南方医科大学学报

南方医科大学学报 ›› 2025, Vol. 45 ›› Issue (10): 2062-2070.doi: 10.12122/j.issn.1673-4254.2025.10.02

• • 上一篇    

丹酚酸B通过抑制Sirt1蛋白降解促进心肌细胞线粒体功能稳态和改善缺血再灌注小鼠的心脏功能

李思蒙1,4(), 陈建宁4, 申思满4, 刘望龙4, 于丽丽1,2,3(), 张良清1,4()   

  1. 1.澳门科技大学医学部中医药学院，澳门 999078
    2.澳门中药机制与质量全国重点实验室，澳门 999078
    3.澳门科技大学珠海澳科大科技研究院，广东 珠海 519099
    4.广东医科大学附属第二医院，广东 湛江 524000
  • 收稿日期:2025-05-01 出版日期:2025-10-20 发布日期:2025-10-24
  • 通讯作者: 于丽丽,张良清 E-mail:3230005101@student.must.edu.mo;llyu@must.edu.mo;zhangliangqing@gdmu.edu.cn
  • 作者简介:李思蒙，在读博士研究生，E-mail: 3230005101@student.must.edu.mo
  • 基金资助:
    国家自然科学基金(82370281);广东省自然科学基金(2024A1515013119)

Salvianolic acid B promotes mitochondrial homeostasis and improves cardiac function in mice with ischemia-reperfusion injury by inhibiting Sirt1 protein degradation

Simeng LI1,4(), Jianning CHEN4, Siman SHEN4, Wanglong LIU4, Lili YU1,2,3(), Liangqing ZHANG1,4()   

  1. 1.Faculty of Chinese Medicine, Medical Sciences Division, Macau University of Science and Technology, Macau 999078, China
    2.The State Key Laboratory of Mechanism and Quality of Chinese Medicine of the Macau University of Science and Technology, Macau 999078, China
    3.Macau University of Science and Technology Zhuhai MUST Science and Technology Research Institute, Zhuhai 519099, China
    4.Second Affiliated Hospital of Guangdong Medical University, Zhanjiang 524000, China
  • Received:2025-05-01 Online:2025-10-20 Published:2025-10-24
  • Contact: Lili YU, Liangqing ZHANG E-mail:3230005101@student.must.edu.mo;llyu@must.edu.mo;zhangliangqing@gdmu.edu.cn
  • Supported by:
    National Natural Science Foundation of China(82370281)

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摘要：

目的 探讨丹酚酸B（Sal-B）对缺氧复氧（HR）小鼠HL-1心肌细胞线粒体功能稳态和小鼠心肌缺血再灌注（IR）诱导心肌损伤调控的分子机制。 方法 体外实验：在验证Sal-B对缺氧复氧小鼠HL-1心肌细胞影响的线粒体表型实验中，将细胞分为对照组（Ctrl组）；缺氧复氧组（HR组；缺氧12 h，复氧4 h）；Sal-B预处理组（Sal-B+HR组；5 μmol/L Sal-B，预处理30 min）；敲降对照组（HR+Sal-B+sh-Ctrl组）；敲降Sirt1组（HR+Sal-B+sh-Sirt1组）。采用 ATP检测试剂盒测定HL-1细胞ATP生成能力；Mito-Sox染色测定HL-1细胞线粒体超氧阴离子水平；使用氧耗率OCR检测试剂盒测定HL-1细胞底物氧化水平。体内实验：在验证Sal-B对小鼠在体IR心功能影响的实验中，将36只8~10周龄 C57BL/6J小鼠随机分为3组（12只/组）：假手术组（Sham组）；缺血再灌注组；Sal-B预处理缺血再灌注组（Sal-B+IR；50 mg/kg，IR前24 h尾静脉注射）。在验证Sal-B对Sirt1-cKO小鼠在体缺血再灌注心功能影响的挽救实验中，将60只8~10周龄C57BL/6J小鼠随机分为5组（12只/组）：假手术组；缺血再灌注组（IR组；缺血45 min，再灌注6 h）；Sal-B预处理缺血再灌注组（Sal-B+IR；50 mg/kg，尾静脉注射）；Sirt1敲除对照组（IR+Sal-B+Sirt1f1/fl）；心脏特异性Sirt1基因敲除组（IR+Sal-B+cKO-Sirt1）。采用HE染色观察心肌的组织损伤程度；通过心脏超声评估小鼠的心脏射血分数和短轴缩短率。 结果 Sal-B处理可以明显提高HR条件下的HL-1心肌细胞ATP生成能力（P<0.01）；Sal-B可以降低HR处理的HL-1心肌细胞线粒体超氧阴离子水平（P<0.01）；Sal-B可以提高HR处理的HL-1心肌细胞的耗氧率（P<0.05）。Sal-B处理显著改善IR处理的心肌的组织结构紊乱，小鼠心脏超声结果提示Sal-B可以提高IR小鼠的心脏射血功能（P<0.05）。放线菌酮的使用联合Western blotting分析，以及Sirt1-IP后泛素化的Western blotting分析显示Sal-B明显抑制HL-1心肌细胞Sirt1蛋白降解（P<0.05）。HL-1心肌细胞在加入Sal-B的基础上，敲降Sirt1逆转了Sal-B对HL-1细胞的促进ATP生成作用（P<0.01）；逆转了Sal-B对HL-1细胞的抑制线粒体超氧阴离子水平（P<0.01），逆转了Sal-B对HL-1细胞耗氧率的促进作用（P<0.05）。在小鼠体内注射Sal-B的基础上，Sirt1-cKO组相较于对照组明显逆转了Sal-B的提高IR小鼠的心脏射血功能的作用（P<0.05），Sirt1-cKO组相较于对照组明显逆转了Sal-B的改善IR处理的心肌组织结构紊乱作用。 结论 Sal-B可通过抑制Sirt1蛋白降解从而促进HR处理的HL-1心肌细胞的线粒体功能稳态提升和改善IR小鼠的心功能。

关键词: 丹酚酸B, Sirt1, 心肌缺血再灌注损伤, 心脏, 线粒体功能稳态

Abstract:

Objective To investigate the molecular mechanism by which salvianolic acid B (Sal-B) modulates mitochondrial functional homeostasis and alleviates myocardial ischemia-reperfusion (I/R) injury in mice. Methods Mouse cardiomyocyte HL-1 cells were pretreated with 5 μmol/L Sal-B with or without sh-Sirt1 transfection before exposure to hypoxia-reoxygenation (HR), and the changes in ATP production, mitochondrial superoxide activity, substrate oxidation level were evaluated. In the animal experiment, 36 C57BL/6J mice were randomized into 3 groups (n=12) for sham operation or ligation of the left anterior coronary artery to induce myocardial I/R injury with or without intravenous injection of Sal-B+I/R (50 mg/kg). In the rescue experiment, 60 adult C57BL/6J mice were randomized into 5 groups (n=12): sham-operated group, myocardial I/R group, Sal-B+I/R group, I/R+Sal-B+Sirt1fl/fl group, and I/R+Sal-B+cKO-Sirt1 group. Myocardial injury was evaluated with HE staining, and cardiac function was assessed by measurement of the ejection fraction and fractional shortening using echocardiography. Results In HL-1 cells with HR injury, Sal-B pretreatment significantly increased cellular ATP production, reduced mitochondrial superoxide anion levels, and enhanced oxygen consumption level. In the mouse models of myocardial I/R injury, Sal-B pretreatment markedly ameliorated I/R-induced structural disarray of the cardiac myocytes and improved cardiac ejection. Cycloheximide chase with Western blotting and ubiquitination assays after Sirt1-IP showed that Sal-B significantly inhibited Sirt1 degradation in HL-1 cells. Sirt1 knock-down reversed Sal-B-induced increases in ATP production, reduction in superoxide, and elevation of OCR in HL-1 cells. Cardiomyocyte-specific Sirt1 knockout obviously reversed Sal-B-mediated improvement in cardiac ejection function and myocardial structure damage in mice with myocardial I/R injury. Conclusion Sal-B promotes mitochondrial functional homeostasis in cardiomyocytes with HR injury and improves cardiac function in mice after myocardial I/R by inhibiting Sirt1 protein degradation.

Key words: salvianolic acid B, Sirt1, myocardial ischemia-reperfusion injury, heart, mitochondrial homeostasis