Laboratory and Clinical Research Institute for Pain, Department of Anaesthesiology, The University of Hong Kong, Hong Kong, China.
Laboratory and Clinical Research Institute for Pain, Department of Anaesthesiology, The University of Hong Kong, Hong Kong, China.
Neurosci Lett. 2019 Sep 14;709:134379. doi: 10.1016/j.neulet.2019.134379. Epub 2019 Jul 16.
Deficiency of deleted in liver cancer 2 (DLC2), a novel domain to inhibit RhoA activity, plays an important role in inflammatory pain. However, the underlying mechanisms remain unclear. This study investigated the role of DLC2 and its downstream cascade of RhoA/ROCK in formalin-induced inflammatory pain using DLC2-knockout (DLC2) mice and compared them with DLC2 wild-type (DLC2) mice. Mechanical allodynia and thermal hyperalgesia were evaluated using von Frey filament aesthesiometer and Hargreaves test, respectively. The spinal cord dorsal horn (L3-L5) was selected for molecular and cellular identification by Western blot and immunofluorescence. DLC2 mice showed increased mechanical allodynia and thermal hyperalgesia. Expression of ROCK1, ROCK2 and IL-1β was significantly higher in DLC2 mice. Intrathecal administration of RhoA inhibitor (C3 exoenzyme) or ROCK inhibitor (Y27632) significantly attenuated formalin-induced inflammatory hyperalgesia in DLC2 mice. ROCK2 and IL-1β expression were reduced by C3 exoenzyme or Y27632. Spinal p38 activation was also inhibited by C3 exoenzyme or Y27632. Double-labelling immunofluorescence demonstrated co-localization of DLC2 with spinal dorsal microglia. The number of activated microglia in the spinal dorsal horn was significantly higher in DLC2 mice, but was reduced by Y27632. These findings indicate that DLC2 deficiency increased formalin-induced inflammatory hyperalgesia through regulating RhoA/ROCK2, and IL-1β may be a downstream effector. Our results also suggest that RhoA/ROCK enhanced p38 activation plays an important role in formalin-induced inflammatory pain. The finding that DLC2 attenuated inflammatory pain through inhibiting RhoA/ROCK2 suggests that the DLC2/RhoA/ROCK2/p38/IL-β pathway may be a potential therapeutic target to reduce inflammatory pain.
肝癌缺失基因 2（DLC2）缺失，一种新型的抑制 RhoA 活性的结构域，在炎性疼痛中发挥重要作用。然而，其潜在机制尚不清楚。本研究利用 DLC2 敲除（DLC2）小鼠和 DLC2 野生型（DLC2）小鼠，研究 DLC2 及其下游 RhoA/ROCK 级联在福尔马林诱导的炎性疼痛中的作用，并进行比较。采用 von Frey 纤维测痛仪和 Hargreaves 测试分别评估机械性痛觉过敏和热痛觉过敏。通过 Western blot 和免疫荧光法选择脊髓背角（L3-L5）进行分子和细胞鉴定。DLC2 小鼠表现出机械性痛觉过敏和热痛觉过敏增加。DLC2 小鼠中 ROCK1、ROCK2 和 IL-1β 的表达明显升高。鞘内给予 RhoA 抑制剂（C3 外毒素）或 ROCK 抑制剂（Y27632）可显著减轻 DLC2 小鼠福尔马林诱导的炎性痛觉过敏。C3 外毒素或 Y27632 可降低 ROCK2 和 IL-1β 的表达。C3 外毒素或 Y27632 还抑制脊髓 p38 的激活。双标免疫荧光显示 DLC2 与脊髓背角小胶质细胞共定位。DLC2 小鼠脊髓背角激活的小胶质细胞数量明显增加，而 Y27632 可减少小胶质细胞数量。这些发现表明，DLC2 缺失通过调节 RhoA/ROCK2 增加了福尔马林诱导的炎性痛觉过敏，而 IL-1β 可能是下游效应物。我们的研究结果还表明，RhoA/ROCK 增强 p38 的激活在福尔马林诱导的炎性疼痛中起重要作用。DLC2 通过抑制 RhoA/ROCK2 减轻炎性疼痛的发现表明，DLC2/RhoA/ROCK2/p38/IL-β 通路可能是减少炎性疼痛的潜在治疗靶点。
