室旁核中嘌呤能受体P2X2参与腰椎间盘突出症模型大鼠痛觉高敏的机制

Mechanism of purine receptor P2X2 in the paraventricular nucleus involved in hypersensitivity in lumbar disc herniation model rats

目的探讨腰椎间盘突出症(LDH)模型大鼠室旁核中嘌呤能受体P2X2参与疼痛中枢敏化的机制。方法采用简单随机分组将大鼠40只构建LDH模型组(20只)和假手术组(sham组,20只);运用免疫荧光技术检测室旁核(PVN)中原癌蛋白(c-fos)、P2X2与神经元共表达情况,蛋白质印迹法(Western blot)实验测定不同组别之间室旁核(PVN)中P2X2蛋白表达差异性变化;通过脑立体定位PVN注射P2X2小干扰RNA(siRNA),观察LDH大鼠疼痛行为的变化。两样本间的比较采用t检验。结果免疫荧光结果显示为LDH组大鼠PVN脑区神经元细胞c-Fos表达高于sham组[(23.20±3.21)%比(6.70±1.29)%,t=36.020,P<0.01,n=3],实时定量反转录聚合酶链反应(RT-qPCR)和Western blot结果提示在LDH大鼠PVN中P2X2的mRNA[(2.280±0.605)%比(0.518±0.278)%,t=5.293,P<0.01]和蛋白表达量[(1.419±0.432)%比(0.041±0.034)%,t=6.354,P<0.01]均高于sham组。脑立体定位PVN注射P2X2-siRNA,P2X2-siRNA组大鼠机械疼痛阈值高于NC-siRNA组(4.887±3.079比3.075±1.402,t=1.328,P<0.05)。结论LDH诱导PVN中嘌呤受体P2X2蛋白水平表达增加,产生中枢敏化,最终引发病理性疼痛。

Objective To investigate the mechanism of purine receptor P2X2 in paraventricular nucleus(PVN)involved in hypersensitivity in rats with lumbar disc herniation(LDH).Methods The rat LDH model group(n=20)and sham group(n=20)were established.The co-expression of c-fos,P2X2 and neurons in the PVN was detected by immunofluorescence technology,and the difference in P2X2 protein expression in the PVN between two groups was detected by Western blotting.LDH rats were injected with P2X2 small interfering RNA(siRNA)by stereotaxic PVN to observe the changes of pain behavior.The t-test was used for comparison between the two groups.Results Immunohistochemical results showed that the expression of c-Fos in neurons in the PVN brain area of rats in the LDH group was higher than that in the sham group.The results of real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR)[(2.280±0.605)%vs.(0.518±0.278)%,t=5.293,P<0.01]and Western blotting[(1.419±0.432)%vs.(0.041±0.034)%,t=6.354,P<0.01]showed that the expression of P2X2 mRNA and protein in the PVN of LDH rats was higher than that in the sham group.After stereotactic injection of P2X2-siRNA into the PVN in the brain,the rats in the P2X2-siRNA group had a higher mechanical pain threshold than those in the NC-siRNA group(4.887±3.079 vs.3.075±1.402,t=1.328,P<0.05).Conclusion LDH induces increased expression of purine receptor P2X2 protein in PVN,resulting in central sensitization,and ultimately pathologic pain.