食慾素與內生性大麻酯在緊張所致止痛之貢獻：小鼠福馬林疼痛模式之研究

食慾素 (orexin) 系統由兩種神經胜肽 (食慾素A和食慾素B) 和兩種Gq蛋白偶合受體 (Gq-protein-coupled receptors; GqPCRs)，食慾素1受體 (orexin 1 receptor; OX1R) 和食慾素2受體 (orexin 2 receptor; OX2R)。食慾素，也稱做下視丘素(hypocretin)，是種下視丘胜肽，已經被研究出有止痛效果。先前我們已經發現在中腦環導水管灰質腹外側區 (ventrolateral periaqueductal gray; vlPAG) 活化食慾素1受體，能夠藉由內生性大麻酯之一的2-arachidonoylglycerol (2-AG)作用於γ-氨基丁酸 (γ-aminobutyric acid; GABA) 神經終端突觸上的大麻酯1受體 (cannabinoid 1 receptor; CB1R) 而抑制GABA釋放，於同個核區內進行逆行性去抑制 (disinhibition) 現象，進而活化下行性疼痛抑制路徑，產生止痛效果。緊張所致止痛 (stress induced analgesia; SIA) 是一種哺乳類的演化保護機制，用來處理緊張的狀況。各式各樣的壓力源可以抑制生物體對傷害性疼痛刺激的反應。急性束縛壓力 (acute restraint stress)，引發止痛的壓力源之一，已經被報導可以經由食慾素調節機制來止痛。在我們先前的研究中，發現對小鼠進行三十分鐘的束縛壓力可以在熱板實驗上呈現止痛效果，而這樣的緊張所致止痛現象可以被vlPAG內注射的OX1R和CB1R拮抗劑所阻斷。在本篇論文中，我們研究在由周邊發炎和接續的中樞促敏感性調控的福馬林疼痛實驗上，對小鼠進行三十分鐘的束縛壓力是否依然可呈現止痛效果，而此效果是否依然由位於vlPAG的食慾素和內生性大麻酯調控。首先，對小鼠進行三十分鐘的束縛壓力在福馬林實驗第二期呈現止痛效果，依此推測出急性束縛壓力可以在發炎疼痛模式的中樞促敏感期引發緊張所致止痛。第二，在腹腔或PAG注射選擇性拮抗劑去阻斷OX1R和CB1R可以抑制束縛壓力所致止痛。然而在PAG阻隔OX2R不會影響這種緊張所致止痛。這些結果推測出OX1R和CB1R被涵蓋在急性束縛壓力所引發的止痛效果，但OX2R則沒有。此外，我們也發現在未受束縛壓力的小鼠腹腔注射CB1R拮抗劑造成過度疼痛的狀況，但在PAG注射則沒有這樣的狀況。第三，naloxone在束縛壓力所致止痛沒有顯著影響，這表示內生性鴉片也許與緊張所致止痛無關。最後，對小鼠PAG注射食慾素A可減少福馬林實驗的第二期疼痛反應，而在PAG內注射OX1R和CB1R拮抗劑可以阻斷此現象。這表示在福馬林實驗中，vlPAG內的食慾素A可以和束縛壓力一樣，經由OX1R和CB1R產生止痛效果。我們先前的免疫組織化學實驗表示，相同的三十分鐘束縛壓力可以增加側下視丘的活化食慾素神經細胞數量。依這些結果可推測出在福馬林實驗中，側下視丘的活化食慾素神經細胞所釋放出的食慾素造成急性束縛壓力所致止痛。綜合本篇論文和先前的電生理結果，可推測出在小鼠福馬林實驗第二期中，急性束縛壓力可引發緊張所致止痛，而此現象是經由活化側下視丘的食慾素神經細胞進而釋放食慾素去活化vlPAG內的OX1R，並透過位於vlPAG內生性大麻酯調控的逆行性去抑制機制，活化下行性疼痛抑制路徑。

關鍵字

食慾素；內生性大麻酯；止痛；小鼠；福馬林；疼痛；束縛壓力

並列摘要

The orexin system consists of two neuropeptides (orexin A and orexin B) and two Gq-protein-coupled receptors (GqPCRs), orexin 1 receptor (OX1R) and orexin 2 receptor (OX2R). Orexin, also called hypocretin, the hypothalamic neuropeptides, has been known to be antinociceptive. Previously, we have found that activation of OX1R in the ventrolateral periaqueductal gray (vlPAG) can induce antinociception through the endocannabinoid, 2-arachidonoylglycerol (2-AG), engaging on the cannabinoid 1 receptor (CB1R) of presynaptic GABAergic terminals to inhibit GABA release, producing retrograde disinhibition in the PAG, which activates the descending pain inhibitory pathway. Stress induced analgesia (SIA) is an evolutional protection mechanism for mammals in coping with stressful conditions. A wide variety of stressors can inhibit the responses of organisms to noxious painful stimuli. Acute restraint stress, one of the stressors to induce analgesia, has been reported to induce analgesia through an orexin-mediated mechansim. In our previous study, we have found that a 30-min restraint stress in mice induces analgesia in the hot-plate test and this SIA effect can be blocked by intra-vlPAG injection of the OX1R or CB1R antagonist. In this study, we examined if in the formalin test, a pain model mediated by peripheral inflammation followed by a central sensitization mechanism, a 30-min restraint stress in mice can also induce analgesia, and this SIA is also regulated by orexins and endocannabinoids in the PAG. First, 30-min restraint stress in mice induced analgesic effect in the second phase of the formalin test, suggesting acute restraint stress can induce SIA in the central-sensitization phase of an inflammatory pain model. Second, blockade of OX1Rs or CB1Rs, either by i.p. or i.pag. injection of the selective antagonist suppressed restraint SIA. However, i.pag. blockade of OX2Rs did not affect this SIA. These suggest that OX1Rs and CB1Rs, but not OX2Rs, are involved in acute restraint stress-induced analgesic effect. Besides, we also found that i.p. but not i.pag., injection of the CB1R antagonist caused hyperalgesic effect in unrestrained mice. Third, naloxone had no significant effect on restraint SIA, suggesting endogenous opioids might not play a role in this SIA. Finally, i.pag. injection of orexin A in mice reduced the phase 2 nociceptive response in the formalin test and this effect was blocked by i.pag. injection of the OX1R or CB1Rs antagonist. This suggests that orexin A in the vlPAG can induce antinociception in the formalin test via OX1Rs and CB1Rs as did restraint stress. Our previous immunohistochemical study has shown that the same 30-min restraint stress can increase the number of activated orexin neurons in the lateral hypothalamus (LH). These results suggest that acute restraint SIA in the formalin test is caused by the orexins released from activated orexin neurons in the LH. Taken together, the results in this study and previous eletrophysiological results, it is suggested that acute restraint stress can induce SIA in the second phase of the formalin test in mice, and this SIA is mediated through activating the LH orexin neurons to release orexins that activate the OX1Rs in the vlPAG, resulting in activation of the descending pain inhibitory pathway through an endocannabinoid-mediated retrograde disinhibition mechanism in the vlPAG.

並列關鍵字

Restraint ； Stress-Induced Analgesia ； Mouse ； Formalin Test ； Orexin ； Endocannabinoid ； SIA ； stress ； PAG

參考文獻

Agarwal N et al. (2007) Cannabinoids mediate analgesia largely via peripheral type 1 cannabinoid receptors in nociceptors. Nature neuroscience 10:870-879.

Akil H, Mayer DJ, Liebeskind JC (1976) Antagonism of stimulation-produced analgesia by naloxone, a narcotic antagonist. Science (New York, NY) 191:961-962.

Amitani M, Umetani Y, Hosoi R, Kobayashi K, Abe K, Inoue O (2005) Changes in in vivo [(3)H]-Ro15-4513 binding induced by forced swimming in mice. Synapse 58:23-29.

Azhdari-Zarmehri H, Semnanian S, Fathollahi Y (2014) Orexin-A microinjection into the rostral ventromedial medulla causes antinociception on formalin test. Pharmacology, biochemistry, and behavior 122:286-290.

Azhdari Zarmehri H, Semnanian S, Fathollahi Y, Erami E, Khakpay R, Azizi H, Rohampour K (2011) Intra-periaqueductal gray matter microinjection of orexin-A decreases formalin-induced nociceptive behaviors in adult male rats. The journal of pain : official journal of the American Pain Society 12:280-287.

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食慾素與內生性大麻酯在緊張所致止痛之貢獻：小鼠福馬林疼痛模式之研究

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