橙皮素衍生物-XIV调控PTCH1基因甲基化对佐剂性关节炎大鼠FLS炎症的影响  

Effect of Hesperidin derivative-XIV regulating methylation of PTCH1 gene on FLS inflammation of adjuvant arthritis rats

作　　者：孙正浩 刘彦会[1,2,3] 刘骏达[1,2,3] 徐丹丹[1,2,3] 李小枫[1,2,3] 张义龙[1,2,3] 孟晓明[1,2,3] 黄成[1,2,3] 李俊[1,2,3] 

机构地区：[1]安徽医科大学药学院,安徽省重大自身免疫性疾病重点实验室,安徽省创新药物产业共性技术研究院 [2]抗炎免疫药物教育部重点实验室 [3]安徽医科大学肝病研究所,安徽合肥230032

出　　处：《中国药理学通报》2017年第6期781-787,共7页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 81273526,81473268);安徽省教育厅重点基金项目(No KJ2016A364,KJ2016A365);安徽省自然科学基金面上项目(No21408085MKL31)

摘　　要：目的研究5-(4-氯苯乙基)亚胺基-7-氧-乙酰-4-氯苯乙胺基橙皮素,即橙皮素衍生物XIV号(hesperidin derivative-XIV,HDND-XIV)对佐剂性关节炎(adjuvant arthritis,AA)大鼠成纤维滑膜细胞(fibroblast-like synoviocytes,FLS)炎症的影响及分子机制。方法弗氏完全佐剂(Freund's complete adjuvant,FCA)诱导Sprague Dawley(SD)大鼠AA模型(FCA,0.01 ml·kg^(-1)),造模后24 d处理并提取原代FLS;MTT法检测HDND-XIV的半抑制浓度(half maximal inhibitory concentration,IC_(50));酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)测定细胞培养基中FLS分泌的白细胞介素-6(interleukin-6,IL-6),肿瘤坏死因子-α(tumor necrosis factor,TNF-α)和白介素-1β(interleukin-1β,IL-1β)水平;Lipofectamine2000转染试剂盒将过表达质粒GV230-MeCP2转染入AA FLS;Western blot法检测MeCP2、PTCH1、Gli1、Shh蛋白的表达;焦磷酸测序法定量检测各组PTCH1基因的甲基化程度。结果 MTT法检测HDND-XIV在FLS上的IC_(50)值为161μmol·L^(-1);ELISA筛选出HDND-XIV最佳抗炎浓度为10μmol·L^(-1);Western blot结果显示经HDND-XIV刺激后,AA FLS中PTCH1表达升高,MeCP2、Gli1和Shh表达下降;焦磷酸测序显示经HDND-XIV刺激后,AA FLS中PTCH1基因甲基化程度明显降低;在AA FLS中过表达MeCP2后加HDND-14刺激,Western blot结果显示成功过表达MeCP2后,PTCH1蛋白表达下降,而Gli1和Shh蛋白表达升高,ELISA结果显示IL-6、IL-1β、TNF-α水平也升高。结论 HDND-XIV对于FLS有较明显的抗炎活性,其机制可能与抑制MeCP2的表达,降低PTCH1基因甲基化程度有关。Aim To explore the effect of 5-( 4-Chlorophenethyl) imino-7-O-acetyl-4-chlorophenethylamine hesperetin( HDND-XIV) on fibroblast-like synoviocytes( FLS) inflammation in adjuvant arthritis( AA) rats and the underlying molecular mechanism. Methods AA rats were induced by Freund' s complete adjuvant( FCA,0. 01 ml·kg^(-1)),after 24 d,the rats were killed and the synovial tissue was used to extract primary cells; MTT was used to detect the half maximal inhibitory concentration( IC50) of HDND-XIV; Enzyme linked immunosorbent assay( ELISA) was used to detect the level of interleukin-6( IL-6),tumor necrosis factor-α( TNF-α),interleukin-1β( IL-1β) in cell culture. Lipo-fectamine2000 transfection kit was used to display the overexpression of MeCP2 in AA FLS.Western blot was used to detect the expression of MeCP2,PTCH1,Gli1 and Shh. Pyrosequencing was used to detect the methylation level of the PTCH1 gene quantificationally. Results The IC50 of HDND-XIV on FLS was 161 μmol·L^(-1) through MTT. The most significant anti-inflammatory concentration was 10 μmol·L^(-1)through ELISA. In AA FLS,the expression of PTCH1 was increased and MeCP2,Gli1,Shh were reduced in response to HDND-XIV. According to pyrosequencing,the methylation level of the PTCH1 gene was significantly decreased in response to HDND-XIV.Overexpression of MeCP2 in AA FLS followed by treating with HDND-14,the expression of PTCH1 was reduced,the expression of Gli1,Shh was increased and the level of IL-6,IL-1β,TNF-α rose. Conclusion HDND-XIV has prominent anti-inflammatory activity in FLS,the mechanism of which may be associated with inhibiting the expression of MeCP2 to reduce the methylation status of PTCH1 gene.

关 键 词：HDND-XIV SD大鼠 佐剂性关节炎 甲基化 HEDGEHOG 成纤维滑膜细胞 过表达 炎症 

分 类 号：R-332[医药卫生] R285.5