香椿皮醇提物对溃疡性结肠炎小鼠黏膜屏障和肠道菌群的作用及机制

Effects of Toona sinensis bark alcohol extract on mucosal barrier and gut microbiota in mice with ulcerative colitis

目的:基于结肠黏膜屏障和肠道菌群探究香椿皮醇提物(TAE)对葡聚糖硫酸钠(DSS)诱导的小鼠溃疡性结肠炎(UC)的作用及机制。方法:60只C57BL/6J小鼠随机分为正常组、模型组、美沙拉嗪(0.2 g/kg)组、香椿皮醇提物组(低、中、高剂量分别相当于2.3、4.6和9.2 g/kg生药剂量)。采用自由饮用2.5%DSS造模,同时根据分组相应干预给药10 d。通过小鼠体质量、疾病活动度指数(DAI)、结肠长度、脾脏重量和结肠组织病理学改变评估小鼠UC严重程度;TUNEL法检测结肠上皮细胞凋亡水平,Western blot法检测结肠组织相关蛋白表达;ELISA法检测结肠组织炎症因子水平;生化法检测结肠组织总超氧化物歧化酶(T-SOD)、过氧化氢酶(CAT)活性及丙二醛(MDA)含量;16SrRNA测序技术分析小鼠肠道菌群变化。结果:与正常组相比,模型组体质量明显下降(P<0.01),结肠长度明显缩短(P<0.05),DAI、脾脏指数显著升高(P<0.01),且结肠黏膜损伤严重(P<0.01),肠上皮凋亡细胞明显增多(P<0.01),紧密连接蛋白occludin、claudin-1表达显著降低(P<0.01),炎症因子IL-6、IL-1β和TNF-α水平明显升高,IL-10水平降低(P<0.05),T-SOD、CAT活性显著降低(P<0.01),MDA含量明显升高(P<0.01),模型组肠道菌群丰度和多样性降低(P<0.05)。与模型组比较,香椿皮醇提物干预后,上述指标均有显著改善(P<0.05),Proteobacteria、Escherichia-Shigella等有害菌丰度降低(P<0.05),Bacteroidota、Muribaculaceae等有益菌丰度上调(P<0.05),菌群丰度和多样性增加。结论:TAE通过减少肠上皮细胞凋亡、抑制炎症反应和减轻氧化应激反应来减轻结肠黏膜损伤,并通过调节肠道菌群丰度,维持肠道菌群稳态,从而改善结肠黏膜屏障功能。

AIM:To investigate the effects and underlying mechanism of Toona sinensis bark extract(TAE)on the colon mucosal barrier and gut microbiota in mice with ulcerative colitis(UC)induced by dextran sulfate sodium(DSS).METHODS:Sixty C57BL/6J mice were randomly assigned to control,model,and mesalazine(0.2 g/kg)groups,as well as TAE groups(low,medium,and high-doses equal to crude drug concentrations of 2.3,4.6 and 9.2 g/kg,respectively).The UC model was induced by drinking of 2.5%DSS,and mean while the drugs were administered for 10 days.The mice were then evaluated in terms of weight,disease activity index(DAI),colon length,spleen index,and pathological changes in the colon tissues.In addition,the level of apoptosis in colon tissues was assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling(TUNEL)fluorescence staining,and the expression of related proteins was evaluated by Western blot,levels of inflammatory factors were determined by enzyme-linked immunosorbent assays(ELISA),and the activities of total superoxide dismutase(T-SOD)and catalase(CAT)and malondialdehyde(MDA)content were assessed by biochemical assays.Furthermore,the constitution and diversity of the gut microbiota were investigated by 16S rRNA gene sequencing.RESULTS:Compared with the control group,mice in the model group showed significantly reduced body weights(P<0.01),and the colon length was shortened significantly(P<0.05).Marked increases in the DAI and spleen index were observed(P<0.01),along with severe damage to the colon mucosa(P<0.01).Mechanistically,the level of intestinal epithelial cell apoptosis was significantly raised(P<0.01).The model group showed markedly reduced expression of occludin and claudin-1(P<0.01),the level of IL-10,and activities of T-SOD and CAT in the colon tissues(P<0.01).While the levels of IL-6,IL-1β,TNF-α,and the MDA content were increased significantly(P<0.05).The abundance and diversity of the gut microbiota were decreased in the model group(P<0.05).Compared with the model group,all these indicators were ameliorated by the administration of TAE(P<0.05).The abundance of pathogenic bacteria,including Proteobacteria and Escherichia-Shigella,was decreased remarkably(P<0.05),while that of probiotics,including Bacteroidota and Muribaculaceae,were increased significantly(P<0.05).The abundance and diversity of the gut microbiota were increased.CONCLUSION:Taken together,Toona sinensis bark alcohol extract can alleviate damage to the intestinal mucosa by suppressing the apoptosis of intestinal epithelial cell,reducing the inflammatory response,and mitigating oxidative stress.Treatment with TAE could also maintain the homeostasis of the gut microbiota by regulating the abundance,ultimately meliorate the function of intestinal mucosal barrier.