【作者】 李威；

【导师】 王锐；

【作者基本信息】 兰州大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 第一部分：神经肽S镇痛活性研究神经肽S（NPS）是最近发现的生物活性肽，具有调节觉醒、焦虑、运动和摄食的功能。NPS受体（NPSR）分布于下行控制系统，如中脑导水管周围灰质（periaqueductal gray，PAG）、中缝核（raphe nuclei）、侧臂旁核（lateral parabrachial nucleus，PBN）等，暗示了NPS-NPSR系统在痛觉调节中的可能作用。我们使用甩尾实验和热板实验首次评价了NPS在脊髓上水平的痛觉调节作用。在两种急性热痛觉模型中，侧脑室注射NPS（0．01-1nmol）能引起明显的镇痛效果。NPS（0．1nmol，i．c．v．）诱导的镇痛活性不被纳洛酮（i．c．v．共注射，10nmol或i．p．提前15分钟注射，10mg／kg）影响。而在相同的剂量条件下，纳洛酮能降低3nmol吗啡诱导的镇痛效果。NPSR的拮抗剂[D-Cys（tBu）⁵]NPS（i．c．v．，3、10nmol）本身没有任何痛觉调节活性。但与NPS共注射后，能明显抑制NPS诱导的镇痛活性。这些结果表明，NPS的镇痛活性，是通过NPS受体而不是阿片受体发挥作用的；NPS-NPSR系统可能是新型止痛药的新靶点。第二部分：[Tyr⁶]γ2-MSH（6-12）在脊髓水平的痛觉调节作用Mrg家族受体（mas-related gene，简称Mrg，也称为sensory neuron-specific receptor，SNSR），特异地分布于脊髓三叉神经节和背根神经节的小直径感觉神经元中，在痛觉传导中发挥重要作用。我们的目的是研究MrgC激活后诱发原痛效果的机制，以及MrgC受体与N／OFQ-NOP系统在调节脊髓痛觉中相互作用。鞘内注射MrgC高效激动剂[Tyr⁶]γ2-MSH（6-12）产生明显的热痛觉过敏效果（小鼠甩尾潜伏期的缩短，0．01-10pmol），和诱发疼痛行为应答（后肢抓挠身体两侧，嘴咬或舔前后肢及尾巴，0．01-10nmol）。这种原痛效果能被NMDA受体拮抗剂非竞争性MK-801、竞争性D-APV和NO合酶抑制剂L-NAME抑制；但不受共注射NK₁受体拮抗剂L-703，606或NK₂受体拮抗剂MEN-10，376影响。在其它实验中，[Tyr⁶]γ2-MSH（6-12）诱发的疼痛行为被N／OFQ双向调节。高剂量N／OFQ（0．01-1nmol）抑制，低剂量N／OFQ（1fmol-3pmol）加强这种疼痛行为应答。另外，NOP受体拮抗剂[Nphe¹]N／OFQ（1-13）-NH₂加强[Tyr⁶]γ2-MSH（6-12）诱发的原痛效果（包括热痛敏效果和疼痛行为应答）。我们的结果表明，[Tyr⁶]γ2-MSH（6-12）诱发的原痛效果是由NMDA-NO系统介导的。MrgC与N／OFQ-NOP系统在调节脊髓痛觉中存在相互作用。更多还原

【Abstract】 PartⅠ: The studies on the antinociceptive effect ofneuropeptide SNeuropeptide S （NPS）, a recently identified bioactive peptide, was reported toregulate arousal, anxiety, motoring and feeding behaviors. NPS receptor （NPSR）mRNA was found in the area related to descending control system of pain, such as theperiaqueductal gray （PAG）, raphe nuclei, and lateral parabrachial nucleus （PBN）,suggesting the possible role of NPS-NPSR system in the regulation of paintransmission. In the present study, we evaluated the effects of NPS in pain modulationat the supraspinal level for the first time, using tail withdrawal test and hot-plate test inmice. NPS （mouse, 0.01-1 nmol） injected intracerebroventricularly （i.c.v.） caused asignificant increase of tail withdrawal latency and paw-licking/jumping latency in thetail withdrawal test and the hot-plate test, respectively. Antinociceptive effect elicitedby NPS （0.1 nmol, i.c.v.） was not affected by naloxone （i.c.v., 10 nmol co-injection ori.p., 10 mg/kg, 10 min prior to NPS） in both tail withdrawal test and hot-plate test.However, at the doses, naloxone significantly inhibited the antinociceptive effectinduced by morphine （i.c.v., 3 nmol）. NPS （0.1 nmol, i.c.v.）-induced antinociceptionwas inhibited by co-injection with 10 nmol, but not 3 nmol [D-Cys（tBu）⁵]NPS, apeptidergic antagonist identified more recently, while [D-Cys（tBu）⁵]NPS （3 and 10nmol） alone induced neither hyperalgesia nor antinociception. These results revealedthat NPS could produce antinociception through NPS receptor, but not opioid receptor,and NPS-NPSR system could be a potential target for developing new analgesic drugs.PartⅡ: The pain-modulating roles of [Tyr⁶]γ2-MSH（6-12） at the spinal levelThe mas-related genes （Mrgs, also known as sensory neuron-specific receptors,SNSRs） are specific expressed in small diameter sensory neurons in the trigeminal anddorsal root ganglia, suggesting an important role of the receptors in pain transmission.The present study aimed to investigate the underlying mechanism of the nociceptiveeffects after activation of MrgC, and the interaction between MrgC and N/OFQ-NOPreceptor system in modulation of nociception in mice. Intrathecal （i.t.） administrationof [Tyr⁶]γ2-MSH（6-12）, the most potent agonist for MrgC receptor, produced asignificant hyperalgesic response as assayed by tail-withdrawal test and a series ofcharacteristic nociceptive responses, including biting, licking and scratching, in adose-dependent manner （0.01-10pmol and 0.01-10nmol, respectively） in mice. These pronociceptive effects induced by [Tyr⁶]γ2-MSH（6-12） were inhibiteddose-dependently by co-injection of competitive NMDA receptor antagonist D-APV,non-competitive NMDA receptor antagonist MK-801, and nitric oxide （NO）synthaseinhibitor L-NAME. However, the tachykinin NK₁ receptor antagonist L-703,606, andtachykinin NK₂ receptor antagonist MEN-10,376. had no influence on pronociceptiveeffects elicited by [Tyr⁶]γ2-MSH（6-12）.In other groups, [Tyr⁶]γ2-MSH（6-12）-induced nociceptive responses werebidirectionally regulated by the co-injection of N/OFQ. N/OFQ inhibited nociceptiveresponses at high doses （0.01-1 nmol）, but potentiated the behaviors at low doses （1fmol-3 pmol）. Furthermore, both hyperalgesia and nociceptive responses wereenhanced after the co-administration with NOP receptor antagonist[Nphe¹]N/OFQ（1-13）-NH₂.These results suggest that intrathecal [Tyr⁶]γ2-MSH（6-12）-induced pronociceptiveeffects may be mediated through NMDA receptor-NO system in the spinal cord, anddemonstrate the interaction between MrgC and N/OFQ-NOP receptor system in paintransmission.更多还原