BACKGROUND Neuropathic pain is a debilitating status that is insusceptible to the existing analgesics. It is important to explore the underlying pathophysiological changes and search for new pharmacological approaches. Transient receptor potential canonical 6 (TRPC6) is a mechanosensitive channel that is expressed by dorsal root ganglia and glial cells. It has been demonstrated that this channel in dorsal root ganglia plays essential roles in the formation of mechanical hyperalgesia in neuropathic pain. Recent pharmacological screening suggests that larixyl acetate (LA), a main constituent of larch resin, is able to selectively inhibit TRPC6 function. But whether LA is effective in treating neuropathic pain remains unknown. We investigated the efficacy of LA in rat neuropathic pain model, examined its effects on central neuroinflammation, and explored the possible molecular mechanisms by targeting the spinal dorsal horn. METHODS Spared nerve injury (SNI) was conducted in Sprague-Dawley rats. Mechanical hypersensitivity and cold allodynia before and after single and multiple i.t. applications of LA at the dose of 3, 10, and 30 μM were evaluated by von Frey filament and acetone tests, respectively. Western blot, immunohistochemical, and immunocytochemical stainings were employed to examine the level and expression feature of ionized calcium-binding adaptor molecule 1 (Iba-1), glial fibrillary acidic protein (GFAP), TRPC6, and phosphorylated p38 kinase. The changes of cytokine concentrations, including that of TNF-α, IL-1β, IL-6, and IL-10, were also assessed by multiplex analysis. TRPC6 antisense strategy was finally adopted to investigate the action mechanisms of LA. RESULTS Single application of LA on day 5 post injury caused dose-dependent inhibition of mechanical allodynia with the ED50 value of 13.43 μM. Multiple applications of LA at 30 μM not only enhanced the analgesic efficacy but also elongated the effective duration without obvious influences on animal locomotor activities. Single and multiple administrations of LA at 30 μM played similar but weaker inhibitory effects on cold allodynia. In addition to behavioral improvements, multiple applications of LA for 6 days dose-dependently inhibited the upregulation of Iba-1, TNF-α, IL-1β, and IL-6, whereas had no obvious effects on the levels of GFAP and IL-10. Combined Western blot and immunostaining assays revealed that the expression of TRPC6 was significantly increased in both spinal dorsal horn after nerve injury and the cultured microglia challenged by LPS, which was however suppressed by the addition of LA at 30 μM or 10 μM, respectively. Further knockdown of TRPC6 with antisense oligodeoxynucleotide produced prominent analgesic effects in rats with SNI, accompanied by the reduced phosphorylation level of p38 in the microglia. CONCLUSIONS These data demonstrate that i.t. applied LA exhibits analgesic and anti-inflammatory action in neuropathic pain. The action of LA involves the suppression of TRPC6 and p38 signaling in the microglia. LA may be thus a promising pharmacological candidate for the treatment of intractable chronic pain.

中文翻译：

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一种有效的TRPC6抑制剂醋酸拉瑞酯在由神经损伤引起的大鼠神经性疼痛模型中的镇痛作用。

背景技术神经性疼痛是对现有止痛药不敏感的使人衰弱的状态。重要的是探索潜在的病理生理变化并寻找新的药理方法。瞬时受体电位规范6（TRPC6）是一种机械敏感通道，由背根神经节和神经胶质细胞表达。已经证实，背根神经节中的该通道在神经性疼痛中的机械性痛觉过敏的形成中起重要作用。最近的药理学筛选表明，落叶松树脂的主要成分乙酸拉尼酯（LA）能够选择性抑制TRPC6的功能。但是，LA是否能有效治疗神经性疼痛尚不清楚。我们研究了LA在大鼠神经性疼痛模型中的功效，研究了其对中枢神经炎症的影响，并探讨了针对脊髓背角的可能分子机制。方法在Sprague-Dawley大鼠中进行稀疏神经损伤（SNI）。通过von Frey灯丝和丙酮测试分别评估了以3、10和30μM的剂量单次或多次施用LA前后的机械性超敏反应和冷异常性疼痛。Western印迹，免疫组化和免疫细胞化学染色被用来检查离子钙结合适配器分子1（Iba-1），胶质纤维酸性蛋白（GFAP），TRPC6和磷酸化的p38激酶的水平和表达特征。还通过多重分析评估了细胞因子浓度的变化，包括TNF-α，IL-1β，IL-6和IL-10的浓度。最终采用TRPC6反义策略研究了LA的作用机制。结果损伤后第5天单次应用LA引起机械异常性疼痛的剂量依赖性抑制，ED50值为13.43μM。30μM的LA的多次施用不仅增强了止痛效果，而且延长了有效持续时间，而对动物运动活性没有明显影响。30μM的LA的单次和多次给药对冷异常性疼痛的抑制作用相似但较弱。除了行为改善外，连续6天多次应用LA剂量依赖性抑制Iba-1，TNF-α，IL-1β和IL-6的上调，而对GFAP和IL-6的水平无明显影响10。结合Western印迹和免疫染色试验表明，神经损伤后脊髓背角和培养的小胶质细胞受到LPS攻击后，TRPC6的表达均显着增加，但是分别加入30μM或10μM的LA抑制了TRPC6的表达。用反义寡脱氧核苷酸进一步敲除TRPC6在SNI大鼠中产生了明显的镇痛作用，并伴随着小胶质细胞中p38磷酸化水平的降低。结论这些数据表明，其应用LA在神经性疼痛中显示出止痛和抗炎作用。LA的作用涉及小胶质细胞中TRPC6和p38信号转导的抑制。因此，洛杉矶可能是治疗顽固性慢性疼痛的有希望的药理学候选人。然而，分别通过添加30μM或10μM的LA抑制了这种情况。用反义寡脱氧核苷酸进一步敲除TRPC6在SNI大鼠中产生了明显的镇痛作用，并伴随着小胶质细胞中p38磷酸化水平的降低。结论这些数据表明，其应用LA在神经性疼痛中显示出止痛和抗炎作用。LA的作用涉及小胶质细胞中TRPC6和p38信号转导的抑制。因此，洛杉矶可能是治疗顽固性慢性疼痛的有希望的药理学候选人。然而，分别通过添加30μM或10μM的LA抑制了这种情况。用反义寡脱氧核苷酸进一步敲除TRPC6在SNI大鼠中产生了明显的镇痛作用，并伴随着小胶质细胞中p38磷酸化水平的降低。结论这些数据表明，其应用LA在神经性疼痛中显示出止痛和抗炎作用。LA的作用涉及小胶质细胞中TRPC6和p38信号转导的抑制。因此，洛杉矶可能是治疗顽固性慢性疼痛的有希望的药理学候选人。施加的LA在神经性疼痛中具有镇痛和抗炎作用。LA的作用涉及小胶质细胞中TRPC6和p38信号转导的抑制。因此，洛杉矶可能是治疗顽固性慢性疼痛的有希望的药理学候选人。施加的LA在神经性疼痛中具有镇痛和抗炎作用。LA的作用涉及小胶质细胞中TRPC6和p38信号转导的抑制。因此，洛杉矶可能是治疗顽固性慢性疼痛的有希望的药理学候选人。