基于Notch1/NF-κB信号通路探讨芍药汤联合美沙拉嗪对溃疡性结肠炎大鼠的治疗作用

Effect of Shaoyao Decoction Combined with Mesalazine on Ulcerative Colitis in Rats via Regulation of Notch1 and NF-κB Signal Pathway

[目的]观察芍药汤(SYD)联合美沙拉嗪(MES)通过调控Notch1/NF-κB信号通路治疗溃疡性结肠炎(UC)大鼠的作用机制。[方法]60只SD大鼠随机分为正常组(N组)、模型组(M组)、SYD组(20 g/kg)、MES组(0.2 g/kg)及SYD+MES组(20 g/kg+0.2 g/kg),每组12只。采用5%葡聚糖硫酸钠(DSS)溶液连续喂养7 d制备UC大鼠模型,造模后连续灌胃给药14 d,给药后第7 d和第14 d进行大鼠疾病活动指数(DAI)、结肠黏膜损伤指数(CMDI)评分;ELISA检测大鼠血浆肿瘤坏死因子-α(TNF-α)、白介素-1β(IL-1β)和白介素-6(IL-6)的水平;HE染色观察结肠病理变化并进行组织损伤指数(TDI)评分;荧光定量PCR检测结肠TNF-α、IL-1β和IL-6 mRNA的表达水平;Western Blot法检测结肠胞浆Notch1、Notch1胞内域(NICD)、TNF-α、IL-1β、IL-6、磷酸化核因子κB抑制因子α(p-IκBα)、磷酸化NF-κB p65(p-NF-κB p65)蛋白表达及胞核内NF-κB p65水平。[结果]与M组比较,SYD组、MES组及SYD+MES组肠黏膜病理损伤明显减轻,TDI、DAI及CMDI评分显著降低,Notch1和NICD表达水平明显升高,胞浆IκBα和NF-κB p65磷酸化及NF-κBp65核转移水平显著下降,炎症因子TNF-α、IL-1β、IL-6的水平明显降低(P<0.05);分别与SYD组及MES组比较,SYD+MES组肠黏膜损伤程度,TDI、DAI、CMDI评分及TNF-α、IL-1β、IL-6的表达水平均进一步下降,Notch1和NICD表达水平进一步升高,同时IκBα和NF-κB p65磷酸化水平及NF-κB p65核转移水平进一步下降(P<0.05)。[结论]芍药汤联合美沙拉嗪对UC的疗效优于单用美沙拉嗪或芍药汤,作用机制可能与其上调Notch1和NICD的表达、抑制NF-κB信号通路活化,进而抑制炎症因子TNF-α、IL-1β、IL-6的表达有关。

[Objective]To observe the effect of Shaoyao decoction(SYD)combined with mesalazine(MES)on ul⁃cerative colitis(UC)in rats via Notch1 and NF-κB signal pathway.[Methods]A total of 60 SD rats were randomly divided into normal group(NG),model group(MG),SYD group(20 g/kg),MES group(0.2 g/kg)and SYD+MES group(20 g/kg+0.2 g/kg),with 12 rats in each group.The UC rat model was induced by feeding 5%dextran sodium sulfate solution,thecontinuous intragastric administration was given for 14 days after modeling.Disease activity index(DAI)and colon mucosa damage index(CMDI)of rats were scored on the 7th and 14th day after administration.The levels of TNF-α、IL-1β、IL-6 in plasma were determined by ELISA.The pathological changes of colon were observed by HE staining and scored by TDI.The mRNA levels of TNF-α,IL-1β,IL-6 were detected by fluorescent quantitative PCR in colon.Notch1,Notch1 intracellular domain(NICD),TNF-α,IL-1β,IL-6,p-IκBα,p-NF-κB p65(cytoplasm)and NF-κB p65(nucleus)were detected by Western blotting in colon.[Results]Compared with MG,in SYD group,MES group and SYD+MES group the pathological damage of intestinal mucosa was significantly alleviated,the scores of TDI,DAI and CMDI were significantly decreased,the expression levels of Notch1 and NICD were significantly increased,the phos⁃phorylation levels of cytoplasmic IκBαand NF-κB p65 and the nuclear transfer level of NF-κB p65 were significantly decreased,and the levels of inflammatory factors TNF-α,IL-1βand IL-6 were significantly decreased(P<0.05).Com⁃pared along with SYD group or MES group,the degree of intestinal mucosal injury,TDI,DAI,CMDI score and the ex⁃pression levels of TNF-α,IL-1βand IL-6 in SYD+MES group were further decreased,the expression levels of Notch1 and NICD were further increased,and the phosphorylation levels of IκBαand NF-κB p65 and the nuclear transfer level of NF-κB p65 were further decreased(P<0.05).[Conclusion]The results show that SYD combined with MES is better than SYD or MES alone in the treatment of UC,and the mechanism may be related to up-regulate Notch1 and NICD expression,and inhibit NF-κB signaling pathway activationand then inhibit inflammatory factorsexpression of TNF-α,IL-1β,IL-6.