在活性氧所致的心肌细胞氧化应激损伤中，抗氧化通路的氧化还原状态失衡是导致心肌细胞凋亡的重要因素。申请者最近发现在心肌细胞中，HSPB1过表达抑制过氧化氢所致的GSH/GSSG比值降低和谷胱甘肽还原酶及硫氧还蛋白的氧化，提示HSPB1对氧化应激损伤所致的氧化还原状态失衡具有重要的调节作用。本项目拟在此基础上，以心肌细胞氧化应激损伤为模型，采用HSPB1基因转染或HSPB1 RNA 干扰技术，利用roGFP 探针实时监测HSPB1过表达或表达下调对细胞氧化还原状态的影响；并使用非还原性蛋白质印迹技术探讨HSPB1过表达或表达下调对两条抗氧化通路的氧化还原状态的影响；在此基础上，进一步从蛋白质的相互作用探讨HSPB1是否与抗氧化通路的重要分子结合而影响他们的活性和氧化还原状态。开展本研究有望深入阐明HSPB1抗心肌细胞氧化应激损伤的细胞机制，为缺血性心血管疾病的防治提供新的线索。

The redox state of intracellular redox regulatory systems is responsible for apoptosis during cardiomyocytes oxidative stress caused by reactive oxygen species. Our preliminary data showed that HSPB1 overexpression markedly inhibited H202-induced decrease of GSH/GSSG ratio, and decreased H202-induced oxidation of glutathione reductase or thioredoxin 1 in neonatal rat cardiomyocytes, indicating that HSPB1 plays a critical role in intracellular redox homeostasis. In the present study, cytosolic or mitochondrial reduction-oxidation sensitive GFP probes will be applied to dynamically monitor the effect of HSPB1 overexpression or knockdown on redox environment of individual organelles in cardiomyocytes oxidative stress models. To address whether HSPB1 overexpression or knockdown regulate redox regulatory signal pathways, redox western blots will be performed to detect the redox state of intracellular redox regulatory systems and the activity of downstream kinases will be measured. Furthermore, coimmunoprecipitation and mass spectrometry will be used to identify the important molecules of redox regulatory systems interacting with HSPB1 in order to investigate how the interaction will regulate the redox homeostasis. Our project will provide new evidences for the anti-apoptosis property of HSPB1 in cardiomyocytes oxidative stress.