The Study On The Mechanism Of AS₂O₃ On The Synoviocytes Apoptosis Of RA In Vitro

Objective: To investigate and analyse the possible mechanism of arsenic trioxide(AS₂O₃) that induces apoptosis of human fibroblastlike synoviocytes (HFLS) byactivating the mitochondria to release Cytochrome C(CytC), then to activate Caspases,on the basis of culturing the synoviocytes in vitro, so intra-cellular apoptosis may bestimulated. Moreover, it can regulate the pathway by affecting Bcl-2 mRNAMethods: Primary synoviocytes were cultured by means of two-enzymatic digestion.Accroding to the foregone research that the cultured cells were identified HFLS byphase-contrast microscope, electron microscope, HE stains, cell immunochemistrydetections and apoptosis could be induced by AS₂O₃, we adopt ELISA to quantitatethe content of cytoplasmic CytC after 6h, 24h and 48h that 5 RA HFLS were addedwith different concentration of AS₂O₃. The expressions of Caspase-3 and Bcl-2mRNA were also examined semiquantitatively by PT-PCR after 48h.Results: 1. Certain concentration of AS₂O₃ could precipitate CytC into cytoplasms inthe early period of apoptosis. After 6h that AS₂O₃ was added, the content ofcytoplasmic CytC, detected by ELISA, was higher than the contrast group, dependentof increasing dose of the drug. After 24h, the content of cytoplasmic CytC washigher significantly, especially among 2-40μmol/L AS₂O₃. After 48h, the content ofcytoplasmic CytC was similar with that at 24h by 20/μmol/L AS₂O₃. Additionally,there were no significant differences between 40/tmol/L and 80/zmol/L AS₂O₃. So inthe condition of high doses of the drug, we presumed that it could cause cells tonecrosis, which displayed disaggregation of cytomembrane, but not mitochondria.2. The expression of Caspase-3 mRNA could be strengthened with 2-40/μmol/LAS₂O₃, detected at48h after medication by means of RT-PCT. And the expression isdose-dependent.3. With AS203 among 2-40/μmol/L, the expression of Bcl-2 mRNA is inhibitedcompared with the contrast group, at 48h after medication by RT-PCR.Conclusion: The apoptosis of HFLS can be induced by AS₂O₃ through activating the mitochondria to release CytC and to activate Caspases. Moreover, it can inhibit theexpression of Bcl-2 mRNA to regulate the pathway. The diversify is dose-dependent,which provides useful help for choosing clinically adequate therapeutic drugs anddoses for RA.