中药苦参水提取物抑制Th17细胞分化的研究  

Inhibition Effect of Sophora flacescens Aqueous Extract on Th17 Differentiation

作　　者：张丽华[1] 陈燕[2] 彭雨舒 周宝珠 危荧靖 黄小玲 郭嘉亮[1,3] ZHANG Lihua;CHEN Yan;PENG Yushu;ZHOU Baozhu;WEI Yingjing;HUANG Xiaoling;GUO Jialiang(Foshan University,Foshan 528000 Guangdong,China;Guangdong No.2 Provincial People's Hospital,Guangzhou 510317 Guangdong,China;Jinan University,Guangzhou 510632 Guangdong,China)

机构地区：[1]佛山科学技术学院,广东佛山528000 [2]广东省第二人民医院,广东广州510317 [3]暨南大学药学院,广东广州510632

出　　处：《中药新药与临床药理》2020年第3期251-256,共6页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：国家自然科学基金项目(81503031,81872832);广东省基础与应用基础研究基金自然科学基金项目(2019A1515010806)。

摘　　要：目的探讨苦参水提取物(Sophora flavescens aqueous extract,SFAE)对Th17细胞分化的影响。方法通过体外分离豚鼠淋巴结获取原代淋巴细胞,采用磁珠分选技术获得初始CD4^+T细胞。采用CCK-8法筛选SFAE对T细胞活力的最佳抑制浓度。通过白细胞介素-6(IL-6)和转化生长因子-β(TGF-β)诱导建立初始CD4^+T细胞向Th17细胞分化的细胞模型,并以SFAE进行干预;采用流式细胞仪检测各组Th17细胞比例,采用qRT-PCR及Western Blot法检测各组细胞中视黄酸相关的孤儿受体γt(RORγt)、信号转导子和转录激活子3(STAT3)、白细胞介素-17A(IL-17A)和白细胞介素-17F(IL-17F)的mRNA及蛋白表达水平。结果与对照组比较,SFAE浓度在0.01 mg·mL^-1时对初始CD4^+T细胞的活力有抑制作用(P<0.01),选择该浓度作为后续实验浓度。与对照组比较,模型组能明显上调CD4^+IL-17A+T细胞的比例(P<0.001),上调RORγt、STAT3、IL-17A、IL-17F mRNA及蛋白的表达(P<0.01,P<0.001)。与模型组比较,SFAE干预后模型处理组的CD4^+IL-17A+T细胞比例明显下调(P<0.05),STAT3、RORγt、IL-17A、IL-17F mRNA及蛋白表达水平均明显下调(P<0.05,P<0.01)。结论SFAE能够抑制Th17细胞分化,降低Th17细胞比例,其作用机制可能是通过抑制转录因子RORγt及STAT3的活性,降低IL-17A、IL-17F的表达。Objective To explore the effects of extract from Chinese medicine Sophora flavescens(SF)on differentiation of Th17(CD4^+IL-17^+T),and provide scientific basis for SF in clinical treatment of Th17 relevant autoimmune disease.Methods The naive T cells were isolated from peripheral lymph nodes with immunomagnetic beads from guinea pig.The CCK-8 assay was used to evaluate the optimum concentration of Sophora flavescens aqueous extract(SFAE)for treatment on naive T cells.Subsequently,the Th17(CD4^+IL-17^+T)cell model was induced by interleukin 6(IL-6)and transforming growth factor beta(TGF-β)in vitro.Then all groups of cells were present or absent with the SFAE in culture mediums.The percentage of CD4^+IL-17^+T cells in different groups was analyzed by flow cytometry.Real-time polymerase chain reaction(qRT-PCR)and Western Blot were used to measure the expression of RAR-related orphan receptor gamma(RORγt),signal transducer and activator of transcription 3(STAT3),interleukin 17 A(IL-17 A)and IL-17 F.Results Compared with the blank control group,the 0.01 mg·mL-1SFAE treatment significantly inhibit the viability of naive CD4^+T cells.Flow cytometry detection showed that the model group of naive T cells treated simultaneously by IL-6 and TGF-βcould increase percentage of CD4^+IL-17^+T cells compare with control(P<0.001).However,the percentage of CD4^+IL-17^+T cells in SF+Model group was significantly declined(P<0.05).Compared with model group,qRT-PCR and Western Blot results showed that the mRNA levels of STAT3,RORγt,IL-17 A and IL-17 F were obviously reduced in SFAE groups(P<0.05,P<0.01),and the expression of STAT3,RORγt,IL-17 A and IL-17 F proteins were also obviously inhibited(P<0.05).Conclusion SFAE not only reduced the ratio of CD4^+T cell differentiating into Th17,but also decreased the inflammatory factor production by inhibiting the transcription factors STAT3 and RORγt.

关 键 词：苦参水提取物 TH17细胞 分化 视黄酸相关的孤儿受体γt 信号转导子和转录激活子3 白细胞介素-17A 白细胞介素-17F 

分 类 号：R285.5[医药卫生—中药学]