Kappa阿片受体激动剂Salvinorin A 缓解蛛网膜下腔出血后脑血管痉挛的作用及机制的实验研究

Cerebral vasospasm is a medical emergency and still a leading cause of morbidity and mortality in patients after subarachnoid hemorrhage (SAH).Unfortunately, there is no optimal drug available to prevent and treat cerebral vasospasm in clinical practice. A safe and effective medication with easy administration is urgently needed. In this proposal, we will explore whether salvinorin A could be an alternative medication based on our recent discoveries that salvinorin A is a potent cerebral vascular dilator and potential neuro-protectant. Salvinorin A is a non-opioid, highly selective opioid receptor agonist from natural resources with easy pass of blood brain barrier as a potential therapeutics. A member of the team, Dr. Renyu Liu at the University of Pennsylvania found that Salvinorin A dilates cerebral vessels in both physiological and pathological conditions (endothelin-induced) via activation of nitric oxide (NO) synthase, adenosine triphosphate-sensitive potassium channel, and the kappa opioid receptor(KOR). Endothelin places critical role in developing cerebral vascular spasm after SAH. Thus, we hypothesized that salvinorin A could be used to manage cerebral vasospasm after SAH via KOR, cGMP and KOR pathway. This program includes three parts: (1)To investigate the effectiveness of salvinorin A and the role of KOR in a rodent cerebral vasospasm model by using salvinorin A and its specific antagonist, Norbinaltorphimine; (2) To investigate the role of nitric oxide(NO) pathway with an eNOS inhibitor(L-NAME);(3)To investigate the role of cGMP with protein kinase G(PKG)specific inhibitor(KT5823)and cAMP with a protein kinase A(PKA)specific inhibitor(H89). To investigate the effect of salvinorin A on the cerebral vasospasm, a sensitive MRI will be used to measure the diameter of basal artery and cerebral blood flow. The molecular microbiological, morphological and microcirculation outcomes after salvinorin A administration will also be investigated.

脑血管痉挛是蛛网膜下腔出血病人致死和致残的主要原因，也是临床和实验研究的热点。Salvinorin A（SA）是一种特殊的强效κ-阿片受体激动剂。最近，美国宾夕法尼亚大学Renyu Liu教授的研究揭示SA对缺血/缺氧后的脑血管具有强烈舒张作用。为此，我们提出合作意向和科学假说：SA有可能通过激活κ-阿片受体后调控多条细胞内的信号传导通道，舒张痉挛的血管，达到缓解脑血管痉挛的作用。研究内容：（1）分别采用SA治疗和KOR抑制剂（Nor-BNI）阻断KOR通道；（2）在SA作用的基础上，采用L-NAME(eNOS抑制剂)剥夺NO作用通道；（3）在SA作用的基础上，用PKG抑制剂（KT5823）阻断cGMP通道，和PKA抑制剂（H89）阻断cAMP通道。采用11.7-T实验用MRI观测大鼠基底动脉管径及脑血流量；采用分子生物学、形态学和微循环方法研究其机制，探索临床应用的前景。

目的：探讨kappa型阿片受体激动剂Salvinorin A（SA）对蛛网膜下腔出血（subarachnoid hemorrhage, SAH）后脑血管痉挛和早期脑损伤的缓解作用及其机制。主要研究内容：雄性SD大鼠（280 ~ 300g），采用动脉刺破法建立大鼠SAH模型，随机分为假手术组、SAH模型组、溶剂对照组和给药组（SA 2、10、50 μg/kg），SA及溶剂DMSO于SAH模型后24 h、48 h、72 h用生理盐水稀释后腹腔注射。SAH后12 h、24 h、48 h、72 h检测大鼠神经功能学评分，SAH后72h，HE染色观察基底动脉血管内径及血管壁厚度，磁共振成像（MRI）观察基底动脉及海马组织，内皮素-1 Elisa试剂盒和一氧化氮试剂盒检测Willis环血管及基底动脉上ET-1浓度和NO含量，Nissl染色及TUNEL染色观察SAH后海马CA1区神经元的形态及神经细胞的凋亡，Western Blot检测目的蛋白的表达。重要结果：（1）SA能够不同程度地升高大鼠SAH后12 h、24 h、48 h、72 h的神经学功能；（2）SA能够增加SAH后基底动脉的血管内径、降低血管壁厚度，并选出SA 10 μg/kg为有效剂量浓度；（3）MRI及双光子显微镜发现SA能够扩张活体状态下的血管；（4）SA能够降低SAH后ET-1的浓度，升高NO的含量；（5）SA能够升高p-PI3K/PI3K、p-Akt/Akt及eNOS蛋白的表达，该作用可以被PI3K抑制剂Wortmannin和eNOS的抑制剂L-NAME所抑制；（6）免疫组织化学染色及免疫荧光染色发现eNOS表达于血管内皮细胞。结论及科学意义：SA能够通过PI3K/Akt/eNOS通路缓解SAH后基底动脉及海马内小血管痉挛，并降低SAH后海马组织神经细胞的凋亡，进而减轻SAH后脑血管痉挛和早期脑损伤。