Effects Of ASIC1a Gene Silencing On The Apoptosis Of Articular Cartilage Cells Of Adjuvant Arthritis Rats

OBJECTIVE To establish the silence cells model of acid sensing ion channels1a(ASIC1a) expression in articular cartilage cells by small interfering RNA(siRNA),and investigate the level of the intracellular calcium ([Ca2+]i) and the expression oftype â…¡ collagen, Bax, and Bcl-2, through which discovering the effects of ASIC1a onthe function of articular cartilage cells.METHODS Lipofectamine2000transfection reagent was used to transfect thesmall interfering RNA (siRNA) with FAM (Carboxyfluorescein) in articular cartilagecells. The expression level of ASIC1a mRNA and protein were detected byfluorescence microscope, flow cytometry, q-RT-PCR (Real-time fluorescentquantitative PCR) and western blotting, respectively. The apoptosis of articularcartilage cell was induced by controlling the pH of cell culture medium at6.0in vitro,based on which the [Ca2+]i level was detected by laser scanning confocal, and theapoptosis of articular cartilage cells were observed by Annexin-V/PI, Hoechst33258and Tunel, and the expression of type â…¡ collagen, Bax,and Bcl-2were detectedthrough immunocytochemistry and q-RT-PCR, respectively. RESULTS1. All the3ASIC1a siRNAs synthesized in the present study could be transfectedinto articular cartilage cells successfully. Detected through q-RT-PCR and Westernblot, the expression of ASIC1a mRNA and protein in transfected articular cartilagecells was lower than that in the normal group;2. After acid stimulus, the range of Ca2+influx in the ASIC1a-silenced cells causedby siRNA-3was lower than that in the model group;3. After acid stimulus, the ratio of apoptosis and the ratio of Bax/Bcl-2in theASIC1a-silenced cells caused by siRNA-3were decreased while the expression of thecollagenâ…¡ in the silencing group was increased compared with the model groupinduced by acid extracellulary.CONCLUSION1. The silence cells model of ASIC1a expression by siRNA in articular cartilagecells is the perfect model to observe the effect on the function of the acid-sensing ionchannels in articular cartilage cells;2. Though the intervene at the gene level to make the acid-sensing ion channel1asilenced, the Ca2+permeability of cell membranes was decreased;3. Knockdown of ASIC1a in articular cartilage cells caused by siRNA-3protectedfrom apoptosis induced by extracellular acid, which may be related with the decreaseof Bax/Bcl-2adjusted by silencing ASIC1a. These suggested that the acid sensing ionchannels1a plays an important role in the function of articular cartilage cells ofadjuvant arthritis rats.