CaM-KII亚硝基化在神经病理性疼痛发病机制中作用的研究

Calcium/calmodulin-dependent kinase II (CaM-KII) act as a key role for the induction of central sensitization in spinal dorsal horn neurons in neuropathic pain. An important property of CaMKII is that, once autophosphorylated on Thr286 by Ca2+ influx, its activity is no longer dependent on calcium-calmodulin. This allows its activity to continue long after the calcium signal has returned to baseline. Continuous phosphorylation of CaMKII might induce the phosphorylation of a variety of receptors known to be present on spinal dorsal horn neurons, including the NMDA, AMPA （GluR1）receptors, and transcription factors, such as cAMP-responsive element-binding proteinand（CREB） subsequently cause neuronal plastic change and contribute to central sensitization. It is important and difficult to try to inhibit the activity of autophosphorylated CaM-KII. We have recently reported that NO is a potent inhibitor of both inactived CaMKII and autophosphorylated CaMKII through S-nitrosylation of Cys6 of CaMKII. Our previous studies demonstrated that protein S-nitrosylation could been detected in spinal dorsal horn in a rat model of neuropathic pain, a work supported by another project for our group. Together with the evidence that both nNOS and CaM-KII translocated to cell membrane and make a co-localization through PSD-95 under the pathological conditions such as neuropathic pain, in this project, we will test the hypothesis that CaM-KII can be S-nitrosylated on spinal dorsal horn during neuropathic pain, this will inhibit the activity of autophosphorylated CaM-KII, subsequently prevent central sensitization， thereby play a protection role in neuropathic pain. Additionally the role of PSD-95 within CaM-KII s-nitrosylation will be further examined. Our project will give novel insight into regulation of neuropathic pain through s-nitrosylation induced by NO, and may open a new area of research, this will lead to therapeutically useful strategies for this terrible condition.

CaM-KII在神经病理性疼痛脊髓中枢敏化过程中起到关键的作用。细胞内钙离子浓度升高，可致CaM-KII Thr286自磷酸化而被激活，此后其活性不受钙离子水平控制，可长时间保持。这使其对下游信号系统的作用长期维持，如磷酸化CREB、GluR1等，导致中枢敏化。如何抑制自磷酸化后的CaM-KII活性，是研究神经病理性疼痛的重要问题。鉴于：(1)我们已发表的论文证实NO介导的蛋白质巯基亚硝基化可以降低Thr286自磷酸化状态下的CaM-KII活性；(2)我们上一个自然科学基金项目发现在神经病理性疼痛时，脊髓背角有蛋白质发生了亚硝基化反应；(3)已知nNOS和CaM-KII在钙离子内流后同时向细胞膜聚集，通过PSD-95而紧邻。本课题拟证实神经病理性疼痛时脊髓背角的CaM-KII发生了亚硝基化反应，活性降低、抑制了中枢敏化，因而具有保护作用；PSD-95可能参与了CaM-KII的亚硝基化过程。

一氧化氮（NO）在神经病理性疼痛发生及维持中的发挥了重要作用。早期的研究认为NO有致痛作用，并且与一氧化氮/环单磷酸鸟苷（cGMP）-蛋白激酶G（PKG）相关，也就是通过NO/cGMP-PKG作用机制。而本课题证实，除NO/cGMP-PKG途径外，NO也能通过使蛋白质巯基发生亚硝基化反应参与神经病理性疼痛的发生及维持。.钙离子/钙调蛋白与钙调蛋白激酶II（CaMKII）在神经病理性疼痛脊髓中枢敏化过程中起到关键的作用。细胞内钙离子浓度升高，可导致CaMKII Thr286自磷酸化而被激活有催化活性。此后其活性不受钙离子浓度调节，即使细胞内钙离子浓度恢复正常，CaMKII仍然具有自主活性，可长时间发挥作用。这使其对下游信号系统的作用长期维持，如磷酸化GLuR1等，引起神经重塑进而导致中枢敏化。本课题研究发现：（1）首次在神经病理性疼痛过程中，在动物体内发现到CaMKII能够发生亚硝基化反应，并且亚硝基化水平主要有nNOS介导；（2）在神经病理性疼痛早期，CaMKII能够在T286磷酸化水平不变的情况下发生亚硝基化，从而使自身活性水平增高，增加下游GluR1磷酸化水平从而产生致痛作用； （3）在神经病理性疼痛中期或晚期，CaMKII的亚硝基化对神经病理性疼痛产生何种作用尚需继续研究。