The biophysical properties of a tetrodotoxin resistant (TTXr) sodium channel, Na(V)1.8, and its restricted expression to the peripheral sensory neurons suggest that blocking this channel might have therapeutic potential in various pain states and may offer improved tolerability compared with existing sodium channel blockers. However, the role of Na(V)1.8 in nociception cannot be tested using a traditional pharmacological approach with small molecules because currently available sodium channel blockers do not distinguish between sodium channel subtypes. We sought to determine whether small interfering RNAs (siRNAs) might be capable of achieving the desired selectivity. Using Northern blot analysis and membrane potential measurement, several siRNAs were identified that were capable of a highly-selective attenuation of Na(V)1.8 message as well as functional expression in clonal ND7/23 cells which were stably transfected with the rat Na(V)1.8 gene. Functional knockdown of the channel was confirmed using whole-cell voltage-clamp electrophysiology. One of the siRNA probes showing a robust knockdown of Na(V)1.8 current was evaluated for in vivo efficacy in reversing an established tactile allodynia in the rat chronic constriction nerve-injury (CCI) model. The siRNA, which was delivered to lumbar dorsal root ganglia (DRG) via an indwelling epidural cannula, caused a significant reduction of Na(V)1.8 mRNA expression in lumbar 4 and 5 (L4-L5) DRG neurons and consequently reversed mechanical allodynia in CCI rats. We conclude that silencing of Na(V)1.8 channel using a siRNA approach is capable of producing pain relief in the CCI model and further support a role for Na(V)1.8 in pathological sensory dysfunction.

译文

河豚毒素抗性 (TTXr) 钠通道Na(V)1.8的生物物理性质及其对周围感觉神经元的限制性表达表明,与现有的钠通道阻滞剂相比,阻断该通道可能在各种疼痛状态下具有治疗潜力,并且可能提供改善的耐受性。然而,Na(V)1.8在伤害感受中的作用不能使用传统的小分子药理学方法来测试,因为目前可用的钠通道阻滞剂不能区分钠通道亚型。我们试图确定小干扰rna (sirna) 是否能够实现所需的选择性。使用Northern印迹分析和膜电位测量,鉴定出几种sirna能够高度选择性地衰减Na(V)1.8信息以及在用大鼠Na(V)1.8基因稳定转染的克隆ND7/23细胞中的功能表达。使用全细胞电压钳电生理证实了通道的功能性敲除。在大鼠慢性收缩神经损伤 (CCI) 模型中,评估了一种显示出Na(V)1.8电流的鲁棒敲除的siRNA探针在逆转已建立的触觉异常性疼痛方面的体内功效。通过留置硬膜外套管递送到腰背根神经节 (DRG) 的siRNA导致腰4和5 (L4-L5) DRG神经元中Na(V)1.8 mRNA表达的显着降低,因此在CCI大鼠中逆转了机械性异常性疼痛。我们得出的结论是,使用siRNA方法沉默Na(V)1.8通道能够在CCI模型中产生疼痛缓解,并进一步支持Na(V)1.8在病理性感觉功能障碍中的作用。

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