尼古丁对血管外周脂肪组织的影响在动脉粥样硬化中的作用及机制研究

Many manuscripts and preliminary studies found that the probability of coexistence of smoking and obesity significantly increases in young patients with coronary artery disease. It suggests that the interaction between tobacco and adipose tissue may affect the occurrence and development of atherosclerosis(AS). However the role and mechanism remains to be explored. Nicotine impacts the activity and expression of NF-?B, and promotes the expression of inflammatory chemokines, cell surface receptors and cell chemotaxis, migration and proliferation through its characteristic nAChRs nicotine receptors on endothelial cells, macrophages and vascular smooth muscle cells. Perivascular adipose tissue associated with AS, however, whether adipocyte nicotine nAChRs-NF-?B regulation axis exists and affects the atherosclerotic process has not been reported. The Hypothesis would be that nicotine induces peripheral vascular adipose tissue and regulates their synthesis, secretion and other functions through nAChRs-NF-?B axis, then impacts atherosclerotic formation. To evaluate the hypothesis, this study uses gene knockout and RNAi technologies to observe the impact of nicotine-induced adipocyte nAChRs-NF-?B axis on AS, the expression of adipocyte on inflammatory chemokines, lipoprotein receptors and proteases of endothelial cells, macrophages and vascular smooth musclecells induced by nicotine, and the impact on macrophages and smooth muscle cell chemotaxis and phenotype in vitro and vivo. Besides, the hypothesis is verified by clinical samples. The project will clarify the effects of nicotine-induced perivascular adipose tissue on the functions and mechanism of AS, providing help for clinical medicine and targeted therapy.

年轻冠心病患者吸烟和超重/肥胖同时暴露率显著升高，提示烟草和脂肪组织相互作用可能影响了动脉粥样硬化（AS）启动和进展，但该作用及机制尚待探讨。尼古丁通过nAChRs受体作用于内皮细胞、巨噬细胞和血管平滑肌细胞，影响NF-kB活性和表达，促进炎症趋化因子和表面受体表达及细胞趋化迁移。血管外周脂肪与AS相关，但脂肪细胞是否存在尼古丁nAChRs-NF-kB调控轴并影响AS过程未见报道。由此推测，尼古丁诱导血管外周脂肪组织，通过nAChRs-NF-kB轴调控其合成分泌等功能，影响AS形成。为验证该假设，体内体外观察尼古丁诱导血管外周脂肪组织nAChRs-NF-kB轴对AS影响和尼古丁诱导脂肪细胞对巨噬细胞和平滑肌细胞的炎症趋化因子、脂蛋白受体等表达，及对平滑肌细胞等趋化和表型的影响，并用临床样本验证。本项目将阐明尼古丁诱导血管外周脂肪组织影响AS的作用和机制，为临床药物和靶向治疗提供帮。

动脉粥样硬化（atherosclerosis， AS）相关性心脑血管病（如冠心病、中风、下肢动脉疾病）是危害人类健康的常见病和多发病，也是全球致残、致死的主要原因。吸烟和肥胖是AS的主要危险因素。为了明确吸烟（主要有害成分尼古丁）和肥胖与AS和冠心病的关系及其机制：一、有研究表明自噬可调控AS的进展，但尼古丁诱导自噬在AS发生过程中作用及机制尚未明确。二、尼古丁可刺激巨噬细胞产生外泌体，其是否会通过旁分泌作用影响血管平滑肌细胞功能，尚未明确。三、分析吸烟、肥胖和外周血淋巴细胞在冠心病的分布及意义。因此，我们通过以下部分验证上述假设：一、Apoe-/-小鼠高脂饲养12周建立小鼠动脉粥样硬化模型，同时给予皮下注射尼古丁，观察12周后，HE染色和油红O染色检测斑块形成情况，α-SMA和CD68免疫组化染色检测斑块内血管平滑肌细胞和巨噬细胞量。体外实验中用尼古丁干预血管平滑肌细胞，Western blot和GFP-LC3荧光显像检测自噬标志蛋白LC3II/I、P62变化和自噬小体的形成。应用自噬激活剂/抑制剂确定自噬在尼古丁诱导血管平滑肌细胞中的作用。二、尼古丁干预巨噬细胞，通过超速离心法收集外泌体，进行鉴定及干预血管平滑肌细胞功能（增殖、迁移功能）。并且，进行高通量测序筛选差异miRNA，进行下游功能验证。三、收集稳定性心绞痛患者和心肌梗死患者的临床资料及血样，记录吸烟情况，测量体重、体脂数据，应用冠状动脉造影明确冠脉病变，流式细胞学分析淋巴细胞亚群百分比。通过实验，我们总结出以下结论：一、尼古丁可通过烟碱受体/氧化应激/NF-κB信号通路促进血管平滑肌细胞的表型转换及迁移、分泌功能，进而加重动脉粥样硬化。二、尼古丁刺激巨噬细胞产生的外泌体可促进血管平滑肌细增殖和迁移，其中miRNA-21-3p在尼古丁刺激巨噬细胞的外泌体内含量增多，且可促进血管平滑细胞功能改变。三、吸烟和超重/肥胖对冠心病影响甚大，其机制可能是通过影响免疫淋巴细胞的炎症 反应。这些研究结果都为进一步明确尼古丁致AS的机制提出了新的方向，也为治疗AS提供新的靶点。

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