基于核受体Nur77探讨景虎通脉方介导M1/M2巨噬细胞极化抗动脉粥样硬化的研究

Atherosclerosis (AS) has been proved to be a chronic inflammatory disease and macrophages are closely related to its formation and progression. Because of the diverse roles in AS, the polarization of M1 and M2 macrophages can affect its development and outcome. How to make good use of the plasticity and pluripotency of macrophages has vast importance for the AS prevention and treatment. The current studies showed that the orphan nuclear receptor, Nur77, might regulate and control the specific expressions of phenotypes of macrophage subsets which can induce the differentiation and polarization of macrophage by activating toll-like receptor (TLR) signal pathway. Jin-hu-tong-mai Prescription is an experienced prescription from professor Zhao-chun, a famous expert in Traditional Chinese Medicine in Yunnan Province. During our previous work, it has been confirmed that Jin-hu-tong-mai Prescription, which is an experienced prescription from professor Zhaochun a can reduce the expressions of inflammatory factors and chemokines and then alleviate the formation of plaques on carotid atherosclerosis rabbit models, which might be effective through TLR4 signal pathway. Using methods such as immunohistochemistry, flow cytometry, Western Blot, ELISA, real-time quantitative PCR, and Laser scanning confocal etc., the study is to explore the intervention of Jin-hu-tong-mai Prescription on the expressions of phenotypes and functions of M1 and M2 macrophage of Apolipoprotein E (ApoE) knockout AS mice model by Nur77 to protect the blood vessels and stabilize the plaques. In the experiment in vitro, we also plan to study if the serum containing Jin-hu-tong-mai Prescription can regulate the polarization of M1 and M2 THP-1macrophages transfected with the constructed Nur77 Fluorescence expression vector. Our project will provide the possible mechanisms of Jin-hu-tong-mai Prescription in AS intervention and bring new idea and more evidence for the AS prevention and treatment with important value of science and broad application prospect. .

M1和M2型巨噬细胞分型极化与动脉粥样硬化（AS）的发生发展密切相关，核受体Nur77可通过TLR4信号通路以调控巨噬细胞特异性表型表达从而介导巨噬细胞分型极化。景虎通脉方是云南省名中医赵淳教授经验方，我们前期工作证实，景虎通脉方可通过抑制TLR4信号通路降低白介素-6、细胞间黏附分子-1的表达以改善颈AS形成。本课题在此基础上，通过体内和体外试验，采用免疫组化、流式细胞、Western Blot、ELISA、实时定量PCR法、激光共聚焦等方法，观察景虎通脉方能否通过Nur77影响ApoE-/-AS小鼠斑块内M1/M2型巨噬细胞分化进而达到保护血管、稳定斑块的作用；构建Nur77荧光表达载体并转染至巨噬细胞，研究景虎通脉方含药血清对M1/M2型巨噬细胞分化的作用及对Nur77表达的影响，深入探讨景虎通脉方干预AS的可能机制，为AS的防治提供实验依据，有重要科学价值及较广阔的应用前景。

M1和M2型巨噬细胞分型极化与动脉粥样硬化（AS）的发生发展密切相关，核受体Nur77可通过TLR4信号通路以调控巨噬细胞特异性表型表达从而介导巨噬细胞分型极化。前期工作证实，景虎通脉方可通过抑制TLR4信号通路降低白介素-6、细胞间黏附分子-1的表达以改善颈AS形成。本课题在此基础上，通过体内和体外试验，采用病理染色、免疫组化、流式细胞、Western Blot、ELISA等方法，研究发现：动物实验中，景虎通脉方及其成分红景天苷可能通过增加ApoE-/-AS小鼠主动脉斑块内Nur77表达，从而抑制TLR4信号通路，抑制小鼠炎性因子TNF-ɑ、IL-1β、IL-6、MCP-1、IL-12p70、iNOS等的分泌，抑制炎症反应，调控巨噬细胞从M1型向M2型转变，促进IL-10、IL-13、CD206的分泌，从而起到稳定斑块、保护血管抗ATH的作用；细胞实验中，通过对Nur77荧光表达载体并转染至巨噬细胞，研究发现景虎通脉方含药血清及红景天苷可以减少巨噬细胞内脂质的沉积，抑制泡沫细胞的形成；通过抑制炎性介质NO及M1型细胞因子IL-6、TNF-α及IL-1β的分泌，抑制巨噬细胞向M1型极化；且可能是通过抑制CD36、CD204的表达来减少泡沫细胞的形成，从而延缓动脉粥样硬化的发生和发展。本研究探讨了景虎通脉方及其成分红景天苷干预AS的可能机制，为AS的防治提供实验依据和研究思路。