PKG通路介导交感维持性痛的信号机制研究

Sympathetically maintained pain (SMP)is one of the intractable pain in clinic.The therapy limitation of sympathectomy and sympathetic ganglion block make it imperative to understand the mechanism that could elucidate the modulation function of sympathetic nerve on peripharal receptor. The stimulation given by sympathetic nerve and norepinephrine on dorsal root ganglion (DRG) would further enhance the neuronal excitability,resulting in sympathetic sensitivity. A previously published report suggested that the agonist of cGMP-PKG system could strengthen the excitability of DRG small neurons. Our preliminary investigation using pharmocological approach as well as the conditional PKG-1 knockout mouse, confirmed that cGMP-PKG system mediates the sympathetic sensitivity on DRG small neurons. Moreover, the functional alteration of potassium channel was also involved. Present study is going to explore the possible phosphorylation effect of PKG on two types of potassium channels, the ATP sensitive channel (KATP) and the calcium dependent potassium channel (KCa). We hypothesize that the resulting inhibition of KATP would depolarize the membrane potential and decrease the threshold for evoked action potential. Collectively, inhibition of SK3 (one subtype of KCa) would decrease the afterhyperpolarization (AHP) and thus increase the repetitive firing. Both effects would reinforce the excitability of the DRG small neurons, which is related to the sympathetic sensitivity. The results will explain the peripheral mechanism of SMP at the molecular level, and establish the precise target of strong pain-suppressing with small side-effects, thus providing sufficient proofs for analgesic strategy for the SMP.

交感维持性痛(SMP)是一种临床常见难治性疼痛，交感神经节阻滞或切断治疗的局限性使得对交感神经调控外周痛感受功能详细机制的阐述势在必行。在背根神经节(DRG)水平，交感神经及其递质可进一步增强细胞兴奋性，产生交感敏化。文献报道cGMP-PKG通路激动剂可以加强损伤DRG小神经元的超兴奋。我们在前期实验中利用药理学手段和条件性PKG-1敲除小鼠,初步证实cGMP-PKG信号通路参与介导DRG小细胞交感敏化，而且提示与钾离子通道功能变化有关。本项目拟利用该敲除动物通过分子生物学和整节膜片钳技术,深入阐明PKG可能通过磷酸化效应抑制KATP通道使膜发生去极化、或者抑制SK3通道的开放使后超极化减小，分别通过阈值减小和放电频率增加促使DRG小细胞兴奋性的进一步增强。研究结果将在分子水平深刻揭示SMP发生的外周机制，确立特异分子靶点，为制定镇痛作用专一性强、副作用小的SMP镇痛策略提供充分依据。

交感维持性痛(SMP)是一种临床常见难治性疼痛，交感神经节阻滞或切断治疗的局限性使得对交感神经调控外周痛感受功能详细机制的阐述势在必行。本课题主要研究内容包括PKG介导交感敏化过程的确认及交感敏化¬PKG作用下游靶通道电流的确定。重要结果如下：尾神经主干切断（CTT）模型中，腹腔注射胍乙啶后翻转了模型的痛过敏表现；小直径DRG神经元电生理记录发现，诱发的放电反应可以被NE进一步增强；术后两周的CTT动物进行络氨酸羟化酶(TH)阳性纤维的检测，发现阳性纤维数量显著高于假手术组。故而，CTT模型术后，在行为学表现、细胞电生理反应及组织化学水平均支持交感维持性痛的存在。故而后期实验多数在该模型展开。在CTT模型中具有交感敏化的小DRG神经元，电生理记录指标显示应用PKG的阻断剂KT5823可以减弱NE的兴奋效应，而 cGMP的激动剂剂Sp-cGMP、 cGMP的类似物8-Br-cGMP均可增强NE的兴奋效应。从而从药理学手段对cGMP-PKG通路介导交感敏化这一胞内过程给予了支持。此外，在细胞培养与实时定量PCR的基础上，选定“5’- UUUACAAGAGUCUUGACGGTT -3’”作为干扰序列，并合成慢病毒进行动物体内注射，观察慢病毒干扰PKG后对NE兴奋效应的影响。Western检测显示包装了干扰蛋白的慢病毒（LV―Prkg shRNA）组，PKG蛋白受到了显著抑制；而对照组（LV-Control）则没有变化。同时，NE的兴奋效应在包装了LV―Prkg shRNA的实验组，也不再出现。在CTT模型具有交感敏化表现的小神经元，进行细胞全钾电流的测定。发现神经元在加入NE后全钾电流减小，而使用ATP敏感钾通道电流阻断剂glibenclamide（格列本脲）可阻断该总钾电流的减小，提示NE的作用通过ATP敏感钾电流实现。进一步实验发现，NE诱导的钾电流减小可以被cGMP的类似物8-Br-cGMP和cGMP的激动剂Sp-cGMP进一步增强，PKG的阻断剂KT5823可以翻转NE的兴奋效应，而单独使用这些药物对总钾电流没有影响。此外，在CTT模型注射LV―Prkg shRNA病毒后，小细胞总钾电流在NE作用后也未检测出电流幅值的改变。本项目研究结果对SMP发生的外周分子机制进行了深入揭示，以此为特异分子靶点，将为制定镇痛作用专一性强、副作用小的SMP镇痛策略提供重要依据