基于Bcl-2/Bax信号通路探讨湿生扁蕾乙酸乙酯提取物治疗溃疡性结肠炎作用机制

Mechanism of Ethyl Acetate Extract of Gentianopsis paludosa in Treatment of Ulcerative Colitis Based on Bcl-2/Bax Signaling Pathway

目目的的:探讨藏族医药湿生扁蕾乙酸乙酯提取物通过B细胞淋巴瘤-2(Bcl-2)与Bcl-2相关X蛋白(Bax)介导的凋亡通路防治复发型大鼠大肠湿热型溃疡性结肠炎(UC)的作用机制。方法:采用“病证结合”法构建复发型大鼠大肠湿热UC模型,将70只SPF级SD雄性大鼠随机分为空白组、模型组、湿生扁蕾乙酸乙酯提取物高、中、低剂量组(150、75、37.5 mg·kg^(-1))及美沙拉嗪组(135 mg·kg^(-1)),连续灌胃给药14 d,记录大鼠一般状态,观察大鼠结肠长度及肠黏膜损伤情况,测定结肠湿质量指数,计算肝脏、脾脏、胸腺脏器系数。酶联免疫吸附测定法(ELISA)检测各组大鼠血清中白细胞介素-6(IL-6)和白细胞介素-1β(IL-1β)的水平,苏木素-伊红(HE)染色观察结肠病理变化,原位末端标记法(TUNEL)检测结肠上皮细胞的凋亡情况,蛋白免疫印迹法(Western blot)检测结肠组织中Bcl-2、Bax、胱天蛋白酶-3(Caspase-3)、胱天蛋白酶-9(Caspase-9)、闭锁连接蛋白-1(ZO-1)、紧密连接蛋白3(Claudin3)、闭合蛋白(Occludin)的表达,免疫组化法(IHC)观察结肠上皮细胞中Bax、Caspase-3的表达。结果:与空白组比较,模型组大鼠疾病活动指数(DAI)评分、结肠黏膜损伤指数(CMDI)、肠上皮凋亡、肝脏、脾脏指数及血清中炎症因子IL-1β和IL-6水平均显著升高(P<0.01),肠黏膜保护蛋白ZO-1、Claudin3、Occludin表达显著降低(P<0.01),促凋亡蛋白Bax、Caspase-3、Caspase-9表达显著升高(P<0.01),抑制凋亡蛋白Bcl-2表达显著降低(P<0.01);与模型组比较,湿生扁蕾乙酸乙酯提取物高、中、低剂量均能够显著改善大鼠的一般状态,减轻结肠病变,降低肠上皮细胞凋亡、肝脏、脾脏指数,上调ZO-1、Claudin3、Occludin及Bcl-2蛋白表达,下调Bax、Caspase-3、Caspase-9蛋白表达,以湿生扁蕾高、中剂量效果明显(P<0.05,P<0.01)。结论:湿生扁蕾乙酸乙酯提取物能够通过调节Bcl-2/Bax信号通路,减轻肠黏膜损伤,发挥治疗UC作用。

Objective:To investigate the mechanism of ethyl acetate extract of Tibetan medicine dampness bud Gentianopsis paludosa in the prevention and treatment of recurrent ulcerative colitis(UC)in rats with dampness-heat in large intestine syndrome based on the apoptotic pathway mediated by the B-cell lymphoma-2(Bcl-2)and Bcl-2-associated X protein(Bax).Method:Using the disease-syndrome combination method,a recurrent UC model of dampness-heat in large intestine syndrome was constructed in rats.Seventy SPF-grade male SD rats were randomly divided into control group,model group,high-,medium-,and low-dose ethyl acetate of G.paludosa groups(150,75,37.5 mg·kg^(-1)),and mesalazine group(135 mg·kg^(-1)).The rats were orally administered with respective drugs for 14 days.The general conditions of the rats were recorded,and colon length and mucosal damage were observed.The colon wet weight index and organ coefficients of the liver,spleen,and thymus were calculated.Enzyme-linked immunosorbent assay(ELISA)was used to measure the levels of interleukin-6(IL-6)and interleukin-1β(IL-1β)in the serum of each group.Hematoxylin-eosin(HE)staining was performed to observe pathological changes in the colon.Terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)was used to detect apoptosis in colonic epithelial cells.Western blot was used to measure the expression levels of Bcl-2,Bax,Caspase-3,Caspase-9,Zona Occludens-1(ZO-1),Claudin3,and Occludin in colonic tissue.Immunohistochemistry(IHC)was used to observe the expression of Bax and Caspase-3 in colonic epithelial cells.Result:Compared with the control group,the model group showed significant increases in the disease activity index(DAI)score,colonic mucosal damage index(CMDI),intestinal epithelial apoptosis,liver and spleen indexes,and levels of inflammatory factors IL-1βand IL-6 in the serum(P<0.01),decreased expression of intestinal mucosal protective proteins ZO-1,Claudin3,and Occluding(P<0.01),increased expression of pro-apoptotic proteins Bax,Caspase-3,and Caspase-9(P<0.01),and decreased expression of anti-apoptotic protein Bcl-2(P<0.01).Compared with the model group,the high-,medium-,and low-dose ethyl acetate of G.paludosa groups all significantly improved the general condition of the rats,reduced colonic lesions,decreased intestinal epithelial cell apoptosis,reduced liver and spleen indexes,upregulated the expression of ZO-1,Claudin3,Occludin,and Bcl-2 proteins,and downregulated the expression of Bax,Caspase-3,and Caspase-9 proteins,with the high-and medium-dose ethyl acetate of G.paludosa groups showing the superior effects(P<0.05,P<0.01).Conclusion:Ethyl acetate of G.paludosa can alleviate colonic mucosal damage and exert a therapeutic effect on UC by regulating the Bcl-2/Bax signaling pathway.