Proteomic analysis reveals ginsenoside Rb1 attenuates myocardial ischemia/reperfusion injury through inhibiting ROS production from mitochondrial complex I

Abstract

Rationale: Reactive oxygen species (ROS) burst from mitochondrial complex I is considered the critical cause of ischemia/reperfusion (I/R) injury. Ginsenoside Rb1 has been reported to protect the heart against I/R injury; however, the underlying mechanism remains unclear. This work aimed to investigate if ginsenoside Rb1 attenuates cardiac I/R injury by inhibiting ROS production from mitochondrial complex I. Methods: In in vivo experiments, mice were given ginsenoside Rb1 and then subjected to I/R injury. Mitochondrial ROS levels in the heart were determined using the mitochondrial-targeted probe MitoB. Mitochondrial proteins were used for TMT-based quantitative proteomic analysis. In in vitro experiments, adult mouse cardiomyocytes were pretreated with ginsenoside Rb1 and then subjected to hypoxia and reoxygenation insult. Mitochondrial ROS, NADH dehydrogenase activity, and conformational changes of mitochondrial complex I were analyzed. Results: Ginsenoside Rb1 decreased mitochondrial ROS production, reduced myocardial infarct size, preserved cardiac function, and limited cardiac fibrosis. Proteomic analysis showed that subunits of NADH dehydrogenase in mitochondrial complex I might be the effector proteins regulated by ginsenoside Rb1. Ginsenoside Rb1 inhibited complex I- but not complex II- or IV-dependent O₂ consumption and enzyme activity. The inhibitory effects of ginsenoside Rb1 on mitochondrial I-dependent respiration and reperfusion-induced ROS production were rescued by bypassing complex I using yeast NADH dehydrogenase. Molecular docking and surface plasmon resonance experiments indicated that ginsenoside Rb1 reduced NADH dehydrogenase activity, probably via binding to the ND3 subunit to trap mitochondrial complex I in a deactive form upon reperfusion. Conclusion: Inhibition of mitochondrial complex I-mediated ROS burst elucidated the probable underlying mechanism of ginsenoside Rb1 in alleviating cardiac I/R injury.

Keywords: Ginsenoside Rb1; Mitochondrial complex I; Myocardial ischemia/reperfusion injury; Proteomic analysis; Reactive oxygen species.

Figure 1

Ginsenoside Rb1 reduces succinate-driven ROS production upon reperfusion. A, ROS production assessed by MitoSOX Red mitochondrial superoxide indicator in adult primary cardiomyocytes subjected to hypoxia/reoxygenation treatment. B, ROS production in primary cardiomyocytes subjected to dimethyl succinate (Suc) and oligomycin (Oligo) treatment for 2 h. C, Inhibitory effect of ginsenoside Rb1 on ROS generation as assessed by MitoB oxidation 15 min post-reperfusion in vivo (n = 5, 6). D, Immunohistochemistry examinations of 8-hydroxyguanosine (8-OHG) in hearts from mice subjected to ischemia (30 min) followed by 15 min reperfusion (n = 4). Scale bar, 20 µm. E-F, Effect of ginsenoside Rb1 pretreatment on heart ROS levels at days 14 (E) or 28 (F) post-reperfusion (n = 6). Data were expressed as mean ± SD. ^*p < 0.05: vs. H/R or I/R only treatment; ^#p < 0.05: vs. indicated treatment. H/R, hypoxia/reoxygenation; I/R, ischemia/reperfusion; Rb1, ginsenoside Rb1.

Figure 2

Attenuation of ROS burst by ginsenoside Rb1 protects cardiomyocytes from acute I/R injury. A-C, Adult primary cardiomyocytes were treated with ginsenoside Rb1 (10 µM) and subjected to hypoxia (1% O₂) for 1 h followed by 1 h reoxygenation (H/R). Mitochondrial permeability transition pore (A), representative images and quantification of mitochondrial potential (Δψm) from four independent experiments (B, bar: 20 µm), and cell viability (C) were assayed. D, Representative images and quantification of TUNEL staining of hearts from mice subjected to ischemia (30 min) followed by 24 h reperfusion (n = 4). Scale bar, 50 µm. E, Myocardial infarct size in mice subjected to cardiac ischemia and reperfusion injury with ginsenoside Rb1 administered prior to ischemia, at the onset of reperfusion, or 15 min post-reperfusion (n = 4). Representative photographs of TTC staining 24 h post-reperfusion. Data were expressed as mean ± SD. ^*p < 0.05: vs. H/R only treatment or I/R only treatment; ^#p < 0.05: vs. indicated group; ns. no significance: vs. I/R only treatment. H/R, hypoxia/reoxygenation; I/R, ischemia/reperfusion; Rb1, ginsenoside Rb1; TMRE, tetramethylrhodamine ethyl ester; TTC, 2,3,5-triphenyltetrazolium chloride.

Figure 3

Ginsenoside Rb1 administration before reperfusion contributed to improving cardiac function during the recovery process. A, Heart function measured 14 days post-I/R insult (n = 7-10). B, Mitochondrial structure of the heart assayed 14 days post-I/R insult (n = 3). Scale bar, 200 nm. C, Cardiac gene profiling measured 14 days post-I/R insult (n = 3). D, Representative images and quantification of Masson's trichrome staining in hearts from mice 28 days post-I/R insult (n = 3). Scale bars, 1 mm (left) and 300 µm (right). Data were expressed as mean ± SD. ^*p < 0.05: vs. I/R only treatment; ^#p < 0.05: vs. indicated group. I/R, ischemia/reperfusion; EF, ejection fraction; FS, fractional shortening; LV vol; d, left ventricular end-diastolic volume; LV vol; s, left ventricular end-systolic volume; Rb1, ginsenoside Rb1.

Figure 4

Ginsenoside Rb1 regulates mitochondrial complex I as revealed by proteomics. C57BL/6J mice were given ginsenoside Rb1 (50 mg/kg, i.p.) and subjected to cardiac ischemia (30 min) followed by 15 min of reperfusion. Mitochondrial proteins of the heart tissue were extracted for proteomic analysis. A, Western blot assay to check the purity of enriched mitochondrial fractions obtained from heart tissues (n = 3). B, Significantly changed proteins identified from comparison of ischemia/reperfusion (I/R) and sham groups. C, Principal component analysis (PCA) of sham group (Sham), ischemia/reperfusion group (I/R), and I/R plus ginsenoside Rb1 treated group (I/R+Rb1) based on identified proteins (n = 3). D, Cluster analysis of significantly changed proteins in Sham, I/R, and I/R+Rb1 groups based on their change tendencies. Each fold line indicated a change tendency of one protein. Proteins positioned in Clusters 1 and 2 were recognized as rescued or regulated by ginsenoside Rb1. E, Functional categorization of proteins rescued or regulated by ginsenoside Rb1 using the Gene Ontology (GO) database. F, Protein-protein interaction analysis of proteins rescued or regulated by ginsenoside Rb1 using the STRING database. Red and blue indicated degree of protein rescued or regulated by ginsenoside Rb1. G, Schematic diagram of the distribution of proteins positioned in mitochondrial complex I rescued or regulated by ginsenoside Rb1 treatment.

Figure 5

Ginsenoside Rb1 selectively inhibits the activity of mitochondrial complex I but not complexes II and IV. A, Oxygen consumption rate (OCR, % baseline) of H9c2 cells in the presence of ginsenoside Rb1 (10 µM), FCCP (2.5 µM), pyruvate (Pyr, 10 mM), and antimycin A (AA, 1 µM). B, OCR traces of H9c2 cells permeabilized by digitonin (3 µg/mL) and then stimulated by ADP (1 mM), pyruvate (5 mM), malate (5 mM) and Rb1 (10 µM). C, Mitochondrial complex I activity in H9c2 cells treated with ginsenoside Rb1 (10 µM) or rotenone (1 µM). D, OCR traces of permeabilized H9c2 cells exposed to ADP (1 mM), glutamate (5 mM) plus malate (5 mM), and succinate (5 mM). Here, H9c2 cells were pretreated with ginsenoside Rb1 (10 µM) or rotenone (1 µM) for 1 h prior to OCR measurements. Then ginsenoside Rb1 or rotenone was then either left on for further assay, or washed out with replacement of drug-free assay buffer. E, OCR (% baseline) of permeabilized H9c2 cells in the presence of succinate (5 mM), rotenone (0.5 µM), and ginsenoside Rb1 (10 µM). F, OCR (% baseline) of H9c2 cells injected with FCCP (2.5 µM), TMPO (0.4 mM) plus ascorbate (0.4 mM), ginsenoside Rb1 (10 µM), and azide (5 mM). Data were expressed as mean ± SD. ^*p < 0.05: vs. untreated control. AA, antimycin A; Dig, digitonin; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; G, glutamate; M, malate; Pyr or P, pyruvate; Rb1, ginsenoside Rb1; Rot or R, rotenone; S, succinate; TMPD, tetramethyl-p-phenylene diamine.

Figure 6

Ginsenoside Rb1 reversibly inhibits NADH dehydrogenase of mitochondrial complex I. A, mRNA expressions of Ndufv1, Ndufv2, Ndufs1, Ndufs4, Ndufs6, and Ndufa12 in H/R-treated cardiomyocytes. B-C, ROS level (B) and cell viability (C) of H9c2 cells transfected with Ndufv1, Ndufv2, Ndufs1, Ndufs4, Ndufs6, or Ndufa12 siRNA. D, NADH dehydrogenase activity in adult primary cardiomyocytes with ginsenoside Rb1 or rotenone treatment. E, NADH dehydrogenase activity in adult primary cardiomyocytes upon hypoxia (1 h) followed by 15 min of reperfusion. F, Adult primary cardiomyocytes were pretreated with ginsenoside Rb1 (10 µM) for 1 h. Then ginsenoside Rb1 was then either left on for the further assay, or washed out. The culture was continued for another 1 h. NADH dehydrogenase activity was measured. G, Ndi1 protein expression in H9c2 cells transfected with Ndi1 plasmid. Oxygen consumption rate (OCR, % baseline) in Ndi1-expressing cells in the presence of ginsenoside Rb1 (10 µM), FCCP (2.5 µM), pyruvate (Pyr, 10 mM) and antimycin A (AA, 1 µM). H, Ndi1 protein expression. ROS levels in Ndi1-expressing cells in response to H/R injury. I, Ndi1 protein expression. ROS production in Ndi1-expressing cells subjected to dimethyl succinate and oligomycin for 2 h. Data were expressed as mean ± SD. ^*p < 0.05: vs. the untreated control, H/R only treatment or suc plus oligo treatment; ^#p < 0.05: vs. indicated treatment; ns. no significance: vs. untreated control. H/R, hypoxia/reoxygenation; Oligo, oligomycin; Rb1, ginsenoside Rb1; Rot, rotenone; Suc, succinate.

Figure 7

Ginsenoside Rb1 controls the active/deactive transition of mitochondrial complex I to regulate NADH dehydrogenase activity. A, Representative images of the mitochondrial location of mito-SypHer. Scale bar, 20 µm. Mitochondrial pH (480/420 ratio) in H9c2 cells stably transfected with mito-SypHer. B, Schematic diagram of BODIPY-TMR labeling of mitochondrial complex I. C, BODIPY-TMR signal (left) of mitochondrial proteins in the hearts of mice upon I/R injury (n = 4). D, Binding mode predicted by AutoDock between ginsenoside Rb1 and the mitochondrial complex I subunits involved in the active/deactive transition (ND3, ND1, and Ndufa9). E, SPR analysis of ginsenoside Rb1 binding to human recombinant ND3 protein. F, Mitochondrial complex I activity in H9c2 cells treated with ginsenoside Rb1 (10 µM) and subjected to hypoxia for 1 h with and without subsequent reoxygenation for 15 min. Data above were expressed as mean ± SD. ^#p < 0.05: vs. indicated group. H/R, hypoxia/reoxygenation; I/R, ischemia/reperfusion; K_D, equilibrium dissociation constant; Rb1, ginsenoside Rb1.