Expression And Some Function Research Of Acid Sensing Ion Channels In Rat Articular Chondrocytes With Adjuvant Arthritis

Rheumatoid arthritis(RA) is one of the autoimmune diseases characterized by massive synovial proliferation,thickening of the lining and subintimal infiltration of inflammatory cells,cartilage and bone tissue damage,which lead to the functional disturbance joint deformity and dysfunction.Cartilage and bone destruction are the main reason for the disability in the RA patients.Therefore,it is important to actively seek to the pathophysiological changes of articular cartilage damage and bone tissue.Metabolism of articular chondrocytes plays a key role in the regulation of extraccllular matrix(ECM).Metabolism of articular cartilage is effected by tissue acidosis.Extremes of extracellular acidity have been shown to inhibit matrix metabolism and may also modify the activity of degradative enzymes.However,at present,the sensors of articular chondrocytes how to adjust these pH changes are largely unknown.The study showed that the body senses extracellular pH changes through acid sensitive ion channels(ASICs).ASICs are ligand-gated cation channels activated by extracellular protons.In recent years,because of extensive and important biological function of ASICs,studies of subunit of ASICs have achieved great progresses.Tissue acidosis is an important feature of cerebral ischemia,epilepsy and tumor.The reason of RA is the joint inflammation leading to local tissue acidification.This is a similar Path-feature with cerebral ischemia.It is not clear wheather the existence of ASICs or not in articular cartilage cell.Is ASICs playing a role in the process of the articular cartilage damage of RA? Tissue acidosis is a common phenomenon in patho-inflammation.However,it is still not clear whether ASICs are involved in the pathophysiological process of joint cartilage injury induced by tissue acidification,or whether an inhibition/blockage of ASICs can protect the joint cartilage from inflammatory injuries.To further explore the role of ASICs in the articular cartilage cell of RA,the model of rat adjuvant-induced arthritis deemed as the object of this study,we will study that expression and some function research of acid sensing ion channels in rat articular chondrocytes with adjuvant arthritisThe main contents were divided into some sections as follows:1.Expression of acid sensing ion channels in rat articular cartilageRT-PCR and Immunoblotting analyses were used to detect ASICs in rat articular cartilage.RT-PCR analysis showed that ASIC1a,ASIC2a and ASIC3 mRNAs were present in the tissue of articular cartilage total RNA extract from the tissue of articular cartilage,and the corresponding protein could detect its expression.Semi-quantitative analysis showed that the production of ASIC1 amRNA is more than other subunit.The results showed that ASICs may be involved in cases of articular cartilage cell metabolism2.Expression of acid sensing ion channels in rat articular cartilage with adjuvant arthritisCFA-induced arthritis was stimulated by injection of 100μl CFA emulsion intradermally into the right hind paw;morphological changes of articular tissues was observed by light microscope;Real-time Polymerase Chain Reaction and Immunoblotting analyses were used to detect ASICs in rat articular cartilage with adjuvant arthritis.The results showed that ASIC1a,ASIC2a and ASIC3 were present in the normal and model group tissue of articular cartilage,Semiquantitative analysis of the ASICsmRNA and protein levels in model group were higher than in the normal group(P＜0.01).The results showed enhanced expression of ASICs in AA articular cartilage and may be involved in pathological cases of articular cartilage cell metabolism3.Effects of several anti-rheumatoid arthritis drugs on expression of acid-sensitive ion channels in articular chondrocytes in rats with adjuvant arthritisThe rats were randomly divided into the normal group,AA model group,aspirin (50 mg.kg^-1) group,dexamethasone(0.2 mg.kg^-1) group,tripterygium wilfordii(50 mg.kg^-1) group and levamisole(10 mg.kg^-1) group.The AA rat model was induced by CFA on d0.The volume of rat hind paw was measured by means of Volume Meter.The secondary inflammation of AA rats appeared on the d10 after intradermal injection of CFA.Intraperitoneal injection of dexamethasone at this time,other drug groups were gavaged corresponding experimental drugs,and the model group and the normal group were administered with the corresponding capacity of sterile water,lasting for seven days.The morphological changes of articular cartilage were observed by light microscope.The quantitative PCR was used to detect the ASICs and aggrcan mRNA expression in rat articular cartilage.The immunoblotting analyses were used to detect the ASICs protein expression in rat articular cartilage.The expression of typeâ…¡collagen was measured by immunohistochemical method.The results showed that the secondary paw swelling was inhibited significantly in AA rats.Aspirin obviously inhibited the expression of ASICs of articular cartilage cells in AA rats,but the inhibitory effects of other drugs were not obvious.The typeâ…¡collagen and aggrcan expression of cell matrix of articular cartilage was increased significantly by four kinds of drugs,especially aspirin,in AA rats.These results suggest that inhibiting expression of ASICs in the articular cartilage has a protective effect to the articular cartilage, ASICs may be involved in RA articular cartilage damage. 4.Effects of amiloride on articular cartilage injury in AA rat modelThe rats were randomly divided into the normal group,AA model group,amiloride (5 mg.kg^-1) group,aspirin(50 mg.kg^-1) group+amiloride(5 mg.kg^-1) group,aspirin (50 mg.kg^-1) group.Hind paw volume was determined with volume meter,the HE staining showed:articular cartilage surface irregularities,defects,structural damage, decrease in the number of cartilage cells and fiber hyperplasia could be seen by AA group,cartilage structure in clear and obvious damage mitigation could be seen by amiloride group;amiloride decreased ASIC1a,ASIC2a and ASIC3 mRNA gene overexpression in AA rats articular cartilage,especially aspirin+amiloride group.Typeâ…¡collagen and PG expression of cell matrix of articular cartilage can be increased significantly by aspirin,amiloride and aspirin+amiloride,especially aspirin+amiloride, in AA rats(P＜0.01).In vitro,we used Fura-3/AM probe and laser scanning microscope to detect intracellular Ca²⁺ concentration.The results showed that extracellular pH(6.5) can decrease articular cartilage cells Ca²⁺ level of short-term increase.Amiloride could block intracellular Ca²⁺ level by extracellular acid(pH 6.5) induced.These results suggest that ASICs blocker amiloride provides the effects of protection in articular cartilage damage,which their mechanism may be that blocking the acid sensitive ion channels affect Ca²⁺ overloading.