低强度脉冲声波对大鼠骨关节炎软骨细胞凋亡的作用及机制研究

Effect and mechanism of low intensity pulsed acoustic wave on apoptosis of chondrocyte in osteoarthritis rats

目的探讨低强度脉冲声波(low intensity pulsed ultrasound,LIPUS)对大鼠骨关节炎(osteoarthritis,OA)中软骨细胞凋亡的作用及作用机制。方法健康雄性SD大鼠40只,随机数字表法分为4组(各10只):假手术组(Sham)、模型组(OA)、30 mW/cm^2组、60 mW/cm^2组。Sham组仅切开关节囊;OA组接受右侧后肢内侧半月板撕裂(MMT)处理;30 mW/cm^2组及60 mW/cm^2组接受与OA组同样手术处理,术后3 d接受低强度脉冲声波治疗,除治疗强度不同外,其他治疗参数均一致。治疗4周后,处死实验大鼠,取右后肢胫骨平台。Elisa检测各组软骨组织中IL-1β、IL-6及TNF-α水平;番红固绿染色评估各组胫骨平台关节软骨病理学改变;TUNEL染色检测各组软骨组织凋亡水平;RT-PCR及免疫组化检测各组软骨组织Bax、Caspase-3表达水平;Western blot法检测各组软骨组织p53蛋白表达水平。结果番红固绿染色表明,行MTT法处理后,OA组较Sham组胫骨平台关节软骨损伤明显加重,关节软骨排列紊乱,软骨表面严重不平整,软骨严重丢失,经LIPUS治疗后,关节软骨损伤减轻,关节软骨排列紊乱减轻,软骨表面不平整程度减轻,且与30 mW/cm^2组相比,60 mW/cm^2组治疗效果更佳。TUNEL染色表明,LIPUS可抑制软骨细胞凋亡;与Sham组比较,OA组IL-1β、IL-6、TNF-α、Bax、Caspase-3及p53水平明显升高(P<0.05);与OA组比较,低强度脉冲声波治疗组(30 mW/cm^2组及60 mW/cm^2组)IL-1β、IL-6、TNF-α、Bax、Caspase-3及p53水平明显下降(P<0.05);与30 mW/cm^2组比较,60 mW/cm^2组IL-1β、IL-6、TNF-α、Bax、Caspase-3及p53水平更低。结论LIPUS可通过抑制促凋亡细胞因子、调控凋亡基因表达及相关信号转导等机制减轻软骨细胞凋亡。

Objective To investigate the effect and mechanism of low intensity pulsed ultrasound(LIPUS)on chondrocyte apoptosis in osteoarthritis(OA)rats.Methods:40 healthy male SD rats were randomly divided into 4 groups(10 rats per group):sham group(Sham),model group(OA),30 mW/cm^2 group and 60 mW/cm^2 group.In Sham group,only the switch capsule was cut off.In OA group,the right posterior medial meniscus tear(MMT)was treated.In 30 mW/cm^2 group and 60 mW/cm^2 group,the same surgical treatment was performed as OA group,and after 3 days,the treatment parameters were the same except for the different intensity of treatment.After 4 weeks of treatment,the rats were executed and the tibial plateau of the right hind limb was taken.The levels of IL-1beta,IL-6 and TNF-alpha in cartilage tissue of each group were detected by Elisa,and the pathological changes of tibial plateau articular cartilage were evaluated by safranine O staining.Also,the apoptotic level of cartilage tissue was detected by TUNEL staining.The expression levels of Bax and Caspase-3 in cartilage tissue of each group were detected by RT-PCR and immunohistochemistry,and the expression level of p53 protein in cartilage tissue of each group was detected by Western blot.Result:Safranine O staining showed that the tibial plateau articular cartilage injury in OA group was significantly worse than that in Sham group.The disorder of articular cartilage arrangement,the serious irregularity of cartilage surface and the serious loss of cartilage were observed.After LIPUS treatment,the damage of articular cartilage,the disorder of articular cartilage arrangement and the degree of cartilage surface irregularity were alleviated.Compared with 30 mW/cm^2 group,the treatment effect in 60 mW/cm^2 group is better.TUNEL staining showed that LIPUS could inhibit chondrocyte apoptosis.Compared with Sham group,the levels of IL-1beta,IL-6,TNF-alpha,Bax,Caspase-3 and p53 in OA group were significantly higher(P<0.05).Besides,compared with OA group,the levels of IL-1beta,IL-6,TNF-alpha,Bax,Caspase-3 and p53 in low intensity pulsed sound wave treatment group(30 mW/cm^2 group and 60 mW/cm^2 group)were significantly lower(P<0.05).Compared with 30 mW/cm^2 group,the levels of IL-1beta,IL-6,TNF-alpha,Caspase-3 and p53 were significantly lower in 60 mW/cm^2 group.Conclusion:LIPUS can alleviate chondrocyte apoptosis by inhibiting apoptotic cytokines,regulating apoptotic gene expression and related signal pathway.