Novel Therapeutic Effects of Pterosin B on Ang II-Induced Cardiomyocyte Hypertrophy

Figures

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  Figure 1. Effects of Pterosin B on H9c2 cell viability. (A) Cell viability was measured by WST assay, and (B) cytotoxicity was measured by LDH assay. Cell viability (CCK) and cytotoxicity (LDH) were measured after treatment with 0–100 µM Pterosin B for 48 h in the presence of Ang II. Quantitative data were expressed as the mean ± SEM of three independent experiments. *** p < 0.001.

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  Figure 2. Anti-hypertrophic Effect of Pterosin B on Ang II-stimulated H9c2 cells. H9c2 cells were treated with 10–50 µM Pterosin B for 48 h in the presence of 300 nM ANG II. (A) Expression levels of cardiomyocyte hypertrophy-related mRNAs (ANP, BNP, and NFATc1) were estimated by reverse transcriptase PCR. (B–D) The ANP, BNP, and NFATc1 mRNA expression levels were normalized by GAPDH. * p < 0.05, ** p < 0.01. n = 3. (E) Texas-Red X-tagged phalloidin was stained for F-actin in H9c2 cells. Representative image was detected by confocal microscopy. P50 = Pterosin B (50 μM). Scale bar = 50 µm. (F) Average cell size was measured in each group and analyzed by Image J software. ** p < 0.01. n = 6. (G) Protein synthesis of H9c2 cells was determined using Click-iT® HPG Alexa Fluor® 488 Protein Synthesis Assay Kit. Green fluorescence indicates synthesis of new intracellular proteins and blue fluorescence (DAPI) indicates nucleus. P50 = Pterosin B (50 μM). Scale bar = 100 µm. (H) Quantification of the fluorescence intensity of protein synthesis. ** p < 0.01, *** p < 0.001. Scale bar = 100 µm. n = 4.

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  Figure 3. Effects of Pterosin B on Ang II-activated HMGB1 secretion. H9c2 cells were treated with 300 nM Ang II with or without Pterosin B. (A) Protein expression levels of AT1R and RAGE in H9c2 cells-treated with 300 nM Ang II with or without Pterosin B for 48 h. (B,C) Quantification of AT1R and RAGE expression levels shown in (A). The protein levels were normalized to GAPDH. * p < 0.05, ** p < 0.01, *** p < 0.001. n = 6. (D) H9c2 cells were treated with Ang II for 10 min and HMGB1 expression level was detected by Western blot from cell lysate and culture medium. (E) Quantification of intracellular HMGB1 protein levels and amount of secreted HMGB1 shown in (D). * p < 0.05, ** p < 0.01, *** p < 0.001. NS vs. control. n = 4. (F) H9c2 cells were treated with Ang II with or without Pterosin B for 48 h. Western blot was performed to detect protein expression levels of TLR2, TLR4, and MyD88. (G–I) Quantification of protein levels shown in (F). * p < 0.05, *** p < 0.001. TLR2 (n = 6), TLR4 (n = 6), and MyD88 (n = 3).

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  Figure 4. Pterosin B attenuates PKC-ERK activation in Ang II-induced cardiomyocyte hypertrophy. (A) H9c2 cells were treated with 300 nM Ang II for 10 min with or without Pterosin B and protein levels were analyzed by western blotting. (B) The values of the relative p-PKC (n = 6) and p-ERK (n = 7) were normalized by PKC and ERK, respectively. * p < 0.05, ** p < 0.01, *** p < 0.001. (C) H9c2 cells were treated with 300 nM Ang II for 2 h in the presence and absence of Pterosin B, and protein levels of NF-κB and p-NF-κB were examined by western blotting. (D) Quantification of protein levels shown in (C). * p < 0.05. n = 3.

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  Figure 5. Anti-oxidant effect of Pterosin B in Ang II-treated H9c2 cells. (A) H9c2 cells were treated with 300 nM Ang II for 2 h with or without Pterosin B. The intracellular ROS levels were detected by CM-H2DCFDA (upper panel) and phase-contrast image was obtained to confirm cell morphology (lower panel). P50 = Pterosin B (50 μM). Scale bar = 100 µm. (B) Quantification of the fluorescence intensity of H9c2 cells in each condition. n = 6. (C) H9c2 cells were treated with 300 nM Ang II with or without Pterosin B for 2 h and the protein expression of NOX2, NOX4, and GAPDH was examined by western blotting. (D–E) Quantification of protein levels shown in (C). The levels of protein were normalized to GAPDH expression. * p < 0.05, ** p < 0.01. n = 3.

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  Figure 6. Schematic diagram with roles of Pterosin B in Ang II-induced cardiomyocyte hypertrophy.