靶向抑制Fyn介导的NMDA受体磷酸化治疗炎性疼痛的实验研究

The N-methyl-D-aspartate (NMDA) receptor, a subtype of ionotropic glutamate receptors, play a critical role in central sensitization of chronic pain. The method of blocking NMDA receptors display good analgesic performance in animal models of chronic pain,but their clinical use is limited by numerous intolerable side effects due to the essential functional contribution of NMDA receptors throughout the central nervous system. Exploring novel ways of modulating NMDA receptor activity without receptor blockade may be an even better analgesic strategy. Recent studies have shown that Fyn (a member of SFKs family) serve as a convergent point of multiple, diverse signaling pathways that regulate NMDA receptor function by phosphorylating NMDA receptor subunits. We have found that a family of postsynaptic density (PSD) proteins PSD-93 is required for Fyn-mediated tyrosine-phosphorylation of the NMDA receptors. Acturely, Fyn may bind its SH2 domain to a region N-terminal of the PDZ3 domain of PSD-93 or 95 directly, and thereby associate with NMDA receptors to mediate NMDA receptor tyrosine phosphorylation. More detailed studies have shown that Fyn kinase-mediated phosphorylation of NMDA receptors in spinal cord dorsal horn is essential for development and maintenance of chronic pain such as inflammatory pain. Thus we propose that disrupting Fyn and PSD-93/95 interaction looks likely to be a good approach to downregulate NMDA receptor tyrosine phosphorylation and then treat inflammatory pain which not only avoids blocking NMDA receptor function but also avoids impairing catalytic activity of Fyn. To test the hypothesis ,we will synthesis a peptde containing the Fyn binding motif of PSD-93/95 and conjugates it with a Tat protein transduction domain(become a cell-permeable short peptide).We will then investigate the effects of this cell-permeable peptide on Fyn-PSD93/95 interaction and NMDA receptor tyrosine phosphorylation. In addition, we will test analgesic roles of the peptide in rats with inflammatory pain and determin wether it can not only avoids blocking NMDA receptor function but also avoids impairing catalytic activity of fyn.In conclusion, we will demonstrate that disrupting Fyn-PSD93/95 interaction can inhibit NMDA receptor tyrosine phosphorylation and treat inflammatory pain and, thus, identify a novel approach to treat chronic inflammatory pain.

慢性炎性疼痛的维持依赖于酪氨酸蛋白激酶Fyn催化的NMDA受体（NR）磷酸化，因此，抑制Fyn介导的NR磷酸化治疗炎性疼痛成为疼痛治疗的新策略，然而，常用的方法如Fyn抑制剂影响Fyn活性因而副作用大。我们前期研究及新的进展表明：Fyn催化的NR磷酸化由突触后致密物(PSD)蛋白与Fyn的独特结合介导，这使我们设想，特异抑制Fyn与PSD蛋白的相互作用可以抑制Fyn介导的NR磷酸化因而可以治疗炎性疼痛。为此，本研究利用课题组已成功表达的Tat转导结构域短肽，设计合成一种穿膜肽特异抑制Fyn与PSD蛋白相互作用从而靶向抑制Fyn介导的NR磷酸化，进而治疗炎性疼痛。这种方法可避免对Fyn活性及NR基础生理功能的影响因而优势明显。本研究试图证实特异抑制Fyn与PSD蛋白的相互作用可抑制炎性疼痛时NR的磷酸化活化，旨在为慢性疼痛如炎性疼痛的治疗提供新靶点新手段。

NMDA受体（NR）的过度活化在慢性疼痛的发生中至关重要。然而，NR阻断剂等治疗手段阻断NR基础活性，副作用大，临床应用受到限制。本项目依据慢性炎性疼痛的维持依赖于酪氨酸蛋白激酶Fyn催化的NR磷酸化活化，结合课题组前期研究及新的进展：Fyn催化的NR磷酸化活化由突触后致密物(PSD)蛋白与Fyn的独特结合介导,因此我们设想抑制Fyn与PSD蛋白的相互作用可特异抑制炎性痛大鼠的NR磷酸化活化，进而对炎性痛具有治疗作用。本方法抑制NR过度活化却不阻断NR，也不影响Fyn活性（特异性强）。为此，本研究基于新兴的多肽阵列技术及课题组已经掌握的Tat蛋白结构域转导技术，设计合成可抑制Fyn与PSD蛋白相互作用的穿膜肽，然后观察此穿膜肽对炎性痛大鼠NR磷酸化、痛行为及其对Fyn活性、NR基础活性等的影响。本研究证实了抑制Fyn与PSD蛋白的相互作用可特异抑制炎性痛时NR的过度活化，为慢性疼痛如炎性痛的治疗提供了新的思路或途径。