大黄素对重症急性胰腺炎小鼠Treg/Th17平衡的影响及机制研究

Effect of emodin on regulatory T cell/T helper 17 cell balance in mice with severe acute pancreatitis and related mechanism

目的:探讨大黄素对重症急性胰腺炎小鼠 Treg/Th17 平衡的影响及可能的机制。方法:将 60 只 C57BL/6 小鼠按照随机数表法分为 3 组:正常对照组(n=20)、模型对照组(n=20)和药物组(n=20),采用雨蛙素联合脂多糖法制备重症急性胰腺炎小鼠模型。模型构建成功后,药物组腹腔注射 2.5 mg/(kg·d)大黄素,模型对照组和正常对照组均给予同等剂量的生理盐水。给药后48 h,将小鼠麻醉后处死。 ELISA 法检测血清中淀粉酶、脂肪酶、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、二胺氧化酶(diamine oxidase,DAO)和白细胞介素-10(interleukin-10,IL-10)水平;流式细胞术法检测脾脏组织中调节性 T 细胞(regulatoryT-cell,Treg)及辅助性 Th17 细胞(T helper cell 17,Th17)比例;q-PCR 法检测脾脏组织叉头状/翼状螺旋转录因子 3(forkhead orwinged helix transcription,FOXP3)、视黄酸相关的孤儿受体γt(retinoid acid related orphan receptor gamma t,ROR γt)和 Toll 样受体 4(Toll-like receptor 4,TLR4)mRNA 的表达。结果:与正常对照组比较,模型对照组血清脂肪酶、血清淀粉酶、TNF-α、DAO、IL-10 水平及 Foxp3、RORγt 和 TLR4 mRNA 表达量明显升高;Treg 及 Th17 细胞数均升高,但 Th17 细胞上升比例高,Treg/Th17降低。与模型对照组比较,药物组血清脂肪酶、血清淀粉酶、TNF-α、DAO、IL-10 水平及 Foxp3、RORγt 和 TLR4 mRNA 表达量明显降低。 Treg 及 Th17 细胞数均有所降低,Treg/Th17 升高,均具有统计学意义(P<0.05)。结论:大黄素可明显降低 TNF-α、DAO和 IL-10 的表达,缓解炎症应激和氧化应激反应,同时可调节 Treg/Th17 平衡,改善免疫紊乱,从而发挥治疗重症急性胰腺炎的作用。

Objective:To investigate the effect of emodin on regulatory T cell(Treg)/T helper 17 cell(Th17) balance in mice with se- vere acute pancreatitis and the possible mechanism. Methods:A total of 60 C57BL/6 mice were divided into normal control group, model control group,and drug group using a random number table,with 20 mice in each group. Cerulein combined with lipopolysac- charide was used to establish a mouse model of severe acute pancreatitis. After the model was established successfully ,the mice in the drug group were given intraperitoneally injected with emodin at a dose of 2.5 mg/kg/day,and those in the model control group and the normal control group were given normal saline at the same dose. The mice were sacrificed after anesthesia at 48 hours after ad- ministration. ELISA was used to measure the serum levels of amylase,lipase,tumor necrosis factor-α(TNF-α),diamine oxidase (DAO),and interleukin-10(IL-10);flow cytometry was used to measure the percentages of Treg cells and Th17 cells in the spleen;q-PCR was used to measure the mRNA expression of forkhead or winged helix transcription factor (Foxp3),retinoid acid-related or- phan receptor gamma t(RORγt),and Toll-like receptor 4(TLR4) in the spleen. Results:Compared with the normal control group,the model control group had significant increases in the serum levels of lipase,amylase,TNF-α,DAO,and IL-10,the mRNA expression of Foxp3,RORγt,and TLR4,and the number of Treg and Th17 cells,as well as a significantly greater increase in the percentage of Th17 cells and a significant reduction in Treg/Th17 ratio(all P< 0.05). Compared with the model control group,the drug group had significant reductions in the serum levels of lipase,amy- lase,TNF-α,DAO,and IL-10,the mRNA expression of Foxp3,RORγt,and TLR4,and the number of Treg and Th17 cells and a significant increase in Treg/Th17 ratio(all P<0.05). Conclusion: Emodin can significantly reduce the expression of TNF -α,DAO,and IL -10 and relieve inflammatory stress and oxidative stress response. It can also regulate Treg/Th17 balance,improve immune disorders,and thus play a role in the treatment of severe acute pancreatitis.