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

发布时间：2018-03-14 18:33

本文选题：HDND-XIV　切入点：SD大鼠　出处：《中国药理学通报》2017年06期 　论文类型：期刊论文

【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。
[Abstract]:Objective to study the effects of 5-chlorophenethyl) amino-7-oxy-acetyl-4-chlorobenzeneethylaminyl hesperidin. The effect and molecular mechanism of hesperidin derivative-XIV-HDND-XIV) on the inflammation of rat synovial fibroblast cells with adjuvant adjuvant arthritis AAA and its molecular mechanism. Methods Freunds complete adjuvant FCA (Freunds complete adjuvant FCA) was used to induce Sprague Dawley SD) rat model FCA 0.01 ml 路kg ~ (-1) and 24 days after establishment of the model, the rat model of FCA0.01 ml 路kg ~ (-1) was induced by Freunds complete adjuvant (Freunds complete adjuvant FCA). Detection of half maximal inhibitory concentration of HDND-XIV and enzyme linked immunosorbent assay-ELISA-MEASURMENT of interleukin-6 interleukin-6 (IL-6), tumor necrosis factor- 伪 necrosis factor-TNF- 伪 (TNF- 伪) and interleukin-1 尾 interleukin-1 尾 interleukin-1 尾 IL-1 尾 (interleukin-1 尾 -interleukin-1 尾 -interleukin-1 尾 -IL-1 尾) in cell culture medium for transfection of Lipofectamine2000 by Lipofectamine2000, the levels of interleukin-6 interleukin-6 (IL-6), tumor necrosis factor- 伪 necrosis factor-TNF- 伪 (TNF- 伪) and interleukin-1 尾 -interleukin-1 尾 -interleukin-1 尾 (IL-1 尾) were detected by Elisa. The overexpression plasmid GV230-MeCP2 was transfected into AA FLSG-Western blot assay to detect the expression of Gli1hprotein. Results the value of HDND-XIV on FLS by MTT assay was 161 渭 mol 路L ~ (-1). The best anti-inflammatory concentration of HDND-XIV was 10 渭 mol 路L ~ (-1) PTCH1 blot. The results showed that the expression of PTCH1 in AA FLS increased after HDND-XIV stimulation and the expression of Shh was decreased. The results of pyrosequencing showed that the degree of methylation of PTCH1 gene in AA FLS was significantly decreased after stimulation by HDND-XIV, and the expression of MeCP2 in AA FLS was decreased after MeCP2 was overexpressed by HDND-14 stimulated by HDND-14. The results of Elisa showed that the level of IL-6 and IL-1 尾 -TNF- 伪 was also increased. Conclusion HDND-XIV has obvious anti-inflammatory activity on FLS, and its mechanism may be related to inhibiting the expression of MeCP2 and reducing the degree of PTCH1 gene methylation.
【作者单位】：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;抗炎免疫药物教育部重点实验室;安徽医科大学肝病研究所;
【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)
【分类号】：R965

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