摘要
目的:探讨燮理汤对葡聚糖硫酸钠(DSS)诱导的溃疡性结肠炎(UC)小鼠的保护作用及其机制.方法:采用DSS复制UC小鼠模型,将60只小鼠随机分为正常组、模型组、柳氮磺吡啶肠溶片(0.6g·kg-1)组和燮理汤低、中、高剂量组(1.67、3.34、6.68 g·kg-1),用药42 d取材,记录小鼠结肠长度,计算疾病活动指数(DAI)评分,采用酶联免疫吸附测定法(ELSIA)检测血清肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)和白细胞介素-10(IL-10)含量,苏木素-伊红(HE)染色观察结肠组织病理形态学变化,蛋白免疫印迹法(Western blot)检测肝组织法尼醇X受体(FXR)、小异二聚体伴侣(SHP)、肝受体同源物-1(LRH-1)、胆固醇7α羟化酶(CYP7A1)、成纤维生长因子受体4(FGFR4)和回肠组织FXR、钠离子依赖型胆汁酸转运体(ASBT)、成纤维细胞生长因子15(FGF15)蛋白表达水平,16S rRNA测序法分析肠道菌组成结构,超高效液相色谱串联质谱测定胆汁酸含量.结果:与正常组比较,模型组小鼠结肠长度、IL-10含量、α多样性指数、厚壁菌门和乳杆菌丰度及去氧胆酸(DCA)和石胆酸(LCA)含量均显著降低(P＜0.01),DAI评分、IL-6、TNF-α含量、拟杆菌门丰度及胆酸(CA)、鹅去氧胆酸(CDCA)和牛磺胆酸(TCA)含量均明显升高(P＜0.05,P＜0.01),肝组织FXR、SHP、FGFR4及回肠组织FXR、ASBT和FGF15蛋白表达水平均显著下调(P＜0.01),肝组织LRH-1和CYP7A1蛋白表达水平显著上调(P＜0.01),结肠黏膜结构破坏,出现炎性细胞浸润;与模型组比较,燮理汤各剂量组小鼠结肠长度、IL-10含量、α多样性指数、厚壁菌门和乳杆菌丰度及DCA和LCA含量均明显升高(P＜0.05,P＜0.01),DAI评分、拟杆菌门丰度及IL-6、TNF-α、CA、CDCA和TCA含量均显著降低(P＜0.01),肝组织FXR、SHP、FGFR4及回肠组织FXR、ASBT和FGF15蛋白表达水平均显著上调(P＜0.01),肝组织LRH-1和CYP7A1蛋白表达水平显著下调(P＜0.01),结肠黏膜病理损伤明显减轻.结论:燮理汤对UC小鼠有较好的治疗作用,其作用机制可能与调节"肠道菌-胆汁酸"轴,改善肠道菌群失调,维持胆汁酸稳态有关.
Abstract
Objective:To investigate the protective effect of Xielitang on dextran sulfate sodium(DSS)-induced ulcerative colitis(UC)mice and its possible mechanism.Methods:DSS was used to establish UC model.Sixty mice were randomly divided into a normal group,a model group,a sulfasalazine group(0.6 g·kg-1),and low-,medium-,and high-dose Xielitang groups(1.67,3.34,6.68 g·kg-1).After treatment for 42 d,the colon length was recorded,and the disease activity index(DAI)score was calculated.Enzyme-linked immunosorbent assay(ELISA)was used to detect the serum levels of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),and interleukin-10(IL-10).Hematoxylin-eosin(HE)staining was used to observe the pathomorphological changes of colon.Western blot was used to detect the protein expression of farnesoid X receptor(FXR),small heterodimer partner(SHP),liver receptor homolog-l(LRH-1),cholesterol 7α-hydroxylase(CYP7A1),and fibroblast growth factor receptor 4(FGFR4)in liver and FXR,sodium-dependent bile acid transporter(ASBT),and fibroblast growth factor 15(FGF15)in ileum.16S rRNA sequencing was used to analyze the intestinal flora.Moreover,ultra-high performance liquid chromatography-tandem mass spectrometry was used to detect the bile acid content.Results:Compared with the normal group,the model group showed significantly decreased colon length,IL-10 content,α-diversity index,abundance of Firmicutes and Lactobacillus,and content of deoxycholic acid(DCA)and lithocholic acid(LCA)(P＜0.01),significantly increased DAI score,IL-6 and TNF-α content,abundance of Bacteroidetes,and the content of cholic acid(CA),chenodeoxycholic acid(CDCA),and taurocholic acid(TCA)(P＜0.05,P＜0.01),significantly down-regulated protein expression of FXR,SHP,and FGFR4 in liver and FXR,ASBT,and FGF15 in ileum(P＜0.01),and significantly up-regulated protein expression of LRH-1 and CYP7A1 in liver(P＜0.01).In addition,the structure of colonic mucosa was destroyed,and inflammatory cells infiltrated in the model group.Compared with the model group,Xielitang could significantly increase the colon length,IL-10 content,α-diversity index,the abundance of Firmicutes and Lactobacillus,and DCA and LCA content(P＜0.05,P＜0.01),decrease DAI score,abundance of Bacteroidetes,and the content of IL-6,TNF-α,CA,CDCA,and TCA(P＜0.01),up-regulate the protein expression of FXR,SHP,and FGFR4 in liver and FXR,ASBT,and FGF15 in ileum(P＜0.01),and down-regulate the protein expression of LRH-1 and CYP7A1 in liver(P＜0.01).The pathological damage of colonic mucosa was obviously alleviated.Conclusion:Xielitang protects against UC probably by regulating the"intestinal microbiota-bile acid"axis,regulating intestinal flora imbalance,and maintaining bile acid homeostasis.
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