物質P引起的止痛作用在肌肉痛覺神經細胞的電生理與分子特性

Abstract

先前的研究發現酸會造成物質P的釋放，進而導致肌肉的止痛作用，而一實驗室研發的止痛藥物T1-11被發現無法在第三型酸敏感通道剔除且經過週期性冷刺激小鼠上產生作用，又發現T1-11引起的向外電流可以被物質P受體的拮抗劑減低，因次我認為物質P的止痛作用會受到剔除第三型酸敏感通道影響，首先，我利用單一細胞逆轉錄聚合酶鏈反應探討第三型酸敏感通道與瞬態感受器陽離子通道V1與物質P是否共同表現在同一個肌肉傳入的背根神經節神經細胞，結果發現，部分第三型酸敏感通道與瞬態感受器陽離子通道V1都有與物質P共同表現在同一顆細胞，其結果說明第三型酸敏感通道與瞬態感受器陽離子通道V1都有可能造成物質P的釋放。我接下來利用電生理的方式檢驗物質P與T1-11引起的向外電流是否會受到少了第三型酸敏感通道影響，結果顯示，物質P所引起的向外電流的細胞比例以及電流大小並不會受到剔除第三型酸敏感通道的影響，然而，在未經過週期性冷刺激小鼠，T1-11所引起的向外電流比例會受到剔除第三型酸敏感通道而顯著下降，但經過週期性冷刺激小鼠不會受到影響。除此之外，我發現剔除第三型酸敏感通道會顯著增加肌肉傳入的背根神經節神經細胞產生動作電位的閥值，其代表著第三型酸敏感通道有控制細胞神經活性的功能。另一方面，物質P所引起的向外電流會因為經過週期性冷刺激而增加，其說明物質P所引起的內生性止痛作用會受到週期性冷刺激而增強。我同時也發現部分對物質P沒有反應的細胞，其動作電位的過衝率也會受到週期性冷刺激而增加，因為較大的過衝率一般被認為是痛覺細胞的神經特性，我認為經過週期性冷刺激的肌肉傳入的背根神經節細胞會產生可塑性變化而趨向於痛覺神經元的特性。總結，我發現在未經過週期性冷刺激小鼠中，第三型酸敏感通道會顯著降低T1-11肌肉肌肉傳入的背根神經節神經細胞引起的向外電流的比例，此結果可能為T1-11無法在剔除第三型酸敏感通道中產生作用的原因。

Previous study demonstrated that acid trigger substance P (SP) release and produced antinociceptive effect in muscle nociceptors, by which SP specifically enhances M-type potassium current and attenuates ASIC3-induced inward current on most gastrocnemius muscle (GM) afferent dorsal root ganglion (DRG) neurons Furthermore, previous study also indicated that the analgesia effect of T1-11, an active ingredient from Gastrodia elata for pain management, is ineffective in ASIC3 KO mice after intermittent cold stress (ICS) treatment and T1-11-induced outward current can be attenuated by NK1 antagonist. Therefore, I aimed to probe whether ASIC3 contributes to SP-mediated antinociception. Firstly, I identified the molecular identity of muscle afferent DRG neurons by use of single cell RT-PCR. The co-expression of SP with ASIC3 or TRPV1 implied the possibility that ASIC3 and TRPV1 serve as the antinociceptive acid sensors that release SP. Besides, I aimed to know whether loss of ASIC3 affects SM-SP-, a synthesized peptide analog of SP, and T1-11-induced outward current (ISM-SP-O and IT1-11-O) in muscle afferent DRG neurons by use of whole-cell patch clamp recording. The result demonstrated that deletion of ASIC3 did not affect neuronal population and average peak current of ISM-SP-O. However, I found diminished neuronal population of IT1-11-O after loss of ASIC3 in naïve but not ICS-treated mice. Furthermore, I found increased action potential (AP) threshold after deletion of ASIC3 and revealed a role of ASIC3 in setting the AP threshold. On the other hand, increased average peak current of ISM-SP-O was found in muscle afferent DRG neurons after ICS treatment, which suggests an enhanced SP-mediated antinociception. Besides, elevated AP overshoot was observed after ICS treatment in a subset of neurons that do not respond to SM-SP. Because large overshoot has been implicated as a feature of nociceptors, elevated overshoot after ICS treatment may indicate a pronociceptive neuronal plasticity occurred in these neurons.