自拟加味白头翁汤通过调节Drp1/LC3改善急性UC小鼠结肠上皮细胞线粒体功能的研究

Self-made Modified Baitouweng Decoction Improves the Mitochondrial Function of Colonic Epithelial Cells in Acute Ulcerative Colitis Mice through Drp1/LC3

目的观察自拟加味白头翁汤对急性溃疡性结肠炎(UC)小鼠结肠上皮细胞线粒体Drp1/LC3的影响。方法3.5%葡聚糖硫酸钠自由饮水7 d复制急性UC模型,48只C57BL/6小鼠随机分为模型组、美沙拉嗪组(100 mg/kg,ASA),加味白头翁汤高、中、低剂量组(108、54、27 g/kg),连续灌胃治疗7 d,每天1次,正常小鼠为对照组。治疗结束后,统计各组小鼠结肠长度;观察小鼠结肠组织黏膜、腺体、隐窝的变化;酶联免疫吸附法测定小鼠结肠组织超氧化物歧化酶(SOD)、白细胞介素-6(IL-6)、线粒体呼吸链复合物Ⅰ/Ⅲ水平;透射电子显微镜观察结肠上皮细胞线粒体分裂及自噬小体形态;蛋白免疫印迹法(Western blotting)、免疫组织化学法(IHC)、免疫荧光法(IF)检测结肠上皮组织中线粒体Drp1、LC3蛋白表达。结果与模型组比较,美沙拉嗪组和加味白头翁汤高剂量组结肠上皮SOD升高,而IL-6水平降低,腺体结构改善,炎症细胞浸润显著减少(P<0.05);ATP、线粒体呼吸链复合物Ⅰ/Ⅲ活性恢复(P<0.05);加味白头翁汤高剂量组线粒体分裂蛋白Drp1和自噬蛋白LC3表达水平均升高(P<0.05),且使用Rap增加自噬后,Drp1表达水平不受影响。结论加味白头翁汤高剂量组可能通过增加结肠上皮线粒体Drp1、LC3的表达,激活线粒体分裂从而促进自噬,改善急性UC小鼠结肠上皮线粒体功能,减轻炎症损伤。

Objective:To observe the effect of self-made Modified Baitouweng Decoction in regulating the Drp1/LC3 and improving mitochondrial function of colonic epithelial cells in acute ulcerative colitis(UC)mice.Methods:48 C57BL/6 mice were randomized into groups,each group of eight.Except for the control group,the other groups duplicated by free drinking 3.5%dextran sodium sulfate(DSS)7 days for acute UC model,and grouped into the model group,mesalamine group(100 mg/kg),high(108 g/kg),middle(54 g/kg)and low dose(27 g/kg)Modified Baitouweng Decoction groups.The mice in each group were given the corresponding drugs by gavage,while those in the control group and model group received the same volume of normal saline,once per day,for 7successive days.The mice body weight,blood in stool and DAI scoring were recorded.After treatment,the colon length was measured in each group,the intestinal epithelial tissue was stained with hematoxylin-eosin(HE)for observing the changes in mucous membrane,gland and crypt.Enzyme-linked immunosorbent assay(ELISA)was used to determine superoxide dismutase(SOD),interleukin-6(IL-6)and mitochondrial Ⅰ/Ⅲ level in colon tissues of mice.Mitochondrial division and autophagosome morphology of colon epithelial cells were observed by transmission electron microscopy.Drp1 and LC3 protein expression were detected by Western blotting,immunohistochemistry,and immunofluorescence.Results:Compared with model group,mesalazine and Modified Baitouweng Decoction high-dose group increased SOD level and decreased IL-6 level in colon epithelium.The glandular structure of pathological tissue was improved and the infiltration of inflammatory cells was significantly reduced(P<0.05).The activities of ATP and mitochondrial respiratory chain complex Ⅰ/Ⅲ were restored(P<0.05).The expression levels of mitochondrial mitogenic protein Drp1 and autophagy protein LC3 were increased in the highdose group of Modified Baitouweng Decoction(P<0.05),and the expression levels of Drp1 were not affected after the use of RAP to increase autophagy.Conclusion:The high-dose group of Modified Baitouweng Decoction can effectively improve the mitochondrial function of colonic epithelium in acute UC mice and reduce inflammatory injury via increased expression of Drp1 and LC3 in colonic epithelial mitochondria.