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

doi:10.12122/j.issn.1673-4254.2025.10.02

Abstract

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

Simeng LI, Jianning CHEN, Siman SHEN, Wanglong LIU, Lili YU, Liangqing ZHANG. Salvianolic acid B promotes mitochondrial homeostasis and improves cardiac function in mice with ischemia-reperfusion injury by inhibiting Sirt1 protein degradation[J]. Journal of Southern Medical University, 2025, 45(10): 2062-2070.

Figures/Tables 5

Fig.1 Sal-B reverses mitochondrial dysfunction in HL-1 cardiomyocytes following hypoxia-reoxygenation (HR) injury. A: ATP production levels in different groups. B, C: Mito-SOX fluorescence staining for detecting superoxide levels in HL-1 cardiomyocytes and statistical results in different groups. D, E: Oxygen consumption rates (OCR) in different groups. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Fig.2 Sal-B alleviates myocardial I/R injury in mice. A-C: Echocardiographic assessment of cardiac ejection fraction and fractional shortening in mice in different groups. D: HE staining showing myocardial tissue disorganization in the mice from different groups. *P<0.05, **P<0.01.

Fig.3 Sal-B inhibits degradation of Sirt1 protein in HL-1 cardiomyocytes with HR injury. A: Molecular docking diagram of Sal-B and Sirt1. B, C: Western blotting showing the effects of different concentrations of Sal-B on Sirt1 protein expression in the cells. D, E: Western blotting showing the effects of 5 μmol/L Sal-B treatment on Sirt1 protein expression at different time points under the same HR conditions with cycloheximide (CHX) treatment. F: Western blotting showing ubiquitination levels of Sirt1 protein in HL-1 cells treated with 5 μmol/L Sal-B before HR injury. *P<0.05, **P<0.01.

Fig.4 Knockdown of Sirt1 partially reverses homeostatic effects of Sal-B on mitochondrial function in HL-1 cells under HR conditions. A: ATP production levels in different groups. B, C: Mito-SOX fluorescence staining for detecting superoxide levels in HL-1 cardiomyocytes in different groups. D, E: OCR detection in different groups. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Fig5 Cardiac-specific Sirt1 gene knockout partially abrogates the protective effect of Sal-B on cardiac function in mice with myocardial I/R injury. A-C: Echocardiographic assessment of cardiac ejection fraction and fractional shortening in mice from different groups. D: HE staining of the myocardial tissue in mice from different groups. *P<0.05, **P<0.01.

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