【作者】 谢艳华；

【导师】 王四旺； 徐仓宝；

【作者基本信息】 第四军医大学 ， 药理学， 2010， 博士

【摘要】 1研究目的吸烟是动脉粥样硬化、冠心病、中风、外周血管疾病等心血管疾病的重要危险因素之一。大量研究表明,吸烟能导致血管壁损伤及血管内皮的局部功能紊乱,并能增强动脉粥样斑块形成。血管内皮细胞和血管平滑肌细胞(VSMC)表面及细胞内分布着不同类型的受体,其中分布在细胞膜上的G蛋白耦联受体(GPCR)家族是最重要的一类受体,与心血管疾病关系密切。大量实验与临床结果报道了生理或病理条件下GPCR的调控,GPCR对于血管收缩起重要作用,GPCR的活化可调节血流和血压水平,GPCR介导的VSMC收缩和增殖异常是血管疾病发展的重要因素。然而,对于吸烟伴随心血管疾病发病率增高的分子机制仍未完全阐明。本研究的目的旨在探讨器官培养及香烟烟雾提取物(DSP)对肾动脉血管平滑肌GPCR的影响,阐明其可能的细胞内信号转导机制,由此对于揭示吸烟相关GPCR紊乱所致的心脑血管疾病的发病机理、发现新的药物作用靶点具有重要意义。2方法采用大鼠肾动脉体外器官培养模型,将分离得到的大鼠肾动脉,去除内皮,制成1~1.5mm的血管环进行器官培养。将特异性或非特异性的细胞内信号通路抑制剂加到培养基中,以检验不同信号通路的参与情况。培养结束后,用灵敏的肌张力描记技术记录血管环的张力变化,依次在血管环浴槽中加入选择性受体激动剂以观察血栓素A2受体(TP)、内皮素受体A (ETA)和内皮素受体B(ETB)等GPCR介导的血管收缩;以实时在线反转录聚合酶链式反应(real-time RT-PCR)技术定量检测受体的mRNA表达;用免疫组织化学染色法结合激光扫描共聚焦显微镜测定受体的蛋白表达,阐明其可能的细胞内信号转导机制。在单纯器官培养的基础上,将吸烟这一心血管疾病的危险因子以香烟烟雾提取物DSP的形式引入培养体系,按上述方法从血管收缩功能、受体的mRNA表达量以及受体的蛋白表达水平三个方面探讨DSP对肾动脉血管平滑肌细胞TP、ETA和ETB受体等GPCR的影响及机制。3结果大鼠肾动脉经过24 h的培养,平滑肌细胞中TP下调。不同通路抑制剂使用后发现TP的下调未被非选择性翻译抑制剂CHX影响,但几乎完全被非选择性转录抑制剂ACD所消除,RT-PCR和免疫组化实验结果也显示TP mRNA表达和TP蛋白表达水平下降,提示TP下调可能与细胞内转录机制有关。进一步使用MAPK通路抑制剂后的研究表明,TP介导的收缩反应未被JNK和p38通路抑制剂SP600125和SB203580影响,但被ERK1/2通路抑制剂U0126部分程度地消除,说明TP的下调可能与激活ERK1/2信号通路有关;蛋白酶体抑制剂MG-132和与NF-κB信号通路抑制剂BMS345541可以明显消除器官培养所致的TP下调,显示ERK1/2的下游通路NF-κB也参与TP下调,IκBα和IκBβ信号通路均与此有关。器官培养导致ETA和ETB受体上调。ERK1/2通路抑制剂U0126和p38通路抑制剂SB203580抑制了ETB受体介导的这种增强的收缩反应;而对于ETA受体介导的收缩反应,U0126,SP600125和SB203580均没有明显影响。ETB受体上调可能与ERK1/2及P38信号通路及下游NF-κB途径有关,而ETA受体的上调可能与MAPK信号通路及下游NF-κB途径无关。此外,蛋白酶体抑制剂MG-132消除了器官培养所导致的TP下调或ETA和ETB受体上调,但磷酸二酯酶并未参与其中。器官培养24h后,Il-6和Il-1β的mRNA表达增高明显。肾动脉血管环在与DSP共同培养后,ETB、ETA和TP受体介导的平滑肌收缩反应明显增强,且这种增强与DSP的浓度在一定范围内(0.1～0.4μl﹒mL-1)呈浓度-效应依赖关系;此外,ETB受体介导的平滑肌收缩反应增强的程度和与DSP共同培养的时间在一定范围内(0～24h)呈时间-效应依赖关系。ETB受体的mRNA表达水平在与DSP共同培养后明显增加,但DSP的加入并未使ETA和TP受体的mRNA表达水平发生变化。免疫组织化学的实验结果显示与DSP共同培养后,在肾动脉血管平滑肌细胞中TP、ETB和ETA受体的蛋白表达水平明显增高。细胞内信号通路研究结果显示,DSP所致ETB受体水平的上调可以被NF-κB信号通路抑制剂BMS345541完全消除,而TP和ETA受体上调却不被BMS345541所影响。以上结果提示DSP引起的ETB受体改变可能与转录机制有关,而TP和ETA受体可能与转录后机制相关。尽管较高浓度的内毒素(LPS)表现出上调ET受体的作用,但LPS和尼古丁可能均不是DSP中引起上述受体改变的关键成分。4结论器官培养能够引起大鼠肾动脉血管平滑肌细胞TP水平下调, ETB和ETA受体水平上调。TP下调可能与细胞内ERK1/2的下游NF-κB通路有关,ETB受体上调可能与ERK1/2及P38信号通路及下游NF-κB途径有关,而ETA受体的上调可能与MAPK信号通路及下游NF-κB途径无关。DSP能够引起大鼠肾动脉血管平滑肌细胞ETB、ETA和TP受体水平上调。DSP引起ETB受体水平上调的机制可能与激活转录因子NF-кB途径有关;ETA和TP受体的上调则可能与转录后机制有关。LPS和尼古丁可能均不是DSP中引起上述受体改变的关键成分。更多还原

【Abstract】 ObjectiveCigarette smoking exposure is a well-known risk factor for hypertension, atherosclerosis (AS), coronary heart disease (CHD), stroke, myocardial infarction, aortic aneurysm, peripheral vascular disease and other cardiovascular diseases. There is plenty of evidences show that smoking may induce damage of vessel wall and disorder of blood vessel endothelium, also enhance the form of atheromatous plaque. There is different receptor subtypes located on or inside vascular endothelial cell and smooth muscle cells, among them, G-protein coupled receptors (GPCR) which located in the surface of the cell are most capital. Mountains of experimental and clinical studies have demonstrated that the GPCR in vascular smooth muscle (VSMC) play an important role on blood vessel contraction. The activation of GPCR can regulate blood flow and the level of blood pressure. The dysfunction of contraction and proliferation mediated by GPCR in vascular smooth muscle is an important cause for the development of vascular diseases. However, the underlying molecular mechanisms that cigarette smoke leads to higher morbidity of cardiovascular disease are not fully understood.The present study was designed to examine if organ culture or DSP regulate TP, ETA and ETB receptors in renal arteries, and which intracellular signal pathways are involved in the organ culture or DSP’s effects. Understanding the intracellular signal mechanisms behind cigarette smoke-induce renal arterial dysfunction and damage may provide a new target for developing therapies for hypertension and cardiovascular disease.MethodsRat renal arteries were dissected and after removal of endothelium, they were cut into about 1~1.5mm long cylindrical segments and subjected to organ culture. Specific intracellular signal pathway inhibitors were added to the culture medium to test the involvement of different signal pathways. A sensitive myograph system was used to record isometric tension of the segments, and selective agonists were added to tissue baths in turn to evaluate the TP, ETA and ETB receptor-mediated vessel contractility. The receptor mRNA expression was quantified by real-time reverse transcription PCR, while the receptor protein expression was examined using a laser scanning confocal microscope after immunohistochemistry staining.Based on the result of organ culture, the ring segments of renal arteries from rats were exposed to DSP (0.1~0.4 ml﹒L-1), nicotine or LPS, in the organ culture for up to 24 h. The alteration of GPCR caused by DSP was studied by three levels: functional evaluation, mRNA analysis and protein localization.ResultsWe observed down-regulation of the TP in rat renal artery after organ culture for 24h. The down-regulation of the TP was partly abolished by general transcriptional inhibitor ACD, but not abolished by general translational inhibitor CHX. The results of RT-PCR and immunohistochemistry showed that the mRNA and protein expression of TP decreased after organ culture. This suggests that the down-regulation of TP involves a transcriptional mechanism. Furthermore, the decreased contraction caused by organ culture was partly abolished by U0126 (a ERK1/2 pathway inhibitor), but not by SP600125 (a JNK pathway inhibitor) and SB203580 (a p38 pathway inhibitor). This suggests that ERK1/2 signal pathway involves the down-regulation of TP. The NF-κB pathway inhibitor BMS345541, co-incubated with the segments during the organ culture, revealed that organ culture-induced down-regulation of the TP significantly was attenuated. This means the down-regulation of TP receptor occurs through the NF-κB pathway. Additionally, we observed that the down-regulation of the TP was abolished by MG-132. This suggests that the down-regulation of TP receptor was activated through the IκB pathway; both IκBαand IκBβwere involved.ETA and ETB receptors were up-regulated after organ culture. The enhanced contraction mediated by ETB receptor was inhibited by U0126 and SB203580, while the enhanced contraction mediated by ETA receptor was not affected by U0126,SP600125 and SB203580. Suggesting that the up-regulated of ETB receptor maybe caused by the activation of ERK1/2and P38 pathway, while MAPK do not involve in the up-regulation of ETA receptor. Furthermore, MG-132 abolished the up- up-regulated of ETB and ETA receptor, but the inhibitors of PDE had no effects. After the segments organ culture for 24h, the mRNA expression of Il-6 and Il-1βincreased.The contractions mediated by TP, ETB and ETA receptors were significantly increased after the arterial segments exposed to DSP in a concentration- dependent (0.1~0.4 ml﹒L-1) manner and ETB receptor also presented in a time-dependent (0～24h) manner, compared to control (DMSO). This was in parallel with enhanced mRNA expression for ETB receptor, but not ETA and TP receptors. The results of immunohistochemistry showed that the protein expression of TP, ETA and ETB receptor increased after the segments cultured with DSP for 24h. BMS345541 totally abolished ETB receptor up-regulation, but not ETA and TP receptor up-regulations. Our results suggest that DSP transcriptionally up-regulated ETB receptor expression in rat renal artery via NF-кB signal pathways, whereas up-regulation of ETA and TP receptor-mediated contraction may involve post-transcriptional mechanisms. Both nicotine and LPS were unlikely involved in DSP effects, although LPS at the higher concentrations had such effects.ConclusionsTP was down-regulated after organ culture, while ETA and ETB receptors were up-regulated. The down-regulation of TP receptor occurs through the ERK1/2 and the downstream NF-κB pathway. The up-regulated of ETB receptor maybe caused by the activation of ERK1/2, P38 and NF-κB pathway, while MAPK and NF-κB pathway do not involve in the up-regulation of ETA receptor.DSP transcriptionally up-regulated ETB receptor expression in rat renal artery via NF-кB signals pathways, whereas up-regulation of ETA and TP receptor-mediated contraction may involve post-transcriptional mechanisms. Both nicotine and LPS were unlikely involved in DSP effects.更多还原