翻白草调控线粒体自噬途径治疗UC的作用机制研究

Mechanism of Potentilla discolor in treating UC by regulating mitochondrial autophagy

研究翻白草对三硝基苯磺酸(2,4,6-trinitrobenzensulfonic acid, TNBS)诱导的实验性溃疡性结肠炎(ulcer colitis, UC)大鼠的治疗作用,并通过线粒体自噬、免疫细胞及细胞因子阐明其疗效机制。采用TNBS-乙醇灌肠法复制UC大鼠模型,实验分为对照组、模型组、柳氮磺吡啶(阳性药)组以及翻白草醇提物高、中、低剂量组。连续给药14 d后,进行疾病活动指数(di-sease activity index,DAI)和苏木素-伊红(hematoxylin and eosin, HE)染色评价,通过透射电镜观察线粒体的形态结构,qRT-PCR法检测线粒体自噬相关基因的表达,流式细胞仪检测大鼠血清中免疫细胞的分化,蛋白芯片检测大鼠结肠组织中细胞因子的表达。结果显示,与模型组比较,翻白草各剂量组均能显著降低UC模型大鼠DAI和结肠组织炎症细胞浸润的程度,提高血清中的T细胞、Th细胞的表达,降低Tc细胞的表达。通过透射电镜观察发现给药组结肠组织中存在线粒体与溶酶体的融合现象,进一步观察到翻白草给药组结肠组织中线粒体自噬相关基因NF-κB、p62、parkin表达均显著升高。蛋白芯片结果显示,与模型组相比,翻白草高剂量组能显著调控模型大鼠的细胞因子表达。综上所述,该研究提供证据支持翻白草对TNBS诱导的急性UC有较好的治愈效果,其作用途径可能与调控线粒体自噬途径及调节细胞免疫炎症因子表达相关。

To evaluate the therapeutic effect of Potentilla discolor on 2,4,6-trinitrobenzensulfonic acid(TNBS)-induced experimental ulcerative colitis(UC) in rats and to determine its therapeutic mechanism through mitochondrial autophagy, immune cells, and cytokines. A rat model of UC was established by TNBS-ethanol enema. Rats were divided into six groups: control, UC model, sulfasalazine(positive drug), and high-dose, moderate-dose, and low-dose ethanol extract groups. After 14-day continuous administration of the corresponding drugs, the disease activity index(DAI) and hematoxylin and eosin(HE) were evaluated. The morphological structure of mitochondria was observed by using transmission electron microscope(TEM), mitophagy-related mRNA expression was detected by using Real-time quantitative polymerase chain reaction(qRT-PCR), immune cell differentiation in rat serum was detected by using flow cytometry(FCM), and cytokine expression in colon tissues of rats was detected by protein microarray. The results showed that compared with the model group, each dose group of P. discolor could significantly reduce the DAI of UC model rats, and decrease the degree of inflammatory cells infiltration in the colon tissue of UC model rats. Meanwhile the expressions of T cells and Th cells in the serum increased significantly, the expression of Tc cells in the serum decreased significantly. Transmission electron microscope found that there was fusion of mitochondria and lysosomes in the colon tissue of the administration group. The expressions of mitochondrial autophagy related genes NF-κB, p62 and parkin were significantly increased in colon tissues. The results of protein chip showed that compared with the model group, the high dose group of P. discolor could significantly regulate the expression of cytokines. In conclusion, these results suggested that P. discolor improved TNBS-induced acute ulcerative colitis in rats by regulating the mitochondrial autophagy and the inflammatory factor expression.