黄芩汤对溃疡性结肠炎小鼠肠道菌群的影响及肠黏膜屏障的保护作用机制

Effect of Huangqintang on Intestinal Flora in Mice with Ulcerative Colitis and Its Protective Mechanism on Intestinal Mucosal Barrier

目的:研究黄芩汤对溃疡性结肠炎(UC)模型小鼠的药效,并探究黄芩汤在UC中是否能调节肠道菌群,发挥屏障保护作用。方法:雄性Balb/c小鼠按体质量随机分为正常组、模型组、黄芩汤高(20 g·kg^(-1))、中(10 g·kg^(-1))、低(5 g·kg^(-1))剂量组、菌群干扰组、菌群干扰模型组、菌群干扰黄芩汤组(黄芩汤,20 g·kg^(-1))。灌胃抗生素(杆菌肽200 mg·kg^(-1)、万古霉素200 mg·kg^(-1))8 d构建菌群干扰模型,自由饮用3%葡聚糖硫酸钠(DSS)溶液7 d构建UC模型,黄芩汤给药治疗7 d。实验结束后处死小鼠,取血、结肠及粪便,苏木素-伊红(HE)染色观察结肠病变,酶联免疫吸附测定法(ELISA)检测血清白细胞介素-4(IL-4)、白细胞介素-6(IL-6)、白细胞介素-10(IL-10)、肿瘤坏死因子-α(TNF-α)含量,实时荧光定量聚合酶链式反应(Real-time PCR)及蛋白免疫印迹法(Western blot)检测结肠组织中紧密连接蛋白1(Claudin1)、黏蛋白1(MUC1)、咬合蛋白(Occludin)、闭锁小带蛋白-1(ZO-1)mRNA和蛋白的表达,提取小鼠粪便DNA进行高通量测序分析。结果:与正常组比较,模型组小鼠血清IL-4、IL-6、TNF-α含量明显升高(P<0.05,P<0.01),IL-10明显降低(P<0.05);与模型组比较,黄芩汤给药组IL-4、IL-6、TNF-α含量下降(P<0.05,P<0.01),IL-10含量显著上升(P<0.01),Claudin1、MUC1、Occludin、ZO-1 mRNA和蛋白表达明显上升(P<0.05,P<0.01)。菌群干扰后,小鼠肠道菌物种多样性及丰度降低,变形菌门(Proteobacteria)增多(P<0.01),厚壁菌门(Firmicutes)、拟杆菌门(Bacteroidetes)减少(P<0.01);DSS诱发UC后,拟杆菌门和柔壁菌门(Tenericutes)明显减少(P<0.05);黄芩汤高、中、低3个剂量组中拟杆菌门、柔壁菌门明显增多(P<0.05,P<0.01),厚壁菌门减少(P<0.05),乳酸菌属(Lactobacillus)、毛螺菌属(Lachnospiraceae NK4A136 group)、大肠埃希菌志贺属(Escherichia-Shigella)、螺杆菌(Helicobacter)的丰度与黄芩汤剂量呈正比。结论:黄芩汤可以抑制UC小鼠的炎症反应,回调失衡的肠道菌群,修复损伤的肠黏膜屏障。

Objective: To evaluate the pharmacodynamic effect of Huangqintang(HQT)on ulcerative colitis(UC)model mice and investigate its protective effect against UC by regulating intestinal flora. Method: Male Balb/c mice were randomly divided into control group,model group,high-,medium-,and low-dose HQT groups(20,10,5 g·kg^(-1)),flora interference group,flora interference model group,and flora interference-drug treatment group(HQT,20 g·kg^(-1)). The flora interference model was constructed through intragastric administration of antibiotics(200 mg·kg^(-1)bacitracin and 200 mg·kg^(-1)vancomycin)for 8 d,and the UC model was constructed by allowing mice with free access to 3% dextran sulfate sodium(DSS)solution for 7 d. HQT was administered for 7 d. After the experiments,the mice were sacrificed,and blood,colon,and feces were collected. Hematoxylin-eosin(HE)staining was performed to observe the colonic lesions. The serum levels of interleukin(IL)-4,IL-6,IL-10,and tumor necrosis factor(TNF)-α were detected by enzyme-linked immunosorbent assay(ELISA). The mRNA and protein expression of Claudin1,MUC1,Occludin,and zonula occludens-1(ZO-1)in colon tissues was detected by Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)and Western blot,respectively. The fecal DNA of mice was extracted and analyzed by high-throughput sequencing. Result: Compared with the normal group,the model group showed increased serum content of IL-4,IL-6,and TNF-α(P<0.05,P<0.01)and decreased IL-10(P<0.05). Compared with the model group,the HQT groups displayed decreased serum levels of IL-4,IL-6,and TNF-α(P<0.05,P<0.01),increased IL-10 content(P<0.01),increased mRNA and protein expression levels of Claudin1,MUC1,Occludin,and ZO-1(P<0.05,P<0.01). After flora interference,the diversity and abundance of intestinal bacteria decreased. To be specific,Proteobacteria increased(P<0.01),and Firmicutes and Bacteroidetes decreased(P<0.01). After UC induction by DSS,Bacteroidetes and Tenericutes decreased(P<0.05). The high-,medium-,and low-dose HQT groups showed increased Bacteroidetes and Tenericutes(P<0.05,P<0.01)and decreased Firmicutes(P<0.05). Additionally,the abundance of Lactobacillus,Lachnospiraceae NK4A136group,Escherichia-Shigella,and Helicobacteris was positively proportional to the dose of HQT. Conclusion: HQT can inhibit the inflammatory response of UC mice,restore the imbalance of intestinal flora,and repair the damaged intestinal mucosal barrier.