緊張所致止痛的全新機轉︰食慾素、物質P和第5型代謝性麩胺酸受體之貢獻

Abstract

物質P (substance P) 已經被研究出在脊髓給予時可以發揮止痛效果並且和緊張所致止痛 (stress-induced analgesia; SIA) 有關，主要作用於神經激肽-1受體 (neurokinin-1 receptor; NK1R) 上，然而，它的實際作用機轉仍不清楚，但內生性大麻酯 (endocannabinoid) 被認為有參與其中的機制。在先前的研究中我們發現活化食慾素1受體 (orexin 1 receptor; OX1R) 能夠在中腦環導水管灰質腹外側區 (ventrolateral periaqueductal gray; vlPAG) 進行內生性大麻酯逆行性訊息 (retrograde signaling) 產生止痛效果，而這個效果是藉由食慾素A (orexin A) 所誘導且和緊張所致止痛有關。此外，過去有電生理的研究已經提出物質P會活化中腦環導水管灰質 (periaqueductal gray; PAG) 的麩胺酸神經元 (glutamatergic neuron)， 並釋放出大量麩胺酸 (glutamate) 作用在突觸周圍的第5型代謝性麩胺酸受體 (type 5 metabolic glutamate receptor; mGluR5) 產生內生性大麻酯，並在同個核區內藉由作用於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，出現逆行性去抑制 (disinhibition) 現象。 因此，我們在這篇論文中使用藥理學的方法驗證食慾素A活化在vlPAG內含有神經激肽的神經 (neuroknin-containing neuron) 釋放物質P經由麩胺酸-mGluR5-內生性大麻酯-CB1R的訊息路徑來產生止痛現象，而且此止痛機轉是藉由食慾素A所誘導。 首先，我們發現在vlPAG內注射物質P可以顯著增加縮腳潛伏期 (withdrawal latency) 的時間，而此止痛效果可以被MPEP和AM251所阻斷，代表mGluR5及CB1R皆涉及物質P的止痛效果。第二，在vlPAG內注射食慾素A能夠產生顯著的止痛效果，且能被L-703,606和MPEP所阻斷，指出NK1R和mGluR5皆參與在食慾素A的止痛作用中。第三，我們過去已經建立一個對小鼠使用束縛壓力 (restraint stress) 所造成之緊張所致止痛的模式，即將小鼠束縛在50 ml的離心管後其縮腳潛伏期會顯著增長，而在實驗中這樣的止痛模式可以藉由先投與L-703,606或MPEP被抑制掉，而其使用的劑量並不會影響老鼠的活動力。第四，經過束縛壓力處理過後的小鼠相較於控制組其vlPAG均質液內物質P蛋白質量顯著增加。第五，先在vlPAG內注射SB-334867能夠倒轉因為壓力而上升的物質P。 本篇論文的實驗結果和先前的電生理結果可以推測出物質P所產生的止痛是在vlPAG內透過活化麩胺酸神經釋放麩胺酸進而活化mGluR5，產生內生性大麻酯逆行性去抑制。既然緊張所致止痛可以被NK1R、mGluR5和OX1R拮抗劑所抑制，由此證明緊張所致止痛是在束縛壓力時釋放食慾素A產生止痛效果並在vlPAG內經由釋放物質P透過mGluR5-內生性大麻酯機制造成去抑制現象。

Substance P has been known to be antinociceptive when given at the supraspinal level and is involved in stress-induced analgesia (SIA), primarily through the neurokinin-1 receptor (NK1R). However, its action mechanism(s) remain unclear while may involve endocannabinoids. Previously, we have found that activation of orexin 1 receptors in the ventrolateral periaqueductal gray (vlPAG) can induce antinociception through the endocannabinoid retrograde signaling, and this effect may contribute to SIA induced by orexin A. Besides, an electrophysiological study showed that substance P can activate glutamatergic neurons in the PAG to release massive glutamate that activates perisynaptic type 5 metabotropic glutamate receptor (mGluR5), yielding endocannabinoid that engages on the cannabinoid 1 receptor (CB1R) of presynaptic GABAergic terminals to inhibit GABA release, producing retrograde disinhibition in the PAG. We, therefore, validated a hypothesis in this study that orexin A activates neurokinin-containing neurons in the vlPAG to release substance P and induce antinociception through the glutamate-mGluR5-endocannabinoid-CB1R signaling, and this mechanism may contribute to orexin A-induced SIA using the pharmacological approach. The SIA model was induced by giving mice a 30-min restraint stress and the antinociceptive response was accessed by the withdrawal latency of mice in the hot-plate test in mice. All drugs were administered by intra-vlPAG (i.pag.) microinjection. Besides, the level of substance P in the homogenate of the vlPAG in restrained un-restrained mice was also measured by enzyme immunoassay (EIA). First, we found that i.pag. microinjection of substance P significantly increased the withdrawal latency. This analgesic effect was blocked by MPEP, an mGluR5 antagonist and AM251, a CB1R antagonist, suggesting CB1Rs and mGluR5s are involved in the analgesic effect of substance P. Second, i.pag. orexin A produced a significant antinociceptive effect, in a manner blocked by i.pag. L-703,606, an NK1 receptor antagonist, and MPEP. This suggests that NK1R and mGluR5 are involved in the analgesic effect of orexin A. Third, we have established a SIA model induced by acute restraint stress in mice, i.e. the withdrawal latency of the mouse restrained in a 50-ml centrifuge tube was significantly longer than in the unrestrained group. This SIA was significantly prevented by i.pag. pretreatment with either L-703,606 or MPEP at the dose that did not affect the locomotor activity. Fourth, the substance P protein level in the vlPAG homogenate was significantly higher in restrained mice, as compared to the unrestrained control group. Fifth, stress-induced elevation of substance P in the vlPAG was significantly reversed by i.pag. pretreatment of SB-334867, a selective orexin OX1 receptor antagonist. The results in this study and previous electrophysiological results suggest that substance P produces supraspinal analgesia through activating glutamatergic neurons in the vlPAG to release glutamate that activates mGluR5, resulting in endocannabinoid retrograde disinhibition in the vlPAG. Since SIA can be blocked by NK1R, mGluR5 and OX1R antagonists, it is suggested that SIA is mediated orexins that are released during restraint stress to induce analgesia via releasing substance P that produces disinhibition in the vlPAG via the mGluR5-endocannabinoid mechanism.