酪氨酸磷酸酶Shp-1调节外周TRPV1受体去磷酸化参与大鼠慢性炎症痛的机制研究

Transient receptor potential vanilloid 1 (TRPV1) receptor is expressed in nociceptive neurons of rat dorsal root ganglion (DRG) and mediates inflammatory pain. TRPV1 activity is regulated mainly by its phosphorylation on amino acid residues like serine/threonine or tyrosine, in which the phosphorylation is counter-balanced by their protein kinases and phosphatases, respectively. On the basis of our and many other previous researches, the present grant application will investigate a new mechanism about the role of TRPV1 in inflammatory pain: Src homology 2 domain-containing tyrosine phosphatase 1 (Shp-1), a tyrosine phosphatase, dephosphorylates TRPV1 in DRG neurons, and thus limits the development of inflammatory pain..In a rat model of CFA-induced inflammatory pain, we will carry out the following experiments: .(1) With immunofluorescent staining and Western blotting, whether Shp-1, TRPV1 and tyrosine-phosphorylated TRPV1 exist and co-localize in nociceptive DRG neurons, and whether the content of them increase in the CFA-induced inflammatory pain rats..(2) With co-immunoprecipitation (co-IP) method, direct evidence about Shp-1 binding with TRPV1 or the interaction between Shp-1 and TRPV1 will be examined..(3) With in vitro single patch recording technique on acutely-dissociated DRG neurons, whether the intracellular application of Shp-1 inhibitor (for example, sodium stibogluconate, SSG) increases the capsaicin (a specific TRPV1 agonist) responses will be examined..(4) With in vivo experiment, whether the intrathecal application of Shp-1 inhibitors induce thermal hyperalgesia in normal rats..(5) In the CFA-induced inflammatory pian rats, whether intrathecal application of Shp-1 inhibitor aggravate the thermal hyperalgesia..(6) With molecular and cellular methods, tyrosine residues of TRPV1 receptors by Shp-1 dephosphorylation will be examined..Eventually, we would like to verify the following hypothesis: In rat DRG neurons, Shp-1 dephosphorylates TRPV1 and inhibits its function, maintaining normal thermal nociceptive thresholds in normal rats. As a complementary mechanism, Shp-1 increases in DRG neurons in rats with CFA-induced chronic inflammatory pain, protecting against excessive thermal hyperalgesia.

本项目欲从酪氨酸位点磷酸化调节的角度，提出外周TRPV1（瞬时感受器电位香草酸受体1或辣椒素受体）参与慢性炎性痛的新机制：酪氨酸磷酸酶Shp-1降低DRG神经元中TRPV1酪氨酸磷酸化，从而限制炎症痛的发展而起到保护作用。.内容主要包括：1.在急性分离的大鼠背根神经节(DRG)中，Shp-1与TRPV1酪氨酸磷酸化的TRPV1含量及在CFA慢性炎症痛时是否上调。2.Shp-1和TRPV1是否直接相互结合。3.离体培养的DRG神经元上，胞外给予Shp-1抑制剂可否增强神经元对TRPV1激动剂的反应。4.DRG局部应用Shp-1抑制剂后热痛敏的变化，以及TRPV1拮抗剂预处理是否阻断这种热痛敏。5.在DRG神经元中过表达Shp-1是否减轻模型大鼠炎症痛。6.Shp-1导致TRPV1去磷酸化的酪氨酸位点。.意义在于探索Shp-1调节TRPV1去酪氨酸磷酸化限制慢性炎性痛这种新机制。

瞬时感受器电位香草酸受体1（transient receptor potential vanilloid 1, TRPV1）是非选择性的阳离子通道，在大鼠背根神经节（dorsal root ganglion, DRG）伤害性神经元中有丰富的表达，介导完全弗氏佐剂（complete Freund’s adjuvant，CFA）诱发的大鼠炎症痛的发生。大量研究显示，TRPV1通道的丝氨酸/苏氨酸磷酸化水平增加，参与痛敏。比较而言，TRPV1的酪氨酸磷酸化被关注得较少。Src homology 2 domain-containing tyrosine phosphatase 1（Shp-1）是含有Src同源序列的酪氨酸磷酸酶。本项目在于探索Shp-1调节TRPV1去酪氨酸磷酸化限制慢性炎性痛这种新机制。项目证实：（1）Shp-1与TRPV1共表达于大鼠DRG伤害性神经元中，并且Shp-1和TRPV1相互结合。胞外给予Shp-1抑制剂可敏化TRPV1通道，增强神经元对TRPV1特异性激动剂辣椒素（capsaicin）的反应。在正常大鼠，蛛网膜下腔注射Shp-1抑制剂，DRG中酪氨酸磷酸化的TRPV1蛋白质含量及TRPV1总蛋白质的含量增加，大鼠出现明显的热痛敏；TRPV1的特异性拮抗剂预处理可阻断Shp-1抑制剂引起的热痛敏现象。说明Shp-1使TRPV1去磷酸化，抑制TRPV1通道活性并维持机体正常的热痛阈。（2）在CFA炎症痛7d时，Shp-1、TRPV1、酪氨酸磷酸化的TRPV1蛋白质含量及Shp-1与TRPV1共表达的神经元比例，均有明显上调。蛛网膜下腔注射Shp-1抑制剂增加痛敏程度。DRG神经元中过表达Shp-1，大鼠炎症痛程度减轻。提示Shp-1限制CFA炎症痛中的发展，起着保护作用。因此，Shp-1是调节TRPV1酪氨酸磷酸化的重要磷酸酶。Shp-1抑制TRPV1维持机体正常的热痛阈，并在CFA炎症痛中代偿性增加以限制炎症痛的发展。本项目为慢性疼痛的基础研究与临床治疗开辟了新思路。本项目共发表SCI论文24篇，包括Cell Reports, Science Advances等高水平SCI期刊，参编论著2部。