CD95/CD95L Promoted Proliferation And Inflammation On Rheumatoid Arthritis Synovial Fibroblasts

ObjectivesCD95(Fas/Apo-1) triggers cell apotosis, cell proliferation, migration and cytokine production following engagement by CD95ligand or by agonistic anti-CD95antibodies. CD95-mediated apoptosis in RA synovium is defective. Our aim is to study intracellular CD95signaling on cell proliferation and pro-inflammatory response, and evaluate the role of PI3K/Akt pathways in this process. We try to reveal the molecular mechanisms of RA pathogenesis and to find new targets for gene therapy provide a theoretical basis and experimental evidence.MethodsFor immunohistochemical staining, the expression of CD95was examined using anti-CD95monoclonal antibody; Apoptosis was detected using the TUNEL method in situ apoptosis detection kit; After RASFs were isolated, RASFs from passages three to seven were used. The expression of CD95on the surface of RASFs and the cell apoptosis rates were assessed by flow cytometry. The RASFs were treated with agonistic anti-CD95antibody、CD95siRNA or PI3K inhibitor LY294002, then the proliferation was detected by CCK-8and inflammatory cytokines were detected by RT-PCR, the total Akt and pAkt protein expression were analyzed by western blot.Results1. A significant increase of CD95was found in RA synovial tissues compared with OA; the mRNA and protein lever of CD95in RASFs were higher than OA; while apoptosis was not obvious in RA synovial tissues and RASFs.2. Low concentrations of agonistic anti-CD95antibody (Ong/ml,50ng/ml,100 ng/ml,500ng/ml,750ng/ml) could promote RASFs growth and inflammatory cytokines IL-6expression (P<0.05), while high concentrations (1000ng/ml)could induce apoptosis(P<0.05). These results also could be inhibited by CD95siRNA.3. The Akt phosphorylation was induced and maximal at30minutes by agonistic anti-CD95antibody in RASFs; moreover CD95-mediated promotion of proliferation and inflammation were significantly inhibited in the presence of PI3K inhibitor LY294002(P<0.05).ConclusionCD95/CD95L could promote rheumatoid arthritis synovial fibroblasts proliferation and inflammatory cytokines production, and activation of the PI3K/Akt signaling pathway might be the potential mechanism.