High-density lipoprotein ameliorates palmitic acid-induced lipotoxicity and oxidative dysfunction in H9c2 cardiomyoblast cells via ROS suppression

Fig. 1. PA increased oxidative stress and induced cell death in H9c2 cells. a H9c2 cells were treated with PA at different concentrations for 24 or 48 h. Cells viability was measured by MTT assay. b H9c2 cells were incubated with PA (0.5 mM) for 24 h. Cells were stained with 4,6-diamidino-2-phenylindole (DAPI) and terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL) assay. H9c2 cells were treated with PA (0.5 mM) for 24 h followed by 1 h incubation with fluorescent probe (c) DCF-AM (10 μM) (d) DHE (10 μM) (e) MitoSOX™ (5 μM). Fluorescence intensity of cells was measured by flow cytometry. H9c2 cells were treated with PA (0.5 mM) for indicated time. f The levels of NADPH oxidase (Nox2-gp91, p47phox, Rac) and (g) antioxidant enzymes (SOD1, SOD2) were measured by Western blot. Data showed the means ± SEM of 3 independent analyses.*p < 0.05 and **p < 0.01 compared with the control

Fig. 2. PA leads to unstability of mitochondria member potential triggered cell apoptosis through mitochondria dependent pathway and NFκB signaling pathway. a H9c2 cells were treated with PA (0.5 mM) for 24 h. △Ψm was assessed with signal from monomeric and J-aggregate JC-1 fluorescence. JC-1 fluorescence was measured by flow cytometry. Left: control, Right: PA. b H9c2 cells were treated with PA (0.5 mM) for indicated time. p-Akt, Bcl-2, Bax, caspase 3 expression was estimated by immunoblotting.(c) H9c2 cells were treated with PA (0.5 mM) for 24 h and the cell lysates were fractionated into cytosolic and mitochondrial proteins. Cytochrome c was analyzed by immunoblotting. β-actin and COX IV served as the cytosolic and mitochondrial loading controls. d H9c2 cells were treated with PA (0.5 mM) for indicated time. The expression of MAPK family (p-ERK, p-JNK, p-P38) was analyzed by immunoblotting. e H9c2 cells were incubated with PA (0.5 mM) for 0-2 h. The expression of NFκB, and IκB was analyzed by immunoblotting. β-actin and PCNA served as the cytosolic and nuclear loading controls. f Cells were transfected with a luciferase NFκB reporter construct. After transfection and treatment with PA…

Fig. 3. HDL attenuated PA-induced ROS production and cell apoptosis. H9c2 cells were incubated with PA (0.5 mM) in the absence or presence of different concentrations of HDL (25-100 μg/ml) for 24 h. a Cell viability was determined via MTT assay. b Flow cytometry profile represents Annexin-V-FITC staining in x axis and PI in y axis. The number represents the percentage of early apoptotic cells in each condition. c Fluorescence images showed the cells stained with 4,6-diamidino-2-phenylindole (DAPI) (upper panel) and stained using terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL) assay (middle panel), and photomicrographs were from phase-contrast microscopy (bottom panel). d TUNEL positive cell was determined via flow cytometric analysis. e Cellular ROS was determined via MitoSOX™ (5 μM). f Fluorescence intensity of cells was measured by phase-contrast microscopy. g SOD1 and SOD2 expression was estimated by immunoblotting. h Neonatal cardiomyocytes were treated with HDL 100 μg/ml for 2 h and then incubated with 0.5 mM PA for an additional 24 h, and followed by 1 h incubation with MitoSOX™ (5 μM). Fluorescence intensity of cells was measured by phase-contrast…

Fig. 4. HDL stabilized on mitochondrial transmembrane permeability transition (△Ψm) and downregulated PA-triggered mitochondrial dependent pathway, JNK phosphorylation and NFκB activity in H9c2 cells. a Cells were incubated with HDL 100 μg/ml for 2 h and then incubated with 0.5 mM PA for an additional 24 h. The change in mitochondrial membrane poteneial was assessed based on the signal intensity from monomeric (green) and J-aggregate (red) JC-1 fluorescence. No treatment (left); PA (middle); and PA + HDL (right). b H9c2 cells were pretreated with the indicated concentrations of HDL (25-100 μg/ml) for 2 h followed by PA (0.5 mM) treatment for 24 h. p-Akt, Bcl2, Bax, Caspase 3 expression was estimated by immunoblotting. c p-JNK, p-NFκB, MMP3, COX2 was estimated by immunoblotting. d Cells were transfected with a luciferase NFκB reporter construct. After transfection and treatment with PA and indicated concentrations of HDL (25-100 μg/ml), the cells were assayed for luciferase activity. Data showed the means±SEM of 3 independent analyses. # p < 0.05 vs. control; *p < 0.05 and **p < 0.01vs. palmitic acid alone treatment

Fig. 5. The proposed mechanism of high-density lipoprotein ameliorates palmitic acid-induced lipotoxicity and oxidative dysfunction