Effects Of NGF On ASIC1a Expression And Matrix Turnover In Rat Articular Chondrocytes In Acidic Condition

In physiolocial conditions, the pH value of synovial fluid is maintained at 7.4 through various H+ transporting mechanism. However, in inflammatory diseases such as rheumatoid arthritis, pH values in inflamed joints are dropped to 6.0 or even lower. Acid-sensing ion channels (ASICs) are a family of ligand-gated cation channels, which belong to the degenerin/epithelial Na+ channel (DEG/ENaC) superfamily[6]. They are Na+-selective and Ca2+-permeant, and are transiently activated by extracellular acid. Results of our previous research showed that ASIC1a was expressed at high level in the knee cartilage cells of rheumatoid arthritis and played important roles in cartilage damage and the production of matrix components. Never growth factor (NGF) is a significant regulatory role in the expression ASIC1a in nerve tissue. Results of our research has shown that NGF in serum and synovial fluid is highly expressed in pathological process of rheumatoid arthritis. However, it is uncertain whether such effect is mediated directly via the high expression of ASICla. Therefore, rat articular chondrocytes were chosen in the present study to further investigate the effects of NGF on ASIC1a expression and matrix turnover under acidic condition.Objective:To investigate the effect of NGF on ASICla expression and matrix turnover in rat cartilage cells in acidic condition.Content:1:To investigate the expression of NGF receptors and ASICla induced by NGF in rat cartilage cells in acidic condition.2:To investigate the role of ERK1/2 pathway in the expression of ASIC la induced by NGF.3:To investigate the effect of NGF on matrix turnover of rat cartilage cells.Methods:1:Cartilage cells were incubated in acidic condition for 0,8,16,24h hours, andexpression of NGF receptors were measured by western blotting; Cartilage cells were treated with extracellular solutions (pH 6.0),16 h later, the cells were stimulated by NGF (0,2,10,50ng/ml) for 8h, and the mRNA and protein expression of ASICla were detected using real-time fluorescent quantitative polymerase chain reaction (RT-PCR) and western blotting, respectively.2:After treatment with extracellular solutions for 16h, cartilage cells were stimulated by 10ng/ml NFG for different time(0,0.5,1,2,4,8h), the time dependent effect of NFG on the expression ERK1/2 and c-fos were measured by western blotting. Moreover, the changes of c-fos and ASIC1a expression were also tested by western blotting after inhibiting ERK pathway by PD98059(30μM).3:Cartilage cells were divided into control group, NGF(2ng/mL) group, NGF(10ng/mL) group, NGF(50ng/mL) group, anti-NGF antibody(1:1000) group, and NGF+ASICla siRNA group. After treatment with acid for 16h, the supernatants were collected. Then the cells were treated with NGF in medium with normal medium. Following incubation in 24h and 72h, total supernatant fluid and protein were extracted according to the manufacturer’s instructions. The expression of MMP-9 was measured by enzyme linked immunosorbent assay (ELISA) and western-blotting. The expression of COL-Ⅱ was measured by immunohistochemistry. The content of HYP was tested by Chloramine-T.Results:1:After treatment with extracellular solutions, the expression of NGF receptor was 3 times higher than that in the normal with a time dependent inductive effect, and attain the maximum at 24h. The expression of ASIC1a was depended on the concentration of NGF, which was stable at 10ng/ml.2:The expression of ERK and c-fos were at maximum level 4h after treated with NGF. Results of western blotting showed that inhibition of ERK was able to down-regulate the expression of c-fos and ASICla.3:The levels of Col-Ⅱ and MMP-9 in cartilage cell were decreased after treatment with NGF, and the effect could be inhibited by anti-NGF antibody (1:1000) or specific ASIC1a knockdown via SiRNA.Conclusion:1:NGF played a role in the expression of ASIC1a.2:The effect of NGF on the expression of ASIC1a might be involved with mediating the ERK signal transduction pathway.3:The effect of NGF on matrix turnover in cartilage cells, specifically for the col-Ⅱ and MMP-9, might be related to the expression of ASIC1a in acidic condition.