Department of Physiology, Zunyi Medical College, Zunyi, Guizhou province, 563006, China.
J Neuroinflammation. 2017 Sep 12;14(1):185. doi: 10.1186/s12974-017-0960-0.
More evidence suggests that dorsal spinal cord microglia is an important site contributing to CB2 receptor-mediated analgesia. The upregulation of P2Y and P2Y purinoceptors in spinal dorsal horn microglia is involved in the development of pain behavior caused by peripheral nerve injury. However, it is not known whether the expression of P2Y and P2Y receptors at spinal dorsal horn will be influenced after CB2 receptor activation in neuropathic pain rats.
Chronic constriction injury (CCI) and intrathecal ADPbetaS injection were performed in rats to induce neuropathic pain. The paw withdrawal latency (PWL) was used to evaluate thermal hyperalgesia in neuropathic rats. The expression of P2Y and P2Y receptors, p-p38MAPK, and NF-kappaBp65 was detected with RT-PCR and western blotting analysis.
Treatment with AM1241 produces a pronounced inhibition of CCI-induced thermal hyperalgesia and significantly inhibited the increased expression of P2Y and P2Y receptors at the mRNA and protein levels, which open up the possibility that P2Y and P2Y receptor expression are downregulated by CB2 receptor agonist AM1241 in CCI rats. Western blot analysis demonstrated that AM1241 reduced the elevated expression of p-p38MAPK and NF-κBp65 in the dorsal spinal cord induced by CCI. After administration with either SB203580 (p38MAPK inhibitor) or PDTC (NF-kappaB inhibitor), the levels of P2Y receptor expression in the dorsal spinal cord were lower than those in the CCI group. However, in CCI rats, the increased expression of P2Y receptor was prevented by intrathecal administration of PDTC but not by SB203580. In addition, minocycline significantly decreased the increased expression of P2Y and P2Y receptors. The similar results can be observed in ADPbetaS-treated rats. Intrathecal injection of ADPbataS causes thermal hyperalgesia and increased expression of P2Y and P2Y receptors in the dorsal spinal cord of naive rats. Moreover, intrathecal injection of AM1241 alleviates pain response and reduces the elevated expression of P2Y and P2Y receptors, p-p38MAPK, and NF-κBp65 in the dorsal spinal cord of ADPbetaS-treated rats. Intrathecal injection of SB203580 significantly inhibited the ADPbetaS-induced P2Y receptor expression, without affecting P2Y receptor expression. However, treatment with either SB203580 or PDTC effectively inhibited P2Y receptor expression compared to ADPbetaS-treated rats.
In CCI- and ADPbetaS-treated rats, AM1241 pretreatment could efficiently activate CB2 receptor, while inhibiting p38MAPK and NF-kappaB activation in the dorsal spinal cord. CB2 receptor stimulation decreased P2Y receptor expression via p38MAPK/NF-kappaB signaling. On the other hand, CB2 receptor activation decreased P2Y receptor expression via p38MAPK-independent NF-kappaB signaling pathway.
越来越多的证据表明，背根脊髓小胶质细胞是参与 CB2 受体介导的镇痛的重要部位。脊髓背角小胶质细胞中 P2Y 和 P2Y 嘌呤能受体的上调参与了外周神经损伤引起的疼痛行为的发展。然而，在神经病理性疼痛大鼠中，CB2 受体激活后，脊髓背角 P2Y 和 P2Y 受体的表达是否会受到影响尚不清楚。
在大鼠中进行慢性缩窄性损伤（CCI）和鞘内 ADPβS 注射，以诱导神经病理性疼痛。用足底缩足潜伏期（PWL）评估神经病理性大鼠的热痛觉过敏。用 RT-PCR 和 Western blot 分析检测 P2Y 和 P2Y 受体、p-p38MAPK 和 NF-κBp65 的表达。
用 AM1241 治疗可显著抑制 CCI 诱导的热痛觉过敏，并显著抑制 P2Y 和 P2Y 受体在 mRNA 和蛋白水平上的表达增加，这表明 P2Y 和 P2Y 受体的表达可能通过 CB2 受体激动剂 AM1241 在 CCI 大鼠中下调。Western blot 分析表明，AM1241 降低了 CCI 诱导的脊髓背角 p-p38MAPK 和 NF-κBp65 的升高表达。用 SB203580（p38MAPK 抑制剂）或 PDTC（NF-κB 抑制剂）给药后，脊髓背角 P2Y 受体的表达水平低于 CCI 组。然而，在 CCI 大鼠中，鞘内给予 PDTC 可阻止 P2Y 受体表达的增加，但 SB203580 则不能。此外，米诺环素可显著降低脊髓背角 P2Y 和 P2Y 受体的表达增加。在 ADPβS 处理的大鼠中也可以观察到类似的结果。鞘内注射 ADPβtaS 可引起大鼠脊髓背角热痛觉过敏和 P2Y 和 P2Y 受体表达增加。此外，鞘内注射 AM1241 可减轻疼痛反应，并降低 ADPβS 处理大鼠脊髓背角升高的 P2Y 和 P2Y 受体、p-p38MAPK 和 NF-κBp65 的表达。鞘内注射 SB203580 可显著抑制 ADPβS 诱导的 P2Y 受体表达，而不影响 P2Y 受体表达。然而，与 ADPβS 处理的大鼠相比，SB203580 或 PDTC 治疗可有效抑制 P2Y 受体的表达。
在 CCI 和 ADPβS 处理的大鼠中，AM1241 预处理可有效激活 CB2 受体，同时抑制脊髓背角 p38MAPK 和 NF-κB 的激活。CB2 受体的刺激通过 p38MAPK/NF-κB 信号通路降低 P2Y 受体的表达。另一方面，CB2 受体的激活通过 p38MAPK 非依赖性 NF-κB 信号通路降低 P2Y 受体的表达。
