芍药汤通过抑制HIF-1α调节Th17/Treg平衡治疗溃疡性结肠炎

Shaoyaotang Treats Ulcerative Colitis by Inhibiting HIF-1α and Regulating Th17/Treg Balance

目的:探讨缺氧诱导因子-1α(HIF-1α)在溃疡性结肠炎辅助性T细胞17(Th17)/调节性T细胞(Treg)平衡中的作用及芍药汤的干预机制。方法:将48只SD大鼠随机分为正常组,模型组,美沙拉嗪组(0.42 g·kg^(-1)),芍药汤组(11.1 g·kg^(-1)),抑制剂组(2-甲氧基雌二醇,0.015 g·kg^(-1)),芍药汤+抑制剂组,采用2,4,6-三硝基苯磺酸(TNBS)诱导溃疡性结肠炎模型,各组分别对应给予生理盐水,美沙拉嗪,芍药汤,抑制剂及芍药汤+抑制剂治疗7 d,观察各组大鼠一般情况和疾病活动指数,采用苏木素-伊红(HE)染色观察结肠组织病理学改变,酶联免疫吸附测定(ELISA)法检测血清中白细胞介素-10(IL-10),IL-17,IL-23水平;蛋白免疫印迹法(Western blot)检测结肠组织叉头状/翼状螺旋转录因子P3(FoxP3),维甲酸相关孤儿受体(RORγt),HIF-1α蛋白表达水平。结果:与正常组比较,模型组大鼠疾病活动指数(DAI)评分,肠黏膜病理学评分显著升高(P<0.01),血清中的IL-10水平显著降低(P<0.01),IL-17和IL-23显著升高(P<0.01),结肠RORγt及HIF-1α蛋白水平显著升高(P<0.01),FoxP3蛋白表达显著降低(P<0.01);与模型组比较,芍药汤组DAI评分,肠黏膜病理学评分降低(P<0.05,P<0.01),血清中IL-10水平升高(P<0.01),IL-17,IL-23降低(P<0.01),结肠RORγt及HIF-1α蛋白水平降低(P<0.01),FoxP3蛋白表达升高(P<0.01);与抑制剂组比较,芍药汤组及芍药汤+抑制剂组RORγt,FoxP3及HIF-1α蛋白表达差异有统计学意义(P<0.05,P<0.01)。结论:芍药汤可以有效治疗溃疡性结肠炎,其作用机制可能与抑制HIF-1α来调节Treg/Th17的重新平衡相关。

Objective: To explore the effect of hypoxia-inducible factor(HIF)-1α on T helper 17(Th17)/regulatory T cell(Treg)balance in ulcerative colitis and the intervention mechanism of Shaoyaotang.Method: Forty-eight SD rats were randomly divided into normal group(normal saline), model group,mesalazine group(0.42 g·kg^(-1)),Shaoyaotang group(11.1 g·kg^(-1)),inhibitor group [2-methoxyestradiol(2 ME2),0.015 g·kg^(-1)],and Shaoyaotang+inhibitor group. The ulcerative colitis model was induced by 2,4,6-trinitrobenzene sulfonic acid(TNBS). The rats in all groups received corresponding treatments for 7 d,and the general condition and disease activity index(DAI)were observed. Hematoxylin-eosin(HE)staining was used to observe histopathological changes of the colon. Enzyme-linked immunosorbent assay(ELISA)was employed to detect serum levels of interleukin(IL)-10,IL-17,and IL-23 in rats. Western blot was used to detect the expression levels of forkhead box protein 3(FoxP3),retinoic acid-related orphan receptor γt(RORγt),and HIF-1α proteins in the colon tissue. Result: Compared with the normal group,the model group showed elevated disease activity index(DAI)score and pathological score for intestinal mucosa(P<0.01),reduced serum IL-10 level(P<0.01),up-regulated IL-17 and IL-23 levels(P<0.01),increased RORγt and HIF-1αexpression(P<0.01),and decreased FoxP3 protein expression(P<0.01). Compared with the model group,the Shaoyaotang group displayed diminished DAI score and pathological score for intestinal mucosa(P<0.05,P<0.01),increased serum IL-10 level(P<0.01),decreased IL-17 and IL-23 levels(P<0.01),dwindled protein levels of RORγt and HIF-1α(P<0.01),and up-regulated expression of FoxP3(P<0.01). Compared with the inhibitor group,the Shaoyaotang group and the Shaoyaotang+inhibitor group exhibited significant differences in the expression of RORγt,FoxP3,and HIF-1α proteins(P<0.05,P<0.01). Conclusion: Shaoyaotang could effectively treat ulcerative colitis,and the underlying mechanism of action might be related to the regulation of Th17/Treg rebalance by inhibiting HIF-1α.