丹皮酚致兔膝骨性关节炎软骨细胞凋亡及相关蛋白Bcl-2、Bax mRNA表达的时间与剂量效应研究

摘要

目的:研究丹皮酚致兔膝骨性关节炎(OA)软骨细胞凋亡及相关蛋白Bcl-2、Bax mRNA表达的时间与剂量效应.方法:将60只大耳白兔随机分为正常(生理盐水)组,模型(生理盐水)组,丹皮酚高、中、低剂量组和曲安奈德(阳性药物)组,每组10只.除正常组外,其余各组白兔采用右膝前交叉韧带切断及内侧半月板前1/3切除复制OA模型,模型建立后各组白兔右膝关节内注射不同剂量丹皮酚(分别是0.8、0.4、0.2 mg/kg)、曲安奈德0.2 mg/kg,每周2次.分别于给药后的4、8周分批取材,电镜下观察关节软骨细胞超微病理结构变化;原位末端标记法观察软骨细胞凋亡情况,并计算凋亡指数;原位杂交技术检测各组兔关节软骨组织凋亡相关基因Bcl-2、Bax mRNA表达.结果:与正常组比较,4、8周时模型组细胞分别呈凋亡早期和中晚期形态学改变,且细胞凋亡指数明显升高,细胞中Bcl-2、Bax mRNA表达上调(P<0.01);给药4、8周时,与模型组比较,丹皮酚各剂量组细胞凋亡指数降低,细胞中Bcl-2 mRNA表达上调、Bax mRNA表达下调(P<0.05或P<0.01);电镜超微病理观察显示,丹皮酚高、中剂量组细胞均处于凋亡早期形态学改变.结论:丹皮酚可呈时间与剂量依赖性地延缓OA模型兔的关节软骨退变和破坏,其机制可能与上调Bcl-2基因表达、下调Bax基因表达有关.%OBJECTIVE:To study the time-effect and dose-effect of paeonol on the apoptosis of knee osteoarthritis(OA)artic-ular chondrocyte in rabbits and the mRNA expression of its related protein Bcl-2 and Bax. METHODS:60 big-ear rabbits were ran-domly divided into normal (normal saline) group,model (normal saline) group,paeonol high-dose,medium-dose and low-dose groups and triamcinolone acetonide(positive drug)group,with 10 rabbits in each group. Except for normal group,OA model was induced by right knee anterior cruciate ligament (ACLT) and the medial meniscus 1/3 resection in those groups. After modeling, different doses of paeonol(0.8,0.4,0.2 mg/kg),triamcinolone acetonide 0.2 mg/kg were injected into right articular cavity twice a week. 4 weeks and 8 weeks after administration,articular cartilage specimens were collected. Ultrapathological structure changes of articular chondrocytes were observed by electron microscope. Apoptosis of cartilage cell was observed by TUNEL and apoptotic index was calculated. mRNA expression of apoptosis related genes of Bcl-2 and Bax in articular cartilage tissue of rabbits were de-tected by in situ hybridization technique. RESULTS:Compared with normal group,articular chondrocyte of model group showed early and middle stage apoptosis morphology change after 4 and 8 weeks,and apoptosis index increased significantly and the mRNA expression of Bcl-2 and Bax was up-regulated (P<0.01);4 and 8 weeks later after administration,compared with model group,apoptosis index decreased and mRNA expression of Bax was down-regulated in paeonol groups,while mRNA expression of Bcl-2 was up-regulated(P<0.05 or P<0.01). Electron microscopy ultrastructural observation showed articular chondrocyte of pae-onol high-dose and middle-dose groups were in early stage of apoptosis.CONCLUSIONS:Paeonol can slow down articular chondro-cyte degeneration and destroy in OA model rabbits in time and dose dependently. Its mechanism may be associated with expression up-regulation of Bcl-2 and expression down-regulation of Bax.