缺氧心肌微血管内皮细胞来源的外泌体 miR-27b-3p 通过 Foxo1/GSDMD 信号抑制氧化应激诱导的细胞焦亡缓解大鼠心肌缺血/再灌注损伤。

Exosomal miR-27b-3p Derived from Hypoxic Cardiac Microvascular Endothelial Cells Alleviates Rat Myocardial Ischemia/Reperfusion Injury through Inhibiting Oxidative Stress-Induced Pyroptosis via Foxo1/GSDMD Signaling.

机构信息

Cardiac Care Unit, Affiliated Hospital of Traditional Chinese Medicine, Xinjiang Medical University, Urumqi City, Xinjiang Uygur Autonomous Region, China.

Department of Cardiology/Cardiac Catheterization Lab, Second Xiangya Hospital, Central South University, Changsha, Hunan, China.

出版信息

Oxid Med Cell Longev. 2022 Jul 6;2022:8215842. doi: 10.1155/2022/8215842. eCollection 2022.

DOI:10.1155/2022/8215842

PMID:35847592

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9279077/

Abstract

BACKGROUND

Exosomes derived from cardiac microvascular endothelial cells (CMECs) under hypoxia can mediate cardiac repair functions and alleviate pyroptosis and oxidative stress during ischemia-reperfusion (I/R) injury. This study is aimed at investigating the effect and mechanism of miR-27b-3p underlying hypoxic CMECs-derived exosomes against I/R injury.

METHODS

CMECs were isolated from the left ventricle of Sprague-Dawley rats, followed by culturing under hypoxic conditions or pretreatment with the miR-27b-3p inhibitor. CMECs-derived exosomes were added into H9C2 cells before hypoxia/reoxygenation (H/R) or injected into the rat heart before I/R injury. An I/R injury model was established by ligating and releasing the left anterior descending coronary artery. Expression of pyroptosis-related factors was detected using Western blot, and heart infarcted size was determined by the 2,3,5-triphenyl-2H-tetrazpinolium chloride staining method. Dual-Luciferase Reporter assays were performed to analyze the interactions of nmiR-27b-3p-forkhead box O1 (Foxo1) and Gasdermin D- (GSDMD-) Foxo1. Chromatin-immunoprecipitation (ChIP) assays were performed to validate the interactions between forkhead box O1 (Foxo1) and Gasdermin D (GSDMD) and Foxo1-mediated histone acetylation of GSDMD.

RESULTS

CMECs were successfully identified from left ventricle of Sprague-Dawley rats. The expressions of Foxo1 and pyroptosis-related proteins (GSDMD, NLPR3, cleaved caspase 1, IL-1 and IL-18) were upregulated in the rat heart after I/R injury. Treatment of CMEC-derived exosomes, especially that under hypoxic conditions, significantly reduced pyroptosis in the rat heart. miR-27b-3p was significantly upregulated in CMEC-derived exosomes under hypoxic conditions, and miR-27b-3p inhibition in exosomes alleviated its cytoprotection and inhibited oxidative stress in H9C2 cells. Treatment with Foxo1 overexpression plasmids aggravated H/R and I/R injury by upregulating pyroptosis-related proteins. Further experiments validated that miR-27b-3p negatively targeted Foxo1, which bound to the promoter region of GSDMD.

CONCLUSIONS

These results demonstrated a great therapeutic efficacy of miR-27b-3p overexpression in hypoxic CMEC-derived exosomes in preventing the development of myocardial damage post I/R injury through inhibiting Foxo1/GSDMD signaling-induced oxidative stress and pyroptosis.

摘要

背景

缺氧诱导的心肌微血管内皮细胞（CMECs）衍生的外泌体可介导心脏修复功能，并减轻缺血再灌注（I/R）损伤过程中的细胞焦亡和氧化应激。本研究旨在探讨缺氧 CMECs 衍生的外泌体中 miR-27b-3p 对 I/R 损伤的作用及机制。

方法

从 Sprague-Dawley 大鼠左心室分离 CMECs，然后在缺氧条件下培养或用 miR-27b-3p 抑制剂预处理。在缺氧/复氧（H/R）前将 CMECs 衍生的外泌体加入 H9C2 细胞中，或在 I/R 损伤前将其注入大鼠心脏。结扎并释放左前降支冠状动脉建立 I/R 损伤模型。采用 Western blot 检测细胞焦亡相关因子的表达，采用 2,3,5-三苯基-2H-四唑氯化物染色法测定心肌梗死面积。采用双荧光素酶报告基因检测分析 nmiR-27b-3p-forkhead box O1（Foxo1）和 Gasdermin D-（GSDMD-）Foxo1 之间的相互作用。采用染色质免疫沉淀（ChIP）检测验证 Foxo1 和 Gasdermin D（GSDMD）之间的相互作用以及 Foxo1 介导的 GSDMD 组蛋白乙酰化。

结果

成功从 Sprague-Dawley 大鼠左心室中鉴定出 CMECs。I/R 损伤后，大鼠心脏中 Foxo1 和细胞焦亡相关蛋白（GSDMD、NLPR3、cleaved caspase 1、IL-1 和 IL-18）的表达上调。CMEC 衍生的外泌体，特别是缺氧条件下的外泌体，可显著减轻大鼠心脏的细胞焦亡。缺氧条件下 CMEC 衍生的外泌体中 miR-27b-3p 显著上调，外泌体中 miR-27b-3p 的抑制可减轻其对细胞的保护作用，并抑制 H9C2 细胞中的氧化应激。Foxo1 过表达质粒的处理通过上调细胞焦亡相关蛋白加重 H/R 和 I/R 损伤。进一步的实验验证了 miR-27b-3p 负靶向 Foxo1，Foxo1 与 GSDMD 启动子区域结合。