Comparison of the Protective Effects of Nebivolol and Metoprolol against LPS-Induced Injury in H9c2 Cardiomyoblasts

Figures

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  Figure 1. Neb and Met reduced LPS-induced cytotoxicity and ROS generation in H9c2 cells: (A). The cytotoxicity effect of increasing LPS concentrations on H9c2 using cell titer assay. (B). The cytotoxic effect of 5 µg/mL LPS for 24 and 48 h obtained by cell titer assay. (C). Cell viability results were obtained by cell titer assay following treatment with Neb 1 µM, and Met 10 µM in the presence or absence of LPS. (D,E) ROS generation was evaluated by DCFDA fluorescence. LPS treatment raised DCF fluorescence, which was markedly reduced by neb and met pretreatment. (F) mRNA levels of NOX2 were downregulated by pretreatment with both Neb and Met. (G,H) The immunoblot and corresponding histogram illustrate the protein expression of NOX2, β-actin was used as an internal control. δ p < 0.02, δδ p < 0.01, ¶ p < 0.05 vs. untreated (Con), ππ p < 0.02, π p < 0.05 vs. LPS. Values are presented as means ± SEM N ≥ 6 for each treatment group.

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  Figure 2. Neb but not Met reduced the gene and protein expressions of inflammatory markers in LPS-stimulated H9c2 cells: Downregulation of mRNA expression levels of NF-κB (A), TNF-α (B) and iNOS (C) by Neb but not Met. (D–F). The immunoblots and corresponding histograms illustrate the expression of NFkB, TNFα and iNOS respectively. β-actin was used as an internal control. ¶ p < 0.05 vs. Con, ππ p < 0.02, π p < 0.05 vs. LPS. and NS nonsignificant vs. LPS. Values are presented as means ± SEM N ≥ 6 for each treatment group.

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  Figure 3. Effects of Neb and Met on LPS-stimulated gene expression related to mitochondrial biogenesis in H9c2 cells. mRNA levels of PGC-1α (A), NRF1 (B), and TFAM (C) were upregulated by both Neb and Met. δ p < 0.05 vs. Con, ππ p < 0.02, π p < 0.05 vs. LPS. Values are presented as means ± SEM N ≥ 6 for each treatment group.

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  Figure 4. Effects of Neb and Met on mitochondrial dynamics and generation of mitochondrial ROS in H9c2 cells. Expression of genes associated with fusion MFN2 (A), OPA1 (B), and fission DRP1 (C) and FIS1 (D). (E) Representative images of H9c2 cells stained with MitoTracker Green and MitoSOX red. Increase in MitoSOX red fluoresence that co-localizes with MitoTracker Green, suggest an increase in mitochondrial ROS production in LPS treated cells. (F) Histogram showing the relative fluorescent intensity of MitoSOX red normalized to MitoTracker green. δ p < 0.05 vs. untreated (Con), ππ p < 0.02, π p < 0.05 vs. LPS and NS, nonsignificant vs. LPS. Values are presented as means ± SEM N ≥ 6 for each treatment group.

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  Figure 5. Effects of Neb and Met on antioxidant defense system in H9c2 cells. Expression of genes associated with cellular antioxidant defense system CAT (A), MnSOD (B) GPx (C). δ p < 0.05 vs. untreated (Con), π p < 0.05 vs. LPS.4.