The Effects Of Sinomenine On TLRs And Core Regulatory Gene Of JAk-STAT Signaling Pathway On Rheumatoid Arthritis Model Rats

Objective:1. To confirm the effectiveness of Sinomenine (SN) on AIA model rats.2. To explore the target and immune regulatory mechanism of SN in treating rheumatoid arthritis.Methods:1. The AIA rats model were established by injecting FCA prepared with BCG in SD rats, then administrated by gavage with SN (the reagent) and Methotrexate (MTX, the positive control medicinal) for3weeks. The general condition, body weight and articular swelling were observed. All rats were killed after administration, and then blood routine test, blood biochemistry, joint histopathological examination, IL-1, IL-6, and TGF-β were tested to verify effectiveness of AIA.2. The expression location and level of TLR2、TLR3、TLR4were tested by immunohistochemistry to explore immune regulatory mechanism of SN on articular tissues of AIA model rats.3. The target and immune regulatory mechanism of SN was explored by testing total gene expression of PBMC and analyzing key signaling pathway JAK-STAT and core regulatory gene with single channel expression profile chip.Results:1. Evaluation on effectiveness of SN in anti-AIASN could improve foodintake, activity, hairs and weight, and down-regulate CPR to relieve swelling of joints. SN could inhibit proinflammatory cytokines IL-1and IL-6, while promote TGF-β, cytokine inhibiting inflammation. Low-dose and medial-dose SN relieved hyperplasia of synovial pannus and destruction of articular cartilage.2. Effect of SN on TLR2, TLR3, and TLR4Low-dose and medial-dose SN significantly decreased expression of TLR2, TLR3and TLR4in hyperplastic synovial cells, neonatal vascular endothelial cells, DC (dendritic cells) and chondrocytes.3. Regulating action of SN on PBMC gene expression of AIA model rats SN can up-regulated645genes and down-regulated202genes. The differential expressed genes mainly distributed in signal pathways network consist of12associated signal transduction pathways participating in immune regulation and apoptosis regulation.JAK-STAT and TLRs signaling transduction pathways were the core in the network, especially the JAK-STAT. SN could regulate the pathway by functioning on genes of some cytokines and their receptors, signal transduction and transcription factors, cell cycle, anti-apoptosis and ubiquitin. SN could significantly down-regulate mRNA expression of AP-1, P38(MAPK11), IL-17and CSF2which are close related with JAK-STAT and TLRs signaling transduction pathways.Conclusions:1. SN has exact effect on AIA model rats by inhibiting IL-1and IL-6, the proinflammatory cytokines, and promoting TGF-β, the inflammation inhibiting cytokine, and relieving hyperplasia of synovial pannus and destruction of articular cartilage.2. The possible target and mechanism of SN is to control inflammatory process by affecting signal pathways network with TLRs/JAK-STAT signaling transduction pathway at the core and their regulatory genes (AP-1, P38), and regulate the expression of inflammatory cytokines.3.The action mechanism of SN manifests as nolinear and interacting three-dimensional network, which is in accordance with basic TCM theories of mutual rooting of yin and yang, waxing and waning of yin and yang, generation cycle, restriction cycle, over-restriction and counter-restriction of the five phases, and the treatment concept of harmonizing yin and yang to achieve balance.