香连止泻片对恶唑酮诱导溃疡性结肠炎小鼠的治疗作用及其机制

Therapeutic effect of Xianglian Zhixie tablet on oxazolone-induced ulcerative colitis in mice

目的探讨香连止泻片对恶唑酮诱导溃疡性结肠炎(UC)小鼠的治疗作用,并基于Th1/Th2免疫轴探讨其作用机制。方法选择雄性BALB/c小鼠48只,随机分为空白组、模型组、美沙拉嗪组以及香连止泻低、中、高剂量组,每组8只。模型组、美沙拉嗪组以及香连止泻低、中、高剂量组采用恶唑酮诱导UC模型,空白组正常饲养,不予恶唑酮诱导UC模型。自建模第1天开始,美沙拉嗪组予美沙拉嗪0.31 g/kg灌胃,香连止泻低、中、高剂量组分别予香连止泻片0.68、1.36、2.72 g/kg灌胃,空白组和模型组予等量超纯水灌胃,连续灌胃7天。灌胃期间,每日评估疾病活动指数(DAI)。末次灌胃结束,称量体质量,麻醉后剪开腹腔,分离脾脏并称质量,计算脾脏指数;分离结肠,测量整段结肠长度。取部分结肠组织,过碘酸-雪夫染色,显微镜下观察结肠组织病理形态变化,计数结肠黏膜杯状细胞数量;取部分结肠组织,冰上匀浆,离心留取上清液,采用ELISA法检测IL-1β、TNF-α含量;取部分结肠组织剪碎,组织裂解液冰上充分裂解,提取组织总蛋白,采用Western blotting法检测IFN-γ、IL-4蛋白表达;末次灌胃结束,麻醉后取血,采用流式细胞术检测全血Th1、Th2细胞比例,计算Th1/Th2。结果与空白组比较,模型组DAI评分、脾脏指数,结肠组织IL-1β、TNF-α含量和IFN-γ蛋白表达,全血Th1细胞比例以及Th1/Th2,均显著升高(P均<0.05);结肠黏膜杯状细胞数量减少、排列不规则,内含糖原物质减少;结肠长度显著缩短,结肠组织IL-4蛋白表达和全血Th2细胞比例均显著降低(P均<0.05)。与模型组比较,美沙拉嗪组和香连止泻高剂量组DAI评分、脾脏指数,结肠组织IL-1β、TNF-α含量,全血Th1细胞比例以及Th1/Th2,均显著降低(P均<0.05);结肠黏膜杯状细胞数量增多、形态完整,内含糖原物质基本趋于正常;结肠长度缩短受到显著抑制,结肠组织IL-4蛋白表达显著上调(P均<0.05)。结论不同剂量香连止泻片对恶唑酮诱导UC小鼠均有一定治疗作用,以香连止泻片高剂量(2倍的临床等效剂量)效果最佳;纠正Th1/Th2免疫轴失衡,减轻肠道炎症,可能是香连止泻片的作用机制之一。

Objective To investigate the therapeutic effect of Xianglian Zhixie tablet on oxazolone-induced ulcerative colitis(UC)in mice,and to explore its mechanism of action based on the Th1/Th2 immune axis.Methods Forty-eight male BALB/c mice were selected and were randomly divided into the blank control group,model group,mesalazine group,low-dose Xianglian Zhixie tablet group,medium-dose Xianglian Zhixie tablet group,and high-dose Xianglian Zhixie tablet group,with eight mice in each group.The mice in the model group,mesalazine group,low-dose,medium-dose,and high-dose Xianglian Zhixie tablet groups were treated with oxazolone to induce UC.The mice in the blank control group were not treated.From the first day of the establishment of UC model,the mice were given intragastric administration once a day for seven days.Mice in the mesalazine group were given mesalazine at the dosage of 0.31 g/kg;mice in the low-dose Xianglian Zhixie tablet group,medium-dose Xianglian Zhixie tablet group,and high-dose Xianglian Zhixie tablet group were given Xianglian Zhixie tablets at the dosage of 0.68,1.36,and 2.72 g/kg,respectively;mice in the blank control group and the model group were given an equal amount of purified water.During the administration,the disease activity index(DAI)scores were performed daily.At the end of last administration,the body mass was weighted.After anesthesia,the mouse's abdominal cavity was opened,the spleen was separated and weighed,and the spleen index was calculated.The colon was separated,and the length of the entire colon segment was measured.The part of colon tissues was taken and stained by peri-odic acid-schiff.The pathological changes of the colon were observed,and the number of goblet cells of the colonic mucosa were counted by periodic acid-schiff(PAS)staining.The levels of interleukin(IL)-1βand tumor necrosis factor-α(TNF-α)in colon were detected by ELISA.The protein expression levels of interferon(IFN)-γand IL-4 in colon were detected by Western blotting.The proportions of Th1 and Th2 cells in the plasma was detected by flow cytometry,and Th1/Th2 was cal-culated.Results Compared with the blank control group,DAI score,spleen index,the levels of IL-1βand TNF-α,the protein expression level of IFN-γ,whole blood Th1 cell ratio,and Th1/Th2 significantly increased,the number of goblet cells of the colonic mucosa decreased,the arrangement was irregular,the glycogen content,colon length,protein expres-sion level of IL-4,and whole blood Th2 cell ratio significantly decreased in the model group(all P<0.05).Compared with the model group,DAI score,spleen index,the levels of IL-1βand TNF-α,the proportion of whole blood Th1 cells,and Th1/Th2 significantly decreased,the number of goblet cells of the colonic mucosa significantly increased,the morphology was intact,and the glycogen content tended to be normal,the shortening of colon length was significantly inhibited,and the protein expression level of IL-4 was significantly up-regulated in the Xianglian Zhixie tablet groups and mesalazine group(all P<0.05).Conclusions Different dosages of Xianglian Zhixie tablet have a certain therapeutic effect on oxazolone-induced UC in mice,and the effect of high dose(twice the clinical equivalent dose)of Xianglian Zhixie tablet group is bet-ter.Regulating the Th1/Th2 immune axis,thereby reducing intestinal inflammation,may be one of the mechanisms of action of Xianglian Zhixie tablets.