PhKv 是一种从蜘蛛毒液中分离出来的毒素，通过抑制胆碱酯酶激活胆碱能系统来诱导镇痛,Scandinavian Journal of Pain

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PhKv 是一种从蜘蛛毒液中分离出来的毒素，通过抑制胆碱酯酶激活胆碱能系统来诱导镇痛
Scandinavian Journal of Pain Pub Date : 2024-02-26 , DOI: 10.1016/j.sjpain.2017.09.019
Flavia Karine Rigo ₁ , Mateus Fortes Rossato ₂ , Gabriela Trevisan ₁ , Samira Dal-Toé De Prá ₁ , Rafael Porto Ineu ₃ , Mariane Bernardo Duarte ₁ , Célio José de Castro Junior ₄ , Juliano Ferreira ₅ , Marcus Vinicius Gomez ₄

Affiliation

背景和目的胆碱能药物通过模仿脊髓胆碱能神经释放乙酰胆碱 (ACh) 来引起镇痛作用。PhKv 是从武装蜘蛛的毒液中分离出来的肽黑风鳅。它具有抗心律失常活性，涉及增强乙酰胆碱的释放。本研究的目的是调查 PhKv 是否对小鼠具有镇痛作用。方法本研究使用雄性白化瑞士小鼠（25-35 克）。PhKv 毒素是从 PhTx3 级分中纯化出来的。黑风鳅蜘蛛的毒液。由于其肽性质，PhKv 不可口服，而是通过鞘内 (it) 途径直接递送至中枢神经系统。使用足底测试仪和上下法评估 PhKV 对热和机械敏感性的影响。在神经性疼痛 (CCI) 和外周辣椒素试验中研究了 PhKv 的镇痛作用。为了测试PhKv是否干扰胆碱能系统，用阿托品（5mg/kg，腹腔注射）或美加明（0.001mg/kg，腹腔注射）和PhKv毒素（30pmol/位点）或新斯的明预处理小鼠(100 pmol/位点)在足底内施用辣椒素(1 nmol/爪)之前15分钟施用。为了研究 PhKv 对 AChE 活性的作用，进行了体外和离体AChE 测定。为了体外实验中，小鼠脊髓组织匀浆上清液 (1 mg/ml) 用作 AChE 活性来源。AChE 测定在 FlexStation 3 多功能酶标仪 (Molecular Devices) 中于 37 °C 405 nm 下监测 10 分钟。结果 PhKv（30 和 100pmol/位点，it）对热或机械灵敏度阈值没有影响。然而，在慢性压迫性疼痛损伤模型中，PhKv（10pmol/位点，it）可显着减少机械撤退，并具有持续 4 小时的镇痛作用。在足底注射辣椒素（1 nmol/爪）前 15 分钟，用 PhKv（30pmol/位点，it）或新斯的明（100pmol/位点，it）对小鼠进行预处理，可产生最大的镇痛效果，分别为 69.5 ± 4.9% 和 85 ± 2.5% ，分别。用阿托品对小鼠进行预处理；5 mg/kg，腹膜内注射或美加明 0.001 mg/kg，腹膜内注射可抑制新斯季明和 PhKv 诱导的镇痛作用，表明存在胆碱能机制。PhKv 抑制脊髓乙酰胆碱酯酶并导致 ED507.6（4.6–12.6 pmol/位点，it）。PhKv 还抑制体外EC 脊髓匀浆的 AChE 活性5020.8 (11.6–37.3 nM)，Km 值从 0.06 mM 变为 18.5 mM，表征 PhKv 对 AChE 活性的竞争性抑制。结论据我们所知，我们的研究结果提供了第一个证据，证明 PhKv 会抑制 AChE，它增加了神经元突触的 ACh 含量，导致胆碱能系统的激活和抗伤害反应。影响关于伤害感受机制和确定疼痛治疗潜在靶点的研究已成为首要任务。PhKv 通过刺激胆碱能受体毒蕈碱和烟碱系统来减轻疼痛，它可能是控制疼痛过程的替代方法。

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PhKv a toxin isolated from the spider venom induces antinociception by inhibition of cholinesterase activating cholinergic system

Background and aims Cholinergic agents cause antinociception by mimicking the release of acetylcholine (ACh) from spinal cholinergic nerves. PhKv is a peptide isolated from the venom of the armed spider Phoneutria nigriventer. It has an antiarrythmogenic activity that involves the enhanced release of acetylcholine. The aim of this study was to investigate whether PhKv had an antinociceptive action in mice. Methods Male albino Swiss mice (25–35 g) were used in this study. The PhKv toxin was purified from a PhTx3 fraction of the Phoneutria nigriventer spider’s venom. Because of its peptide nature, PhKv is not orally available and it was delivered directly into the central nervous system by an intrathecal (i.t.) route. PhKV on the thermal and mechanical sensitivity was evaluated using plantar test apparatus and the up-and-down method. The analgesic effects of PhKv were studied in neuropathic pain (CCI) and in the peripheral capsicin test. In order to test whether PhKv interfered with the cholinergic system, the mice were pre-treated with atropine (5mg/kg, i.p.) or mecamylamine (0.001 mg/kg, i.p.) and the PhKv toxin (30 pmol/site i.t.) or neostigmine (100 pmol/site) were applied 15 min before the intraplantar capsaicin (1 nmol/paw) administrations. To investigate PhKv action on the AChE activities, was performed in vitro and ex vivo assay for AChE. For the in vitro experiments, mice spinal cord supernatants of tissue homogenates (1 mg/ml) were used as source of AChE activity. The AChE assay was monitored at 37 °C for 10 min in a FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices) at 405 nm. Results PhKv (30 and 100pmol/site, i.t.) had no effect on the thermal or mechanical sensitivity thresholds. However, in a chronic constriction injury model of pain, PhKv (10pmol/site, i.t.) caused a robust reduction in mechanical withdrawal with an antinociceptive effect that lasted 4 h. A pretreatment in mice with PhKv (30pmol/site, i.t.) or neostigmine (100pmol/site, i.t.) 15min before an intraplantar injection of capsaicin (1 nmol/paw) caused a maximal antinociceptive effect of 69.5 ± 4.9% and 85 ± 2.5%, respectively. A pretreatment in mice with atropine; 5 mg/kg, i.p. or mecamylamine 0.001 mg/kg, i.p. inhibited a neostigimine and PhKv-induced antinociception, suggesting a cholinergic mechanism. Spinal acetylcholinesterase was inhibited by PhKv with ED50 of 7.6 (4.6–12.6 pmol/site, i.t.). PhKv also inhibited the in vitro AChE activity of spinal cord homogenates with an EC50 of 20.8 (11.6–37.3 nM), shifting the Km value from 0.06 mM to 18.5 mM, characterizing a competitive inhibition of AChE activity by PhKv. Conclusions Our findings provide, to our knowledge, the first evidence that PhKv caused inhibition of AChE, it increased the ACh content at the neuronal synapses, leading to an activation of the cholinergic system and an antinociceptive response. Implications Studies regarding the nociceptive mechanisms and the identification of potential targets for the treatment of pain have become top priorities. PhKv, by its action of stimulating the cholinergic receptors muscarinic and nicotinic system, reduces pain it may be an alternative for controlling the pain processes.

更新日期：2024-02-26

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