Zuo Bo, Zhu Sha, Wang Guisong, Li Zhengpeng
Department of Cardiology, Cardiovascular Centre, Beijing Friendship Hospital, Capital Medical University, Beijing, China.
Department of Cardiology, Peking University Third Hospital, Beijing, China.
Front Cardiovasc Med. 2023 May 10;10:1089151. doi: 10.3389/fcvm.2023.1089151. eCollection 2023.
Remote ischemic postconditioning (RIPostC) induced by brief episodes of the limb ischemia is a potential therapeutic strategy for myocardial ischemia/reperfusion injury, achieved by reducing cardiomyocyte death, inflammation and so on. The actual mechanisms underlying cardioprotection conferred by RIPostC remain unclear. Exploring gene expression profiles in myocardium at transcriptional level is helpful to deepen the understanding on the cardioprotective mechanisms of RIPostC. This study aims to investigate the effect of RIPostC on gene expressions in rat myocardium using transcriptome sequencing.
Rat myocardium samples from the RIPostC group, the control group (myocardial ischemia/reperfusion group) and the sham group were performed transcriptome analysis using RNA sequencing. The levels of cardiac IL-1β, IL-6, IL-10 and TNFα were analyzed by Elisa. The expression levels of candidate genes were verified by qRT-PCR technique. Infarct size was measured by Evans blue and TTC staining. Apoptosis was assessed by TUNEL assays and caspase-3 levels were detected using western blotting.
RIPostC can markedly decrease infarct size and reduce the levels of cardiac IL-1β, IL-6 and increase the level of cardiac IL-10. This transcriptome analysis showed that 2 genes were up-regulated (Prodh1 and ADAMTS15) and 5 genes (Caspase-6, Claudin-5, Sccpdh, Robo4 and AABR07011951.1) were down-regulated in the RIPostC group. Go annotation analysis showed that Go terms mainly included cellular process, metabolic process, cell part, organelle, catalytic activity and binding. The KEGG annotation analysis of DEGs found only one pathway, amino acid metabolism, was up-regulated. The relative mRNA expression levels of ADAMTS15, Caspase-6, Claudin-5 and Prodh1 were verified by qRT-PCR, which were consistent with the RNA-seq results. In addition, the relative expression of ADAMTS15 was negatively correlated with the level of cardiac IL-1β ( = -0.748, = 0.005) and positively correlated with the level of cardiac IL-10 ( = 0.698, = 0.012). A negative correlation statistical trend was found between the relative expression of ADAMTS15 and the level of cardiac IL-6 ( = -0.545, = 0.067).
ADAMTS15 may be a potential inflammation-related gene in regulation of cardioprotection conferred by remote ischemic postconditioning and a possible therapeutic target for myocardial ischemia reperfusion injury in the future.
肢体短暂缺血诱导的远程缺血后处理（RIPostC）是心肌缺血/再灌注损伤的一种潜在治疗策略，可通过减少心肌细胞死亡、炎症等实现。RIPostC赋予心脏保护作用的实际机制尚不清楚。在转录水平探索心肌中的基因表达谱有助于加深对RIPostC心脏保护机制的理解。本研究旨在通过转录组测序研究RIPostC对大鼠心肌基因表达的影响。
对RIPostC组、对照组（心肌缺血/再灌注组）和假手术组的大鼠心肌样本进行RNA测序转录组分析。通过酶联免疫吸附测定法（Elisa）分析心脏白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）、白细胞介素-10（IL-10）和肿瘤坏死因子α（TNFα）的水平。通过定量逆转录聚合酶链反应（qRT-PCR）技术验证候选基因的表达水平。通过伊文思蓝和氯化三苯基四氮唑（TTC）染色测量梗死面积。通过末端脱氧核苷酸转移酶介导的缺口末端标记法（TUNEL）检测细胞凋亡，并使用蛋白质免疫印迹法检测半胱天冬酶-3水平。
RIPostC可显著减小梗死面积，降低心脏IL-1β、IL-6水平，并提高心脏IL-10水平。该转录组分析表明，RIPostC组中有2个基因上调（脯氨酸脱氢酶1（Prodh1）和含血小板反应蛋白基序的解聚素样金属蛋白酶15（ADAMTS15）），5个基因（半胱天冬酶-6（Caspase-6）、紧密连接蛋白5（Claudin-5）、琥珀酰辅酶A连接酶（Sccpdh）、受体酪氨酸激酶4（Robo4）和AABR07011951.1）下调。基因本体（Go）注释分析表明，Go术语主要包括细胞过程、代谢过程、细胞部分、细胞器、催化活性和结合。差异表达基因（DEG）的京都基因与基因组百科全书（KEGG）注释分析发现只有一个途径，即氨基酸代谢上调。通过qRT-PCR验证了ADAMTS15、Caspase-6、Claudin-5和Prodh1的相对信使核糖核酸（mRNA）表达水平，与RNA测序结果一致。此外，ADAMTS15的相对表达与心脏IL-1β水平呈负相关（r = -0.748，P = 0.005），与心脏IL-10水平呈正相关（r = 0.698，P = 0.012）。在ADAMTS15的相对表达与心脏IL-6水平之间发现了负相关统计趋势（r = -0.545，P = 0.067）。
ADAMTS15可能是远程缺血后处理赋予心脏保护作用调控中一个潜在的炎症相关基因，未来可能是心肌缺血再灌注损伤的一个治疗靶点。
