Dampness-hot Pattern Of Rheumatoid Arthritis In Traditional Chinese Medicine:the Mechanism Of DNA Methylation In Epigenetics And The Preliminary Validation For Anti-inflammation Of BaiHu-GuiZhi Decoction

ObjectiveTo research the of dampness-hot environmental factors on rheumatoid arthritis in DNA methylation and the effect of BaiHu-GuiZhi decoction, we would primary reveal the epigenetic mechanism of RA and the preliminary validation for anti-inflammation of BaiHu-GuiZhi decoction.MethodIn this study, our works mainly concentrate on the influence of dampness and hot environmental factors on RA. We choose adjuvant arthritis (AA) rats as subjects, set them in the environment of 37.5 Celsius and relative humidity 70%-80%. Ankle swelling, pathology, serum IL-6, TNF-α, RF and ACPA are observed as basal evaluations of the disease. Globe genome methylation data of RA synovial tissue have been measured by MeDIP-seq. In order to research the difference among normal, CFA rats and CFA rats with dampness-hot environmental factors, we have analyzed the basal evaluations and MeDIP-seq database. These three datasets reveal the environment and epigenetic contributing to the development of RA. Especially, dampness-hot environmental factors can aggravate pathology, and represent a different methylation status of RA. Through the analysis of the different expression of DNA methylation of RA, we have researched the swelling, pathology, serum(IL-6, TNF-α, IL-1β, IL-12P70, MCP-1, IP-10, VEGF) level, and reveal the anti-inflammatory mechanism of BaiHu-GuiZhi decoction from the perspective of cytokines.Results1.The influence of dampness-hot environmental factors on serum inflammatory factors levels and pathology of adjuvant arthritis ratsCompared with the control group, the paw swelling, pathology, serum IL-6, TNF-α, RF and ACPA levels of CFA group were significantly increased (P< 0.05). Compared with CFA group, the serum IL-6, TNF-a, RF and ACPA levels of CFA group with dampness-hot environmental factors (P< 0.05). The paw swelling and pathology have no significant, but aggravate the severity of RA.2.The study of distinct DNA methylation signatures in adjuvant arthritis rats with dampness-hot environmentalCompared with the control group, CFA group’s DNA methylation level have decreased significantly, and there are 4485 differentially expressed genes were significantly enriched in 17 pathways(P< 0.05), such as T cell receptor signaling pathway, B cell receptor signaling pathway, MAPK signaling pathway, Wnt signaling pathway, focal adhesion and primary immunodeficiency. And there are 52 significant genes such as Zap 70, PAK7, Tab1, Pla2g6, Map3kl4, Nfatc3, Fas etc. Compared with CFA group, the methylation level is slightly higher than that of CFA group, but significantly lower than the control group, there were 2721 differentially methylated gene (1380 down genes and 1341 up genes), and which are enriched in KEGG pathway (P＜0.05), including 10 common prediction such as calcium signal pathway transduction pathway, Wnt signaling pathway, adherens junction, vascular smooth muscle contraction, tight junction, leukocyte transendothelial migration, chemokine signaling pathway and MAPK signaling pathway,7 different prediction pathway such as dopamine synapses, transcription regulation, metabolism, cell adhesion molecules (CAMs),B cell receptor signaling pathway, adhesion and the NF-κB signaling pathway.15 genes like Ssx2ip, Tjapl, Vwf, Hgf, Collal, Col6a3, Plcb4, Atp1b4, Csnk2a2, Mpdz, Epb4112, Ddx58、Ripkl、Ifitm2 and IKKα are significantly enriched in these KEGG pathways.3.The influence of Baihu-Guizhi decoction on inflammatory-related cytokines of adjuvant arthritis rats with dampness-hot environmental factorsCompared with the control group, model group’s rats paw swelling, pathology, serum proinflammatory cytokines IL-6, TNF-α, IL-1β and IL-12P70, chemotactic factors MCP-1, IP-10 and growth factor VEGF levels have significantly increased (P＜0.05). Compared with the model group, Baihu-Guizhi decoction has significantly improved rat paw swelling, pathology, serum proinflammatory cytokine IL-6, TNF-a, IL-lp and IL-12P70, chemotactic factors MCP-1, IP-10 and growth factor VEGF levels (P< 0.05).But IL-4, IL-17A, IFNr, EGF of the three groups have not been detected.Conclusion1.Through the difference between CFA group and CFA group with dampness-hot environmental factors’ paw swelling, pathology and serum cytokines, we have found the dampness-hot environmental factors can increase the joint synovial tissue lesions of RA rats. And it has demonstrated the importance of dampness-hot environmental factors for the pathogenesis of diseases.2.1n this study, we have analyzed the basal evaluations (paw swelling, pathology and serum cytokines) and MeDIP-seq database. These three datasets reveal the environment and epigenetic contributing to the development of RA. Especially, dampness-hot environmental factors can aggravate pathology, and represent a different methylation status of RA. The CpG island methylation level of is low. There are 1380 down and 1341 up genes. These genes are enriched in biological adhesion, immune system processes, cell, cell proliferation and killing of 27 biological processes. And also significantly enriched in the adhesion, calcium signaling pathway, Wnt signaling pathway, adherens junction, vascular smooth muscle contraction, tight junction, leukocyte transendothelial migration, chemokine signaling pathway, MAPK signaling pathway, B cell receptor signaling pathway, transcriptional misregulation of dopamine synapses, cancer, metabolic pathway, cell adhesion molecules (CAMs),and the latter 7 pathways are specific expression.Finally,15 molecular signatures like Ssx2ip, Tjap1, Vwf, Hgf, Colla1, Col6a3, Plcb4, Atp1bM, Csnk2a2, Mpdz, Epb4112, Ddx58、Ripk1、Ifitm2 and IKKa have been identified from RA rats with dampness-hot environmental factors.3.Through the improvement of rats paw swelling, pathology, serum proinflammatory cytokines IL-6, TNF-α, IL-1β and IL-12P70, chemotactic factors MCP-1, IP-10 and growth factor VEGF levels by Baihu-Guizhi decoction, we think Baihu-Guizhi decoction has good anti-inflammatory effect,which may influence the inflammatory medium and inhibit the pannus of RA.