Th17/Treg细胞在健脾方防治克罗恩大鼠中的作用机制

摘要

目的:观察Th17/Treg细胞平衡在健脾方防治TNBS诱导的克罗恩大鼠中的作用机制;方法:24只清洁级雄性SD大鼠随机分为:1)正常对照组NG(n =8):从造模第2周起双蒸水灌胃,1次/d,灌胃量按1 mL/100 9,共3周;2)TNBS模型组MG(n =8):从第1周开始TNBS灌肠,灌肠剂量(mL)=体重(g) ×0.003 mL/g,1次/周,造模持续共4周;3)模型+健脾方防治组JP(n =8):从造模第2周开始,在TNBS灌肠后的第2天新增中药健脾方灌胃治疗,灌胃量按1 mL/100 9,1次/d,灌胃持续3周;采用ELISA、免疫组织化学、real-time PCR等技术观察各组大鼠血液、结肠黏膜中与Th17、Treg分化和功能密切相关的IFN-γ、IL-17、RORγt、FoxP3蛋白与基因的表达差异;结果:健脾方能通过下调Th1细胞释放的IFN-γ,进而来调节Th1/Th2的平衡,同时也能通过下调Th17细胞分泌IL-17、RORγt炎性反应因子的分泌和促进Treg细胞分泌的抗炎因子FoxP3来调节Th17/Treg的平衡,进而达到防治TNBS诱导的大鼠克罗恩病的作用.结论:健脾方能够通过调节Th 17/Treg细胞平衡中多个关键细胞因子和转录因子的方式,达到预防和治疗TNBS诱导的大鼠克罗恩病炎性反应程度之目的.%Objective:To observe the mechanism of TH17/Treg cells in the prevention and treatment for trinitrobenzene sulfonic acid (TNBS)-induced rats with Crohn's disease using a spleen-invigorating prescription (SIP).Methods:A total of 24 SPF grade male Sprague-Dawley (SD) rats were randomly divided into 3 groups:(1) the normal rat control group (NG;n =8) received intragastrically administered double-distilled water once daily at 1 mL/100 g for 3 weeks;(2) the TNBS model group (MG;n =8) received enemas with TNBS once weekly at a dosage of body weight (g) × 0.003 ml/g for 4 weeks;and (3) the TNBS model + SIP and treatment group (JP;n =8) was treated with an additional Chinese SIP 1 week after undergoing TNBS enema.The Chinese medicine was administrated intragastrically once daily for another 3 weeks at a dosage of 1 mL/100 g.Multiple techniques,including enzyme-linked immunosorbent assay(ELISA),immunohistochemistry,and real-time PCR,were used to observe the differential expression of particular genes and proteins in blood and colonic mucosa of rats which belong to different groups.This study focused on genes and proteins that are highly associated with Thl7 and Treg cell differentiation and function,including interferon (INF)-γ,IL-17,retinoid-related orphan nuclear receptor (ROR) γt,and FoxP3.Results:SIP effectively adjusted Th1/Th2 homeostasis by downregulating the Th1-secreted cytokines INF-γ.By downregulating Thl7-secreted inflammatory factors,IL-17 as well as RORγt and suppressing the secretion of FoxP3,SIP also promoted Th17/Treg homeostasis.Then,the symptoms of TNBS-induced rat colonitis were successfully prevented and treated.Conclusion:SIP prevented and treated inflammation associated with TNBS-induced rat with Crohn's disease by regulating multiple key cytokines and transcription factors related to Th1/Th2 and Th17/Treg cell homeostasis.