BRD4 Silencing Protects Angiotensin II-Induced Cardiac Hypertrophy by Inhibiting TLR4/NF-κB and Activating Nrf2-HO-1 Pathways

Figures

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  Figure 1. siBRD4 inhibits Ang II-induced cardiomyocyte hypertrophy. Cultured H9c2 cells were treated by vehicle (control) or Ang II (1 μM) for 12, 24, and 48 hours. (a) The mRNA expression level of BRD4 in Ang II-induced H9c2 cells. (b) The protein expression level of BRD4 in Ang II-induced H9c2 cells. (c) The BRD4 expression was silenced using siRNA in H9c2 cells and validated using a Western blot. (d) H9c2 cells were stained with α-actinin (green) and DAPI (blue) to show morphology and size; representative photographs are shown (scale bar: 10 μm). (e) Cardiomyocyte hypertrophy was evaluated by the surface area. mRNA expressions of the ANP (f), BNP (g), and β-MHC (h) genes were evaluated using a real‐time PCR. All data are expressed as mean ± SD, and the experiment was performed three times, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. control group; ###P < 0.001 vs. Ang II + siNC group.

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  Figure 2. siBRD4 protects H9c2 cells from Ang II-induced apoptosis. H9c2 cells were transfected with siBRD4 or siNC and treated with Ang II (1 μM) for 48 hours. (a) H9c2 cells were stained with TUNEL to show the apoptotic cells. (b) The number of TUNEL + cells was counted. (c) The caspase-3 activity was determined. Each experiment was performed in triplicate and repeated at least three times. ∗∗∗P < 0.001 vs. control group; ##P < 0.01 vs. Ang II + siNC group.

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  Figure 3. siBRD4 suppresses Ang II-induced oxidative stress in H9c2 cells by upregulating Nrf2 and HO-1. Respective commercial kits examined the MDA level (a) and SOD activity (b) in the control group, Ang II + siNC group, and Ang II + siBRD4 group. (c) The nuclear Nrf2 and total HO-1 protein levels were determined using a Western blot, and the representative protein bands are shown. siBRD4 increased the nuclear Nrf2 (d) expression and HO-1 (e) in H9c2 cells with Ang II stimulation. Each experiment was performed in triplicate and repeated at least three times. ∗∗P < 0.01, ∗∗∗P < 0.001 vs. control group; ##P < 0.01, ###P < 0.001 vs. Ang II + siNC group.

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  Figure 4. siBRD4 attenuates the Ang II-induced inflammatory response in H9c2 cells. A real-time PCR was performed to detect the mRNA levels of IL-1β (a), IL-6 (b), and IL-10 (c) in H9c2 cells. ELISA was performed to detect the secreted IL-1β (d), IL-6 (e), and IL-10 (f) in the cell supernatant of H9c2 cells. Each experiment was performed in triplicate and repeated at least three times. ∗∗P < 0.01, ∗∗∗P < 0.001 vs. control group; ##P < 0.01, ###P < 0.001 vs. Ang II + siNC group.

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  Figure 5. siBRD4 inhibits the TLR4-NF-κB pathway in Ang II-induced H9c2 cells. Real-time PCR was performed to measure the mRNA level of TLR4 (a). Western blot was carried out to determine the TLR4 (b), phosphorylated NF-κB p65(c), total NF-κB p65(d), protein expression in H9c2 cells (e). Each experiment was performed in triplicate and repeated at least three times. ∗∗∗P < 0.001 vs. control group; ##P < 0.01, ###P < 0.001 vs. Ang II + siNC group.

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  Figure 6. Overexpression of TLR4 reduces siBRD4-mediated protection against Ang II-induced cardiac hypertrophy. The induced Ang II cells were used as a control in the experiments. (a) A Western blot confirms the efficiency of TLR4 overexpression after 48 hours. ∗∗∗P < 0.001 vs. control group. (b) H9c2 cells were co-transfected with BRD4-specific siRNA (siBRD4) and pcDNA-TLR4 or vector and then subjected to Ang II for 48 hours. A Western blot was performed to determine NF-kB p65 (b), (c) and nuclear Nrf2 (d) in H9c2 cells. Immunofluorescence was performed to evaluate the hypertrophic responses (e), and the cell surface area was compared (f). The mRNA expression of ANP was detected using a real-time PCR (g). Each experiment was performed in triplicate and repeated at least three times. ∗∗P < 0.01, ∗∗∗P < 0.001 vs. vector + siNC group; ##P < 0.01, ###P < 0.001 vs. vector + siBRD4 group.

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  Figure 7. Overexpression of TLR4 attenuates the siBRD4-induced inhibition on oxidative stress and inflammatory cytokine. Ang II-induced cells were used as a control. Respective commercial kits examined the MDA level (a) and SOD activity (b) in cells of siBRD4 and/or pcDNA-TLR4. ELISA measured the secreted IL-1β (c), IL-6 (d), and IL-10 (e) in the cell supernatant. Each experiment was performed in triplicate and repeated at least three times. ∗∗P < 0.01, ∗∗∗P < 0.001 vs. vector + siNC group; ##P < 0.01, ###P < 0.001 vs. vector + siBRD4 group.