Cardiovascular diagnosis and therapy · 2026
Ginsenoside Rb2 alleviates myocardial ischemia/reperfusion injury through IKKα lactylation regulation of macrophage polarization.
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An Peng, Zhang Meiqi, Cheng Mengshi, Wu Weihua, Zhang Wei, Yu Zhenfei, Liu Rui
Аннотация
BACKGROUND: Myocardial ischemia/reperfusion (I/R) injury (MIRI) is a major contributor to cardiovascular morbidity, characterized by inflammatory responses and cardiomyocyte death. Ginsenoside Rb2 (Rb2), a bioactive compound from Panax ginseng, has shown potential cardioprotective effects, but its mechanisms in I/R injury remain unclear. Macrophage polarization refers to the process by which macrophages dynamically shift between pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes in response to microenvironmental signals. This study aimed to investigate whether Rb2 could participate in the progression of MIRI by regulating macrophage polarization.
METHODS: In vitro, THP-1-derived macrophages were co-cultured with AC16 cardiomyocytes under hypoxia/reoxygenation (H/R) conditions and treated with Rb2 (0, 25, 50, or 100 µM) for 24 h. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay. Macrophage polarization (CD86/CD206) was evaluated by immunofluorescence (IF), and apoptosis by terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) staining. Gene expression of inflammatory markers [interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), IL-6, arginase-1 (Arg-1), IL-10, transforming growth factor-β (TGF-β)] was analyzed by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Inhibitor of kappa B kinase (IKKα) lactylation was assessed by immunoprecipitation (IP) and Western blot. In vivo, a murine MIRI model was established in male C57BL/6 mice by transient occlusion of the left anterior descending (LAD) coronary artery for 30 min, followed by 24 h of reperfusion. Rb2 was administered intraperitoneally at 10 mg/kg daily for 14 consecutive days prior to I/R induction. Cardiac function was assessed by echocardiography, infarct size by triphenyltetrazolium chloride (TTC) staining, and macrophage markers (CD86, CD206) by immunohistochemistry (IHC). Serum cardiac troponin T (cTnT) levels were measured by enzyme-linked immunosorbent assay (ELISA), and inflammatory gene expression in cardiac tissues was analyzed by RT-qPCR. IKKα lactylation in cardiac tissues was evaluated by IP and Western blot. Molecular docking, surface plasmon resonance (SPR), and bioinformatics were used to validate Rb2-IKKα interactions.
RESULTS: Rb2 treatment enhanced cell viability (104.2±3.1 vs. 88.7±2.8, P<0.001), promoted M2 polarization (P<0.001), reduced apoptosis (29.6±2.1 vs. 10.1±1.8, P<0.001), and improved cardiac function post-I/R [cTNT, 532±53 vs. 125±22, P<0.001; left ventricular ejection fraction (LVEF), 41±6 vs. 61±4, P<0.001; left ventricular fractional shortening (LVFS), 20±3 vs. 31±2, P<0.01; Infarct size, 63±4 vs. 20±4, P<0.001]. Mechanistically, Rb2 directly bound to IKKα, inhibited its lactylation at K617 site, and destabilized the protein. Overexpression of IKKα reversed Rb2's protective effects.
CONCLUSIONS: Rb2 attenuated MIRI by modulating macrophage polarization via IKKα lactylation inhibition, offering a potential therapeutic strategy for MIRI treatment.
METHODS: In vitro, THP-1-derived macrophages were co-cultured with AC16 cardiomyocytes under hypoxia/reoxygenation (H/R) conditions and treated with Rb2 (0, 25, 50, or 100 µM) for 24 h. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay. Macrophage polarization (CD86/CD206) was evaluated by immunofluorescence (IF), and apoptosis by terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) staining. Gene expression of inflammatory markers [interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), IL-6, arginase-1 (Arg-1), IL-10, transforming growth factor-β (TGF-β)] was analyzed by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Inhibitor of kappa B kinase (IKKα) lactylation was assessed by immunoprecipitation (IP) and Western blot. In vivo, a murine MIRI model was established in male C57BL/6 mice by transient occlusion of the left anterior descending (LAD) coronary artery for 30 min, followed by 24 h of reperfusion. Rb2 was administered intraperitoneally at 10 mg/kg daily for 14 consecutive days prior to I/R induction. Cardiac function was assessed by echocardiography, infarct size by triphenyltetrazolium chloride (TTC) staining, and macrophage markers (CD86, CD206) by immunohistochemistry (IHC). Serum cardiac troponin T (cTnT) levels were measured by enzyme-linked immunosorbent assay (ELISA), and inflammatory gene expression in cardiac tissues was analyzed by RT-qPCR. IKKα lactylation in cardiac tissues was evaluated by IP and Western blot. Molecular docking, surface plasmon resonance (SPR), and bioinformatics were used to validate Rb2-IKKα interactions.
RESULTS: Rb2 treatment enhanced cell viability (104.2±3.1 vs. 88.7±2.8, P<0.001), promoted M2 polarization (P<0.001), reduced apoptosis (29.6±2.1 vs. 10.1±1.8, P<0.001), and improved cardiac function post-I/R [cTNT, 532±53 vs. 125±22, P<0.001; left ventricular ejection fraction (LVEF), 41±6 vs. 61±4, P<0.001; left ventricular fractional shortening (LVFS), 20±3 vs. 31±2, P<0.01; Infarct size, 63±4 vs. 20±4, P<0.001]. Mechanistically, Rb2 directly bound to IKKα, inhibited its lactylation at K617 site, and destabilized the protein. Overexpression of IKKα reversed Rb2's protective effects.
CONCLUSIONS: Rb2 attenuated MIRI by modulating macrophage polarization via IKKα lactylation inhibition, offering a potential therapeutic strategy for MIRI treatment.