藿香正气口服液对DSS诱导结肠炎小鼠肠黏膜屏障的保护作用及机制研究

Study on the Protective Effect and Mechanism of Huoxiang Zhengqi Oral Liquid on Intestinal Mucosal Barrier in Colitis Mice Induced by DSS

目的探讨藿香正气口服液对葡聚糖硫酸钠(DSS)诱导结肠炎小鼠肠黏膜屏障的保护作用及机制。方法将32只C57BL/6小鼠随机分为正常对照组、DSS模型组、藿香正气口服液组(5 mL·kg^(-1))和柳氮磺吡啶组(SASP,1.5 g·kg^(-1))。灌胃给药,给药体积10 mL·kg^(-1),每日1次,连续8 d。给药第2天开始,采用2%DSS连续干预7 d建立急性结肠炎小鼠模型。给药干预后,测定小鼠体质量、结肠长度、疾病活动指数(DAI);采用HE染色法观察结肠组织的病理学变化并计算病理评分;采用ELISA法测定血清中炎症因子肿瘤坏死因子α(TNF-α)、白细胞介素(IL)1β(IL-1β)、IL-6及IL-10的含量;采用偶氮基质显色法测定血清中脂多糖(LPS)的含量;采用异硫氰酸荧光素(FITC-D)标记的右旋葡聚糖法以及肠系膜淋巴结细菌移位率来检测结肠黏膜通透性;采用免疫荧光染色法检测结肠组织中紧密连接蛋白Claudin-1和ZO-1蛋白的表达。结果与正常对照组比较,DSS模型组小鼠的体质量从第3天开始持续降低(P<0.05,P<0.01),结肠长度明显缩短(P<0.01),DAI评分明显升高(P<0.01);结肠组织病理评分显著升高(P<0.01);血清TNF-α、IL-1β及IL-6水平明显升高(P<0.01),而IL-10水平明显降低(P<0.01);血清LPS含量、结肠黏膜FITC-D渗透率以及细菌移位率均显著升高(P<0.01);结肠组织中紧密连接蛋白Claudin-1和ZO-1蛋白表达减弱。与DSS模型组比较,藿香正气口服液组小鼠的体质量有所回升(P<0.05,P<0.01),结肠长度明显延长(P<0.05),DAI评分明显降低(P<0.05);结肠组织病理评分显著降低(P<0.01);血清TNF-α、IL-1β及IL-6水平明显降低(P<0.05,P<0.01),而IL-10水平明显升高(P<0.01);血清LPS含量、结肠黏膜FITC-D渗透率及细菌移位率均明显降低(P<0.05,P<0.01);结肠组织中紧密连接蛋白Claudin-1和ZO-1蛋白表达增强。结论藿香正气口服液可降低DSS诱导的结肠炎模型小鼠肠黏膜的通透性,对结肠炎小鼠的肠黏膜屏障具有保护作用。

Objective To study the protective effect and mechanism of Huoxiang Zhengqi oral liquid on intestinal mucosal barrier in colitis mice induced by DSS. Methods 32 C57 BL/6 mice were randomly divided into normal control group,DSS model group,Huoxiang Zhengqi oral liquid group (5 mL·kg^(-1))and salazosulfapyridine group(SASP, 1.5 g · kg^(-1)). Intragastric administration was performed with the dosage of 10 mL · kg^(-1) once a day for 8 consecutive days. Acute colitis mouse model was established by 2% DSS intervention for 7 d. The body mass,colon length and disease activity index(DAI)of mice were measured after the intervention. Pathological changes of colon tissues were observed by HE staining method and pathological scores were calculated. The levels of tumor necrosis factor-α(TNF-α),interleukin 1(IL-1),IL-6 and IL-10 were determined by ELISA. The content of lipopolysaccharide(LPS)in serum was determined by azo matrix chromogenic method. The permeability of intestina mucosa was measured by FITC-labelled dextran method and bacterial translocation. The protein expression levels of Claudin-1 and ZO-1 were measured by immunofluorescence staining. Results Compared with the normal control group,the body mass of the DSS model group decreased continuously from the 3 rd day(P<0.05,P<0.01),thecolon length was significantly shortened(P<0.01), and DAI score was significantly increased(P<0.01). The histopathological score of colon was significantly increased(P<0.01). The serum TNF-α,IL-1β and IL-6 levelswere significantly increased(P<0.01),while IL-10 levels were significantly decreased in colon tissues(P<0.01).The serum LPS content, colonic mucosa FITC-D permeability and bacterial translocation rate were significantly increased(P<0.01). The expression of Claudin-1 and ZO-1 proteins in colon tissue was decreased. Compared with the DSS model group,the body mass of mice in the Huoxiang Zhengqi oral liquid group was improved(P<0.05,P<0.01),the colon length was significantly increased(P<0.05),and DAI score was significantly decreased(P<0.05). The serum TNF-α,IL-1β and IL-6 levels were significantly decreased(P<0.05,P<0.01),while IL-10 levels were significantly increased(P<0.01). The serum LPS content, colonic mucosa FITC-D permeability and bacterial translocation rate were significantly reduced(P<0.05,P<0.01). The expression of Claudin-1 and ZO-1 proteins was increased in colon tissue. Conclusion Huoxiang Zhengqi oral liquid can reduce the permeability of intestinal mucosa in colitis mice induced by DSS,and has a protective effect on the intestinal mucosal barrier in mice with colitis.