To explore the role of P2X4 receptor in opioid-induced hyperalgesia (OIH). Methods: A total of 30 Sprague-Dawley (SD) male rats were randomly divided into 5 groups: a saline (N0) group, a remifentanil at 0.5 μg/(kg.min) (R1) group, a remifentanil at 1.0 μg/(kg.min) (R2) group, a remifentanil at 1.5 μg/(kg.min) (R3) group, and a remifentanil at 5.0 μg/(kg.min) (R4) group. The paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were measured at follow time points to optimize the dosages: the day before treatment (T1), 30 min after tail intravenous catheterization (T2), and 30 min (T3), 1 h (T4), 2 h (T5), 24 h (T6) after withdrawal from remifentanil. Then, the rats were randomly divided into 2 groups: a saline group (N group), a remifentanil at 1.0 μg/(kg.min) group (R group). The PWMT and PWTL were measured at follow time points: T1, T2, and T4. The lumbar enlargement of spine was selected at 1 h after withdrawal from remifentanil, and the expression of P2X4 receptor mRNA and protein was examined in OIH. Additional male rats were selected and randomly divided into 2 groups: a plantar incision surgery followed by saline treatment group (I+N group), a plantar incision surgery followed by remifentanil treatment group (I+R group). The PWMT and PWTL were measured at follow time points: T1, T2, T3, T4, T5, T6, 48 h (T7) and 72 h (T8) after withdrawal from remifentanil. The lumbar enlargement of spine was selected at 1 h after withdrawal from remifentanil, the expression of P2X4 receptor mRNA and protein was examined by PCR and Western blotting, and the microglial activation in spine 1 h after withdrawal from remifentanil were assessed by immunofluorescence. Results: The pain thresholds including PWMT and PWTL in different groups were as follows: R4 group<R3 group<R2 group<R1 group at T3 and T5 (all P<0.05). At T4, the PWMT and PWTL in the R4 group were lower than those in the R2 group, while in the R4 group they were lower than those in the R3 group (both P<0.05). Meanwhile, the PWMT and PWTL in the R2 group, the R3 group and the R4 group at T4 were lower than those in the other time points (all P<0.05). Compared with the N group, the rats in the R group had a lower PWMT and PWTL at T4 (both P<0.05), but there was no significant difference in P2X4 receptor protein and mRNA expression in the lumbar enlargement of spine between the R group and the N group. Compared with the I+N group, the pain threshold including PWMT and PWTL at T4, T5, and T6 in the I+R group was significantly decreased (all P<0.05). Meanwhile, compared with the I+N group, the expressions of P2X4 receptor mRNA and protein in rat spinal cord in the I+R group were significantly up-regulated, and the microglia was activated (all P<0.05). Conclusion: Remifentanil pumped via tail intravenous can result in the hyperalgesia, but the spinal P2X4 receptor might be not relevant to remifentanil-induced hyperalgesia. Remifentanil incision pain can cause significant hyperalgesia in rats, and the spinal P2X4 receptors and microglial activation is involved in the remifentanil-induced postoperative hyperalgesia.
目的：探究P2X4受体在阿片类药物诱导的痛觉过敏中的作用。方法：成年雄性Sprague-Dawley(SD)大鼠随机分为尾静脉泵注生理盐水组(N0组)、瑞芬太尼0.5 μg/(kg.min)组(R1组)、瑞芬太尼1.0 μg/(kg.min)组(R2组)、瑞芬太尼1.5 μg/(kg.min)组(R3组)、瑞芬太尼5.0 μg/(kg.min)组(R4组)。于处理前1 d(T1)，尾静脉置管后30 min(T2)，停药后30 min(T3)，1 h(T4)，2 h(T5)，24 h(T6)测量大鼠机械缩足阈值(paw withdrawal mechanical threshold，PWMT)和热缩足潜伏期(paw withdrawal thermal latency，PWTL)，获得诱导痛觉过敏最佳的瑞芬太尼输注速度；随后取成年雄性SD大鼠，分为尾静脉置管后泵入生理盐水组(N组)、尾静脉置管后泵入瑞芬太尼1.0 μg/(kg.min)组(R组)。于上述处理时间点T1，T2，T4测量大鼠PWMT和PWTL，并于停药后1 h选取大鼠脊髓腰膨大检测P2X4受体的mRNA和蛋白表达。另取成年雄性SD大鼠，并将其分为切口痛模型并尾静脉置管后泵入生理盐水组(I+N组)和切口痛模型并尾静脉置管后泵入瑞芬太尼1.0 μg/(kg.min)组(I+R组)。于上述处理时间点T1，T2，T3，T4，T5，T6以及8 h(T7)和72 h(T8)测量大鼠PWMT和PWTL，于停药后1 h取大鼠脊髓腰膨大组织检测P2X4受体的mRNA和蛋白表达，免疫荧光检测小胶质细胞激活的状态。结果：T3和T5时间点PWMT和PWTL的趋势均为R4组<R3组<R2组<R1组，差异均有统计学意义(均P<0.05)；T4时间点R2，R3，R4组的PWMT和PWTL趋势为R4组<R2组、R4组<R3组，差异均有统计学意义(均P<0.05)；R2，R3，R4组在T4时间点的PWMT和PWTL低于其他时间点，差异均有统计学意义(均P<0.05)。与N组相比，R组大鼠T4时间点的PWMT和PWTL均明显下降，差异均有统计学意义(均P<0.05)；但R组与N组两组大鼠脊髓腰膨大的P2X4受体蛋白和mRNA表达差异无统计学意义(均P>0.05)。与I+N组相比，I+R组大鼠在T4，T5，T6时间点的PWMT和PWTL明显降低，差异均有统计学意义(均P<0.05)，同时I+R组大鼠脊髓P2X4受体mRNA和蛋白表达水平均明显上调，差异均有统计学意义(均P<0.05)。此外，I+R组大鼠脊髓背角的小胶质细胞的活化较I+N组明显增强。结论：静脉注射瑞芬太尼可致大鼠痛觉过敏，单纯静脉注射瑞芬太尼导致的痛觉过敏可能与P2X4受体无关；瑞芬太尼可导致切口痛大鼠出现更明显的痛觉过敏，其中小胶质细胞活化及P2X4受体可能参与瑞芬太尼诱发术后痛觉过敏的形成机制。.