Ginsenoside Rb2 alleviates myocardial ischemia/reperfusion injury through IKKα lactylation regulation of macrophage...

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.

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.

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.

Rb2 attenuated MIRI by modulating macrophage polarization via IKKα lactylation inhibition, offering a potential therapeutic strategy for MIRI treatment.