PICK1-ICA69复合物调控腺苷A1受体介导的针刺镇痛机制

Acupuncture is widely practiced in pathological pain clinically, exist studies suggest that endogenous opioid peptides have a relevance with anti-nociceptive effects of acupuncture. Now days, a growing number of researchers set out to explore the mechanism of adenosine and its receptors mediated analgesic effects of acupuncture. Goldman N et al. discovered that adenosine1 receptors (A1R) around certain acupuncture points were involved in local anti-nociceptive effects of acupuncture (Goldman N, et al. Nat Neurosci, 2013, 13(7):883-8). However, the underling regulation mechanism of adenosine A1 receptors’ anti-nociceptive effects is still unknown. PICK1 (Protein Interact with C-Kinase 1), a member of transfer proteins, which can form heteromeric complexes with Islet cell auto antigen 69 kDa (ICA69) in cells, has a certain association with adenosine A1R. Our group observed that acupuncture at zusanli enhanced the expression of A1R in spinal, and ICA69 was involved in the anti-nociceptive effects of acupuncture. Thus, we hypothesized that inner ICA69 could form heteromeric complexes with PICK1, which made it stuck on the membrane and inhibit PICK1 from transferring to plasma membrane, decreasing the phosphorylation of A1R on membrane, then increased the number of A1R on membrane, so that it could regulate A1R mediated analgesic effects of acupuncture. We made inflammatory pain model in wild type mice (WT), ICA69 knockout mice (ICA69 KO) and PICK1 knockout mice (PICK1 KO), respectively, each group received acupuncture at zusanli equally, then we observed the threshold of mechanical allodynia and thermal hyperalgesia, the number of activated spinal gliocytes and A1R on membrane, the expression of phosphorylated A1R, the quantity of complexes PICK1-ICA69 were examined. We utilized RNA transfection technique, cultivating originated spinal neuron of wild type mice, PICK1 knockout mice and ICA69 knockout mice, aiming to explore how complexes PICK1-ICA69 affect A1R. Our study is aim to elucidate the inner associations among acupuncture’s analgesic effects, complexes PICK1-ICA69 and A1R, to reveal whether complexes PICK1-ICA69 regulate A1R mediated anti-nociceptive effects of acupuncture from whole body, tissue, cell, molecular and genetic aspects, the results may offer a scientific clue for the mechanism study of acupuncture’s analgesic effects.

针刺广泛用于疼痛治疗，腺苷受体介导镇痛已成为针刺机制研究的热点。Goldman发现腺苷A1受体（A1R）参与针刺镇痛，但其机制不明。PICK1是转运蛋白，在胞内与ICA69形成PICK1-ICA69复合体，并与A1R相互联系。我们发现针刺可增加脊髓A1R表达，且ICA69参与针刺镇痛。因此，提出科学假设：细胞内ICA69与PICK1形成复合物，滞留胞浆内，抑制PICK1上膜，减少膜上A1R磷酸化，增加A1R表达，从而调控A1R介导的针刺镇痛。项目采用WT、ICA69 KO和PICK1 KO小鼠炎性痛模型，针刺足三里，运用免疫荧光、WB、细胞培养和转染等技术，测定小鼠痛阈、A1R与PICK1-ICA69含量,阐明镇痛效应、PICK1-ICA69和A1R之间的联系。从整体、细胞、基因等层面,论证PICK1-ICA69复合物是否调控腺苷A1R介导针刺镇痛,其研究将为针刺治疗疼痛提供科学依据。

电针(EA)被广泛应用于临床以减轻炎症性疼痛。胰岛细胞自身抗原69 (ICA69)具有调节小鼠长期痛觉过敏的作用。ICA69基因敲除(KO)导致脊髓背角(SDH)中相互作用蛋白1(PICK1)蛋白表达减少，并增加了谷氨酸受体亚基2 (GluR2)的磷酸化。在本研究中，我们通过调节SDH中的GluR2和PICK1来评价ICA69在EA的抗痛觉过敏作用中的作用及其机制。用足底皮下注射完全弗氏佐剂(CFA)诱发小鼠痛觉过敏，引起炎症性疼痛。每隔一天给予EA 30分钟。与CFA组相比，CFA+EA组的缩足频率明显降低。CFA+EA组中同侧Ica1 mRNA表达及ICA69蛋白水平显著升高。ICA69表达在第3天左右达到峰值。更重要的是，ICA69的缺失破坏了EA的镇痛效应以及EA对GluR2-p的抑制作用，而PICK1的缺失不能。给ICA69- KO小鼠鞘内注射ICA69肽，模拟了EA镇痛作用，可以同样抑制GluR2-p。EA对PICK1和GluR2的总蛋白无影响。EA可以增加ICA69-PICK1复合物的形成，减少PICK1-GluR2复合物的数量。我们的研究结果表明，ICA69通过PICK1调节小鼠脊髓中的GluR2，介导EA对CFA诱导的炎性疼痛的镇痛作用。