鞘内注射p38MAPK抑制剂SB203580对CCI大鼠脊髓背角多种嘌呤受体表达的影响

Influence of intrathecal injection of p38MAPK inhibitor SB203580 on the expression of multiple P2 receptors in dorsal spinal cord in CCI rats

目的:观察鞘内注射p38MAPK磷酸化抑制剂SB203580对慢性坐骨神经结扎(chronic constriction injury,CCI)大鼠热痛阈及脊髓背角P2X_4、P2X_7、P2Y_(12)、P2Y_(13)受体mRNA表达变化的影响。方法:成年SD大鼠随机分为5组(n=8):假手术组(sham组)、CCI模型组(CCI+鞘内注射0.005%DMSO生理盐水)、CCI+鞘内注射SB203580 1μmol/L组、CCI+鞘内注射SB203580 10μmol/L组、CCI+鞘内注射SB203580 50μmol/L组。CCI模型组及CCI+SB203580处理组大鼠均于鞘内置管7 d后行慢性坐骨神经结扎,连续14 d鞘内注射0.005%DMSO生理盐水或SB203580(1,10,50μmol/L),2次/d,分别在术前1 d、术后第1,3,5,7,10,14 d测定给药1 h后热缩足潜伏期(TWL);并在第3,7,14 d取大鼠脊髓背角进行荧光定量PCR,观察脊髓背角P2X_4、P2X_7、P2Y_(12)、P2Y_(13)受体mRNA的表达变化。结果:CCI术后大鼠即形成稳定的热痛敏,与sham组相比,CCI模型组大鼠术后TWL明显缩短(P<0.05);与CCI模型组相比,CCI+SB203580 10,50μmol/L处理组大鼠随药物剂量增加,TWL延长更为明显(P<0.05)。荧光定量PCR结果显示:与sham组相比,CCI模型组第3,7,14 d,P2X_4、P2X_7、P2Y_(12)、P2Y_(13)受体mRNA的表达上调(P<0.05);给予SB203580 50μmoL/L后,大鼠脊髓背角P2X_7、P2Y_(13)受体mRNA表达有所下调(P<0.05),但P2X_4和P2Y_(12)受体mRNA表达没有明显变化。结论:鞘内注射p38MAPK磷酸化抑制剂SB203580可以明显抑制CCI大鼠热痛敏行为学表现,其机制与抑制脊髓背角P2X_7、P2Y_(13)受体mRNA表达有关。这提示脊髓背角小胶质细胞p38MAPK活化后上调P2X_7和P2Y_(13)受体基因转录水平可能是p38MAPK在脊髓水平参与伤害性信息传递的机制之一。

Objective：To investigate the effect of SB203580,a selective p38 MAPK inhibitor,administered intrathecally on thermal withdrawl latency（TWL） and the gene expression of P2X_4,P2X_7,P2Y_（12） and P2Y_（13） receptor in dorsal spinal cord of rats with chronic constriction injury（CCI） of the sciatic nerve.Methods：SD rats were randomized into five groups（n = 8）：sham group（sham operation,intrathecal 0.005%DMSO in saline）,CCI group（CCI ＋ intrathecal0.005%DMSO in saline）,CCI＋SB203580 1 μmol/L group,CCI＋SB203580 10 μmol/L group,CCI ＋ SB203580 50μmol/L group.For the rats after ligation,SB203580 or 0.005%DMSO in saline（10 μl） were intrathecally administered twice a day for 14 d.TWL were evaluated before the surgey and on 1,3,5,7,10 d and 14 d after surgery.The expression change of P2X_4,P2X_7,P2Y_（12） and P2Y_（13） receptor in dorsal horn was assessed by using realtime fluorescence quantitative PCR（RTFQ-PCR） on 3,7,14 post-operative days.Results：TWL in CCI group were significantly lower than sham group on post-operative days（P〈0.05）.Repeated intrathecal administration of SB203580（10,50 μmol/L）markedly suppressed this decrease in TWL after nerve injury（P〈0.05）.RTFQ-PCR results showed that the expression of P2X_4,P2X_7,P2Y_（12） and P2Y_（13） receptors mRNA were markedly enhanced in the ipsilateral dorsal horn on days 3,7,14（P〈0.01） days after nerve injury.Compared with CCI group,after SB203580（50 μmol/L） adiministration,the expression of P2X_7 and P2Y_（13） receptors mRNA were significantly suppressed on days 3,7,14（P〈0.01）.However,Compared with CCI group,the expression of P2X_4 and P2Y_（12） receptors mRNA was not influenced after SB203580（50 μmol/L） administration.Conclusion;The intrathecal injection of p38 MAPK inhibitor SB203580 can attenuate hyperalgesia in CCI rats through decreasing P2X_7 and P2Y_（13） receptors mRNA expression,which suggests that p38 MAPK signalling plays a crucial role in neuropathic pain via the transcriptional regulation of P2X_7 and P2Y_（13） receptors in spinal microglia after nerve injury.