regulating Inflammatory Response of AKT/NF-κB Signaling Pathway

目的:观察电针“天枢”“足三里”对缓解肠易激模型小鼠AKT/NF-κB信号通路相关炎症因子的调节及对肠屏障蛋白Claudin1、Occludin、ZO-1的影响,探讨电针修复肠黏膜屏障的潜在机制。方法:将30只10周龄雄性c57小鼠随机分为对照组、模型组和治疗组,每组10只。以避水应激法(WAS)造成的肠易激小鼠为模型,结合电针组给予0.5~1.0 m A,2 Hz/15 Hz,疏密波干预双侧“天枢”“足三里”,1次/d,每次15 min,共10 d。观察腹壁回撤反应(AWR)评分,HE染色法观察结肠组织形态,Westen Blot法和荧光定量PCR法检测结肠组织中蛋白激酶B(AKT)、磷酸化(p)-AKT、核因子κBp65(NF-κBp65)、磷酸化(p)-NF-κBp65、IKKα、IKKβ、Claudin1、Occludin、ZO-1蛋白的相对表达量。ELISA法检测血清中的白介素-1β(IL-1β)、白介素-6α(IL-6α)和肿瘤坏死因子-α(TNF-α)表达量。结果:与对照组相比较,模型组AWR评分增高(均P<0.05),AKT、p-AKT、NF-κBp65、p-NF-κBp65、IKKα、IKKβ蛋白和mRNA的相对表达量升高(P<0.01),Claudin1、Occludin、ZO-1 mRNA相对表达量减少(P<0.01),与模型组比较,电针干预后AWR评分降低(均P<0.05),AKT、p-AKT、NF-κBp65、p-NF-κBp65、IKKα、IKKβ蛋白和mRNA的相对表达量升高(P<0.01)。模型组与对照组相比,结肠组织Westen Blot检测AKT、p-AKT、NF-κBp65、p-NF-κBp65、IKKα、IKKβ、Claudin1、Occludin和ZO-1蛋白表达差异具有统计学意义(P<0.01);治疗组与模型组相比,AKT、p-AKT、NF-κBp65、p-NF-κBp65、IKKα、IKKβ、Claudin1和Occludin蛋白表达有所降低,差异具有统计学意义(P<0.05)。小鼠结肠组织荧光定量PCR法检测结果显示,模型组与对照组相比,AKT、p-AKT、NF-κBp65、p-NF-κBp65、IKKα、IKKβ、Claudin1、Occludin和ZO-1 mRNA表达差异具有统计学意义(P<0.001);治疗组与模型组相比,AKT、p-AKT、NF-κBp65、p-NF-κBp65、IKKα、IKKβ、Claudin1、Occludin mRNA表达降低,差异具有统计学意义(P<0.001)。模型组与对照组Elisa结果相比,IL-6α、IL-1β和TNF-α表达差异具有统计学意义(P<0.001);治疗组与模型组相比,IL-6α、IL-1β和TNF-α表达有所降低,差异具有统计学意义(P<0.001)。结论:电针可能通过调节AKT/NF-κB信号通路缓解炎症反应恢复受损的肠黏膜屏障。

Objective:To observe the regulation of inflammatory factors related to AKT/NF-κB signaling pathway and intestinal barrier proteins Claudin1 and Occludin in the model mice with irritable bowel syndrome(IBS)after the intervention of electro-needling Tianshu(ST25)and Zusanli(ST36),thus to investigate the potential mechanism of electro-acupuncture in repairing intestinal mucosal barrier.Methods:30 c57 mice(10-week-old,male)were randomly divided into the control group,the model group and the treatment group,with10 mice in each group.IBS model was established by the method of water avoidance stress(WAS).The treatment group was intervened by electro-needling ST25 and ST36 for 10 days(0.5~1.0 mA,2 Hz/15 Hz,alternative wave,1 time/day,15 min/time).The scores of abdominal wall retraction response(AWR)were evaluated,the colonic tissue morphology was observed by HE staining,the relative expression levels of protein kinase B(AKT),phosphorylated(p)-AKT,nuclear factor-κBp65(NF-κBp65),phosphorylated(p)-NF-κBp65,IKKα,IKKβ,Claudin1,Occludin and ZO-1 in colon tissues were detected by the methods of Westen blot and quantitative fluorescent PCR,and the serum levels of interleukin-1β(IL-1β),interleukin-6 alpha(IL-6α)and tumor necrosis factor-α(TNF-α)were determined by ELISA method.Results:Compared to those in the control group,the scores of AWR were significantly increased(P<0.05),the protein and mRNA relative expressions of AKT,p-AKT,NF-κBp65,p-NF-κBp65,IKKαand IKKβwere significantly increased(P<0.01),and the mRNA relative expressions of Claudin1,Occludin and ZO-1 were significantly decreased in the model group(P<0.01).Compared to those in the model group,the scores of AWR were significantly decreased(P<0.05),and the protein and mRNA relative expressions of AKT,p-AKT,NF-κBp65,p-NF-κBp65,IKKαand IKKβwere significantly increased after electro-acupuncture intervention(P<0.01).Western blot analysis of colon tissue showed that there were statistical differences in the protein expressions of AKT,p-AKT,NF-κBp65,p-NF-κBp65,IKKα,IKKβ,Claudin1,Occludin and ZO-1 between the control group and the model group(P<0.01);the expressions of AKT,p-AKT,NF-κBp65,pNF-κBp65,IKKα,IKKβ,Claudin1 and Occludin were significantly decreased in the treatment group compared to those in the model group(P<0.05).Fluorescent quantitative PCR of colon tissue showed that there were statistical differences in the mRNA expressions of AKT,p-AKT,NF-κBp65,p-NF-κBp65,IKKα,IKKβ,Claudin1,Occludin and ZO-1 between the model group and the control group(P<0.001);the mRNA expressions of AKT,p-AKT,NF-κBp65,p-NF-κBp65,IKKα,IKKβ,Claudin1 and Occludin were significantly decreased in the treatment group compared to those in the model group(P<0.001).ELISA results showed that there were statistical differences in the expressions of IL-6α,IL-1βand TNF-αbetween the model group and the control group(P<0.001);which were significantly decreased in the treatment group compared to those in the model group(P<0.001).Conclusion:Electro-acupuncture may alleviate inflammatory response and restore the damaged intestinal mucosal barrier by regulating AKT/NF-κB signaling pathway.