Mechanism Study Of Geniposidic Anti-acid Against Synovial Cells Inflammation In Rats With Adjuvant Arthritis And Its MAPK Signal Transduction Pathway

Objective: Rheumatoid arthritis(RA) is a multisystem chronic autoimmune disease mainly affecting peripheral joints, which is characterized by inflammatory changes in the synovial tissue and progressive destruction in articular cartilage. The disease exists throughout the world with occurrence in all races. Current medications are mainly used to control and relieve RA symptoms, and there are many adverse reactions, so it is an important direction for future research to find a new drug treatment of RA with efficiency and low toxicity. Gardenia medicine has significant anti-inflammatory and analgesic effects, gardenia extract and its monomer geniposidic RA acid significantly inhibit paw edema in rat, suggesting that the drug has a good effect prospects for the treatment of RA. Yet it is not clear how geniposidic acid acts on RA synovial cells, and further affects the MAPK signal transduction pathways in cells, thereby regulating proinflammatory cytokine secretion and the expression of function proteins, and it is required to reveal its mechanism to provide a theoretical basis for further screening it as an effective drug.Methods: This study is divided into four parts:The first part: establish rat adjuvant arthritis(AA) model, and isolate and culture fibroblast-like synovial cells using complete Freund’s adjuvant;The second part is divided into five groups: control group, geniposide acid high(1×10-5 mol�'L-1), medium(1×10-6 mol�'L-1)and low(1×10-7 mol�'L-1)dose group as well as the positive drug control group of methotrexate. Detect relevant indicators following administration of the drug intervention. Detect cell proliferation with MTT assay, and observe apoptosis with Hoechst33342 and PI double staining fluorescence microscopy.The third part: study the impact of geniposidic acid on cell supernatants cytokines. Respectively determine IL-1β, TNF-α, IL-10 in cell supernatant by ELISA. Explore the treatment mechanism of rheumatoid arthritis with geniposidic acid by detecting changes in the amount of various cytokines;The fourth part: study the impact of geniposidic acid on Ras-MAPKS pathway in fibroblast-like synovial cells. Detect JNK, ERK, P38 m RNA gene expression with RT-PCR method, detect the effect of phosphorylated JNK ERK, P38 protein expression with western blotting, investigate action mechanisms of geniposidic acid in RA cell through signal transduction pathways.Results:It was found in the first part that AA model had been successfully established.It was found in the second part that the geniposidic acid concentration of 10-5 mol. L-1 could inhibit the proliferation of synovial cells,and geniposidic acid concentrations of 10-5 mol. L-1,10-6 mol. L-1 could promote apoptosis of synovial cells. It was found in the third part that after adding geniposide acid drugs, the amount of IL-1β、TNF-α in cell supernatants in 10-5 mol�'L-1,10-6 mol�'L-1 concentration groups among treatment groups was significantly lower compared with that in the control group; the amount of 10-5 mol�'L-1 concentrations group was significantly upregulated.In the fourth part it was shown by the RT-PCR test results that: 10-5 mol�'L-1,10-6 mol�'L-1 geniposidic acid concentrations groups can inhibit m RNA expression of JNK, ERK, P38 gene. It can be seen by the western blotting results that: 10-5 mol�'L-1,10-6 mol�'L-1 geniposidic acid concentrations groups can inhibit the expression of phosphorylated JNK, ERK, P38 protein.Conclusion:1 Geniposidic acid with sufficient concentration can inhibit proliferation of synovial cell and promote apoptosis of synovial cells.2 Geniposidic acid palys a role of suppressing the secretion of IL-1β, TNF-αin synovial cells supernatants as well as stimulating the secretion of IL-10.3 Geniposidic acid can inhibit the expression of phosphorylated JNK, ERK, P38 protein and m RNA.Based on the above results of four parts, it is found that geniposidic acid has a very good therapeutic effect for RA, which can significantly inhibit the proliferation of fibroblast-like synovial cells and promote apoptosis of fibroblast-like synovial cells. Geniposidic acid can inhibit fibroblast-like synovial cells releasing inflammatory IL-1 β, TNF-α, promoting the release of the protective factor of IL-10. It can also inhibit the activation of JNK、ERK、P38-MAPKs signaling pathway in synovial cells among patients with RA.