Tollip与NF-κB在脾肾阳虚型溃疡性结肠炎中的级联表达及温补脾肾法的干预研究

Ulcerative colitis (UC) is a chronic recurrent diseases, which are characterstic of precancerous lesions of colon cancer, and no specific treatment was found untill now. Evidence of pathogenesis of TCM clinical treatment showed that the key pathological mechanisms of UC is Yang deficiency of spleen and kidney. and the treatment on UC by the law of warming and tonifying kidney and spleen proved effective exactly. but syndrome foundations of molecular basis and relevent experimental study made no breakthrough progress in recent years. Our previous study proved that Jiu Xie Ling granules based on the law of warming and recuperating kidney could cure colonic mucosa injury in UC rats. Based on previous rearch foundation, this study is to explore dynamic expressions of key molecular of TLR4 signal pathway downstream around Tollip and NF-κB in UC and cross talk of corresponding inflammation medias such as IL-1, and IL-6, and TNF-a, and IFN-γof UC by using Western blotting,immunohistochemical and RT-PCR methods, furtherly,Jiu Xie Ling granules based on the law of warming and tonifying kidney is to be used to interve UC to discover treatment mechanisms and drug targets from the point of transmembrane signal transduction and inflammatory factors response, and to reveal the scientific connotation of warming and tonifying kidney and spleen in the treatment of UC, and to support key technology to develope superior Chinese medicines for preventing and treating UC.

溃疡性结肠炎（UC）多呈慢性反复发作,是结肠癌的癌前病变，迄今尚无特异性治疗方法。中医临床证实其病机演变多为脾肾阳虚，且温补脾肾类方药治疗UC疗效确切，但其证候基础及相关治则治法实验研究尚未取得突破性进展。我们研究发现,以温补脾肾法为指导的久泻灵（理中丸合四神丸）对 UC大鼠结肠黏膜损伤具有修复作用。故基于UC病机规律和前期基础，本研究拟以脾肾阳虚型UC大鼠为研究对象，运用Western blot、免疫组化和RT-PCR技术观察TLR4信号通路下游Tollip和NF-κB关键分子在UC进程中的动态级联表达及相应炎症介质IL-1、IL-6、TNF-α、IFN-γ的对话响应，在此基础上采用温补脾肾方药久泻灵进行干预，进一步从跨膜信号转导和炎症因子串联响应角度揭示温补脾肾法及其方药防治UC的作用机制和药靶部位，以期揭示温补脾肾法治疗UC的科学内涵，为开发防治UC优势中成药的关键技术提供理论支撑。

观察久泻灵颗粒对脾肾阳虚型UC模型大鼠结肠组织病理形态变化的影响，以及应用PCRArray法筛选久泻灵颗粒对脾肾阳虚型UC的作用靶点，应用RT-qPCR法、免疫组化法、免疫印迹法对部分信号通路和靶点进一步验证。探讨久泻灵颗粒治疗UC的作用及机制，为临床治疗UC用药的探讨提供有效的理论依据。采用大黄水煎液灌胃+肌注氢化可的松+三硝基苯磺酸（TNBS）/乙醇灌肠法复建动物模型，实验一分为空白组、模型7、14、21天组，探讨脾肾阳虚型UC模型大鼠的制备方法；实验二分为空白组、模型组、久泻灵颗粒治疗7、14、21天组及阳性组，探讨中药治疗的时效关系；实验三分为空白组、模型组、久泻灵颗粒治疗高、中、低剂量组及阳性组，探讨中药治疗的量效关系，多方向研究证明。选用病理学方法观察各组大鼠结肠组织病理形态变化、ELISA法检测大鼠血清中炎症因子的含量、PCR Array法筛选出Toll样受体信号通路靶点并进行以下验证，RT-qPCR法、免疫组化法及免疫印迹检测大鼠结肠组织中相关靶点基因和蛋白的表达。复建脾肾阳虚型UC成功：模型建立后大鼠血清中FT3、FT4、T降低。久泻灵颗粒对脾肾阳虚型UC具有较好的治疗作用：久泻灵颗粒可显著降低促炎因子含量，并且提高抑炎因子含量，降低结肠组织炎症损伤。PCR Array结果显示：不同疗程及不同剂量久泻灵颗粒干预后24个指标出现了显著变化，PCR Array筛选出中药靶点，对比PATHWAY检索结果，选择Toll样受体通路中部分靶点用RT-qPCR法、免疫组化法及免疫印迹法进行验证，结果一致。脾肾阳虚是UC反复发病，病情迁延难愈的关键病机，以温补脾肾法为指导的久泻灵颗粒治疗UC疗效显著，为临床用药提供了有效依据。久泻灵颗粒可以明显降低脾肾阳虚型UC模型大鼠血清中炎症因子的含量及相关基因的转录和蛋白的表达，进而抑制炎性细胞浸润，减少炎症活性产物的释放，减轻机体肠粘膜的炎症反应和组织损伤程度，促进肠粘膜修复。