气体分子硫化氢调控蛋白激酶C及其下游信号通路与干预吗啡耐受形成的关系

Morphine is a typical opioid drug mostly used for relieving moderate to serious pain in the clinical. However, repeated or prolonged administration of morphine would develop antinociptive tolerance, thus greatly limiting its clinical application. At present, there is still no effective way to prevent the development of morphine tolerance. Hydrogen sulfide (H2S) is a new gaseous signaling molecule and plays an important role in the central nervous system. In our preliminary experiment, we for the first time confirmed that treatment with exogenous H2S (NaHS) obviously blocked the development of morphine tolerance in mice. On the basis of published data and our work, we propose a hypothesis that H2S may regulate protein kinase C (PKC) and its downstream signaling pathway and further affect the target gene expression related to morphine tolerance and neuron adaptive changes, thus preventing the formation of morphine tolerance. To test this hypothesis, using a SH-SY5Y neuron cells, we plan to study the effect of H2S on PKC and its downstream signaling pathway in detail by up- or down- regulation of H2S level in the cell, including NMDA receptor, nitric oxide synthase (NOS), transcription factor CREB and its target genes. Moreover, we will set up cell or animal models of morphine tolerance to evaluate the role of H2S on the development of morphine tolerance in vitro and in vivo, by treating with H2S donors or promoting the endogenous H2S generation in the cell (H2S synthetase over-expression), and to clarify its relationship with the effect of H2S on the PKC signaling pathway. The findings obtained from this project will help us further elucidate the molecular mechanism of morphine tolerance and provide benefits for its prevention and treatment.

吗啡是临床最常用于治疗中重度疼痛的阿片类药物，长期使用易产生镇痛耐受且缺乏有效防治手段。硫化氢（H2S）是一种新型气体分子且具有重要的中枢调节作用。我们的预实验结果首次证实给予外源性H2S可明显抑制小鼠表现的吗啡耐受形成。在总结前人及自己工作基础上，我们提出假说即H2S可能通过调控蛋白激酶C（PKC）及其下游信号通路，进一步影响吗啡耐受相关靶基因表达及神经元适应性变化，从而干预吗啡耐受形成过程。为验证此假说，我们以SH-SY5Y神经元细胞株为实验对象，通过上调或下调细胞内H2S水平，详尽研究H2S对PKC及其下游信号通路主要环节的调控作用；并且建立吗啡耐受细胞和动物模型，给予外源性H2S或促进内源性H2S生成（促使H2S合成酶过表达），从体内外评价H2S对吗啡耐受形成的干预作用，并进一步阐明其与PKC及下游信号通路调控的关系。本课题可为阐明吗啡耐受分子机制及促进其临床防治提供新的理论依据。

吗啡是临床常用于治疗中重度疼痛的阿片类药物，长期使用容易产生镇痛耐受且缺乏有效防治手段。硫化氢（H2S）是一种新型气体分子且具有重要的中枢调节作用。在总结前人及自己工作基础上，我们提出假说即H2S可能通过调控蛋白激酶C（PKC）及其下游信号通路，进一步影响吗啡耐受相关靶基因表达及神经元适应性变化，从而干预吗啡耐受形成过程。为验证此假说，我们以SH-SY5Y神经元细胞株为实验对象，通过上调（给予外源性H2S）或下调（抑制H2S合成酶CBS表达）细胞内H2S水平，详尽研究H2S对PKC及其下游信号分子的调控作用。我们的研究证实：1、H2S对神经元细胞中PKC蛋白（PKCα、PKCγ 和PKCε）表达及转位、转录因子CREB磷酸化具有上调作用；2、长时程吗啡作用可上调某些特异的PKC亚型分子（PKCα、PKCε）表达及转位、NMDA 受体NR1磷酸化、细胞内一氧化氮（NO）及Ca2+水平，并可抑制转录因子CREB磷酸化；给予外源性H2S可明显逆转吗啡诱导的上述现象；3、长时程吗啡作用可导致内源性H2S水平降低，并代偿性诱导CBS表达上调，该现象与吗啡诱导的PKC信号通路改变并无直接相关性。我们进一步通过建立吗啡耐受细胞和动物模型，给予外源性H2S或抑制内源性H2S生成，探讨H2S对吗啡耐受形成的干预作用，并阐明其与PKC及cAMP信号通路调控的关系。我们的研究证实：1、细胞和动物实验均证实给予外源性H2S可在一定程度上抑制吗啡耐受形成，其机制与抑制PKC蛋白信号通路相关；2、CBS抑制剂氨基氧乙酸（AOAA）可阻断吗啡诱导的cAMP水平升高（吗啡耐受形成特征），其机制与抑制吗啡诱导的腺苷酸环化酶（AC）表达上调相关，并可进一步逆转吗啡诱导的立早基因c-fos及降钙素基因相关肽（CGRP）表达下调。本课题可为深入阐明吗啡耐受分子机制及促进其临床防治提供新的理论依据及治疗靶点。