参苓白术散对脾虚湿困型溃疡性结肠炎大鼠结肠组织NF-κB p65,IκBα,IκKβ蛋白及mRNA表达的影响

Effect of Shenling Baizhusan on Protein and mRNA Expression of NF-κB p65,IκBα,IκKβin Ulcerative Colitis Rats with Syndrome of Dampness Stagnancy Due to Spleen Deficiency

目的:观察参苓白术散对脾虚湿困型溃疡性结肠炎(UC)大鼠结肠组织核转录因子-κB(NF-κB)抑制蛋白激酶(IκK)/NF-κB抑制蛋白(IκB)/NF-κB信号通路的影响,探讨参苓白术散改善UC的作用机制。方法:将48只Wistar大鼠按照随机数字表法分为正常组、模型组、参苓白术散(15.6 g·kg^-1)组、奥沙拉嗪钠(0.68 g·kg^-1)组,每组12只,脾虚湿困型UC大鼠模型采用2,4,6-三硝基苯磺酸(TNBS)/乙醇灌肠结合环境与饮食干预法复制,给药组连续灌胃14 d。采用酶联免疫吸附测定(ELISA)检测各组大鼠血清肿瘤坏死因子-α(TNF-α),白细胞介素-6(IL-6)及白细胞介素-1β(IL-1β)含量,免疫组化和蛋白免疫印迹法(Western blot)检测各组大鼠结肠组织中NF-κB p65,IκBα,IκKβ蛋白表达,实时荧光定量聚合酶链式反应(Real-time PCR)检测各组大鼠结肠组织NF-κB p65,IκBα,IκKβmRNA表达。结果:与正常组比较,模型组大鼠血清TNF-α,IL-6及IL-1β含量均显著升高(P<0.01),结肠组织NF-κB p65,IκKβ蛋白和mRNA表达显著升高(P<0.01),IκBα蛋白和mRNA表达显著降低(P<0.01);与模型组比较,参苓白术散组和奥沙拉嗪钠组大鼠血清TNF-α,IL-6及IL-1β水平均显著下降(P<0.01),结肠组织NF-κB p65,IκKβ蛋白和mRNA均显著下降(P<0.01),IκBα蛋白和mRNA表达显著升高(P<0.01)。结论:参苓白术散能明显下调脾虚湿困型UC大鼠结肠组织NF-κB p65,IκKβ,蛋白和mRNA表达,上调IκBα蛋白和mRNA表达;参苓白术散抑制UC大鼠IκK/IκB/NF-κB信号通路活化是其发挥肠黏膜保护作用的重要机制。

Objective:To observe the effect of Shenling Baizhusan on the protein and mRNA expression of inhibitor of nuclear factor kappa B kinase(IκK)/inhibitor of nuclear factor kappa B(IκB)/nuclear factor kappa B(NF-κB)signaling pathway in the colon of rats with ulcerative colitis(UC)of spleen deficiency and dampness stagnation type,and to explore the mechanism of Shenling Baizhusan in the treatment of UC.Method:The 48 Wistar rats were randomly divided into normal group,model group,Shenling Baizhusan group(15.6 g·kg^-1)and osalazine sodium group(0.68 g·kg^-1),12 rats in each group.The model of UC with spleen deficiency and dampness stagnation was reproduced by trinitrobenzene sulfonic acid(TNBS)/ethanol enema combined with environment and diet intervention.Serum levels of tumour necrosis factor-α(TNF-α),interleukin-6(IL-6)and interleukin-1β(IL-1β)were determined by enzyme-linked immunosorbent assay(ELISA).The expression of NF-κB p65,IκBα,IκKβ protein in colon tissue was measured by Western blot and immunohistochemical method,and the mRNA expression of NF-κB p65,IκBαand IκKβin colon tissue of rats in each group was detected and compared by real time polymerase chain reaction(Real-time PCR).Result:Compared with normal group,the levels of TNF-α,IL-6 and IL-1β in the serum,the protein and mRNA expression of NF-κB p65,IκKβ in colon tissue of the model group was significantly higher than that of normal group(P<0.01),and the protein and mRNA expression of IκBα was significantly lower than that of normal group(P<0.01).Compared with model group,the protein and mRNA expression of NF-κB p65,IκKβ in colon tissue of the Shenling Baizhusan group and osalazine sodium group were significantly decreased(P<0.01),and the protein and mRNA expression of IκBα was significantly increased(P<0.01).Conclusion:Shenling Baizhusan can obviously down regulate the protein and mRNA expression of NF-κB p65,IκKβ,up regulate the expression of IκBαin colon tissue of UC rats with spleen deficiency and dampness stagnation.The inhibition of IκK/IκB/NF-κB signal pathway activation by Shenling Baizhusan is an important mechanism of its role in protecting intestinal mucosa.