Cui Liu-Gen, Wang Shu-Hui, Komal Sumra, Yin Jian-Jian, Zhai Miao-Miao, Zhou Yue-Jiao, Yu Qing-Wen, Wang Cong, Wang Pei, Wang Zhi-Mo, Zafar Aliza Muhammad, Shakeel Muhammad, Zhang Li-Rong, Han Sheng-Na
Department of Pharmacology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou 450001, Henan, China.
Department of Ultrasound, The Affiliated Cancer Hospital of Zhengzhou University & Henan Cancer Hospital, Zhengzhou, 450008, China.
Cell Signal. 2025 Mar;127:111574. doi: 10.1016/j.cellsig.2024.111574. Epub 2024 Dec 20.
Through bioinformatics screening, we previously found that AlkB homolog 5 (ALKBH5) expression, an mA demethylase, was higher in patients with heart failure than in the normal population. This study aimed to investigate the molecular mechanisms by which ALKBH5 regulates heart failure. We established a myocardial infarction (MI)-induced heart failure model in rats in vivo and an in vitro hypoxia model using rat primary cardiac fibroblasts (RCFs). MA sequencing, RNA immunoprecipitation assay, RNA pull-down assay, proximity ligation assay, gain-of-function and loss-of-function experiments, and transcriptomic analysis were performed to confirm the pyroptosis-promoting effects of ALKBH5. The effects of two small-molecule inhibitors (ZINC78774792 and ZINC00546946) on ALKBH5 expression were examined. The expression of mA demethyltransferase ALKBH5 was significantly elevated in hypoxia-induced RCFs. Transcriptional profiling revealed Notch receptor 1 (Notch1) as an mA modification target of ALKBH5, and Notch1 mRNA mA modifications were increased in ALKBH5-deficient RCFs. Moreover, Notch1 and NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) are associated. ALKBH5 knockdown alleviated hypoxia-induced RCF cell pyroptosis by inhibiting Notch1, NLRP3 inflammasome activation, and pyroptosis-associated protein (N-GSDMD), whereas ALKBH5 overexpression had the opposite effect. Targeting ALKBH5 with two small-molecule inhibitors (ZINC78774792 and ZINC00546946) reduced hypoxia-induced RCF pyroptosis, and ZINC00546946 alleviated cell pyroptosis after myocardial infarction in mice. Our results indicate that ALKBH5 promotes cardiac fibroblast pyroptosis after myocardial infarction through the Notch1/NLRP3 pathway. Therefore, inhibiting ALKBH5 may be a strategy for treating cardiovascular diseases.
通过生物信息学筛选，我们先前发现，作为一种mA去甲基化酶的AlkB同源物5（ALKBH5）在心力衰竭患者中的表达高于正常人群。本研究旨在探讨ALKBH5调节心力衰竭的分子机制。我们在体内建立了大鼠心肌梗死（MI）诱导的心力衰竭模型，并使用大鼠原代心脏成纤维细胞（RCF）建立了体外缺氧模型。进行了mA测序、RNA免疫沉淀分析、RNA下拉分析、邻近连接分析、功能获得和功能丧失实验以及转录组分析，以证实ALKBH5的促焦亡作用。检测了两种小分子抑制剂（ZINC78774792和ZINC00546946）对ALKBH5表达的影响。在缺氧诱导的RCF中，mA去甲基转移酶ALKBH5的表达显著升高。转录谱分析显示Notch受体1（Notch1）是ALKBH5的一个mA修饰靶点，并且在缺乏ALKBH5的RCF中Notch1 mRNA的mA修饰增加。此外，Notch1与含NOD、LRR和pyrin结构域的蛋白3（NLRP3）相关。敲低ALKBH5可通过抑制Notch1、NLRP3炎性小体激活和焦亡相关蛋白（N-GSDMD）减轻缺氧诱导的RCF细胞焦亡，而ALKBH5过表达则产生相反的效果。用两种小分子抑制剂（ZINC78774792和ZINC00546946）靶向ALKBH5可减少缺氧诱导的RCF焦亡，并且ZINC00546946可减轻小鼠心肌梗死后的细胞焦亡。我们的结果表明，ALKBH5通过Notch1/NLRP3途径促进心肌梗死后心脏成纤维细胞焦亡。因此，抑制ALKBH5可能是治疗心血管疾病的一种策略。
