CAJ | 학술논문

目的利用一氧化氮(NO)供体硝普钠(SNP)和c-jun氨基末端激酶(JNK)抑制剂SP600125,研究JNK通路对NO诱导的兔关节软骨细胞凋亡的影响.方法采用机械-酶消化法分离3周龄新西兰兔关节软骨细胞,甲苯胺蓝染色鉴定细胞后,SNP和JNK抑制剂SP600125作用于细胞24h,采用AnnexinV-FITC/PI流式细胞术和末端脱氧核苷酸转移酶介导的dUTP缺口末端标记技术(TUNEL)检测软骨细胞凋亡,免疫印迹(Western blot)法测定核因子(NF-κB)p65和p53蛋白的表达水平.结果 SNP作用于软骨细胞后,随着其浓度的增大,细胞早期凋亡率与对照组比较呈浓度依赖性增加,差异有统计学意义(P＜0.05),NF-κB p65和p53的表达分别出现上调,差异有统计学意义(P＜0.05)；加入JNK抑制剂SP600125能抑制这种增加,差异有统计学意义(P＜0.05).结论 JNK信号转导通路在NO诱导的软骨细胞凋亡起着非常重要的作用,JNK的特异性抑制剂SP600125能以浓度依赖方式减少兔关节软骨细胞中NO诱导的凋亡和NF-κBp65及p53蛋白的表达活性.
목적 이용일양화담(NO)공체초보납(SNP)화c-jun안기말단격매(JNK)억제제SP600125,연구JNK통로대NO유도적토관절연골세포조망적영향.방법 채용궤계-매소화법분리3주령신서란토관절연골세포,갑분알람염색감정세포후,SNP화JNK억제제SP600125작용우세포24h,채용AnnexinV-FITC/PI류식세포술화말단탈양핵감산전이매개도적dUTP결구말단표기기술(TUNEL)검측연골세포조망,면역인적(Western blot)법측정핵인자(NF-κB)p65화p53단백적표체수평.결과 SNP작용우연골세포후,수착기농도적증대,세포조기조망솔여대조조비교정농도의뢰성증가,차이유통계학의의(P＜0.05),NF-κB p65화p53적표체분별출현상조,차이유통계학의의(P＜0.05)；가입JNK억제제SP600125능억제저충증가,차이유통계학의의(P＜0.05).결론 JNK신호전도통로재NO유도적연골세포조망기착비상중요적작용,JNK적특이성억제제SP600125능이농도의뢰방식감소토관절연골세포중NO유도적조망화NF-κBp65급p53단백적표체활성.
Objective Tostudytheroleofc-junN-terminalkinase (JNK) signalingpathwayinchondrocyte apoptosis induced by nitric oxide (NO) using NO donor sodium nitroprusside (SNP) and JNK inhibitor SP600125.Methods Articular chondrocytes were separated from New Zealand rabbits aged 3 weeks by mechanical digestion and enzyme digestion and identified by toluidine blue staining,and then the chondrocytes were treated with SNPand SP600125 for 24 h.The cell apoptosis was evaluated by Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) flow cytometry and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL),and the expression levels of nuclear factor-kappa B (NF-κB) p65 and p53 were measured by western blot.Results Compared with those in control group,the early apoptotic rate of SNP-treated chondrocytes increased as the concentration of SNProse,exhibiting a concentration dependency (P＜0.05),and the expression levels of NF-κB p65 and p53 also increased (P＜0.05); JNK inhibitor SP600125 inhibited these increases (P＜0.05).Conclusion JNK signaling pathway plays an important role in NO-induced chondrocyte apoptosis.JNK inhibitor SP600125 can reduce NO-induced apoptosis and expression of NF-κB p65 and p53 in articular chondrocytes of rabbits in a concentration-dependent manner.