香烟烟雾脂溶性组分通过MEK/ERK/5-HT受体途径增强脑动脉收缩性的研究

Cigarette smoking is a major risk factor for stroke which is closely related to cerebral vasoconstriction. We have already demonstrated that cigarette smoke enhanced 5-HT1B receptor-mediated contraction in basilar artery. However, what component of cigarette smoke particles is responsible for the above mentioned alteration and what is the molecular mechanism are still unclear. We speculate that the lipid-soluble smoking particles of cigarette smoke may activate MAPK signal pathway, then upregulate 5-HT1B receptors of cerebral artery and increase the cerebrovascular contractile response. In the present project we will use DMSO-soluble smoking particles (DSP) or water-soluble smoking particles (WSP) to treat cerebral arteries, rats and cells. The present project aims to examine vascular contractile and relaxant response by myograph technique; to determine receptor expression and pathway activation level by qRT-PCT, Western blotting and immunofluorescence; to monitor cerebral blood flow by laser speckle flow monitoring system; to detect cell apoptosis, proliferation and migration by flow cytometer, MTT or cell wound scratch assay; to examine the vascular endothelial cells ring formation ability by light microscope; using MAPK pathway inhibitors or RNA interference technique to explore the involvement of MAPK pathway in 5-HT1B receptor expression and its mediated contraction. The completion of the project may clarify the effects of DSP on cerebrovascular vasoconstriction and reveal the underlying molecular mechanism. It may provide new approach for the prevention and treatment of cigarette smoking-associated cerebrovascular diseases, and provide novel target for the development of new specific drugs.

吸烟是脑卒中的主要危险因素，其与脑血管收缩密切相关。我们前期发现，香烟烟雾能增强脑动脉对5-HT1B受体介导的收缩作用，而其有效组分和机制不明。我们假设，香烟烟雾中脂溶性组分通过活化MAPK信号通路，上调脑动脉5-HT1B受体，进而增强脑血管的收缩性。本项目拟用香烟烟雾颗粒的脂溶性或水溶性溶解物处理离体脑动脉、动物和细胞，用肌动描记技术测定脑动脉的收缩和舒张功能；用qRT-PCR、免疫印迹、免疫荧光研究5-HT1B受体表达及通路活化水平；用HE染色分析脑动脉病理学改变；用激光散斑血流系统监测脑血流变化；用流式细胞仪、MTT、划痕实验检测脑动脉平滑肌细胞凋亡、增殖、迁移能力；用光镜测定内皮细胞血管成环能力；用通路抑制剂及RNAi研究MAPK通路在5-HT1B受体表达及血管收缩中的作用。本项目的完成将阐明香烟烟雾有效组分诱发脑血管收缩的分子机制，为吸烟相关脑血管疾病的防治提供新思路和可能靶点。

吸烟是脑卒中的主要危险因素，其与脑血管收缩密切相关。我们前期发现，香烟烟雾能增强脑动脉对5-HT1B受体介导的收缩作用，而其有效组分和机制不明。我们假设，香烟烟雾中脂溶性组分通过活化MAPK信号通路，上调脑动脉5-HT1B受体，进而增强脑血管的收缩性。本项目拟用香烟烟雾颗粒的脂溶性或水溶性溶解物处理离体脑动脉、动物和细胞，用肌动描记技术测定脑动脉的收缩和舒张功能；用qRT-PCR、免疫印迹、免疫荧光研究5-HT1B受体表达及通路活化水平；用HE染色分析脑动脉病理学改变；用激光散斑血流系统监测脑血流变化；用流式细胞仪、MTT、划痕实验检测脑动脉平滑肌细胞凋亡、增殖、迁移能力；用光镜测定内皮细胞血管成环能力；用通路抑制剂及RNAi研究MAPK通路在5-HT1B受体表达及血管收缩中的作用。本项目的完成将阐明香烟烟雾有效组分诱发脑血管收缩的分子机制，为吸烟相关脑血管疾病的防治提供新思路和可能靶点。.已有的研究提示，香烟烟雾可增强脑血管的收缩性，该现象可能在缺血性脑卒中的发生、发展的机制中起重要作用。然而，香烟烟雾中的哪些组分引起上述变化，其具体分子机制是什么，目前仍不清楚。我们假设，香烟烟雾脂溶性组分通过活化MAPK分子信号通路，上调脑血管平滑肌5-HT1B受体，进而增强脑血管的收缩。本项目拟在以往研究的基础上，将香烟烟雾颗粒分别溶解为DSP和WSP，在整体动物、离体血管、细胞和分子水平上检测脑动脉5-HT1B受体介导的收缩功能、受体表达以及MAPK信号通路活化水平等指标。同时，在离体血管实验中使用通路抑制剂，细胞实验中使用小干扰RNA（siRNA），研究MAPK/5-HT受体途径对香烟烟雾不同组分引起脑血管收缩性改变的调控作用，阐明DSP增强脑动脉收缩性、诱发缺血性脑卒中的机制。本研究将为吸烟相关缺血性脑卒中的预防和治疗提供新思路，也可为药物研发提供新的作用靶点，具有重要的理论和实际意义。