基于PI3K/Akt信号通路探讨电针对膝骨关节炎模型大鼠软骨细胞凋亡的影响

Effect of Electroacupuncture on Chondrocyte Apoptosis in Knee Osteoarthritis Rat Based on PI3K/Akt Signaling Pathway

目的:基于PI3K/Akt信号通路探讨电针内、外膝眼对膝骨关节炎模型大鼠软骨细胞凋亡的影响。方法:选择体质量(200±20) g的8周龄雄性SD大鼠50只,采用随机数字表法分为假手术组、模型组、实验1组、实验2组、实验3组。适应性饲养2周后,假手术组于关节内侧切口,逐层分离肌肉、韧带,暴露关节腔后缝合,其余4组采用改良Hulth法复制膝骨关节炎大鼠模型。假手术组与模型组不予电针干预;实验1组取双侧内、外膝眼直刺3~5 mm,连接电针15 min;实验2组取双侧内、外膝眼直刺3~5 mm,连接电针30min;实验3组取非经非穴向上斜刺3~5 mm,连接电针15 min。3个实验组均每日1次,每周5次,共干预12周。电针参数:选用疏密波模式,其中疏波2 Hz,密波10 Hz;脉冲宽度≤1 ms;输出电压≤10 V;耐受度以局部肌肉开始轻微抖动,大鼠不挣扎为度。干预12周后,取大鼠关节软骨组织和滑膜,采用苏木素-伊红染色及甲苯胺蓝染色法,光镜下观察关节软骨组织形态学改变,改良Mankin’s评分标准评价软骨退变程度;采用酶联免疫吸附实验(ELISA)检测滑膜白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)含量;采用实时荧光定量PCR与蛋白质印迹法(Western blot)检测PI3K、Akt、Bcl-2、Bax的基因和蛋白表达。结果:与假手术组比较,模型组大鼠软骨形态结构紊乱,软骨细胞数量明显减少,基质失染,潮线扭曲且不完整;Mankin’s评分明显升高(P<0.01);滑膜中IL-1β、TNF-α含量明显升高(P<0.01);PI3K、Akt、Bcl-2的基因与蛋白表达均明显降低(P<0.01),Bax的基因和蛋白表达明显升高(P<0.01)。与模型组比较,实验1组、实验2组大鼠软骨形态结构较整齐,软骨细胞数量增多,基质染色加深,潮线较完整;Mankin’s评分明显降低(P<0.01);滑膜中IL-1β、TNF-α含量明显降低(P<0.01);PI3K、Akt、Bcl-2的基因与蛋白表达明显升高(P<0.01),Bax的基因和蛋白表达明显降低(P<0.01)。实验3组形态结构未见改善,各项指标检查结果差异无统计学意义(P>0.05)。结论:电针内、外膝眼可通过调节PI3K/Akt信号通路提高Bcl-2表达,降低Bax表达,减少炎症因子IL-1β、TNF-α生成,从而抑制软骨基质的降解和软骨细胞的凋亡,保护并改善软骨形态结构,延缓膝骨关节炎的发展进程。

Objective:To investigate the effect of electroacupuncture on chondrocyte apoptosis in knee osteoarthritis(KOA)rat based on PI3 K/Akt signaling pathway.Methods:A total of 50 male Sprague Dawley(SD)rats,aged eight weeks,weighing(200±20)g were selected and randomly divided into sham operation group,model group,experiment group 1,experiment group 2 and experiment group 3.After two weeks of adaptive feeding,the sham operation group received incision in the medial aspect of the joint,the muscles and ligaments were separated layer by layer,the joint cavity was exposed and then suturing.Rat models were replicated in the other four groups by the method of modified Hulth.The sham operation group and the model group did not receive electroacupuncture intervention.In the experimental group 1,the bilateral internal and external knee eyes of rats were directly punctured 3-5 mm,once a day,15 minutes each time.In the experimental group 2,the bilateral internal and external knee eyes of rats were directly punctured 3-5 mm,once a day,30 minutes each time.In the experimental group 3,points(non-meridian and non-acupoint)of rats were obliquely pricked upward for 3-5 mm,once a day,15 minutes each time.The three experimental groups received electroacupuncture for 12 weeks,5 times a week.Electroacupuncture parameters:dilatational wave,frequency of sparse wave 2 Hz and dense wave 10 Hz,pulse width less than 1 ms,output voltage less than 10 V,and the extent was that the local muscles began to shake slightly and the rats did not struggle.After 12 weeks of electroacupuncture intervention,the articular cartilage and synovium were harvested,and the changes of histomorphology of articular cartilage were observed under light microscope by hematoxylin-eosin staining and toluidine blue staining.The degree of cartilage degeneration was evaluated by modified Mankin’s scoring standard.The contents of interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)in synovium were detected by immunosorbent assay(ELISA).The expression of PI3 K,Akt,Bcl-2,Bax m RNA and protein were detected by real-time quantitative PCR and Western blot.Results:Compared with the sham operation group,the cartilage morphology of the model group was disordered,the number of chondrocytes was significantly reduced,the matrix staining was lost,the tidal line was distorted and incomplete,Mankin’s score was significantly increased(P<0.01),the expressions of IL-1βand TNF-αin the synovium were significantly increased(P<0.01),the expressions of PI3 K,Akt and Bcl-2 m RNA and protein were significantly decreased(P<0.01),and the expression of Bax m RNA and protein were significantly increased(P<0.01).Compared with the model group,the cartilage morphology of the experimental group 1 and the experimental group 2 was neat,the number of chondrocytes was increased,the matrix staining was deepened,the tidal line was complete,Mankin’s score was significantly decreased(P<0.01),the expressions of IL-1βand TNF-αin the synovium were significantly decreased(P<0.01),the expressions of PI3 K,Akt and Bcl-2 m RNA and protein were significantly increased(P<0.01),and the expression of Bax m RNA and protein were significantly decreased(P<0.01).The morphological structure of the experimental group 3 was not improved,and there was no significant difference in the results of the indicators(P>0.05).Conclusion:Electroacupuncture of the bilateral internal and external knee eyes can improve the expression of Bcl-2,decrease the expression of Bax,and reduce the production of inflammatory factors IL-1βand TNF-αby regulating PI3 K/Akt signaling pathway,thereby inhibiting the degradation of cartilage matrix and apoptosis of chondrocytes,protecting and improving the morphology of cartilage,and delaying the development of knee osteoarthritis.