白虎加桂枝汤对热痹模型大鼠特征性甲基化基因表达的影响

Effect of Baihu Guizhi decoction on characteristic methylation genes expression of pyretic arthralgia rat model

考察湿热环境下热痹模型大鼠特征性甲基化基因及白虎加桂枝汤对热痹特征性甲基化基因的调控作用。采用大鼠足底注射CFA并复合湿热环境制备热痹大鼠模型。造模后第15天给予白虎加桂枝汤干预治疗30 d。造模后每隔4 d检测足容积,第45天采用HE染色法检测大鼠踝关节组织病理学,Me DIP-Seq测序法检测大鼠膝关节滑膜甲基化水平并以取差集的方法筛选热痹模型特征性甲基化基因,悬浮芯片检测血清中IL-1β,IL-17,TNF-α,EGF,IL-12p70,IL-4,IL-6及IFN-γ含量水平,qRT-PCR检测大鼠滑膜特征性甲基化基因mRNA相对表达水平。实验发现,与完全弗氏佐剂性关节炎(AA)模型组比较,热痹模型组足肿胀度和病理严重程度仅轻微增加;与空白组比较,2个模型组全基因Cp G岛均呈低甲基化状态,热痹模型组甲基化水平最低,且AA模型组共705个差异性甲基化基因,热痹模型组共2 418个差异性甲基化基因;与AA模型组比较,热痹模型组存在1 287个差异性甲基化基因,其中共974个差异性基因甲基化下调,这些差异性基因显著分布于32个KEGG Pathway中(P<0.05),此外启动子区上热痹特征性甲基化基因共52个,其中36个甲基化下调基因,16个甲基化上调基因;药物干预后,白虎加桂枝汤显著改善热痹模型组足肿胀度和病理损伤,且明显降低IL-1β,TNF-α,EGF,VEGF,IL-17,IL-12p70的含量水平(P<0.05),还能抑制热痹特征性甲基化下调基因Ahcy及Rpl3 mRNA的表达,促进热痹特征性甲基化上调基因Agxt mRNA的表达水平。可见,热痹模型滑膜基因存在独特的甲基化水平变化,而白虎加桂枝汤可能针对性回调热痹特征性基因的甲基化水平,达到治疗热痹的作用。

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 15 thday 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. Me DIP-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-1 β,IL-17,TNF-α,EGF,IL-12 p70,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 Cp G 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-1 β,TNF-α,EGF,VEGF,IL-17,IL-12 p70,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.