基于PI3K/Akt/mTOR通路探讨补阳还五汤调控Treg外周分化稳定斑块机制

The instability of AS plaque relating to immunological inflammation response is a crucial factor that triggers acute cardiocerebrovascular diseases. Regulatory T cells (Treg) plays an important role in keeping atherosclerotic plaque stable. Studies, however, have shown that Treg decreases in the body of atherosclerosis patients, thus breaking the balance between Treg and effect T cells and amplifying inflammatory response, and consequently the plaque became unstable. The reason is believed to be related to PI3K/Akt/mTOR pathway which inhibits Treg peripheral differentiation. It is thus suggested that it is important to stabilize the plaque through controlling the signaling pathway and increasing Treg’s number. Buyang Huanwu Decoction (BYHWD) is a famous medical formulate of treating cardiocerebrovascular diseases related with AS. However, the mechanism underneath has yet not been sufficiently explored and adequately described. Our research has shown that BYHWD has the function of anti-inflammation and plaque-stabilization. Additionally Astragalus, the monarch drug of the decoction, has a function of increasing Treg’s number. So, the hypothesis currently being made is that BYHWD can be utilized to regulate PI3K/Akt/mTOR pathway to promote Treg differentiation, which would further contribute to inhibiting the inflammatory response and to restoring the balance between Treg and effect T cells, and therefore facilitates the stabilization of the plaque. This study, adopting methods from morphology and molecular biology, takes ApoE-/- mouse model of atherosclerosis and CD4+T cells as the research objects and focuses specifically on vivo and vitro to explore the mechanism of how BYHWD can be used to stabilize plaque from the perspective of immunology, which will offer new insights into the stabilization of the plaque and its pharmacological effects.

免疫炎症反应可导致动脉粥样（AS）斑块不稳定，引发急性心脑血管事件。调节性T细胞（Treg）能通过抑制效应性T细胞毒性，进而控制免疫炎症反应稳定斑块。然而，Treg在AS患者体内却表达减少。研究显示这一现象与PI3K/Akt/mTOR通路活性增强引发Treg外周分化减少有关。因此，选择药物调控该通路增加Treg数量，恢复免疫平衡对稳定斑块有重要意义。补阳还五汤为治疗心脑血管疾病之名方，对AS疗效确切，但其深入机制尚未阐明。课题组前期研究发现补阳还五汤有稳定斑块、抗炎等作用，加之该方君药黄芪能调控Treg数量，故提出“补阳还五汤能调控PI3K/Akt/mTOR通路，增加Treg数量，恢复免疫平衡，进而抑制炎症反应、稳定斑块”假说。以CD4+T细胞和ApoE-/-小鼠AS模型为研究对象，采用形态学和分子生物学等方法，从免疫炎症角度阐明补阳还五汤稳定斑块机制，为临床多靶点入手稳定斑块提供新思路。

动脉粥样硬化(atherosclerosis, AS)斑块不稳定是导致急性心脑血管事件的主要原因。近年来研究显示：斑块内大量巨噬细胞、淋巴细胞聚集引发的免疫炎症反应是导致斑块不稳定的重要因素。其中，CD4+T淋巴细胞在参与AS慢性免疫炎症反应方面发挥巨大作用。具体表现为：促炎反应的效应性T细胞（Teff）增多和抗炎反应的调节T细胞（Treg）减少，两者失衡，导致促炎因子（INF-γ、TNF-α、MMP-2、MMP-9、IL-17等）表达增多，抗炎因子（TGF-β、IL-10等）表达减少，炎症反应放大，斑块易损性增加。因此，增加AS患者体内Treg细胞数量，被认为是一种新的有发展前途的治疗AS的措施之一。补阳还五汤（BYHWD）为补气活血祛瘀之名方，能有效针对AS气虚血瘀、本虚标实的病机，对AS造成的心脑血管疾病疗效确切。本研究利用ApoE基因敲除（AopE-/-）小鼠模型和CD4+T细胞模型探究BYHWD治疗AS机制。首先，高脂饮食干预AopE-/-小鼠并采用补阳还五汤干预，分组如下：对照组（C57BL/6小鼠，正常饮食）、模型组（AopE-/-小鼠，高脂饮食饲养12周）、补阳还五汤组（低、中、高，在模型组基础上灌胃4周）、阳性药物组。采用免疫组化、ELISA、流式等技术明确BYHWD抗AS作用与增加Treg细胞数量及抑制炎症反应的相关性。其次，采用CD4+T细胞模型，在免疫平衡和失衡条件下，采用流式细胞技术检测BYHWD对Treg细胞的调控作用。最后收集两种模型的RNA和蛋白，采用PCR和蛋白印迹技术阐明BYHWD调控Treg细胞数量与细胞分化信号通路的相关性及干预靶点，进而明确BYHWD增加Treg细胞数量，进而稳定斑块的具体机制。结果显示，BYHWD通过TGF-β/Smad2通路调控Treg细胞分化，增加其在AS小鼠中的表达（外周血、脾脏、动脉粥样斑块内），进而抑制炎症反应，发挥抗AS作用，为临床多靶点入手治疗AS提供新思路。