电针对实验性膝骨关节炎大鼠软骨细胞凋亡及软骨基质的影响

Effect of Electroacupuncture on Chondrocyte Apoptosis and Cartilage Matrix of Experimental Rats with Knee Osteoarthritis

目的:观察电针对膝骨关节炎大鼠软骨细胞凋亡及软骨基质的影响,探讨电针治疗膝骨关节炎的作用机制。方法:将2月龄清洁级雄性SD大鼠40只,适应性喂养1周后,采用随机数字表法分为空白组10只和造模组30只。造模组在第1、4、7天双膝关节腔注射0.2 m L 4%木瓜蛋白酶。造模成功后,采用随机数字表法分为模型组、电针15 min组(EA15组)和电针30 min组(EA30组)3组,每组10只。EA 15组给予电针治疗15 min/(次·d),5次/周;EA 30组给予电针治疗30 min/(次·d),5次/周;空白组和模型组不给予任何治疗。8周后,处死动物,腹主动脉采血,ELISA法测定Ⅱ型胶原(collagenⅡ)C端肽(CTXⅡ)和Ⅱ型胶原C前肽(CPⅡ);取大鼠右侧胫骨平台内侧软骨组织于4%多聚甲醛中固定,石蜡包埋,TUNEL法检测软骨细胞凋亡情况;甲苯胺蓝染色观察软骨基质糖胺聚糖的变化;免疫组化染色观察collagenⅡ变化;左侧胫骨平台软骨组织迅速置入液氮中保存,分别用RT-PCR和Western blot检测Bcl-2、Bax mRNA和蛋白表达。结果:与空白组比较,模型组软骨细胞凋亡率明显升高(P<0.05),Bcl-2 mRNA和蛋白表达降低(P<0.05),Bax mRNA和蛋白表达升高(P<0.05);糖胺聚糖含量减少,collagenⅡ蛋白表达降低(P<0.05),血清CTXⅡ显著升高(P<0.05),CPⅡ显著降低(P<0.05)。与模型组比较,电针组均能显著降低软骨细胞凋亡率(P<0.05),增加Bcl-2 mRNA和蛋白表达(P<0.05),降低Bax mRNA和蛋白表达(P<0.05);增加糖胺聚糖含量和collagenⅡ蛋白表达(P<0.05),下调CTXⅡ(P<0.05),上调CPⅡ水平(P<0.05)。结论:电针治疗膝骨关节炎的作用机制是通过降低软骨细胞凋亡和减轻关节软骨基质破坏,进而减缓关节软骨退变。

Objective： To observe the effect of electroacupuncture on chondrocyte apoptosis and cartilage matrix of rats with knee osteoarthritis（KOA）, so as to explore its treatment mechanism on knee osteoarthritis. Methods： After one week of acclimation, 40 clean, two-month-old male Sprague Dawley（SD） rats were randomly divided into blank group（n=10） and model group（n=30）. The knee osteoarthritis rat model was induced by injecting 0.2 m L 4% papain solution on days 1, 4 and 7. After the model were successfully established, the induced knee osteoarthritis rats were randomly divided into three groups： the model group（without any treatment, n=10）, the EA 15 min group（received electroacupuncture treatment for 15 min/once/day, 5 times/week, n=10） and the EA 30 min group（received electroacupuncture treatment for 30 min/once/day, 5 times/week, n=10）. The blank group did not receive any treatment.After 8 weeks, the rats were sacrificed and blood from the abdominal aorta was obtained, and then measured the C-terminal peptide of collagenⅡ（CTX Ⅱ） and collagen Ⅱ C-propeptide（CP Ⅱ） by ELISA. The medial cartilages of right tibial plateau were obtained, and fixed in 4% paraformaldehyde, and embedded in paraffin. TUNEL was performed to assess the chondrocyte apoptosis. Toluidine blue stain and collagenⅡ immunohistochemistry stain was performed to assess the contents of proteoglycan and collagenⅡ, respectively.The cartilages of left tibial plateau were obtained, and saved in liquid nitrogen, and then measured the mRNA and protein levels of Bcl-2 and Bax by RT-PCR and Western blot, respectively. Results： Compared with the blank group, the chondrocyte apoptosis rate in the model group was significantly increased（P0.05）; the mRNA and protein expression of Bcl-2 was decreased（P0.05）, and the mRNA and protein expression of Bax was significantly increased（P0.05）. Toluidine blue stain indicated that there was proteoglycan loss in the model group. Collagen Ⅱ immunohistochemistry stain indicated that there was collagen Ⅱ loss in the model group（P0.05）. CTX Ⅱ in serum was significantly increased（P0.05）, and CPⅡ in serum was significantly decreased in the model group（P0.05）. Compared with the model group, the electroacupuncture treatment group can significantly reduce the chondrocyte apoptosis rate（P0.05）; promote the mRNA and protein expression of Bcl-2（P0.05）; reduce the mRNA and protein expression of Bax（P0.05）; increase proteoglycan and type Ⅱ collagen content（P0.05）; reduce levels of CTXⅡ and raise CPⅡ（P0.05）. Conclusion： Electroacupuncture can delay cartilage degeneration by effectively reducing chondrocyte apoptosis and articular matrix destruction of rats with knee osteoarthritis, this may explain its clinical efficacy in the treatment of KOA.