The Molecular Mechanism Of Calreticulin Participating In Synovitis Of Rheumatoid Arthritis Via Regulating The Expression Of Adhesion Molecules

ObjectiveRheumatoid arthritis(RA) is a systematic autoimmune disease characterized by chronic synovitis and pannus formation, whose pathogenesis is still not fully understood. It has been reported that calreticulin(CRT) may participate in the inflammation of RA synovial membrane. In this study, the levels of CRT and adhesion molecules in the serum and synovial fluid from patients with RA, as well as the expression in the synovium, were determined. And the correlativity between CRT and these adhesion molecules was also analyzed. Next, the regulation of CRT for intercelluar adhesion molecule(ICAM-1) and the related signaling pathway involved in were investigated. This study intended to describe the role of CRT in the pathogenesis of RA from the point of synovium inflammation which induced by adhesion molecules, and finally provide new theory foundation for clinical diagnosis and treatment of RA.Methods1. Serum and synovial fluid levels of calreticulin, ICAM-1, and VCAM-1 were measured by ELISA in patients with established RA. Correlativity between CRT and these adhesion molecules was also analyzed.2. Expression of CRT, ICAM-1, and VCAM-1were detected by Immunofluorescent.3. The expression of ICAM-1 in human umbilical vein endothelial cell(HUVECs) stimulated by CRT or inhibitors was determined by flow cytometry.4. The mRNA levels of ICAM-1was detected by real-time PCR.5. The activation and expression of Akt in HUVECs was analyzed by western blot.Results1. Serum and synovial fluid levels of CRT levels in RA patients were(5.359±2.27) ng/ml and(4.997± 0.826) ng/ml, respectively. The levels of ICAM-1and VCAM-1 in serum were(485.772 ± 197.612) ng/ml and(419.609 ± 188.338) ng/ml. The synovial fluid levels were(579.467± 70.604) ng/ml and(511.368± 101.390) ng/ml.2. Immunofluorescent results showed that CRT and ICAM-1 were highly expressed in endothelial cells and the perivascular areas in RA synovium. CRT was also observed in synovial lining and sublining layer. While the VCAM-1 was mainly expressed in synovial fibroblasts.3. The expression of ICAM-1 in HUVWCs was significantly elevated after CRT stimulation. And this effect was decreased with PI3 K and e NOS inhibitors.4. The phosphor-Akt level was increased when the HUVECs were treated with CRT.Conclusions1. Serum and synovial fluid levels of CRT, ICAM-1 and VCAM-1were elevated, and a significant correlation was observed between CRT and ICAM-1 as well as CRT and VCAM-1.2. CRT and ICAM-1were co-lacated in endothelial cells and the perivascular areas in RA synovium.3. CRT could increase the expression of ICAM-1 in endothelial cells.4. Akt protein in the HUVECs could be activated by CRT, and further induce the activation of e NOS. The increased expression of ICAM-1 mediated by CRT could be influenced by PI3K/Akt/e NOS signaling pathway.