基于“调和脾胃，心无凝滞”探讨健脾祛痰化瘀方对Nox5-ERK1/2通路调控冠脉内皮氧化应激所致炎症反应的作用机制

Atherosclerotic cardiovascular disease is a serious disease that is related to people's livelihood and national economy. Method of 'Jian Pi Qu Tan Hua Yu' is the root of Traditional Chinese Medicine treating coronary heart disease. Our study group has found that 'Jian Pi Qu Tan Hua Yu' prescription may regulate NADPH oxidase 5 (Nox5) gene level, inhibit the oxidative stress-induced inflammatory response in coronary endothelium, and reduce atherosclerosis. However, the mechanism is not yet clear. This study is guided by the theory of 'reconcile spleen and stomach to remove stagnation from heart'. Thus we put forward the following hypothesis that 'Jian Pi Qu Tan Hua Yu' prescription may regulate Nox5-ERK1/2 pathway in intervening oxidative stress-induced inflammatory response in coronary endothelium to mitigate atherosclerosis. We will establish the model of atherosclerosis in Bama miniature pig to explain the mechanism of phlegm and blood stasis which is caused by spleen deficiency strengthening oxidative stress-induced inflammatory response in coronary endothelium. We will apply CRISPR/Cas9 technology to knockout Nox5 gene from human coronary endothelial cell to establish cell model. We will investigate the effects of 'Jian Pi Qu Tan Hua Yu' prescription on coronary endothelial structure, oxidative stress-induced inflammatory response and Nox5-ERK1/2 pathway through the in vitro and in vivo experiment. We will elucidate the mechanism of 'Jian Pi Qu Tan Hua Yu' prescription treating atherosclerosis and the machanism of spleen deficiency-induced phlegm and blood stasis exacerbating atherosclerosis. We will clarify the scientific connotation of the theory 'reconcile spleen and stomach to remove stagnation from heart' from levels of organ, tissue, cell and molecular. This study will provide a scientific basis for treating coronary heart disease from the perspective of spleen.

动脉粥样硬化性心血管疾病是关系国计民生的重大疾病。健脾祛痰化瘀法是中医治疗冠心病的治本之道。通过课题组前期研究发现健脾祛痰化瘀方可调节NADPH氧化酶5（Nox5）基因，抑制氧化应激所致炎症反应，减轻动脉粥样硬化，但作用机制尚不明确。本研究基于“调和脾胃，心无凝滞”理论，提出如下假说：健脾祛痰化瘀方通过Nox5-ERK1/2通路调控冠脉内皮氧化应激所致炎症反应，减轻动脉粥样硬化。本项目拟构建巴马小猪动脉粥样硬化模型，阐述脾虚痰瘀互结增强血管内皮氧化应激所致炎症反应的机制，结合CRISPR/Cas9技术敲除人冠脉内皮细胞Nox5基因构建细胞模型，通过体内及体外实验探讨健脾祛痰化瘀方对冠脉内皮、氧化应激所致炎症反应及Nox5-ERK1/2通路的影响，阐明健脾祛痰化瘀方治疗动脉粥样硬化的效应机制，从器官、组织、细胞、分子水平阐明“调和脾胃，心无凝滞”的科学内涵，为“从脾论治”冠心病提供科学依据。

动脉粥样硬化性心血管疾病是关系国计民生的重大疾病。健脾祛痰化瘀法是中医治疗冠心病的治本之道。本研究基于“调和脾胃，心无凝滞”理论，通过构建ApoE基因敲除小鼠动脉粥样硬化模型、巴马小猪动脉粥样硬化模型、动脉粥样硬化大鼠模型以及人脐静脉内皮细胞AngⅡ损伤模型，阐述了脾虚痰瘀互结增强血管氧化应激所致炎症反应，抑制血管及心肌细胞线粒体自噬的疾病机制。结果发现：动脉粥样硬化动物模型组血脂和细胞因子（VCAM-1、TNF-α、IL-6、IL-10、hs-CRP、ET）水平升高、病理切片显示主动脉平滑肌细胞增殖明显，细胞排列紊乱，弹力纤维层结构不清晰，可以看到具有狭窄管腔和大量空泡的动脉粥样硬化斑块。Nox5、ERK1/2、L-type Ca2+ channel、VCAM-1蛋白和基因表达上调，Pink1、Mfn2、Parkin蛋白和基因表达下调、miRNA-125a表达下调。健脾祛痰化瘀低中高浓度组血脂和细胞因子水平降低，病理切片显示主动脉壁内膜较光滑，结构较完整，有少量炎症细胞浸润。且Nox5、ERK1/2、L-type Ca2+ channel、VCAM-1蛋白和基因表达下调，Pink1、Mfn2、Parkin蛋白和基因表达上调、miRNA-125a表达上调。通过体内及体外实验验证了健脾祛痰化瘀方能够通过Nox5-ERK1/2通路抑制动脉氧化应激所致炎症反应、通过miRNA-125a调控Pink1-Mfn2-Parkin通路增强线粒体自噬，从而治疗动脉粥样硬化的效应机制，从器官、组织、细胞、分子水平阐明“调和脾胃，心无凝滞”的科学内涵，为“从脾论治”冠心病提供科学依据。