內生性下視丘素透過中腦環導水管灰質內生性大麻酯之逆行性訊息貢獻於緊張導致之止痛

Abstract

致痛素(nociceptin)，即orphanin FQ, (N/OFQ)，是ORL1的內生性促效劑。ORL1，全名為類鴉片孤兒受器(opioid receptor-like orphan receptor 1)是第一個去孤兒化的G蛋白偶合受體(G-protein coupled receptor (GPCR)) ， 其後被列入鴉片受器家族， 並重新命名為NOR (N/OFQ peptide receptor)。第二個去孤兒化的G蛋白偶合受體為OX受器，包含了OX1及OX2兩種，其內生性促效劑由orexins A和B組成(或名為hypocretin 1 和2)，合稱為食慾素(orexins)或下視丘素(hypocretins)。N/OFQ 會抑制神經細胞的活性，而orexins則會促進其活性；將N/OFQ直接打入大腦會引起痛覺過敏(hyperalgesia)，給予orexins卻可以止痛。一般認為N/OFQ產生痛覺過敏是因為抑制了腦室內(i.c.v.)注射過程產生的緊張所致止痛 (stress-induced analgesia, SIA)，而有報告指出orexins有貢獻於SIA的產生。SIA是當人體或動物緊張時痛覺感受降低的一種現象，是由於中腦環島水管灰質(periaqueductal gray, PAG)內的腦內啡或腦內大麻素，活化這個負責下行性疼痛抑制訊息的腦區所產生的止痛效果。 我們之前發現在中腦環島水管灰質腹側區中注射orexin A會活化內生性大麻素的訊息傳遞路徑而產生止痛反應。Orexin A可活化OX1，一種Gq蛋白偶合受體，繼而促進phospholipase C (PLC)生成diacylglycerol (DAG)，接著由DAG lipase (DAGL)將DAG轉變為內生性大麻素之一的2-arachidonoylglycerol (2-AG)。2-AG會逆行活化突觸前的CB1受器，進而抑制GABA釋放。抑制中腦環島水管灰質的GABAergic transmission可以活化下行性疼痛抑制路徑，進一步產生止痛反應。已有證據指出緊張反應會活化下視丘的orexin神經細胞，促使orexins釋放，因此我們假設：緊張反應會活化投射到中腦環島水管灰質的下視丘orexin神經細胞，在中腦環島水管灰質釋放的orexin活化了OX1，接著經由PLC-DAGL的酵素路徑產生內生性大麻素所媒介的逆行性抑制反應，最終導致下行性疼痛抑制路徑的活化，產生止痛。另外，我們進一步假設在緊張時，將N/OFQ注射至腦內所觀察到的痛覺過敏是由於N/OFQ引發下視丘orexin神經細胞的過極化，降低orexin的釋放量，使得中腦環島水管灰質內的orexin和其下游內生性大麻素所調控的SIA被抑制，而產生的疼痛反應。 我們得到下列結果：1) 在SIA時，由免疫染色看到下視丘orexin神經細胞被激活；2) 在熱板實驗中，在老鼠上給限制行動之壓力所致SIA可以經由腹腔注射OX1或CB1拮抗劑所抑制；3) 在熱板實驗中，SIA可以經由PAG埋管給OX1或CB1拮抗劑所抑制；4)SIA的表現似乎是透過OX1受體所媒介而不是OX2；5)NOR致活劑似乎參與SIA的動物模式；6)Ro 64-6198具有活動力低下的表現。 因此結論出：緊張反應會活化下視丘orexin神經細胞，在中腦環島水管灰質釋放的orexin活化了OX1，接著經由酵素路徑產生內生性大麻素所媒介的逆行性抑制反應，最終產生止痛。

Nociceptin, also named orphanin FQ, (N/OFQ) is the endogenous agonist of ORL1, opioid receptorlike orphan receptor 1, the 1st de-orphanized G-protein coupled receptor (GPCR) which was renamed as N/OFQ peptide receptor (NOR) and enlisted in the opioid receptor family. Orexins, consisting of orexins A and B (also named hypocretin 1 and 2), are endogenous peptide agonists of the 2nd de-orphanized GPCR family, OX1 and OX2. Nociceptin inhibits, while orexins enhance, neuronal activity. Nociceptin was so named because it induces hyperalgesia, but orexins are analgesic, when given in the brain. The hyperalgesic effect of N/OFQ was believed to be due to its inhibition of stress-induced analgesia (SIA) generated by the i.c.v. procedure. Orexins, however, were reported to contribute to SIA. SIA is a phenomenon of reducing pain sensation in humans or animals during stress and can be mediated by endogenous opioids or cannabinoids in the periaqueductal gray (PAG), a midbrain region for initiating descending pain inhibition. We previously found that microinjection of orexin A in the ventrolateral PAG (vlPAG) induced analgesia through endocannabinoid signaling. Orexin A activates the OX1 receptors, a GqPCR family, leading to phospholipase C (PLC) activation and yielding diacylglycerol (DAG) which can be converted by DAG lipase (DAGL) into 2-arachidonoylglycerol (2-AG), an endocannabinoid. The generated 2-AG can produce retrograde inhibition of GABA release by activating the presynaptic CB1 receptors. Inhibition of GABAergic synaptic neurotransmission in the vlPAG results in activation of the descending pain inhibitory pathway, leading to analgesia. Since hypothalamic orexins are activated during stress and orexins contribute to SIA, we hypothesize that during stress, the orexin neurons in the lateral hypothalamus, which project to the vlPAG are activated; the released orexins then activate the OX1 receptors and initiate the endocannabinoid mediated retrograde disinhibition in the PAG via PLC-DAGL enzymatic pathway. In addition, we also hypothesize that N/OFQ, when given in the brain, will hyperpolarize the hypothalamic orexins to decrease the orexin release during stress, and then produces hyperalgesia through inhibiting orexin-mediated SIA in the PAG, which is endocannabinoid-dependent. We got the results : 1) orexin neurons are activated during SIA by the immunohistochemical approach; 2) SIA was induced by restrain stress in naïve mice but not in the mice pre-injected intraperitoneally with OX1R antagonist (SB 334867) or CB1 antagonist (AM 251) in the hot-plate test; 3) SIA was blocked by intra-PAG microinjection of OX1 antagonist (SB334867) or CB 1 antagonist (AM 251) in the hot-plate test.; 4) SIA behavior seems to be expressed through the OX 1 receptor, but not OX 2 receptor mediated; 5) NOR agonist seems to play a role in the stress-induced analgesia animal model; 6) Ro 64-6198 has hypolocomotor activity effect. According to our present results, we can conclude that: the activation of orexin neurons during the stress releases orexin to activate the OX1 receptor to generate the endocannabinoid by enzyme system to perform the analgesia.