A Study Of ATO’s Regulations On Ras-p38MAPK Signal Transduction Pathway In RA HFLS Induced By TNF-α

Objective:1. To observe the effect of TNF-α on the activity of Ras-p38mitogenactivated protein kinase in fibroblast-like synoviocyte of rheumatoidarthritis.2. To observe the effect of ATO in different concentration on the activityof Ras-p38mitogen activated protein kinase in fibroblast-likesynoviocyte of rheumatoid arthritis induced by ATO.Materials and Methods:1. Knee synovia fluid was obtained from a rheumatoid arthritis (RA)patient. FLS was separated from synovia fluid and culture in vitro byprimary cell culture.Then FLS was purified when cells transferred to3rdgeneration.2. The3rdgeneration RA HFLS was cultured with TNF-α（10μg/L）for30minutes. The expression of Ras and phosphorylation status of p38inthe group of RA HFLS treated with TNF-αand group of blank controlwere detected by Western blot.3. RA HFLS were treated with ATO in concerntration of0.5μmol/L、5μmol/L、25μmol/L for24hours. Then RA HFLS were treated by TNF-α（10μg/L）for30minutes. The expression level of Ras andphosphorylation status of p38were detected by Western blot.Results:1. The primary cultures obtained from knee synovia fluid. FLS wasseparated from synovia fluid and culture in vitro by primary cellculture.Then FLS was purified when cells transferred to3rdgeneration.2. The expression level of Ras and p-p38detected by Western blot inRA FLS could significantly increase after stimulated by TNF-α.（p<0.05）3. Compared with TNF-α group ATO group could decreased theexpression level of Ras and p-p38of Ras-MAPK signaling pathwayin RA FLS induced by TNF-α in a dose-dependent manner.（p<0.05）Conclusions:1. There were plenty of macrophages contaminating FLS in primarycultures. But the macrophages would be eliminated as the generationof cell lines reached to the3rdgeneration.2. Ras-p38MAPK signaling pathway could be activated by TNF-αsignificantly in RA HFLS.3. ATO could inhibit the activation of Ras-p38MAPK signaling pathway dose dependently.