Chen Huan, Ju Shao-Hua, Wei Jiang-Ping, Fu Wen-Jun, Zheng Hang, Xu Shi-Jun
Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China.
Key Laboratory of Systematic Research and Exploitation of Traditional Chinese Medicine Resources in Sichuan Province, Chengdu 611137, China.
Zhongguo Zhong Yao Za Zhi. 2017 Jan;42(2):332-340. doi: 10.19540/j.cnki.cjcmm.20161222.013.
To investigate the characteristic methylation genes of pyretic arthralgia model in hot and dampness environment and the regulation effect of Baihu Guizhi decoction on this characteristic methylation genes. Plantar injection of CFA was used in hot and dampness environment to induce the pyretic arthralgia rat models. From 15th day after modeling, Baihu Guizhi decoction was given for 30 days. Foot volume was detected every 4 days after modeling, and HE staining was used to detect the histopathology of all rats' ankle joint at day 45.MeDIP-Seq sequencing method was used to detect the methylation level of knee joint synovial, and the method of difference sets was used to screen the characteristic methylation genesinpyretic arthralgia models.The contents of IL-1β, IL-17, TNF-α, EGF, IL-12p70, IL-4, IL-6 and IFN-γ in serum were measured by using suspension chips. The mRNA expression level of characteristic methylation genes was measured by qRT-PCR. The results suggested that as compared with adjuvant arthritis rat models(AA), the foot swelling and histopathology inpyretic arthralgia models (PA) were only slightly increased. As compared with normal group (NG), the wholegenome CpG island in both AA and PA groups was kept in a lower methylation state, furthermore, the methylation level was lowest in PA group; with 705 difference methylation genes in AA group and 2 418 difference methylation genes in PA group. As compared with AA, there were 1 287 difference methylation genes, including 974 down-regulated methylation genesand 313 up-regulated methylation genes. This difference methylation genes were mostly enriched in 32 KEGG pathways. Moreover, there were 52 characteristic methylation genes of PA models in promoter region, including 36 down-regulatedmethylation genes and 16 up-regulatedmethylation genes. After drug intervention, Baihu Guizhi decoction improved the foot swelling and pathological injury in PA models, significantly decreased the levels of IL-1β, TNF-α, EGF, VEGF, IL-17, IL-12p70, inhibited the mRNA expression levels of down-regulated methylation genes AHCY and RPL3, and promoted the mRNA expression levels of up-regulated methylation gene Agxt. In conclusion, unique methylation changes of synovial genes were present in PA models, and Baihu Guizhi decoction may adjust the methylation level of PA's characteristic methylation genes to achieve the therapeutic effect of pyretic arthralgia.
为研究湿热环境下热痹模型的特征性甲基化基因以及白虎桂枝汤对该特征性甲基化基因的调控作用。采用在湿热环境下足底注射弗氏完全佐剂（CFA）诱导热痹大鼠模型。造模后第15天起给予白虎桂枝汤灌胃30天。造模后每4天检测足容积，造模后第45天取所有大鼠踝关节进行苏木精-伊红（HE）染色检测组织病理学变化。采用甲基化DNA免疫沉淀测序（MeDIP-Seq）技术检测膝关节滑膜组织的甲基化水平，运用差异集方法筛选热痹模型中的特征性甲基化基因。采用悬浮芯片检测血清中白细胞介素-1β（IL-1β）、白细胞介素-17（IL-17）、肿瘤坏死因子-α（TNF-α）、表皮生长因子（EGF）、白细胞介素-12p70、白细胞介素-4（IL-4）、白细胞介素-6（IL-6）和干扰素-γ（IFN-γ）的含量。采用实时荧光定量聚合酶链反应（qRT-PCR）检测特征性甲基化基因的mRNA表达水平。结果显示，与佐剂性关节炎大鼠模型（AA）相比，热痹模型（PA）的足肿胀及组织病理学变化仅轻度增加。与正常组（NG）相比，AA组和PA组全基因组CpG岛均处于较低甲基化状态，且PA组甲基化水平最低；AA组有705个差异甲基化基因，PA组有2418个差异甲基化基因。与AA组相比，PA组有1287个差异甲基化基因，其中甲基化水平下调的基因有974个，上调的基因有313个。这些差异甲基化基因主要富集于32条京都基因与基因组百科全书（KEGG）通路。此外，PA模型启动子区有52个特征性甲基化基因，其中甲基化水平下调的基因有36个，上调的基因有16个。药物干预后，白虎桂枝汤改善了PA模型的足肿胀及病理损伤，显著降低了IL-1β、TNF-α、EGF、血管内皮生长因子（VEGF）、IL-17、IL-12p70的水平，抑制了甲基化水平下调基因腺苷高半胱氨酸酶（AHCY）和核糖体蛋白L3（RPL3）的mRNA表达水平，促进了甲基化水平上调基因丙氨酸-γ-酮戊二酸转氨酶（Agxt）的mRNA表达水平。综上所述，PA模型滑膜基因存在独特的甲基化变化，白虎桂枝汤可能通过调节PA特征性甲基化基因的甲基化水平而达到治疗热痹的作用。
