基于NF-κB/TGF-β1/Smad3信号通路研究通腑活血方防治肠粘连作用机制

The mechanism of Tongfu Huoxue prescription in preventing intestinal adhesion based on NF-κB/TGF-β1/Smad3 signal pathway

目的:观察通腑活血方对肠粘连模型大鼠盲肠组织中核转录因子-κB(NF-κB)/转化生长因子-β1(TGF-β1)/Smad3通路中相关因子表达的影响,探讨通腑活血方防治术后肠粘连的作用机制。方法:雄性SD大鼠48只,按照随机数字法分为四组:对照组、模型组、通腑活血方组和几丁糖组,每组12只,除对照组外,其余大鼠采用Ellis法制备肠粘连模型,分别给予各组大鼠相应处理。苏木素-伊红(HE)染色观察盲肠组织病理学变化,酶联免疫吸附法检测血清白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)含量,Western blot和RT-PCR法检测盲肠NF-κB/TGF-β1/Smad3信号通路中关键因子的蛋白和mRNA表达水平。结果:与对照组比较,模型组大鼠组织病理学出现显著变化,血清IL-6、TNF-α含量显著升高(P<0.01),盲肠组织中IL-6、TNF-α、p-NF-κBp65、TGF-β1、Smad3 mRNA和蛋白相对表达水平升高(P<0.01),基质金属蛋白酶9(MMP-9)蛋白相对表达水平升高(P<0.01),核转录因子-κB抑制蛋白(IκB)、基质金属蛋白酶抑制剂-1(TIMP-1)蛋白表达水平显著降低(P<0.01);与模型组比较,通腑活血方组和几丁糖组大鼠血清IL-6、TNF-α含量显著降低(P<0.01),盲肠组织中IL-6、TNF-α、p-NF-κBp65、TGF-β1、Smad3 mRNA和蛋白相对表达水平降低(P<0.01)、MMP-9蛋白相对表达水平降低(P<0.01),IκB、TIMP-1蛋白表达水平升高(P<0.01),且盲肠组织病理学均有显著改善,其中以通腑活血方组改善更显著。结论:通腑活血方可有效防止术后肠粘连的发生,其作用机制可能是通过抑制NF-κB/TGF-β1/Smad3信号通路活化,减少炎症因子的释放,调节TIMP-1/MMP-9平衡,防止肠粘连。

Objective:To observe the effect of Tongfu Huoxue prescription on the expression of nuclear transcription factor-κB(NF-κB)/transforming growth factor-β1(TGF-β1)/Smad3 signal pathway in cecum of intestinal adhesion model rats,and to explore the mechanism of Tongfu Huoxue prescription in preventing postoperative intestinal adhesion.Methods:Forty-eight male SD rats were divided into four groups according to random number method:control group,model group,Tongfu Huoxue prescription group and chitose group,with 12 rats in each group.Except for the control group,the rat model of intestinal adhesion was established by Ellis method.The histopathological changes of cecum were observed by HE staining,the contents of serum IL-6 and TNF-αwere detected by ELISA,and the protein and mRNA expression levels of key factors in cecal NF-κB/TGF-β1/Smad3 signal pathway were detected by Western blot and RT-PCR.Results:Compared with the control group,the grade of intestinal adhesion in the model group increased significantly,the histopathology changed significantly,the contents of IL-6 and TNF-αin serum increased significantly(P<0.01),and the expressions of IL-6,TNF-α,p-NF-κBp65,TGF-β1,Smad3 mRNA and protein in cecal tissue were significantly increased(P<0.01),the protein expression level of MMP-9 in cecal tissue were significantly increased(P<0.01).The protein expression level of IκB,TIMP-1 protein decreased significantly(P<0.01).Compared with the model group,the contents of serum IL-6 and TNF-αin the Tongfu Huoxue prescription group and chitose group were significantly decreased(P<0.01).The expression level of IL-6,TNF-α,p-NF-κBp65,TGF-β1,Smad3 mRNA and protein decreased significantly(P<0.01),the level of MMP-9 protein in cecal tissue decreased significantly(P<0.01),while the expression level of IκB,TIMP-1 protein increased significantly(P<0.01).The cecal pathology and intestinal adhesion grade were improved,especially in the Tongfu Huoxue prescription group.Conclusion:Tongfu Huoxue prescription can effectively prevent the occurrence of intestinal adhesion after operation,and its mechanism may be through inhibiting the activation of NF-κB/TGF-β1/Smad3 signal pathway,reducing the release of inflammatory factors and inhibiting the inflammatory reaction of cecal tissue,adjust TIMP-1/MMP-9 balance,so as to prevent the occurrence of intestinal adhesion.