Department of Cardiology, Fuwai Hospital, Chinese Academy of Medical Sciences (Shenzhen Sun Yat-Sen Cardiovascular Hospital), Shenzhen, China.
Shenzhen Health Administrative Center for Cadre and Talent, Shenzhen, China.
Appl Biochem Biotechnol. 2023 Mar;195(3):1999-2014. doi: 10.1007/s12010-022-04208-9. Epub 2022 Nov 19.
Ischemic postconditioning (IPost) represents short periods of nonlethal ischemia-reperfusion performed at the onset of reperfusion. Studies have shown that IPost involves various biological processes such as cell proliferation, apoptosis, and pyroptosis and can activate complex signaling pathways. CCL12 is a critical mediator in the inflammatory process after tissue injury. In the present study, we examined the potential actions of CCL12-mediated signaling pathways in cardioprotection after IPost using a cardiomyocyte model. By applying the bioinformatics analysis, we found that CCL12 was upregulated in the rat heart tissues after I/R injury, and the expression level of CCL12 was restored in rats with IPost. The in vitro studies showed that CCL12 and CCR2 expression levels were upregulated in the hypoxia/reoxygenation (H/R)-induced H9C2 cells, which was attenuated in the H/R + hypoxia post-conditioning (PostC) group. The functional assays showed that H/R treatment reduced cell viability, increased cell apoptosis, and promoted fibrosis and pyroptosis of H9C2 cells, which was attenuated in the H/R + PostC group. Overexpression of CCL12 impaired the protective action of hypoxia post-conditioning in the H9C2 cells. Further mechanistic studies showed that miR-144-5p could directly target the 3' untranslated region of CCL12. Overexpression of miR-144-5p markedly repressed the expression levels of CCL12 and CCR2 in H9C2 cells, while miR-144-5p inhibition had the opposite effects. Furthermore, the inhibition of miR-144-5p reduced the cell viability, increased cell apoptosis, and enhanced fibrosis and pyroptosis of H9C2 cells after H/R or H/R + PostC treatment. In conclusion, CCL12 was downregulated in cardiomyocytes following ischemic postconditioning, and CCL12 overexpression impaired the cardioprotective actions of ischemic postconditioning by reducing cell viability, enhancing cell apoptosis, fibrosis, and pyroptosis. Further mechanistic evidence revealed that CCL12 was a direct target of miR-144-5p, and miR-144-5p/CCL12/CCR2 signaling may represent a critical pathway in mediating the cardioprotective effects of ischemic postconditioning.
缺血后处理（IPost）代表在再灌注开始时进行的短暂非致死性缺血-再灌注。研究表明，IPost 涉及细胞增殖、细胞凋亡和细胞焦亡等多种生物学过程，并能激活复杂的信号通路。CCL12 是组织损伤后炎症过程中的关键介质。在本研究中，我们使用心肌细胞模型研究了 CCL12 介导的信号通路在 IPost 后心脏保护中的潜在作用。通过应用生物信息学分析，我们发现 I/R 损伤后大鼠心脏组织中 CCL12 上调，IPost 后大鼠 CCL12 表达水平恢复。体外研究显示，CCL12 和 CCR2 表达水平在缺氧/复氧（H/R）诱导的 H9C2 细胞中上调，而在 H/R+缺氧后处理（PostC）组中下调。功能测定显示，H/R 处理降低 H9C2 细胞活力，增加细胞凋亡，并促进 H9C2 细胞纤维化和细胞焦亡，而 H/R+PostC 组则减弱。CCL12 的过表达损害了 H9C2 细胞中缺氧后处理的保护作用。进一步的机制研究表明，miR-144-5p 可以直接靶向 CCL12 的 3'UTR。miR-144-5p 的过表达显著抑制 H9C2 细胞中 CCL12 和 CCR2 的表达水平，而 miR-144-5p 抑制则有相反的效果。此外，miR-144-5p 的抑制降低了 H/R 或 H/R+PostC 处理后 H9C2 细胞的活力，增加了细胞凋亡，并增强了纤维化和细胞焦亡。总之，缺血后处理后心肌细胞中 CCL12 下调，CCL12 过表达通过降低细胞活力、增强细胞凋亡、纤维化和细胞焦亡来损害缺血后处理的心脏保护作用。进一步的机制证据表明，CCL12 是 miR-144-5p 的直接靶标，miR-144-5p/CCL12/CCR2 信号可能代表介导缺血后处理心脏保护作用的关键途径。
