Effects of ASIC1a gene silencing by siRNA on apoptosis of articular cartilage cells of adjuvant arthritis rats

Abstract

Abstract: AIM: To estimate the effects of ASIC1a gene silencing by siRNA on apoptosis in articular cartilage cells of adjuvant arthritis rats. METHODS: Lipofectamine 2000 transfection reagent was used to transfect the small interfering RNA(siRNA) with FAM in articular cartilage cells. The expression level of ASIC1a mRNA and protein were detected by fluorescence microscope,flow cytometry(FCM),real-time PCR and Western blotting, respectively. And the apoptotic cell dyed with Annexin-V and propidium iodide were measured with a flow cytometer. RESULTS: ASIC1a siRNA were transfected into articular cartilage cells successfully. The expression of ASIC1a mRNA and protein in transducted chondrocytes was lower than the control chondrocytes (P<0.01). The rate of apoptosis was lower in the ASIC1a-silenced cell than that in the model cell. CONCLUSION: The silence cells model of ASIC1a expression by siRNA in chondrocytes of adjuvant arthritis rats is the perfect model to observe the effect on metabolism of chondrocytes of the acid-sensing ion channels. Transfection siRNA-3 into articular cartilage cells protected from apoptosis by extracellular acid, which may be related with different expression levels of ASIC1a.

Key words: Articular cartilage cells, Acid-sensing ion channels (ASICs), Liposomes, Gene silence, Cell apoptosis

CLC Number:

R965.2

JIANG Sheng, CHEN Fei-hu, CHEN Cun-zhi, RONG Chao, HU Wei, WU Fan-rong, GE Jing-fang, TANG Jie. Effects of ASIC1a gene silencing by siRNA on apoptosis of articular cartilage cells of adjuvant arthritis rats[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2012, 17(3): 256-262.

References

[1] Jasti J, Furukawa H, Gonzales EB, et al. Structure of acid-sensing ion channel 1 at 1.9 A resolution and low pH[J]. Nature, 2007, 449(7160): 316-323.
[2] Xiong ZG, Chu XP, Simon RP. Acid sensing ion channels--novel therapeutic targets for ischemic brain injury[J]. Front Biosci, 2007, 12: 1376-1386.
[3] Yuan FL, Hu W, Lu WG, et al. Targrting interleukin-21 in rheumatoid arthritis[J]. Mol Biol Rep, 2011, 38(3): 1717-1721.
[4] 袁凤来,陈飞虎,李霞,等. 大鼠关节软骨酸敏感离子通道的表达[J]. 安徽医科大学学报,2007, 42(5): 513-516.
[5] 袁凤来,陈飞虎,黄学应,等. 酸敏感离子通道在大鼠佐剂性关节炎关节软骨中的表达[J]. 中华风湿病学杂志,2008, 12(5): 321-324.
[6] 陈飞虎,袁凤来,李霞,等. 几种抗类风湿性关节炎药物对佐剂性关节炎大鼠关节软骨细胞酸敏感离子通道的表达影响及作用[J]. 中国临床药理学与治疗学,2008, 13 (2) :131-137.
[7] 张腾跃,陈飞虎,王念,等. 酸敏感离子通道阻断剂Amiloride对AA大鼠关节软骨细胞凋亡抑制作用的初步研究[J]. 中国临床药理学与治疗学,2010, 15 (1) : 41 -46.
[8] 王颖,蔡刚祥,吴亮. 靶向Survivin基因siRNA诱导肝癌细胞凋亡的实验[J]. 医药导报,2006, 25(7): 648-651.
[9] Liechti LA, Berneche S, Bargeton B, et al. A combined computational and functional approach identifies new residues involved in PH-dependent gating of ASIC1a[J]. J Biol Chem, 2010, 285(21): 16315-16329.
[10] 袁凤来,陈飞虎,陆伟国,等. 酸敏感离子通道阻断剂阿米洛利对佐剂性关节炎大鼠关节软骨损伤的影响[J]. 中国药理学通报,2009, 25(7): 895-900.
[11] Lopes RB, Gangeswaran R,McNeish IA,et al. Expression of the IAP protein family is dysregulated in pancreatic cancer cells and is important for resistance to chemotherapy[J]. Int J Cancer, 2007, 120(11): 2344-2352.
[12] Agrawal N, Dasaradhi PV, Mohmmed A, et al. RNA interference: Biology, mechanism and applications[J]. Microbiol Mol Biol Rev, 2003, 67(4): 657-685.
[13] 朱有凯,林汉良,马怡晖,等. RNA干扰致基因表达沉默细胞模型的建立[J]. 解剖学研究,2005, 27(3): 190-193.
[14] 邹颖刚,薛惠荣,崔满华. 靶向卵巢癌HIF-1a基因siRNA真核表达载体的构建及鉴定[J]. 中国实验诊断学,2010, 14(8):1265-1266.
[15] Yang XH,Sladek TL,Liu X,et al. Reconstitution of caspase 3 sensitizes MCF-7 breast cancer cells to doxorubicin and etoposide-induced apoptosis[J]. Cancer Res, 2001, 61(1): 348-354.
[16] Yuan FL, Chen FH, Lu WG, et al. Inhibition of acid-sensing ion channels in articular chondrocytes by amiloride attenuates articular cartiage destruction in rats with adjuvant arthritis[J]. Inflamm Res, 2010, 59(11): 939-947.
[17] Yuan FL, Chen FH, Lu WG, et al. Acid-sensing ion channel 1a mediates acid-induced increases in intracellular calcium in rat articular chondrocytes[J]. Mol Cell Biochem, 2010, 340(1/2): 153-159.[18] Yu JY, DeRuiter SL, Turner DL. RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells[J]. Proc Natl Acad Sci USA, 2002, 99(9): 6047-6052.