Study On Brucine’s Effect On Proliferation And JNK Regulation In MARK Signal Transduction Pathway On RA HFLS Induced By TNF-α

Background:Rheumatoid arthritis (RA), one of the most commondiseases in Rheumatology, is a kind of connective tissue disease. Thejoint structure damages and function impaired caused by synovialinflammation can restrict patients’ movement and make them hard to takecare of themselves in daily life, which has severe impact on the lifequality for patients and their families. Thus, it is advocated to treatrheumatoid arthritis early and timely. However, on one hand, althoughtraditional therapeutic drug is cheap, the effect is slow and poor; the newtype of drug, on the other hand, has fast and good effect but it is veryexpensive, which cannot be afforded by ordinary patient for long-termuse. There is a lot of proprietary Chinese medicine that has the effect ofanalgesic and strengthening tendons and bones, nux vomica belongs toone of this kind. Research shows that brucine pieces can selectivelyinhibit cellular immunity and has hypersensitivity for immune complex; italso has inhibitory effect on synovial cell in vitro cultured fibroblasts.Therefore, we are going to further study the molecular mechanism ofbrucine.Objective:1. To observe the effect of TNF-α on JNK of MAPK signaling pathway activation levels in HFLS-RA.2. To observe the effect of different concentration of brucine on HFLS-RA proliferation induced by TNF-α.3. To observe the effect of different concentration of brucine on the JNKsignaling pathway of HFLS–RA induced by TNF-α.Method:1. To subculture the recovered HLFS-RA cell to the4th generation, andreserve it.2. Adopt CCK8method to observe the effect on HFLS-RA proliferationwith TNF-α and different concentration of brucine treatment3. Add10μg/l of TNF-α into the4th generation HFLS-RA, incubate30min, then detect and compare the expression level of JNK and P-JNKwith Western blot between TNF-αgroup and the blank control group.4. Add different concentration of brucine（0.125mg/ml;0.25mg/ml;0.5mg/ml；2mg/ml）respectively into4thgeneration HFLS–RA, incubate24h, then abandon the nutrientsolution, further to incubate with10mu g/l of TNF-α for30min. Afterreaction termination, test the expression level of p–JNK and JNK foreach group with Western blotResults:1. HFLS-RA proliferation is significantly increased after TNF-αinduction. High dose of brucine has inhibitory effect on theproliferation of HFLS induced by TNF-α. The effect is dose-independent.2. Detected by Western blot, the expression level of p-JNK issignificantly increased in HFLS-RA after induced by TNF-α (α=0.05，p<0.001)3. Detected by Western blot, p-JNK expression level is increased withlow concentration of brucine group; p-JNK expression level isdecreased with high concentration of brucine group.(α=0.05,p<0.001)Conclusions:1. TNF-α has active proliferation effect on HFLS–RA (α=0.05，p<0.001)2. High dose of brucine has inhibition effect on HFLS–RA proliferation,and it is dose-dependent (α=0.05，p<0.001).3. After induced by TNF-α,the p–JNK expression level of HFLS-RA isincreased compared with blank control group (α=0.05，p<0.001)4. Brucine could inhibit p-JNK expression level of HFLS-RA in thedose dependent manner.