蝎毒致痛的神经机制研究
文献类型：学位论文
| 作者 | 刘通 |
| 学位类别 | 博士 |
| 答辩日期 | 2008-06-03 |
| 授予单位 | 中国科学院上海生命科学研究院 |
| 授予地点 | 上海生命科学研究院 |
| 导师 | 吉永华 |
| 关键词 | 蝎毒 痛相关行为 炎症 外周敏化 中枢敏化 |
| 其他题名 | The neuronal mechanisms of nociception induced by scorpion venom |
| 学位专业 | 神经生物学 |
| 中文摘要 | 本论文工作采用药理学、行为学、组织病理学、免疫组化和免疫印迹等技术和方法对富含特异性钠离子通道调制剂的东亚钳蝎（Buthus martensi Karsch，BmK）毒液致痛相关的外周和中枢敏化的分子细胞机制进行了较系统的研究，主要结果如下： 1. 肥大细胞脱颗粒和组胺部分介导BmK粗毒诱导的痛相关行为和水肿 大鼠经足底注射BmK粗毒和类样蝎毒素多肽BmK I，可诱发肥大细胞脱颗粒。BmK粗毒诱导的自发痛、双侧后足机械痛敏、原发性热痛敏和后足水肿以及脊髓浅层c-Fos的表达可被compound 48/80慢性耗竭肥大细胞以及外周共施加组胺受体H1或H2受体拮抗剂部分压抑。提示肥大细胞脱颗粒和组胺部分介导了BmK粗毒诱导的痛相关行为以及水肿反应。 2. 脊髓谷氨酸受体参与BmK粗毒诱导的痛相关行为的诱导和维持 大鼠经鞘内注射MK-801（NMDA受体拮抗剂），CNQX（非NMDA受体拮抗剂），DL-AP3（I 组mGluR受体拮抗剂），APDC（II组mGluR受体激动剂）等谷氨酸受体拮抗剂或激动剂研究了脊髓谷氨酸受体在BmK粗毒诱导的痛相关行为中的作用。药物前处理实验发现，BmK粗毒诱导的自发痛行为可被MK801和CNQX所压抑，被DL-AP3和APDC轻微压抑，原发性热痛敏可被MK-801或DL-AP3压抑，双侧机械痛敏可被CNQX或DL-AP3压抑，而仅镜像机械痛敏可被APDC压抑。药物后处理实验发现，原发性热痛敏可被MK-801, CNQX, DL-AP3 或APDC所逆转，而双侧机械痛敏仅被APDC所逆转。提示脊髓谷氨酸受体参与了BmK蝎毒诱导的不同痛相关行为。 3. 脊髓一氧化氮参与BmK粗毒诱导的痛相关行为和脊髓c-Fos表达 大鼠足底注射BmK粗毒可诱发双侧脊髓背角浅层（I-II）、深层（V-VI）和中央管附近（VII-X）nNOS阳性神经元数目显著增加，注射同侧比对侧强，但在腰椎固有层（III-IV）没有明显的增加。免疫印迹结果表明，BmK粗毒诱导脊髓nNOS的表达上调。BmK粗毒注射前10分钟用L-NAME（一种NOS抑制剂）预处理，显著性地压抑自发痛行为、原发性热痛敏、双侧机械痛敏以及脊髓背角c-Fos的表达。提示脊髓一氧化氮参与了BmK粗毒诱导的痛相关行为和脊髓c-Fos表达。 4. 脊髓胶质细胞的激活参与BmK诱导的痛相关反应 大鼠足底注射BmK粗毒3天后，脊髓星形胶质细胞标志物GFAP免疫染色强度开始增加，7天达到峰值并在14天后逐渐恢复。免疫印迹结果表明，BmK粗毒可诱导脊髓GFAP表达上调。小胶质细胞标志物OX-42的免疫染色强度在BmK毒素注射4小时后开始增加，峰值出现在1天，并在3到7天消退。鞘内注射胶质细胞抑制剂Fluorocitrate或腹腔注射小胶质细胞特异性抑制剂Minocycline预处理，可以有效地压抑BmK粗毒诱导的自发痛反应、双侧机械痛敏和原发性热痛敏。提示脊髓星形胶质细胞和小胶质细胞的激活参与了BmK粗毒诱导的痛相关反应。 5. 脊髓胞外信号调节激酶（ERK）信号通路激活参与BmK粗毒诱导的痛相关行为 大鼠足底注射BmK粗毒2分钟激活了注射侧L4-L5脊髓节段的ERK，峰值在30-60分钟，4小时后基本消失。免疫印迹结果表明，BmK 粗毒可诱导的脊髓磷酸化ERK表达的增加。预先鞘内注射MEK特异性抑制剂U0126抑制了BmK粗毒诱导的自发痛行为、原发性热痛敏和双侧机械痛敏，且剂量依赖性的抑制脊髓c-Fos的表达。预先鞘内注射MK-801或CNQX显著压抑BmK粗毒诱导的脊髓ERK的激活。以上结果提示，脊髓NMDA受体和非NMDA受体部分介导的胞内ERK信号通路的激活参与BmK粗毒诱导的痛相关行为。 综上所述，本研究发现肥大细胞和组胺参与蝎毒诱导的外周敏化机制，而脊髓谷氨酸受体、一氧化氮通路、胶质细胞激活以及胞内ERK信号通路的激活都被涉及到蝎毒诱导的脊髓中枢敏化机制中。对蝎毒致痛的分子细胞机制的系统解析，将丰富我们对炎症痛的理解，从而有助于寻求更好的镇痛策略用于包括蝎蜇伤在内的多种临床炎症痛的治疗。 |
| 英文摘要 | In the present study, the neuronal mechanisms of pain-related behaviors induced by the venom of BmK (Buthus martensi Karsch) were investigated using behavioral test, histopathological staining, immunohistochemical methods, immunobloting, etc. The results are presented as follows: 1. Mast cells degranulation and histamine are involved in BmK venom-induced pain-related behaviors and paw edema in rats The degranulation of mast cells could be triggered by BmK venom in rat hindpaw. The chronic degranulation of mast cells using compound 48/80 relieved the spontaneous nociceptive responses, the primary thermal and bilateral mechanical hyperalgesia and the rat paw edema, as well as partially reduced c-Fos expression in superficial layers of bilateral spinal cord induced by BmK venom. In addition, individual peripheral co-administration of either 100 nmol chlorpheniramine or 100 nmol pyrilamine (histamine H1 receptor antagonist) or 500 nmol cimetidine (histamine H2 receptor antagonist) and BmK venom suppressed the spontaneous nociceptive responses, partially the primary thermal and bilateral mechanical hyperalgesia and rat paw edema induced by BmK venom. Thus, these results suggest that mast cells degranulation and histamine are involved in BmK venom-induced rat pain-related behaviors and paw edema. 2. Spinal glutamate receptors are involved in BmK venom-induced rat pain-related behaviors MK-801 (a noncompetitive NMDA receptor antagonist), CNQX (a non-NMDA receptor antagonist), DL-AP3 (a group I metabotropic glutamate receptor antagonist) and APDC (a group II metabotropic glutamate receptor agonist) were employed to explore the role of spinal glutamate receptors in BmK venom-induced pain. Upon the intrathecal injection of glutamate receptor antagonists/agonist before BmK venom administration by 10 min, BmK venom-induced spontaneous nociceptive responses could be suppressed by all tested agents. Primary thermal hyperalgesia could be inhibited by MK-801 and DL-AP3, while bilateral mechanical hyperalgesia could be inhibited by CNQX and DL-AP3 and contrlateral mechanical hyperalgesia could be inhibited by APDC. Upon the intrathecal injection of glutamate receptor antagonists/agonist after BmK venom injection by 4.5 hour, primary thermal hyperalgesia could be partially reversed by all tested agents, while bilateral mechanical hyperalgesia could only be inhibited by APDC. The results suggest that the role of spinal glutamate receptors may be different on the various manifestations of BmK venom-induced rat pain-related behaviors. 3. Spinal nitric oxide (NO) contributes to BmK venom-induced pain-related behaviors and spinal c-Fos expression in rats Immunohistochemistrical results showed that the number of neuronal NO synthase (nNOS) positive neurons in bilateral spinal cord significantly increased in superficial (I–II), deep (V–VI) dorsal horn laminae and the ventral gray laminae (VII–X), but not in the nucleus proprius (III and IV) of lumbar spinal cord, after unilateral intraplantar injection of BmK venom from 2 hour to 7 day. This increase on the ipsilateral side to BmK venom injection was always greater than that on the contralateral side. Western blotting results confirmed that spinal nNOS expression was significantly up-regulated after BmK venom administration. In addition, intrathecal delivery of Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME; a NOS inhibitor) before intraplantar injection of BmK venom by 10 min significantly attenuated spontaneous nociceptive responses and prevented the development of primary thermal hyperalgesia and bilateral mechanical hyperalgesia. Spinal c-Fos expression at 2 h following BmK venom injection could be partially inhibited by intrathecal injection of L-NAME. The results suggest that spinal NO is involved in various pain-related behaviors and c-Fos expression induced by BmK venom in rats. 4. Spinal glia activation contributes to BmK venom-induced rat pain-related behaviors Glial fibrillary acidic protein (GFAP) immunoreactivity indicative astrocyte activation in bilateral spinal cord started to increase by 3 d, peaked at 7 d and gradually reversed 14 d following intraplantar injection of BmK venom. The up-regulation of GFAP expression was confirmed by western blotting analysis. In contrast, bilateral spinal increase of OX-42 immunoreactivity indicative of microglial activation began at 4 h, peaked 1 d and gradually reversed at 3 and 7 days after BmK venom administration. Pretreatment with either intrathecal injection of fluorocitrate or intraperitonially injection of minocycline, two glial activation inhibitors, suppressed the spontaneous nociceptive responses, and prevented the primary thermal and bilateral mechanical hyperalgesia induced by BmK venom in rats. Moreover, intraperitonially injection of minocycline partially inhibited spinal c-Fos expression induced by BmK venom but lack of effect on BmK venom-induced paw edema. The current study demonstrated that spinal astrocyte and microglial activation may contribute to BmK venom-induced pain-related behaviors in rats. 5. Spinal ERK activation contributes BmK venom-induced rat pain-related behaviors and c-Fos expression Following intraplantar injection of BmK venom，extracellular signal-regulated kinases (ERK) were activated in ipsilateral L4-L5 spinal cord dorsal horn, which started at 2 min, peaked at 30-60 min and almost disappeared at 4 h following intraplantar injection of BmK venom. Intrathecal injection of U0126 (0.1, 1 and 10 μg), a widely used specific MAP kinase kinase (MEK) inhibitor, suppressed spontaneous nociceptive responses and reduced primary heat hyperalgesia and bilateral mechanical hyperalgesia induced by BmK venom. BmK venom-induced spinal c-Fos expression could be inhibited by U0126 dose-dependently. In addition, intrathecal injection of MK-801 or CNQX suppressed the BmK venom-induced ERK activation in spinal cord. Thus, activation of ERK in spinal cord dorsal horn contributes to BmK venom-induced rat pain-related behaviors and c-Fos expression. Taken together, it was found that mast cell degranulation and histamine were involved in BmK venom-induced peripheral sensitization, while glutamate receptors, NO, glia activation and intracellular ERK signaling activation were involved in BmK venom-induced central sensitization. |
| 语种 | 中文 |
| 公开日期 | 2013-01-05 |
| 页码 | 116 |
| 源URL | [http://ir.sibs.ac.cn/handle/331001/2394]  |
| 专题 | 上海神经科学研究所_神经所(总) |
| 推荐引用方式 GB/T 7714 | 刘通. 蝎毒致痛的神经机制研究[D]. 上海生命科学研究院. 中国科学院上海生命科学研究院. 2008. |
入库方式： OAI收割
来源：上海神经科学研究所
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