基于mTOR/p-S6K1探讨加味附子理中汤干预UC大鼠肠黏膜炎症反应的效应机制

Exploring Mechanism of Modified Fuzi Lizhongtang in Intervening Inflammatory Response of Intestinal Mucosa in Rats with Ulcerative Colitis Based on mTOR/p-S6K1

目的:探讨加味附子理中汤治疗溃疡性结肠炎(UC)模型大鼠的疗效及相关作用机制。方法:选用72只雄性SD大鼠,分为正常组,模型组,柳氮磺胺嘧啶组(0.5 g·kg^-1),加味附子理中汤高、中、低剂量组(23.62,11.81,5.91 g·kg^-1)。运用2,4,6-三硝基苯磺酸(TNBS)-乙醇复合造模法复制UC大鼠模型,连续灌胃治疗2周,观察各组大鼠一般情况,大鼠麻醉后腹主动脉采血,取结肠组织。运用结肠黏膜损伤指数(CMDI)半定量评分,苏木素-伊红(HE)染色观察大鼠结肠组织病理学改变,酶联免疫吸附测定(ELISA)检测大鼠血清白细胞介素(IL)-4,IL-6,IL-10,肿瘤坏死因子-α(TNF-α)含量;免疫组化和蛋白免疫印迹法(Western blot)分别检测结肠黏膜组织哺乳动物雷帕霉素靶蛋白(mTOR)及磷酸化核糖体蛋白S6激酶1(p-S6K1)蛋白表达。结果:与正常组比较,模型组大鼠CMDI评分显著升高(P<0.01),血清IL-4,IL-10含量显著下降,IL-6,TNF-α含量显著上升(P<0.01),结肠黏膜组织mTOR,p-S6K1蛋白表达水平显著上调(P<0.01);与模型组比较,加味附子理中汤高剂量组大鼠CMDI评分明显下降(P<0.05),高、中剂量组大鼠血清促炎因子IL-6,TNF-α显著降低(P<0.01),抗炎因子IL-4,IL-10明显升高(P<0.05,P<0.01);加味附子理中汤高剂量大鼠mTOR,p-S6K1的蛋白表达水平明显下调(P<0.05,P<0.01)。结论:加味附子理中汤高剂量组可显著减轻UC结肠黏膜充血水肿、炎性细胞浸润、腺体扭曲、排列紊乱等病理表现,其机制可能与其下调mTOR/p-S6K1信号、调控炎症因子的分泌有关。

Objectives: To investigate the therapeutic effect and mechanism of modified Fuzi Lizhongtang on ulcerative colitis(UC)model rats. Method: The 72 male SD rats were randomly divided into normal group,model group,sulfasalazine group(0. 5 g·kg^-1),modified Fuzi Lizhongtang high,medium and lowdose group(23. 62,11. 81,5. 91 g·kg^-1). These rats were used to replicate the UC rat model by 2,4,6-trinitrobenzene sulfonic acid(TNBS)-ethanol composite modeling and treated by gavage for 2 weeks. The general condition of rats in each group was observed. After anesthesia,blood was collected from abdominal aorta and colonic tissue was taken. Semi quantitative evaluation by the colon mucosa damage index(CMDI),the pathological changes of colonic tissue were observed by the hematoxylin and eosin(HE)staining. The contents of serum interleukin-4(IL-4),IL-6,IL-10 and tumor necrosis factor-α(TNF-α)were detected by enzyme-linked immunosorbent assay(ELISA). The expressions of mammalian target of rapamycin(mTOR) and phosphorylated ribosomal protein S6 kinase 1(p-S6 K1) in colonic mucosa were detected by immunohistochemistry(IHC)and Western blot. Result: Compared with normal group,the CMDI score of the model group rats was significantly increased(P<0. 01). The contents of IL-4 and IL-10 in serum were significantly decreased,the contents of IL-6 and TNF-α were significantly increased(P<0. 01). The expression levels of mTOR and p-S6 K1 in colonic mucosa were up-regulated(P<0. 01). Compared with model group,the CMDI score of the modified Fuzi Lizhongtang high dose group was significantly decreased(P<0. 05). In modified Fuzi Lizhongtang high and medium dose group,the contents of IL-6 and TNF-α were significantly decreased(P<0. 01) and the contents of IL-4 and IL-10 in serum were significantly increased(P<0. 05,P<0. 01). In the modified Fuzi Lizhongtang high dose group,the expression level of mTOR and p-S6 K1 protein was down-regulated significantly(P<0. 05,P<0. 01). Conclusion: Modified Fuzi Lizhongtang high dose group can significantly reduce the congestion and edema,inflammatory cell infiltration,gland distortion,disorder of arrangement and other pathological manifestations of UC colon mucosa,and its mechanism may be related to its down-regulation of mTOR/p-S6 K1 signal and the regulation of inflammatory factors secretion.