Acid Sensing Ion Channels Blocker Amiloride Protected Articular Cartilage In Adjuvant Arthritis Rat And Its Mer-mechanisms

OBJECTIVE To analysis the protective effects of articular cartilage by acid-sensing ion channels (ASICs) blocker Amiloride and its mechanisms. We attempted to investigate the roles of Amiloride playing in the apoptosis of articular chondrocyte,in the ionic concentration of the extracellular [Ca²⁺]I,in the expression of erzymes which relate to Apoptosis and depend of Ca²⁺ level. We elucidate its relative mechanisms of Acid sensing ion channels blocker Amiloride protecting articular cartilage in adjuvant arthritis rat.METHODS The rats were randomly divided into normal group, AA model group, Amiloride(2.5,5.0,10.0 mg·kg-1·d-1) group, aspirin(50 mg/kg)group.The AA model rats were induced by CFA. The secondary inflammation of AA rats was appeared on the tenth day via intradermal injection CFA.The drug groups were given correspond- -ing experimental drugs, the model group and the normal group were administered with the corresponding capacity of sterile water, lasting for eight days.The right hind paw articular swelling degree was determined with volume meter. The morphological changes of articular cartilage was observed by light microscope.The type II collagen (COII) expressions of extracellular matrix of articular cartilage were determined by using immunohistochemistry.The proteoglycan (PG) contents of the cartilage tissue were detected by Alcian blue staining. The apoptosis of articular cartilage cells were observed by transmission electron microscope,Tunel method. articular chondrocyte from rat knees excised and dispersed with sequential incubation of collagenase type II and trypsin. The cells were exposured to the extracellular solution(pH 6.0) for 3h, then continue to be cultured in the extracellular solutions (pH 7.4) for about 4 h. The apoptosis of articular cartilage cells were observed by Tunel method ang PI simple staining.the [Ca²⁺]_i level was detected by Ca²⁺ imaging. The expression of calpain,calcineurin,iNOS were detected by immuocytochemistry.RESULTS(1)Effects of ASICs blocker Amiloride on articular cartilage injury in AA rats Amiloride have no conspicuous effect on the hind paw swelling with AA rats. The pathological study showed that the surface of articular cartilage was slick, the layer was lampros and the quantity of chondrocyte was incresaed. The COII and PG of extracellular matrix of articular cartilage were significantly increased in Amiloride groups by immunohistochemical method and Alcian blue staining.(2)Effects of ASICs blocker Amiloride on the apoptosis of articular Chondrocyte in AA ratsTransmission electron microscope study showed that condensation and crack of nucleus and apoptotic bodies were appeared in articular cartilage cells in model group,and the condensation of heterochromatin ,the distention of rough endoplasmic reticulum and the swell of bioblast were relieved in Amiloride groups.The apoptosis cell calculation in the experiment groups were fewer than those in the model group(P<0.01) by Tunel method in vivo. PI dyeing showed that the apoptosis rate in Amiloride and PcTx1 groups were fewer than those in the model group. Tunel method showed that the positive cell population in Amiloride and PcTx1 groups were fewer than those in the model group. (3)Effects of ASICs blocker Amiloride on the ionic concentration of the extracellular [Ca²⁺]_i and the expression of erzymes which relate to Apoptosis and depend of [Ca²⁺]_iThe variation of The Ca²⁺ F340/F380 ratios in Amiloride and PcTx1 groups were statistical significance compares to the model group by Ca²⁺ imaging. The expression of calpain,calcineurin,iNOS which relate to Apoptosis and depend of Ca²⁺ level degrade obviously in Amiloride and PcTx1 groups compares to the model group (P<0.01) by immuocytochemistry.CONCLUSIONS1. ASICs blocker Amiloride has no conspicuous inhibitory action to Inflammatory reaction but protection to chondrocyte and extracellular matrix(ECM) in AA rats. ASICs blocker amiloride provides the effects of protection to articular cartilage in AA rats.2. ASICs blocker Amiloride inhibits the Apoptosis of articular chondrocyte in AA rats in vivo and vitro what the articular chondrocyte were treated by acid. Amiloride provides the effects of protection to articular cartilage, through to inhibit the apoptosis of articular chondrocyte by block up ASICs in AA rats.3. ASICs blocker Amiloride inhibits the variation of the Ca²⁺ F340/F380 ratios and the expression of calpain,calcineurin,iNOS which relate to apoptosis and depend of Ca²⁺ level. These results suggest that ASICs blocker amiloride provides the effects of inhibit the Apoptosis of articular chondrocyte,which their mechanism may be that blocking the acid sensing ion channels affect Ca²⁺ overloading,to inhibit the expression of calpain,calcineurin,iNOS which relate to apoptosis and depend of Ca²⁺ level.