The Cellular And Molecular Mechanism Of Lockade Acid-sensing Ion Channel La To Rotect Articular Cartilage In Rats With Djuvant Arthritis

A stable pH is important for normal cellular function. In physiolocial conditions, thepH value of tissues are maintained at7.0to7.3through various H+transportingmechanism.The physiological pH value of the synovial fluid (pH7.2-7.4) is alsoimportant for the integrity of normal articular cartilage. However, in inflammatorydiseases such as rheumatoid arthritis, pH values in inflamed joints are always lowerthan those of normal joints, ranging from pH5.5to pH7.0. Therefore, low pH value isregarded as a hallmark of inflammatory reactions in inflamed joints. Recently, severalstudies have focused on the effect of extracellular pH on articular chondrocytes andtheir functions. Although it was reported that the severity of cartilage damage correlateswith the degree of acidity in arthritic joints, the molecular mechanism through whicharticular chondrocytes are regulated by acidosis remained unknown.Acid-sensing ion channels (ASICs) are the primary acid sensors in throughout centraland peripheral tissues. They are members of the degenerin/epithelial sodium channel(DEG/ENaC) protein superfamily. To date, seven ASIC subunits have been identified:ASIC1a, ASIC1b, ASIC1b2, ASIC2a, ASIC2b, ASIC3, ASIC4. ASICs represent a novelclass of ligand-gated cation channels that are activated by increased acidification.Recent studies have indicated that ASICs is involved in synaptic plasticity, learning andmemory. Activation or sensitization of Ca2+-permeable ASICs has also beendemonstrated to be responsible for acidosis-mediated ischemic brain。Our previous studies indicated that ASICs expressed at high levels in articularcartilage and blockade ASICs by amiloride provide protection for the articular cartilagein adjuvant arthritis rats. However, the molecular mechanism of amiloride protection against cartilage lesions has not been clearly defined. Previous studies have indicatedthat extracellular acidosis has a strong influence on both the production of matixcomponents and the production of agents involved in the breakdown of the matrix. Wetherefore hypothesized ASICs serve as an sensor for extracellualr acidosis and involvedmatrix turnover of articular chondrocytes to acidosis. The main contents are dividedinto three sections, as fellows:1Acid-sensing ion channel1a mediates acid-induced increases in intracellularcalcium in articular chondrocytesIn this study, we investigated the expression of ASIC1a on rat articular chondrocytesand its possible role in the function of articular chondrocytes. We found that the mRNAand protein expression of ASIC1a was expressed abundantly in articular chondrocytes.In cultured chondrocytes, extracellular pH6.0increased intracellular calcium ([Ca2+]i)in the presence of extracellular Ca2+. The ASIC1a-specific blocker PcTx-1venomsignificantly reduced this increase in [Ca2+]i, and inhibited acid-induced articularchondrocyte injury. However, the increase in [Ca2+]i and articular chondrocyte injurywere not observed in the absence of extracellular Ca2+. These findings show thatincreased [Ca2+]i, mediated via ASIC1a, might contribute to acidosis-induced articularchondrocyte injury.2Acid-sensing ion channel1a contributes the effect of acidosis on the matrixmetabolism of articular chondrocytesIn this study, we investigated the effect of acid-sensing ion channel1a on the matrixmetabolism of articular chondrocytes induced by acidosis. Lipofectamine2000transfection reagent was used to transfect ASIC1a siRNA with FAM in articularchondrocytes. The expression levels of ASIC1a protein were detected by Western blot.Results showed the silence cells model of ASIC1a expression was produced by siRNAin chondrocytes. We found that acidosis down-regulate the production of GAG, HYP,MMP-2, MMP-9, TIMP-1and TIMP-2via ASIC1a. However production of MMP-2 and MMP-9by chondrocytes was relatively insensitive to pH than GAG, HYP, TIMP-1and TIMP-2. Blocking ASIC1a signifcantly restored the production of GAG, HYP,TIMP-1and TIMP-2. Our research showed that blocking ASIC1a significantly inhibitthe effect of acidosis on production of GAG, HYP, TIMP-1and TIMP-2and does notelevate the production of agents able to degrade matrix components such as MMP-2and MMP-9from chondrcytes. These results suggest that ASIC1a are involved in theacidosis–mediated effect on matrix metabolism of articular chondrocytes3The role of MAPK pathways in Acid-sensing ion channel1a regulates matrixmetabolism from acid-induced articular chondrocytesOur research has shown that extracellular pH plays an important role in matrixsynthesis and metabolism in articular chondrocytes by activated ASIC1a. However,little is kown about the underlying mechanism of ASIC1a on matrix synthesis andmetabolism in articular chondrocytes. The present study was undertaken to investigatethe mechanism of ASIC1a on matrix synthesis and metabolism in articularchondrocytes by acidosis. Down-regulation of GAG, HYP, MMP-2, MMP-9, TIMP-1and TIMP-2mRNA expression and production was abolished by inhibit acidosis-elicited increase in intracellular Ca2+concentration. Pretreatment with BAPTA-AM(Ca2+chelator) abolished acidosis induced ERK1/2, p38MAPK, c-jun and c-fosactivation. PD98059(ERK1/2inhibitor) attenuate c-jun activation and restored GAG,HYP, TIMP-1and TIMP-2mRNA expression and production. SB203580(p38MAPKinhibitor) inhibit c-fos activation and restored MMP-2and MMP-9mRNA expressionand production. We founded that bolcking ASIC1a inhibit the effect of extracellularacidosis on GAG, HYP, MMP-2, MMP-9, TIMP-1and TIMP-2mRNA expression andproduction in articular chondrocytes. Taken together, our data indicate that blockingacid-sensing ion channel1a inhibit abberant matrix synthesis and metabolism fromacid-induced articular chondrocytes via Ca2+-mediated activation of p38MAPK/c-junand ERK/c-fos pathway.