Yang Yang, Liu Yuanyuan, Chen Xi, Gong Jie, Huang Zhen, Wang Wei, Shi Yuanqi, Wang Yu, Yao Jianting, Shen Zhaoqian, Tian Zhen, Jin Hong, Tian Ye
Department of Cardiology, The First Affiliated Hospital, Cardiovascular Institute, Harbin Medical University, Harbin, China.
Department of Cardiology, Heilongjiang Provincial Hospital, Harbin, China.
Cell Physiol Biochem. 2018;47(1):83-96. doi: 10.1159/000489751. Epub 2018 May 9.
BACKGROUND/AIMS: We and other groups have demonstrated that 5-aminolevulinic acid (ALA)-mediated sonodynamic therapy (ALA-SDT) induces macrophage and foam cell apoptosis and stabilizes atherosclerosis (AS) plaques in animal models. Lymphocytes also play vital roles in the development of AS. The primary purpose of the present study was to investigate the effects of ALA-SDT on T helper (Th) cell fate and function, Th subset differentiation, and atherosclerotic lesion stability.
We utilized ALA-SDT on Western diet-fed apoE-/-mice in vivo and human Jurkat cells in vitro. Hematoxylin and eosin staining and TUNEL assays were used to evaluate the atherosclerotic plaque size and apoptosis within the atheroma. ALA induced cytotoxicity on cultured Jurkat cells was determined with CCK-8 assay. To address the mechanisms, levels of intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and mitochondrial permeability transition pore (MPTP) opening were evaluated by staining with fluorescent probes. Western blot analysis and confocal microscopy were used to analyze the protein levels of caspases, Bax and cytochrome c and the release of cytochrome c. Cell apoptosis and necrosis and phagocytosis were examined by flow cytometry. ELISAs and immunofluorescent staining were used to assess the corresponding cytokine levels and Th subset cell numbers within the atheroma.
Our studies revealed that ALA-SDT significantly enhanced CD4+ cell apoptosis and macrophage-mediated phagocytosis and hence reduced the necrotic core size. ALA-SDT activated the mitochondrial apoptotic signaling pathway with minimal necrosis in Jurkat cells. ALA-SDT inhibited the Th1 response and enhanced the Th2 response. These effects of ALA-SDT were mediated primarily through the generation of ROS.
ALA-SDT alleviates AS by enhancing cytotoxic effects on Th cells, subsequently stimulating efferocytosis and facilitating a shift in the Th1/Th2 balance toward Th2 cells, a discovery that might help elucidate the mechanism underlying SDT as a potential treatment to prevent atherothrombotic events.
背景/目的：我们和其他研究团队已证明，5-氨基酮戊酸（ALA）介导的声动力疗法（ALA-SDT）可诱导巨噬细胞和泡沫细胞凋亡，并使动物模型中的动脉粥样硬化（AS）斑块稳定。淋巴细胞在AS的发展中也起着至关重要的作用。本研究的主要目的是探讨ALA-SDT对辅助性T（Th）细胞命运和功能、Th亚群分化以及动脉粥样硬化病变稳定性的影响。
我们在体内对喂食西方饮食的载脂蛋白E基因敲除（apoE-/-）小鼠以及在体外对人Jurkat细胞使用了ALA-SDT。采用苏木精-伊红染色和TUNEL检测来评估动脉粥样硬化斑块大小和动脉粥样瘤内的细胞凋亡情况。用CCK-8检测法测定ALA对培养的Jurkat细胞的细胞毒性。为探究其机制，用荧光探针染色评估细胞内活性氧（ROS）水平、线粒体膜电位（MMP）以及线粒体通透性转换孔（MPTP）的开放情况。采用蛋白质印迹分析和共聚焦显微镜来分析半胱天冬酶、Bax和细胞色素c的蛋白水平以及细胞色素c的释放。通过流式细胞术检测细胞凋亡、坏死和吞噬作用。采用酶联免疫吸附测定（ELISA）和免疫荧光染色来评估动脉粥样瘤内相应细胞因子水平和Th亚群细胞数量。
我们的研究表明，ALA-SDT显著增强了CD4+细胞凋亡和巨噬细胞介导的吞噬作用，从而减小了坏死核心的大小。ALA-SDT在Jurkat细胞中激活了线粒体凋亡信号通路，且坏死程度最小。ALA-SDT抑制了Th1反应并增强了Th2反应。ALA-SDT的这些作用主要是通过ROS的产生介导的。
ALA-SDT通过增强对Th细胞的细胞毒性作用、随后刺激胞葬作用并促进Th1/Th2平衡向Th2细胞方向转变来减轻AS，这一发现可能有助于阐明SDT作为预防动脉粥样硬化血栓形成事件潜在治疗方法的作用机制。
