乌梅丸对溃疡性结肠炎模型小鼠炎症因子及肠道菌群的影响

Effects of Wumei pill on inflammatory factors and intestinal flora in mice with ulcerative colitis

关键词:  乌梅丸  溃疡性结肠炎  炎症因子  肠道菌群

Key words:  Wumeiwan  ulcerative colitis  inflammatory factors  intestinal flora

摘要:

目的：研究乌梅丸对葡聚糖硫酸钠(Dextran Sulfate Sodium, DSS)诱导的溃疡性结肠炎(Ulcerative colitis, UC)模型小鼠炎症因子和肠道菌群的调控作用。方法：C57BL/6J小鼠40只，随机分为空白组、模型组、美沙拉秦组、乌梅丸低剂量组、乌梅丸高剂量组，每组8只。自由饮用2% DSS复制UC小鼠模型，造模同时治疗组每天灌胃给药，空白组和模型组灌胃等体积生理盐水，持续7 d。期间观察记录小鼠状态、体重、便血情况和粪便性状，计算疾病活动指数(Disease Activity Index, DAI)评分。实验结束后，取血液、处死小鼠，摘取完整结肠，收集粪便，观察结肠黏膜损伤情况，苏木素-伊红(H&E)染色观察结肠病理损伤和肝、肾损伤；阿利新蓝-过碘酸-雪夫(Alcian blue-periodic acid-Schiff, AB-Pas)染色观察结肠杯状细胞；ELISA法检测血清白细胞介素-6(Iinterleukin-6, IL-6)、白细胞介素-10(IL-10)、肿瘤坏死因子-α(Tumor necrosis factor-α, TNF-α)水平，实时荧光定量聚合酶链反应(Real-time PCR)法检测结肠IL-6、IL-10、TNF-α mRNA表达，蛋白免疫印迹法(Western blot)检测p-P65、P65蛋白表达，16S rDNA高通量测序检测小鼠肠道菌群变化。结果：与空白组相比，模型组小鼠便血，体重下降，结肠缩短，结肠表面明显损伤，结肠组织炎性细胞浸润且杯状细胞流失，结肠黏膜损伤指数（Colon mucosa damage index，CMDI）、病理组织学评分（Histological score, HS）升高，IL-6、TNF-α水平升高，IL-10水平降低，P65蛋白磷酸化水平升高，小鼠肠道菌群丰度降低，菌群构成改变。与模型组相比，两个剂量乌梅丸均能延缓小鼠体重下降，缓解便血症状，降低DAI评分，抑制小鼠结肠缩短，缓解结肠表面损伤，减少杯状细胞流失(P < 0.05, P < 0.01)，降低CMDI、HS评分，降低血清和结肠组织IL-6、TNF-α水平，抑制结肠P65磷酸化；乌梅丸高剂量(Wumei Wan, high-dose, WMW H)组小鼠血清和结肠组织IL-10表达升高，各组小鼠均未出现肝肾损伤。在门水平上，厚壁菌门丰度减少，变形菌门、拟杆菌门和放线菌门丰度增加。在属水平上，梭状芽孢杆菌丰度减少，Muribaculacea丰度增加。结论：乌梅丸能减轻DSS诱导的UC模型小鼠结肠炎症，其作用机制可能与调节炎症因子和调控肠道菌群有关。

Abstract:

Objective: To investigate the regulatory effects of Wumei Wan(WMW) on the expression of inflammatory factors and gut microflora in mice with dextran sodium sulfate (DSS)-induced ulcerative colitis (UC) model. Method: Forty C57BL/6J mice were randomly divided into a control group, a model group, a mesalazine group , a Wumei Wan low-dose group, and a Wumei Wan high-dose group, with 8 mice in each group. The UC model was induced in mice by freely drinking a 2% DSS solution, and at the same time, the treatment group received daily oral administration, while the control and model groups received an equal volume of normal saline, over 7 days. The general conditions of mice , body weight, blood in stool, and fecal traits were recorded, the disease activity index(DAI) score was calculated. After the experiments, the mice were sacrificed, and blood, Intact colon, and feces were collected, Colonic mucosa damage was observed. Hematoxylin-eosin(H&E) staining was performed to observe pathological injury of colon, liver and kidney, Alcian blue-periodic acid-Schiff (AB-Pas) staining was performed to observe Goblet cells. The serum levels of interleukin(IL)-6, IL-10, and tumor necrosis factor(TNF)-α were detected by enzyme-linked immunosorbent assay(ELISA). The mRNA expression of IL-6, IL-10, TNF-α in colon was detected by Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR). The protein expression of P65, p-P65 was detected by western blot. The differences in intestinal flora among the groups were analyzed using 16S rRNA sequencing technology. Result: Compared with the control group, the mice in the model group showed hematochezia, weight loss, colon shortening, and obvious damage to the colon surface. Significant inflammatory cell infiltration and goblet cell loss in histopathologic staining of the colon, significantly elevated CMDI, HS scores, elevated levels of TNF-α, IL-6, and decreased IL-10. The phosphorylation level of P65 protein was significantly increased. The abundance of intestinal flora in mice was decreased, and the composition of intestinal flora was changed. Compared with model group, WMW delayed weight loss in mice, alleviated blood in stool, decreased DAI scores, inhibited colon shortening , alleviated colon surface injury, reduced goblet cell loss, and significantly decreased Colon mucosa damage index(CMDI) and Histological score(HS) scores. The levels of IL-6 and TNF-α in serum and colon tissue were significantly decreased, and the phosphorylation of P65 in colon tissue was significantly inhibited. In addition, the expression of IL-10 in serum and colon tissue of UC mice in Wumei Wan high-dose(WMW H) group was increased. The effect of WMW H group was better than that of WMW L group, and there was no liver and kidney injury. At the phylum level, the abundance of Firmicutes decreased and Proteobacteria, Bacteroidetes, and Actinobacteria increased. At the genus level, the abundance of Clostridium decreased and that of Muribaculacea increased. Conclusion: Wumei Wan can alleviate colonic inflammation in DSS-induced UC mice. and its mechanism of action is related to regulating the expression of inflammatory factors and the composition of intestinal flora.