淫羊藿苷调控mTOR和p38MAPK信号通路抑制内皮祖细胞凋亡和自噬修复血管内皮损伤的作用机制研究

Reduced tissue levels of endothelial progenitor cells (EPCs) and functional impairment of endothelium are frequently observed in patients with atherosclerosis and other cardiovascular disease. The vascular endothelium is specifically sensitive to oxidative stress, and this is one of the mechanisms that causes widespread endothelial dysfunction in most cardiovascular diseases and disorders. Hence attention has increasingly been paid to enhance mobilization and differentiation of EPCs for therapeutic purposes. Icariin, a natural bioactive component known from Traditional Chinese Medicine, can induce angiogenic differentiation and inhibit oxidative stress-induced cell dysfunction in bone marrow derived EPCs (BM-EPCs), but the underlying mechanisms are not well understood. We observed that treatment of BM-EPCs with Icariin significantly promoted cell migration and capillary tube formation, substantially abrogated hydrogen peroxide (H2O2)-induced apoptotic and autophagic programmed cell death that was linked to the reduced intracellular reactive oxygen species (ROS) levels and restored mitochondrial membrane potential (MMP). These anti-apoptotic and anti-autophagic effects of Icariin are possibly mediated by restoring the loss of Akt/mTOR/4EBP1 phosphorylation as well as attenuation of p38 MAPK/ATF2 protein levels after H2O2 treatment. We accordingly suppose that Akt/mTOR/4EBP1 and p38 MAPK/ATF2 signaling pathways may play important roles in attenuation of oxidative stress and endoplasmic reticulum stress-induced cell apoptosis and autophagy, therefore contributing to the repairment of endothelial injury. We plan to reveal the underlying mechanisms of angiogenic and protective effect of Icariin via detecting the effect of Icariin on homing and angiogenic differentiation of EPCs in vitro and in vivo, on cell apoptosis and autophagy under the oxidative stress and endoplasmic reticulum stress, and determining the corresponding expression of proteins, thus making Icariin as novel proangiogenic and cytoprotective therapeutic agent with potential applications in the fields of coronary atherosclerotic heart disease.

氧化应激(OS)诱导的内皮损伤和炎症反应是动脉粥样硬化(AS)的两个关键发病机制。淫羊藿苷(ICAR)可以促进内皮祖细胞(EPC)归巢和分化，有效抑制AS形成，但具体机制还不清楚。我们的前期工作发现ICAR可能通过调控mTOR和p38 MAPK信号通路，促进EPC成血管分化，拮抗OS和内质网应激(ERS)诱导的细胞损伤和炎症反应，并能调控细胞凋亡与自噬。据此假设ICAR通过调控mTOR和p38 MAPK信号通路及其下游关键转录因子4EBP1和ATF2，抑制OS和ERS介导的细胞凋亡和自噬性死亡，修复AS内皮损伤。通过检测应激条件下ICAR对EPC在体内的归巢和分化、体外的凋亡和自噬的影响，并阻断相关信号位点后观察细胞损伤情况及相关蛋白差异表达，进一步阐明mTOR和p38 MAPK信号通路在ICAR促进EPC修复AS内皮损伤中的调控机制，为ICAR治疗动脉粥样硬化性心脏病提供新的理论依据。

冠心病的病理基础是动脉粥样硬化，冠状动脉血管发生动脉粥样硬化而引起血管腔狭窄或阻塞，导致心肌缺血缺氧或坏死是导致心脏功能结构改变的最重要、最常见的原因之一，因而预防动脉粥样硬化的形成是冠心病治疗的关键。对动脉粥样硬化发病机制的深入研究揭示，慢性和重复性的动脉壁内皮受损及功能障碍是动脉粥样硬化病灶形成的起始和主要环节，内质网应激诱导的内皮损伤和细胞焦亡是动脉粥样硬化的两个关键发病机制。.在国家自然科学基金项目执行期间，我们对中药单体成分抑制应激反应促进损伤内皮功能恢复的作用及机制，以及内皮祖细胞（EPCs）凋亡与焦亡在参与调控内皮损伤中的作用机制等问题进行了深入研究。研究从内皮损伤保护的角度，以抗氧化应激/内质网应激介导的炎症反应与细胞凋亡，以及NLRP3炎性小体介导的细胞焦亡为切入点，以参与内皮修复的关键细胞EPCs为研究对象，采用细胞生物学、分子生物学等多种研究手段深入探究中药活性单体成分淫羊藿苷 (ICAR)和丹酚酸B (SalB) 拮抗动脉粥样硬化内皮损伤的作用机制，研究发现Akt/mTOR/4EBP1和p38 MAPK/ATF2信号通路介导了ICAR对氧化应激诱导的细胞凋亡和自噬的抑制作用；ICAR抑制EPCs自噬性死亡在其内皮损伤修复中发挥关键作用；SalB能抑制内质网应激介导的EPCs线粒体功能障碍和炎症反应，还能调控Bax/Bcl-xL家族蛋白和Nox4的表达来恢复细胞迁移和管腔形成功能，从而修复内皮损伤。我们的进一步研究还发现SalB能够通过调控AMPK/FoxO4/KLF2和Syndecan-4/Rac1/ATF2信号通路及相关蛋白的表达抑制NLRP3炎性小体活化，抑制caspase-1、ASC、IL-1ß、IL-18等细胞焦亡相关蛋白表达和活化，拮抗内质网应激介导的细胞焦亡，并在调控EPCs血管生成活性中发挥重要作用。.理论上，部分阐明ICAR和SalB防治动脉粥样硬化血管内皮损伤的新机制，即通过调控mTOR/4EBP1和p38 MAPK/ATF2信号通路抑制氧化应激诱导的内皮损伤和炎症反应，并通过AMPK/KLF2和Syndecan-4/ATF2信号轴拮抗内质网应激介导的线粒体功能障碍，调控NLRP3炎性小体介导的细胞焦亡，参与内皮修复。临床上，为探寻ICAR和SalB预防和治疗动脉粥样硬化性心脏病的新作用机制提供实验数据和理论依据。