陈皮酵素调节肠菌稳态及Nrf2/NLRP3通路修复肠黏膜改善小鼠急性溃疡性结肠炎

Xinhui citrus fermentation liquor ameliorates acute ulcerative colitis in mice via regulating intestinal bacteria homeostasis and Nrf2/NLRP3 pathway to repair intestinal mucosa

本研究考察了陈皮酵素对小鼠急性结肠炎的保护作用及机制。动物实验遵循南方医科大学动物伦理委员会规定。C57BL/6小鼠自由饮用3%右旋葡聚糖硫酸钠(dextran sodium sulfate, DSS) 6天诱导急性溃疡性结肠炎,期间灌胃陈皮酵素、陈皮水煎液(均以粗多糖300 mg·kg^(-1)·d^(-1)计)或阳性药5-氨基水杨酸(100 mg·kg^(-1)·d^(-1)),连续9天。收集盲肠内容物进行16S rRNA测序分析;RT-PCR、免疫荧光及Western blot法检测结肠炎症因子、紧密连接蛋白及核因子E2相关因子2/Nod样受体蛋白3 (nuclear factor erythroid 2 related factor 2/Nod-like receptor protein 3,Nrf2/NLRP3)通路相关蛋白水平。结果显示:陈皮酵素及陈皮水煎液均能明显改善急性结肠炎小鼠的体重下降、腹泻、血便和结肠缩短;且陈皮酵素显著降低结肠促炎因子,如白介素6 (interleukin 6, Il-6)、CXC趋化因子配体10(CXC chemokine ligand 10, Cxcl10)及NLRP3炎症小体相关基因或蛋白表达;同时显著增加结肠抗炎因子(Il-10)、紧密连接蛋白[Zo-1 (zonula occludens-1)、occludin、claudin-1]、黏蛋白(mucin 2, Muc2)及Nrf2的基因表达,以及醌氧化还原酶1 [NAD(P)H quinine oxidoreductase 1, NQO1]和血红素氧合酶1 (heme oxygenase 1, HO-1)的基因和蛋白表达。此外,陈皮酵素显著富集Akkermansia并降低Enterococcus、Streptococcus等有害菌丰度;关联分析结果显示,Akkermansia丰度与结肠抗炎因子、紧密连接蛋白水平和Nrf2信号通路呈显著正相关。综上,陈皮酵素可通过调节肠菌稳态及Nrf2/NLRP3通路修复肠黏膜改善小鼠急性溃疡性结肠炎。

This study investigated the protective effect and related mechanisms of Xinhui citrus fermentation liquor on mice with ulcerative colitis.Animal experiments follow the rules of Animal Ethics Committee of Southern Medical University.C57BL/6 mice were given 3% dextran sodium sulfate(DSS) for 6 days to induce acute ulcerative colitis.During this period,Xinhui citrus fermentation liquor,decoction of Citrus reticulata blanco(both orally administrated with 300 mg·kg^(-1)·d^(-1) crude polysaccharide) or positive drug 5-aminosalicylic acid(100 mg·kg^(-1)·d^(-1)) were gavaged continuously for 9 days.Cecal contents were collected for 16S rRNA sequencing analysis.The levels of inflammatory factors,tight junction proteins and nuclear factor erythroid 2 related factor 2/Nod-like receptor protein 3(Nrf2/NLRP3) pathway related proteins in the colon were detected by real-time PCR(RT-PCR),immunofluorescence and Western blot.Our results showed that Xinhui citrus fermentation liquor and Citrus reticulata blanco protected against UC-induced weight loss,diarrhea,bloody stool,and colon shortening.The mRNA and protein levels of pro-inflammatory factors,such as interleukin 6(Il-6) and CXC chemokine ligand10(Cxcl10) and NLRP3 inflammasome were significantly decreased;the mRNA levels of colon anti-inflammatory factor(Il-10),tight junction protein [zonula occludens-1(Zo-1),occludin,claudin-1],mucin 2(Muc2),Nrf2,as well as the mRNA and protein levels of NAD(P)H quinine oxidoreductase 1(NQO1) and heme oxygenase 1(HO-1)were significantly increased.In addition,Xinhui citrus fermentation liquor increased the abundance of Akkermansia and reduced the abundance of harmful bacteria Enterococcus and Streptococcus.The correlation analysis showed that the abundance of Akkermansia was positively correlated with anti-inflammatory factors,tight junction protein and the related genes levels of Nrf2 signaling pathway.In summary,Xinhui citrus fermentation liquor ameliorates acute ulcerative colitis in mice via regulating intestinal bacteria homeostasis and Nrf2/NLRP3 pathway to repair intestinal mucosa.