丹酚酸B激活SIRT3抑制氧化应激与炎症互作改善心肌再灌注损伤机制

心肌缺血再灌注损伤(MIRI)是影响介入治疗预后的难题。SIRT3调控的线粒体蛋白乙酰化可触发MIRI氧化应激和NLRP3炎症小体的激活。丹酚酸B(SalB)是常用的活血化瘀中药成分，抗炎抗氧化作用显著。申请人前期工作证实SalB能上调再灌注导致的SIRT3低表达。然而，SalB能否通过激活SIRT3抑制线粒体蛋白乙酰化，阻断氧化应激与NLRP3炎症小体的互作改善MIRI，值得深入研究。本项目拟在体内结扎再通冠状动脉左前降支建立大鼠MIRI模型和在体外H9c2细胞复制缺氧复氧模型，以SIRT3调控的线粒体蛋白乙酰化为切入点，通过基因干扰抑制或过表达SIRT3，应用线粒体ROS清除剂、NLRP3抑制剂和SalB干预，运用表面等离子共振和生物膜干涉技术，揭示SalB的作用靶点，阐明SalB改善MIRI的药理机制。本研究将有助于完善MIRI的发病机理，为SalB在MIRI领域的应用提供实验依据。

Percutaneous coronary intervention is effective to recover myocardial perfusion, but can not reduce mortality due to myocardial ischemia reperfusion injury (MIRI). Though the significance of limiting MIRI has been recognized for decades, however no effective therapeutic interventions have been applied in clinic to prevent MIRI. Protection of MIRI induced myocardial injury remains a challenge for clinician. Mitochondrial homeostasis is the key link between oxidative stress and inflammatory response in MIRI. Recent evidence confirms that SIRT3-regulated mitochondrial acetylation is involved in maintaining mitochondrial homeostasis, which plays an important role in triggering oxidative stress and NLRP3 inflammasome in MIRI. However, the association between mitochondrial acetylation, oxidative stress, and NLRP3 inflammasome remains unclear. SalB is the main active ingredient of salvia miltiorrhiza, which has significant anti-inflammatory and anti-oxidation effects. The applicant’s previous work has confirmed that SalB could upregulate MIRI-induced low expression of SIRT3, inhibit MIRI-evoked myocardial infarct size and cardiomyocyte apoptosis, finally ameliorating rat MIRI. However, the mechanism underlying the protective effect of SalB against MIRI remains to be clarified. Therefore, the purpose of the present study is to explore the crosstalk between oxidative stress and NLRP3 inflammasome in MIRI mediated by SIRT3-regulated mitochondrial acetylation, and to clarify the target mechanism of SalB on rat MIRI. .In the present study, rat MIRI model will be established by ligating and loosening left anterior descending coronary artery in vivo and hypoxia/reoxygenation (H/R) model will be replicated in H9c2 cardiomyocytes in vitro. Myocardial enzyme detection, histological evaluation, and molecular biological analysis will be performed to determine the effect of SalB on H9c2 H/R and rat MIRI. SIRT3 small interfering RNA (siRNA), SIRT3 overexpression plasmid, SIRT3 inhibitor, mitochondrial ROS (mtROS) scavenger, and NLRP3 inhibitor (MCC950) will be applied to explore the crosstalk between oxidative stress and NLRP3 inflammasome mediated by SIRT3-regulated mitochondrial acetylation. Furthermore, surface plasma resonance assay and biolayer interferometry will be applied to detect the binding affinity of SalB to SIRT3. This study will help to further explore the pathogenesis of MIRI and promote the application of SalB in protecting cardiomyocytes against MIRI.