Yan'an Hospital Affiliated To Kunming Medical University, Kunming, China.
Yan'an Hospital Affiliated To Kunming Medical University, Kunming, China.
Cell Signal. 2024 Dec;124:111423. doi: 10.1016/j.cellsig.2024.111423. Epub 2024 Sep 18.
Our prior research determined that USP7 exacerbates myocardial injury. Additionally, existing studies indicate a strong connection between USP7 and ferroptosis. However, the influence of USP7 on ferroptosis-mediated myocardial infarction (MI) remains unclear. Given these findings, we are particularly interested in USP7's regulatory role in ferroptosis-mediated MI and its underlying mechanisms.
In this study, we established MI models and lentivirus-transfected groups to inhibit USP7 expression both in vivo and in vitro. Cardiac function was detected with Echocardiography. TTC and HE staining were employed to assess myocardial alterations. The expression of ferroptosis markers (PTGS2, ACSL4, GPX4) were analyzed by RT-qPCR and Western blotting. Flow cytometry and ELISA were used for measuring Fe, lipid ROS, GSH, and GSSG levels. TEM and Prussian blue staining were used to observe mitochondrial alterations and iron deposition. RT-qPCR, Western blotting, and immunofluorescence were conducted to analyze Keap1, Nrf2, and nuclear Nrf2 expression in vitro and in vivo.
In the MI model group, USP7 expression significantly increased, worsening ferroptosis-mediated MI. Conversely, in the USP7-inhibited group, activation of the Keap1-Nrf2 signaling pathway improved ferroptosis-mediated MI outcomes. In vitro, the MI model exhibited a marked decline in cardiomyocyte viability and notable mitochondrial damage. However, these issues improved in the USP7-inhibited groups. In vivo, USP7 intensified MI and iron deposition within the MI model group, with decreased values of LVEF, LVFS, SV, LVAWd, and LVPWs, all of which showed improvement in the USP7-inhibited group, except for LVPWd and LVPWs, which showed no significant variation. Importantly, both the in vitro and in vivo experiments revealed analogous results: a reduction in Keap1 expression and an increase in both Nrf2 and nuclear Nrf2 post USP7 inhibition. Additionally, GPX4 expression decreased while PTGS2 and ACSL4 expressions increased. Notably, concentrations of Fe, lipid ROS, GSH, and GSSG significantly decreased.
In vitro and in vivo studies have found that inhibition of USP7 attenuates iron deposition and suppresses oxidative stress, resulting in amelioration of ferroptosis-induced MI.
我们之前的研究表明 USP7 会加重心肌损伤。此外，现有研究表明 USP7 与铁死亡之间存在很强的联系。然而，USP7 对铁死亡介导的心肌梗死（MI）的影响尚不清楚。鉴于这些发现，我们特别关注 USP7 在铁死亡介导的 MI 中的调节作用及其潜在机制。
在这项研究中，我们建立了 MI 模型和慢病毒转染组，以在体内和体外抑制 USP7 的表达。通过超声心动图检测心功能。采用 TTC 和 HE 染色评估心肌变化。通过 RT-qPCR 和 Western blot 分析铁死亡标志物（PTGS2、ACSL4、GPX4）的表达。采用流式细胞术和 ELISA 法检测 Fe、脂质 ROS、GSH 和 GSSG 水平。采用 TEM 和普鲁士蓝染色观察线粒体变化和铁沉积。通过 RT-qPCR、Western blot 和免疫荧光法在体内和体外分析 Keap1、Nrf2 和核 Nrf2 的表达。
在 MI 模型组中，USP7 的表达显著增加，加重了铁死亡介导的 MI。相反，在 USP7 抑制组中，Keap1-Nrf2 信号通路的激活改善了铁死亡介导的 MI 结局。在体外，MI 模型表现出明显的心肌细胞活力下降和显著的线粒体损伤。然而，这些问题在 USP7 抑制组中得到改善。在体内，USP7 加剧了 MI 模型组中的 MI 和铁沉积，LVEF、LVFS、SV、LVAWd 和 LVPWs 值降低，而在 USP7 抑制组中均有所改善，除了 LVPWd 和 LVPWs 外，其值没有明显变化。重要的是，无论是在体内还是在体外实验中，结果都类似：USP7 抑制后，Keap1 表达降低，Nrf2 和核 Nrf2 表达增加。此外，GPX4 表达降低，PTGS2 和 ACSL4 表达增加。值得注意的是，Fe、脂质 ROS、GSH 和 GSSG 的浓度显著降低。
在体内和体外研究中发现，抑制 USP7 可减轻铁沉积并抑制氧化应激，从而改善铁死亡诱导的 MI。
