痛泻要方对肝郁脾虚型溃疡性结肠炎大鼠炎性因子表达量的影响

Effect of Tongxie Yaofang on the expression of inflammatory factors in rats of stagnation of liver and spleen deficiency syndrome

目的建立肝郁脾虚型溃疡性结肠炎(ulcerative colitis,UC)动物模型并评价,分析痛泻要方对炎性反应因子IL-17、IFN-γ、IL-4、IL-10基因和蛋白表达量的影响。方法 40只SPF级SD大鼠随机分为4组,正常组、模型组、痛泻要方干预组和柳氮磺胺吡啶干预组,正常组不进行任何干预,其余各组采用TNBS/乙醇溶液灌肠+束缚法+饮食失节法建立肝郁脾虚型溃疡性结肠炎模型,模型建立后采用旷场试验、高架十字迷宫实验和新环境进食抑制实验进行评价。药物干预组分别采用痛泻要方和柳氮磺胺吡啶进行治疗,剂量分别为20.25g/kg和0.54g/kg,治疗一周后采用ELISA和RTPCR检测结肠黏膜IL-4、IL-10、IL-17、INF-γ等指标的表达量。结果采用TNBS/乙醇溶液灌肠+束缚法+饮食失节法能够成功建立肝郁脾虚型UC动物模型,大鼠表现为精神萎靡、毛发凌乱、喜欢蜷伏角落,挑逗大鼠表现为明显烦躁、易于被激怒、防御姿态明显,大便稀溏等;旷场实验中大鼠运动的总距离、中央区进入次数明显减少(P<0.05),高架十字迷宫实验运动的总距离、双侧开放臂进入的次数、双侧开放臂的停留时间、中央区进入的次数等方面均明显低于正常组(P<0.05);新环境进食抑制实验发现模型组大鼠的进食时间较长(P<0.05)。ELISA和mRNA检测结果显示,与正常组相比,模型组大鼠IL-4和IL-10含量明显降低,而IL-17和IFN-γ含量明显升高,差异具有统计学意义(P<0.01);与模型组相比,痛泻要方干预组和柳氮磺胺吡啶干预组IL-17和IFN-γ降低趋势明显(P<0.01),而IL-4升高趋势明显(P<0.01),IL-10呈现升高趋势(P>0.05)。结论 TNBS/乙醇溶液灌肠+束缚法+饮食失节法能够成功建立肝郁脾虚型UC动物模型,痛泻要方能够通过调节细胞炎性因子的表达量而参与对UC的治疗作用,从而发挥对T细胞介导的炎性平衡的调节作用,进而抑制过度的炎性反应,发挥相应的治疗作用。

Objective To establish and evaluate an animal model of ulcerative colitis（ UC） with liver-depression and spleen-deficiency syndrome,and analyze the effect of Tongxie Yaofang（ TXYF） on the gene and protein expression of inflammatory response factors including IL-17,IFN-γ,IL-4 and IL-10. Methods Forty SPF SD rats were randomly divided into 4 groups：normal group,model group,TXYF treatment group and sulfasalazine treatment group. The normal group received no intervenes;the other groups underwent enema with TNBS/ethanol solution ＋ tight-binding ＋ improper diet to establish a model of UC with liver-depression and spleen-deficiency syndrome. After the establishment,the model was evaluated by using open field test,elevated plus-maze test and feeding inhibition test in new environment. The TXYF treatment group and sulfasalazine treatment group were treated with 20. 25 g/kg TXYF and 0. 54 g/kg sulfasalazine,respectively. One week after treatment,the expression of IL-4,IL-10,IL-17 and INF-γ in the colonic mucosa were detected by ELISA and RT-PCR. Results The rat model of UC with liver-depression and spleen-deficiency syndrome was successfully established by enema with TNBS/ethanol solution ＋tight-binding ＋ improper diet. The rats showed listlessness,messy hair and fancy crouching at the corner. When were teased,the rats presented obvious fidget,irritability and defensive stance and loose stool. In the open field test,the total distance of rat movement and the number of entering central area reduced significantly（ P 〈0. 05）. In the elevated plus-maze test,the total distance of movement,the number of entering with bilateral open arms,the duration of keeping bilateral open arms and the number of entering central area in the model group were significantly lower as compared with the normal group（ P 〈0. 05）. The feeding inhibition test in new environment revealed that the duration of eating was longer in the model group than the normal group（ P 〈0. 05）. ELISA and RT-PCR results demonstrated that compared with the normal group,IL-4 and IL-10 contents decreased significantly while IL-17 and IFN-γ contents increased apparently in the model group（ P 〈0. 01）. Compared with the model group,IL-17 and IFN-γ decreased obviously（ P 〈0. 01） while IL-4 increased evidently（ P 〈0. 01） and IL-10 showed an increasing trend（ P〉 0. 05） in the TXYF treatment group and sulfasalazine treatment group. Compared with the sulfasalazine treatment group,the decrease in Il-17 content was more obvious in the TXYF treatment group（ P 〈0. 01）,while the decrease in IFN-γ was less obvious. Conclusion Enema with TNBS/ethanol solution ＋ tight-binding ＋ improper diet can successfully establish an animal model of UC with liver-depression and spleen-deficiency syndrome. In addition,TXYF can participate in the treatment of UC through regulating the expression of inflammatory cytokines and then T cell-mediated balance of inflammatory cytokines. Furthermore,TXYF play a role in the treatment of UC through inhibiting excessive inflammatory response.