清肠化湿方通过影响IDO1调节Th17/Treg细胞因子表达平衡治疗溃疡性结肠炎

Qingchang Huashi Formula Treating UC by Influencing IDO1 in Regulating Th17/Treg Balance

目的:采用葡聚糖硫酸钠(dextran sulfate sodium,DSS)建立小鼠溃疡性结肠炎模型,观察清肠化湿方对肠炎小鼠的治疗作用,并探讨清肠化湿方通过吲哚胺2,3-双加氧酶1(IDO1)调节溃疡性结肠炎小鼠辅助性T细胞17(Th17)/调节性T细胞(Treg)细胞因子表达平衡的相关机制。方法:将24只健康的SPF级雄性C57BL6/J小鼠随机分为空白组、模型组、清肠化湿方低剂量组[12 g(/kg·d)]和清肠化湿方高剂量组[24 g(/kg·d)]。第1~13天,空白组自由饮用纯水,模型组小鼠给予纯水灌胃,清肠化湿方低剂量组及高剂量组给予中药灌胃。实验第4天,模型组、清肠化湿方低剂量组及高剂量组小鼠给与2.5%DSS自由饮用,每日观察小鼠一般情况,记录小鼠体质量,粪便性状及便血情况,计算疾病活动指数(DAI)评分。采用苏木素-伊红(HE)染色观察结肠组织病理学改变。实时荧光定量(RT-qPCR)法检测结肠组织RORγt、IL-17、IL-22、IL-23、Foxp3、IL-10、TGF-β、IDO1 mRNA表达水平;蛋白免疫印迹(Western Blot)检测结肠组织RORγt、Foxp3、IDO1蛋白表达。结果:清肠化湿方可有效治疗UC小鼠,降低小鼠DAI评分,增加结肠长度,改善病理损伤。qPCR结果显示,与模型组相比,清肠化湿方可有效降低UC小鼠结肠组织RORγt、IL-17、IL-22、IL-23 mRNA及IDO1 mRNA表达水平,促进Foxp3 mRNA表达,升高IL-10、TGF-β mRNA表达。Western Blot结果显示,UC小鼠给与清肠化湿方干预后,低剂量组RORγt、IDO1蛋白表达降低,Foxp3蛋白表达升高;高剂量组RORγt、IDO1蛋白表达降低,Foxp3蛋白表达显著升高。结论:清肠化湿方能够有效治疗溃疡性结肠炎,高剂量组治疗效果更明显,其机制可能与下调IDO1表达,调节Th17/Treg细胞因子表达平衡相关。

Objective:To observe the therapeutic effect of Qingchang Huashi Formula in treating mice model of ulcerative colitis(UC)induced by dextran sulfate sodium(DSS),and to explore the related mechanism of Qingchang Huashi Formula in regulating the cytokine expression balance of helper T cell 17(Th17)/regulatory T cell(Treg)through indoleamine 2,3-Dioxygenase 1(IDO1)in mice with UC.Methods:24 SPF male C57BL6/J mice were randomly divided into the normal group,the model group,the Qingchang Huashi Formula groups of low-dose(QCHS-L,12 g/kg·d)and high-dose(QCHS-H,24 g/kg·d).During the days of 1-13,the mice in the normal group drank pure water freely,the mice in the model group were gavaged with pure water,and the QCHS groups were treated with corresponding concentration of Qingchang Huashi Formula.On the 4th day of the experiment,the mice in the model group and the Qingchang Huashi Formula groups were given 2.5%DSS for free drinking.The general condition of the mice was observed daily,and the body weight,fecal features and hemafecia were recorded,and the disease activity index(DAI)scores was calculated.HE staining was used to observe histopathological changes of the colons in mice.RT-qPCR method was used to detect the mRNA expressions of RORγt,IL-17,IL-22,IL-23,Foxp3,IL-10,TGF-β and IDO1 in colon tissues;Western Blot method was applied to detect the protein expressions of RORγt,Foxp3 and IDO1 in colon tissues.Results:Qingchang Huashi Formula could effectively treat UC mice,reduce the DAI score,increase the length of colons and improve pathological damages.The qPCR results showed that Qingchang Huashi Formula could effectively reduce the mRNA expressions of RORγt,IL-17,IL-22,IL-23 and IDO1 in colon tissues of the UC mice,it could promote the mRNA expressions of Foxp3,IL-10 and TGF-β when compared to the model group.Western Blot results showed that after the intervention of Qingchang Huashi Formula,the protein expressions of IDO1 and RORγt were decreased,and the protein expression of Foxp3 was increased in the QCHS-L group and in the QCHS-H group.Conclusion:Qingchang Huashi Formula can effectively treat UC,and the high-dose group has a more obvious therapeutic effect.Its mechanism may be related to decreasing IDO1 expression and regulating the cytokine expression balance of Th17/Treg.