Qiu Quan, Shen Tao, Yu Xiaoxue, Jia Na, Zhu Kaiyi, Wang Que, Liu Bing, He Qing
Department of Cardiology, The First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, Zhejiang 310006, China.
The Key Laboratory of Geriatrics, Beijing Institute of Geriatrics, Beijing Hospital, National Center of Gerontology, National Health Commission, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing 100730, China.
Biomed Res Int. 2021 Jan 19;2021:8880179. doi: 10.1155/2021/8880179. eCollection 2021.
Regulated necrosis (necroptosis) is crucially involved in cardiac ischaemia-reperfusion injury (MIRI). The aim of our study is to investigate whether shock wave therapy (SWT) is capable of exerting protective effects by inhibiting necroptosis during myocardial ischaemia-reperfusion (I/R) injury and the possible role of autophagy in this process. We established a hypoxia/reoxygenation (H/R) model using HL-1 cells to simulate MIRI. MTS assays and LDH cytotoxicity assay were performed to measure cell viability and cell damage. Annexin V/PI staining was used to determine apoptosis and necrosis. Western blotting was performed to assess the changes in cell signaling pathways associated with autophagy, necroptosis, and apoptosis. Reactive oxygen species (ROS) production was detected using DHE staining. Autophagosome generation and degradation (autophagic flux) were analysed using GFP and RFP tandemly tagged LC3 (tfLC3). HL-1 cells were then transfected with p62/SQSTM1 siRNA in order to analyse its role in cardioprotection. Our results revealed that SWT increased cell viability in the H/R model and decreased receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3 expression. ROS production was also inhibited by SWT. Moreover, SWT decreased Beclin1 expression and the ratio of LC3-II/LC3-I following H/R. Simultaneously, in the tfLC3 assay, the SWT provoked a decrease in the cumulative autophagosome abundance. siRNA-mediated knockdown of p62 attenuated H/R-induced necroptosis, and SWT did not exert additive effects. Taken together, SWT ameliorated H/R injury by inhibiting necroptosis. SWT also relieved the blockade of autophagic flux in response to H/R injury. The restoration of autophagic flux by SWT might contribute to its cardioprotective effect on necroptosis following H/R injury.
程序性坏死（坏死性凋亡）在心肌缺血再灌注损伤（MIRI）中起关键作用。我们研究的目的是探讨冲击波疗法（SWT）是否能够通过抑制心肌缺血再灌注（I/R）损伤期间的程序性坏死发挥保护作用，以及自噬在此过程中的可能作用。我们使用HL-1细胞建立了缺氧/复氧（H/R）模型来模拟MIRI。进行MTS试验和LDH细胞毒性试验以测量细胞活力和细胞损伤。使用膜联蛋白V/PI染色来确定凋亡和坏死。进行蛋白质免疫印迹以评估与自噬、程序性坏死和凋亡相关的细胞信号通路的变化。使用DHE染色检测活性氧（ROS）的产生。使用绿色荧光蛋白和红色荧光蛋白串联标记的LC3（tfLC3）分析自噬体的产生和降解（自噬流）。然后用p62/SQSTM1 siRNA转染HL-1细胞，以分析其在心脏保护中的作用。我们的结果显示，SWT增加了H/R模型中的细胞活力，并降低了受体相互作用丝氨酸/苏氨酸蛋白激酶1（RIPK1）和RIPK3的表达。SWT也抑制了ROS的产生。此外，SWT降低了H/R后Beclin1的表达以及LC3-II/LC3-I的比值。同时，在tfLC3试验中，SWT使累积自噬体丰度降低。siRNA介导的p62敲低减轻了H/R诱导的程序性坏死，并且SWT没有发挥累加效应。综上所述，SWT通过抑制程序性坏死改善了H/R损伤。SWT还缓解了H/R损伤引起的自噬流阻断。SWT恢复自噬流可能有助于其对H/R损伤后程序性坏死的心脏保护作用。
