三硝基苯磺酸/乙醇灌肠液诱导大鼠溃疡性结肠炎TLR4/NF-κB 和 PI3K/AKT/NF-κB 信号通路的表达及电针的干预作用

摘要

目的：探讨三硝基苯磺酸(TNBS)/乙醇灌肠液诱导大鼠溃疡性结肠炎 TLR4/NF-κB 和 PI3K/AKT /NF-κB 信号通路的表达及电针的干预作用。方法雄性 Wistar 大鼠240只,随机分为正常对照组、模型组、电针组、TLR4单克隆抗体(TLR4mAb)组、LY294002组和 TLR4mAb-LY294002联合治疗(T&L)组。以 TNBS/乙醇溶液灌肠诱导大鼠溃疡性结肠炎模型,电针组造模同时给予电针足三里穴治疗,TLR4mAb 组给予 TLR4mAb 腹腔注射,LY294002组则予 LY294002注射液腹腔注射,T&L 组则同时给与上述 TLR4mAb 和 LY294002腹腔注射,共4周,每日行疾病活动指数(DAI)评分。造模4周后处死大鼠取结肠组织标本,评价结肠黏膜损伤指数(CMDI)和组织学损伤指数(TDI)；免疫印迹法测定结肠黏膜磷酸化 AKT(P-AKT)和活化 NF-κB 含量；RT-PCR 法检测结肠组织中 TLR4 mR-NA、PI3K mRNA、AKT mRNA、NF-κB mRNA、TNF-αmRNA 及 IL-1βmRNA 表达。结果与正常对照组比较,模型组结肠组织 TLR4 mRNA、PI3K mRNA、P-AKT、活化 NF-κB 、TNF-αmRNA、IL-1βmRNA 表达和 DAI、CMDI 及TDI 均显著升高(P <0.01)；与模型组比较,TLR4mAb 组 TLR4 mRNA 表达显著下降(P <0.01),LY294002组 PI3K mRNA、P-AKT 表达显著下降(P <0.01),而在电针组和 T&L 组 TLR4 mRNA、PI3K mRNA 及 P-AKT 表达均明显降低(P <0.01)；与上述变化相对应,与模型组相比,各治疗组活化 NF-κB、TNF-αmRNA、IL-1βmRNA 表达和 DAI、CMDI 及 TDI 均明显降低(P <0.05或 P <0.01),电针组和 T&L 组上述6指标优于 TLR4mAb 组和 LY294002组,活化 NF-κB 与 TNF-αmRNA 及 IL-1βmRNA 表达呈正相关(r 1=0.579,P <0.05；r 2=0.561,P <0.05)。结论电针足三里穴能显著降低实验性溃疡性结肠炎大鼠的 NF-κB 活性及 TNF-αmRNA、IL-1βmRNA 表达,从而减轻结肠组织损害程度,这与其阻断 TLR4/NF-κB 和 PI3K/AKT/NF-κB 信号通路有密切关系。%ABSTRACT:Objective To explore the expressions of Toll-like receptor4/NF-κB and PI3K/AKT/NF-κB signa-ling pathways in rat ulcerative colitis (UC)induced by the combined enema of trinitrobenzene sulphonic acid and ethano and the interventional effect of electroacupuncture on them.Methods Totally 240 male Wistar rats were randomly divided into 6 groups:normal control group,model control group,electroacupuncture group,TLR4mAb group,LY294002 group,and TLR4mAb combined with LY294002 (T&L)group.The combined enema of trinitro-benzene sulphonic acid (TNB)and ethanol was intrarectally administered for 4 weeks to induce UC.At the same time of modeling ,Zusanli point was electro-acupunctured in electroacupuncture group while intraperitoneal injec-tion of TLR4mAb and LY294002 was given respectively to the corresponding group.Each rat was treated with the above-mentioned TLR4mAb injection and LY294002 injection in T&L group for 4 weeks.The disease activity index (DAI)of all the rats was evaluated daily.The rats were killed after 4 weeks.The colonic mucosa damage index (CMDI)and tissue damage index (TDI)were evaluated by a pathologic grading system.The expressions of P-Akt and active NF-κB protein in the colon mucosa were determined by Western blotting.TLR4 mRNA,PI3K mRNA, AKT mRNA,NF-κB mRNA,TNF-αmRNA and IL-1βmRNA expressions were measured with RT-PCR.Results Compared with those in normal control group,TLR4 mRNA,PI3K mRNA,P-AKT,active NF-κB,TNF-αmRNA and IL-1βmRNA expressions as well as DAI,CMDI and TDI were all increased obviously in model control group (P <0.01).Compared with those in model control group,TLR4mRNA expression was decreased obviously in TLR4mRNA group (P <0.01),the expressions of PI3KmRNA and P-AKT were decreased obviously in LY294002 group (P <0.01 ).Not only TLR4mRNA expression but also PI3KmRNA and P-AKT expressions were decreased significantly in electroacupuncture group and T&L group (P <0.01 ).Corresponding to the above-mentioned chan-ges,active NF-κB,TNF-αmRNA and IL-1βmRNA expressions as well as DAI,CMDI and TDI were decreased obvi-ously in all the treated groups compared with those in model control group (P <0.05 or P <0.01),but the six inde-xes were better in electroacupuncture group and T&L group than in TLR4mAb group and LY294002 group (P <0.05).There were obvious positive correlations of active NF-κB with TNF-αmRNA and IL-1β mRNA expressions (r 1 =0.579,P <0.05;r 2 =0.561,P <0.05).Conclusion Electroacupuncture can significantly decrease NF-κB activity and TNF-αmRNA and IL-1β mRNA expressions in UC rats,thus alleviating the severity of UC,which is closely correlated to its blocking both TLR4/NF-κB and PI3K/AKT/NF-κB signaling pathways.