心肌缺血/再灌注损伤新机制：USP7介导的心肌细胞凋亡和铁坏死

Apoptosis and ferroptosis are two important types of cell death following myocardial ischemia/reperfusion (I/R). The preliminary data of this project showed that in rat model of myocardial I/R, the expression of ubiquitin specific protease 7(USP7) was up-regulated in myocardial tissue and the USP7 inhibitor could attenuate the I/R injury; silence of USP7 was able to block hypoxia/reoxygenation (H/R) -induced myocardial apoptosis and ferroptosis concomitant with a decrease in p53 and transferrin receptor 1 (TfR1) levels; the decrease in phosphorylation level of myosin regulatory light chain(MLC20) attenuated the expression of USP7. Based on these observations, we hypothesize for the first time that the up-regulation of USP7 promoted myocardial apoptosis and ferroptosis following I/R, which might be related to increase of p53 and TfR1 protein levels; while the expression of USP7might be modulated by MLC20. In the I/R or H/R injury model, combining the methods of drug interference and gene silence, etc., we will mainly focus on the research as follows: confirming the role of USP7 in promotion of myocardial apoptosis and ferroptosis; verifying the mechanisms for USP7-mediated apoptosis and ferroptosis; and elucidating the regulatory mechanisms for USP7 expression. This project will lay a theoretic foundation and provide the new idea for seeking novel drug targets against myocardial ischemia.

凋亡和铁坏死是缺血/再灌注（I/R）心肌细胞死亡的两种重要方式。本项目预实验首次发现：I/R大鼠心肌组织泛素特异性蛋白酶7（USP7）表达上调，USP7抑制剂能减轻I/R损伤；沉默USP7可减少低氧/复氧（H/R）诱导的心肌细胞凋亡和铁坏死，伴随p53和转铁蛋白受体1(TfR1)蛋白水平下降；而降低MLC20磷酸化水平则可阻止I/R诱导的USP7表达。我们因而率先提出：心肌I/R时，USP7表达上调促进了心肌细胞凋亡和铁坏死，其机制可能与升高p53和TfR1蛋白水平有关，USP7表达受MLC20调节。利用I/R（或H/R）大鼠（或心肌细胞）模型，结合药物干预、基因沉默等，本项目拟明确USP7促心肌细胞凋亡和铁坏死作用；确定USP7通过p53和TfR1促凋亡和铁坏死机制；阐明MLC20对USP7的表达调节机制。本项目将有助于阐明心肌I/R损伤新机制，为寻找防治心肌I/R损伤新药提供理论依据。

凋亡和铁坏死是缺血/再灌注（I/R）心肌细胞死亡的两种重要方式。本项目预实验首次发现：I/R大鼠心肌组织泛素特异性蛋白酶7（USP7）表达上调，USP7抑制剂能减轻I/R损伤；沉默USP7可减少低氧/复氧（H/R）诱导的心肌细胞凋亡和铁坏死，伴随p53和转铁蛋白受体1(TfR1)蛋白水平下降，但USP7在心肌缺血/再灌注损伤中的作用及机制尚未明确。本项目主要研究内容如下：1）建立大鼠心肌缺血/再灌注损伤和心肌细胞低氧/复氧损伤模型，检测心肌凋亡和铁死亡相关指标及USP7表达水平，结合药物干预和基因敲低等手段，确定USP7促心肌细胞凋亡和铁死亡作用；2）利用上述动物和细胞模型，检测TfR1、p53等分子表达水平、USP7与TfR1相互作用和TfR1和p53泛素化水平等，结合药物干预和基因敲低等手段，阐明USP7促心肌细胞凋亡和铁死亡机制；3）利用上述动物和细胞模型，检测MLCK 活性、MLC20总蛋白水平和核MLC20磷酸化水平，结合药物干预和基因敲低等手段，阐明USP7表达调节机制。本项主要发现包括：1）铁死亡主要发生在心肌缺血再灌注阶段，而不是发生在缺血阶段；2）USP7通过介导铁坏死促进大鼠心肌缺血/再灌注损伤；USP7通过激活p53/TfR1通路促进缺血/再灌注大鼠心肌铁坏死的发生；3）抗铁死亡药物与抗坏死样凋亡药物联合用药能同时减少铁死亡和坏死样凋亡，产生相加效果；4）USP7表达调节机制可能涉及MLC20磷酸化与去磷酸化。这些发现为心肌缺血/再灌注损伤损伤机制提供了新的理论依据，为寻找新药靶点，开发抗心肌缺血新药提供了新思路。