核肌球蛋白调节轻链在Nox2介导的心肌缺血/再灌注氧化应激损伤中的作用及机制研究

We previously demonstrated that oxidative stress injury induced by myocardial ischemia/reperfusion is involved in up-regulation of NADPH oxidase (Nox2)gene expression. But the underlying mechanisms remain poorly understood. The preliminary data of this project suggest that nuclear myosin regulatory light chain (NMLC20) may participate in the regulation of Nox2 gene expression. It is the first time for this project to explore the role of NMLC20 in regulation of Nox2 gene expression and the underlying mechanisms as follows: 1) Establish a rat model of myocardial ischemia/reperfusion injury and finish the measurements of myocardial injury, expression levels of MLCK, Nox2 and MLCP，and phosphorylation level of NMLC20. Through combining the strategies of drug interference, the role of NMLC20 in regulation of Nox2 gene expression will be evaluated; 2) Establish a cell model of hypoxia/reoxygenation injury and finish the measurements of the associated parameters. Through combining the strategies of drug interference, gene silence or expression, the role of NMLC20 in regulation of Nox2 gene expression will be confirmed; 3) Through using the above-mentioned animal and cell models and combining the strategies of co-immunoprecipitation, chromatin immunoprecitation, agarose-oligonucleotide pull-down assay, luciferase reporter genes, etc, the molecular mechanisms for NMLC20 in regulation of Nox2 gene expression will be elucidated. This project will lay a theoretic foundation and provide the new idea for seeking novel drug targets against myocardial ischemia.

我们前期工作证明心肌缺血/再灌注氧化应激损伤涉及Nox2基因表达上调，但机制不详。本项目预实验首次发现核肌球蛋白调节轻链（NMLC20）可能参与了Nox2基因的表达调控。本项目将率先从以下方面探讨NMLC20对Nox2的表达调控作用及机制：1) 建立大鼠心肌缺血/再灌注损伤模型，检测心肌损伤，MLCK、Nox2 和MLCP 表达水平，NMLC20 磷酸化水平等指标, 结合药物干预手段，初步确定NMLC20 对Nox2 基因的表达调控作用；2）建立心肌细胞低氧/复氧损伤模型，检测相关指标, 结合药物干预手段、基因沉默或表达技术，确定NMLC20对Nox2 的表达调控作用；3）利用上述动物和细胞模型，结合免疫共沉淀、染色质免疫沉淀、寡核苷酸pull-down、荧光素酶报告基因等方法技术，阐明NMLC20对Nox2 表达调控机制。本项目将为寻找新药靶点，开发抗心肌缺血新药提供理论依据和新思路。

氧化应激是心肌缺血/再灌注损伤的重要机制之一。我们前期工作证明心肌缺血/再灌注氧化应激损伤涉及Nox2基因表达上调，但机制不详。本项目主要研究内容如下：1) 建立大鼠心肌缺血/再灌注损伤模型，检测心肌损伤，MLCK、Nox2 和MLCP 表达水平，NMLC20 磷酸化水平等指标, 结合药物干预手段，初步确定NMLC20 对Nox2 基因的表达调控作用；2）建立心肌细胞低氧/复氧损伤模型，检测相关指标, 结合药物干预手段、基因沉默或表达技术，确定NMLC20对Nox2 的表达调控作用；3）利用上述动物和细胞模型，结合免疫共沉淀、染色质免疫沉淀、寡核苷酸pull-down、荧光素酶报告基因等方法技术，阐明NMLC20对Nox2 表达调控机制。本项主要发现包括：1）心肌缺血/再灌注时，肌球蛋白节轻链(MLC20)通过识别结合Nox2基因启动子特定序列“AGCTCC”，促进Nox2表达；2) 非肌肉型肌球蛋白调节轻链(nmMLC20)通过与XO基因启动子下游第一个内含子中的GTCGCC序列结合，对XO表达发挥调节作用；3）法舒地尔通过降低MLC20磷酸化水平，抑制Nox2表达而发挥抗心肌缺血/再灌注损伤作用。这些发现为心肌缺血/再灌注损伤氧化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。