目的考察白虎加桂枝汤对热痹特征性甲基化基因的影响,揭示其治疗热痹的表观遗传学机制。方法采用佐剂性关节炎模型(AA)复合湿热环境(温度37℃、相对湿度70%~80%,2 h/d)制备热痹模型(HB)。第15 d后,连续灌胃给与白虎加桂枝汤(28,14和7 g·kg-1)30 d后取空白组(NG)、AA及HB大鼠膝关节滑膜,采用Me DIP-Seq测序检测滑膜DNA甲基化水平,以取NG vs HB和NG vs AA差集的方式筛选热痹特征性甲基化基因,并采用q RT-PCR检测特征性甲基化基因的表达水平。结果 NG vs AA,NG vs HB及NG vs HB各有差异性甲基化基因分别为705,2418及1287个,这些甲基化差异性基因主要与炎症免疫通路,如T、B细胞受体信号通路、MAPK信号通路及抗原提呈和处理等有关,生物合成与代谢通路,如多种氨基酸代谢途径及糖类、蛋白质代谢途径等有关;热痹特征性甲基化基因共42个,与AA比较,白虎加桂枝汤高、中、低各剂量组均能抑制热痹特征性甲基化下调基因Ahcy(0.57±0.12、0.58±0.32、0.73±0.12)及Rpl3(0.65±0.24,0.65±0.24,0.62±0.30)的表达,而仅高、中剂量组促进热痹特征性甲基化上调基因Agxt(1.35±0.20,1.15±0.23)的表达水平。结论特征性基因甲基化水平变化可能是导致热痹发病的原因,白虎加桂枝汤可通过纠正特征性基因的甲基化水平达到治疗疾病的目的。
OBJECTIVE: To investigate the mechanism underlying anti-inflammatory and immunoregulatory effect of total glucosides of paeony(TGP) based on toll-like receptor 2(TLR2) mediated tumor necrosis factor(TNF) receptor-associated factor 6(TRAF6)/nuclear factor-kappa B(NF-κB) pathway activation in rats with rheumatoid arthritis.METHODS: Adjuvant arthritis(AA) model was developed by complete freund’s adjuvant(CFA) immunization. TGP(100, 50, 25 mg/kg) and celecoxib(2.8 mg/kg) were administered by intragastric administration for 21 d. Right hind paw swelling was assessed every 2 d. After 21 d, synovial changes of the ankle were detected by histopathology. CD4+and CD8+ T cell amounts in peripheral blood were measured by flow-cytometrically. Gene and protein levels of toll-like receptor(TLR)2, TRAF6, tumor necrosis factor ligand superfamily member 6(FASLG)in the spleen were assessed by RT-qPCR and Western Bolt, respectively. Nuclear expression of NF-κB p65 was detected by NF-κB p65 Assay Kit.RESULTS: Paw swelling and synovium lesions were obviously aggravated in AA rats. These symptoms were significantly relieved by TGP.The ratio of CD4+/CD8+ T cell was increased in AA rats, while TGP reduced this increased ratio.Gene and protein levels of splenic TLR2, TFAR6 and FASLG, and nuclear NF-κB p65 in AA rats were significantly increased, but overtly inhibited by TGP.CONCLUSION: These findings suggest that TGP’s anti-inflammatory effect onRA in rats with CFA may be related to the downregulation of TLR2/TRAF6/NF-κB pathway and the regulation of T cell subsets.
