Wang Yu, Wang Wei, Xu Haobo, Sun Yan, Sun Jing, Jiang Yongxing, Yao Jianting, Tian Ye
Department of Cardiology, the First Affiliated Hospital, Cardiovascular Institute, Harbin Medical University, Harbin, China.
Heilongjiang Academy of Medical Sciences, Harbin, China.
Cell Physiol Biochem. 2017;41(6):2432-2446. doi: 10.1159/000475913. Epub 2017 May 3.
Previous studies from our group showed that low-intensity sonodynamic therapy (SDT) has protective effects on atherosclerosis (AS). However, because the intensity of ultrasound passing through tissue is attenuated, the consequences of very low-intensity SDT, referred to as non-lethal SDT (NL-SDT), on atherosclerotic plaques are unclear. The aim of this study was to determine whether NL-SDT affects atherosclerotic plaques and to elucidate the possible underlying mechanisms.
An AS model was established using ApoE-/- mice fed a western diet. En face Oil Red O staining was used to measure atherosclerotic plaque size. Hematoxylin and eosin staining and immunohistochemical staining were used to observe plaque morphology and assess the location of macrophages and heme oxygenase 1 (HO-1). HO-1 mRNA and protein levels in AS plaques were evaluated by real-time PCR and western blotting. Human THP-1 cells and mouse peritoneal macrophages were used in this study. Western blotting was used to investigate the expression of cellular proteins after NL-SDT. Macrophage apoptosis was evaluated by TUNEL assays and flow cytometry with Annexin V/PI double staining. Intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were measured with 2'-7'-dichlorofluorescein diacetate (DCFH-DA) and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl benzimidazolyl carbocyanine iodide (JC-1) staining, respectively.
NL-SDT significantly inhibited AS progression and reduced the necrotic core area. NL-SDT induced HO-1 expression in lesional macrophages and in cultured macrophages. NL-SDT activated the protein kinase B (AKT) and extracellular signal-related protein kinase (ERK) pathways and the transcription factor NF-E2-related factor 2 (Nrf2).NL-SDT significantly reduced oxidized LDL (ox-LDL)-induced macrophage MMP collapse, ROS production and cell apoptosis. Zinc protoporphyrin (ZnPP), a HO-1-specific inhibitor, reversed the protective effects of NL-SDT.
NL-SDT inhibits atherosclerotic plaque progression and increases plaque stability. In vitro, NL-SDT has a protective effect on ox-LDL-induced macrophage impairment via HO-1.
我们团队之前的研究表明，低强度声动力疗法（SDT）对动脉粥样硬化（AS）具有保护作用。然而，由于穿过组织的超声强度会衰减，极低强度SDT（称为非致死性SDT，NL-SDT）对动脉粥样硬化斑块的影响尚不清楚。本研究的目的是确定NL-SDT是否会影响动脉粥样硬化斑块，并阐明可能的潜在机制。
使用喂食西式饮食的ApoE-/-小鼠建立AS模型。采用油红O整体染色法测量动脉粥样硬化斑块大小。用苏木精-伊红染色和免疫组织化学染色观察斑块形态，并评估巨噬细胞和血红素加氧酶1（HO-1）的定位。通过实时PCR和蛋白质印迹法评估AS斑块中HO-1的mRNA和蛋白质水平。本研究使用了人THP-1细胞和小鼠腹腔巨噬细胞。采用蛋白质印迹法研究NL-SDT后细胞蛋白的表达。通过TUNEL检测和Annexin V/PI双染流式细胞术评估巨噬细胞凋亡。分别用2'-7'-二氯荧光素二乙酸酯（DCFH-DA）和5,5',6,6'-四氯-1,1',3,3'-四乙基苯并咪唑基羰花青碘化物（JC-1）染色测量细胞内活性氧（ROS）和线粒体膜电位（MMP）。
NL-SDT显著抑制AS进展并减小坏死核心面积。NL-SDT诱导病变巨噬细胞和培养巨噬细胞中HO-1的表达。NL-SDT激活蛋白激酶B（AKT）和细胞外信号相关蛋白激酶（ERK）通路以及转录因子NF-E2相关因子2（Nrf2）。NL-SDT显著降低氧化型低密度脂蛋白（ox-LDL）诱导的巨噬细胞MMP崩溃、ROS产生和细胞凋亡。HO-1特异性抑制剂锌原卟啉（ZnPP）逆转了NL-SDT的保护作用。
NL-SDT抑制动脉粥样硬化斑块进展并增加斑块稳定性。在体外，NL-SDT通过HO-1对ox-LDL诱导的巨噬细胞损伤具有保护作用。
