脉冲电磁场对椎间盘退行性病变大鼠A2A腺苷受体及p38 MAPK信号通路的影响

Pulsed electromagnetic field activation of the A2A adenosine receptors can retard intervertebral disc degeneration

目的观察脉冲电磁场(PEMF)对椎间盘退行性病变(IDD)大鼠A2A腺苷受体(A2AR)及p38 MAPK信号通路的影响。方法采用随机数字表法将40只SD大鼠分为对照组、IDD模型组(简称模型组)、PEMF组和PEMF联合CGS-21680治疗组(简称观察组)。将模型组、PEMF组及观察组大鼠制成IDD动物模型,PEMF组于制模后给予PEMF干预,观察组则给予PEMF干预并注射A2AR激动剂CGS-21680。于造模8周后采用番红O-快绿染色评估各组大鼠椎间盘病理改变,同时检测各组大鼠椎间盘A2AR、环磷酸腺苷(cAMP)、蛋白激酶A(PKA)、半胱氨酸天冬氨酸蛋白水解酶-3(Caspase-3)、Ⅱ型胶原(Col-Ⅱ)、基质金属蛋白酶-3(MMP3)表达水平。结果模型组大鼠髓核组织皱缩,纤维成分及软骨细胞增多,观察组大鼠髓核形态基本趋于正常,纤维环完整无破裂。模型组椎间盘组织A2AR蛋白表达及mRNA水平均高于对照组,观察组A2AR蛋白表达及mRNA水平均显著高于其他各组(P<0.05)。PEMF组和观察组椎间盘cAMP含量及PKA mRNA表达均较模型组增高,且观察组与模型组间差异具有统计学意义(P<0.05)。模型组大鼠椎间盘p38 MAPK、p-p38 MAPK含量及p-p38 MAPK/p38 MAPK比值均显著高于对照组(P<0.05),PEMF组和观察组p38 MAPK、p-p38 MAPK蛋白含量及p-p38 MAPK/p38 MAPK比值均有不同程度降低,且观察组上述指标数值均显著低于模型组(P<0.05),p-p38 MAPK蛋白含量及p-p38 MAPK/p38 MAPK比值亦显著低于PEMF组(P<0.05)。模型组大鼠Caspase-3蛋白含量及mRNA表达均显著高于对照组,PEMF组及观察组上述指标含量均较模型组明显降低(P<0.05)。模型组大鼠MMP3含量较对照组显著升高,Col-Ⅱ含量则明显下降;PEMF组、观察组MMP3含量均较模型组降低,Col-Ⅱ表达均较模型组增高,并且观察组上述指标含量与模型组间差异均具有统计学意义(P<0.05)。结论炎性因子刺激能活化p38 MAPK信号通路并诱导细胞凋亡,这也是促进IDD大鼠病变的重要原因之一。PEMF联合A2AR激动剂干预能激活A2AR/cAMP/PKA信号通路,进而抑制p38 MAPK磷酸化,减少髓核细胞凋亡,缓解IDD损伤。

Objective To explore any effect of pulsed electromagnetic field(PEMF)stimulation on intervertebral disc degeneration(IDD).Methods Forty Sprague-Dawley rats were randomly divided into a control group,an IDD model group,a PEMF group and an observation group.An IDD model was induced in all except those in the control group.Both the PEMF and observation groups were given PEMF stimulation,while the latter was additionally injected with the A2AR agonist CGS-21680.Eight weeks after the modelling any pathological changes in the morphology of the rats'intervertebral disc tissues were evaluated using saffron solid green staining.The expression of A2AR,cyclic adenosine phosphate(cAMP),protein kinase A(PKA),cysteine aspartate proteolytic enzyme-3(Caspase-3),type Ⅱ collagen(COL-Ⅱ)and matrix metalloproteinase-3(MMP3)in the intervertebral discs were evaluated.Results The nucleus pulposus had shrunk,while fibrous tissues and chondrocytes had increased in the IDD model group.In the observation group the nucleus pulposus was intact and of basically normal shape.A2AR mRNA and protein levels were higher in the intervertebral disc tissue of the model group than among the control group,on average,while the levels in the observation group were significantly higher than in the other groups.In the PEMF and observation groups cAMP and PKA mRNA were significantly higher than in the IDD model group.The p38 MAPK and P-P38 MAPK levels of the IDD model group and its average P-P38 MAPK/p38 MAPK ratio were significantly higher than in the control group.In the PEMF and observation groups those indices had decreased to varying degrees,with those of the observation group significantly lower than among the model and PEMF groups on average,except for the p38 MAPK values.Caspase-3 and its mRNA were significantly higher in the model group than in the control group,on average,and those values were significantly lower in the PEMF and observation groups than in the IDD model group.The average MMP3 contents of the IDD model group had increased significantly compared with the control group,while the Col-Ⅱ level had decreased significantly.Compared with the IDD model group,the MMP3 level had decreased but Col-Ⅱ expression had increased in both the PEMF and observation groups,with significant differences between the IDD model and observation groups.Conclusions The activation of the p38 MAPK signaling pathway by inflammatory factors to induce apoptosis is one of the important reasons for the aggravation of IDD lesions.PEMF combined with A2AR agonists can activate the A2AR/cAMP/PKA signaling pathway,inhibit p38 MAPK phosphorylation,reduce apoptosis of nucleus pulposus cells,and relieve IDD damage.