Fatty acid-binding protein 4 silencing protects against lipopolysaccharide-induced cardiomyocyte hypertrophy and apoptosis by inhibiting the Toll-like receptor 4–nuclear factor-κB pathway

Figures

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  Figure 1.. FABP4 expression in rat cardiomyocytes treated with LPS. H9c2 cells were treated with LPS (10 µg/mL) for 6, 12, 24, and 48 hours. (a) The CCK-8 assay was used to monitor cell viability. (b) Cell injury was detected by a LDH activity assay. Expression levels of cardiomyocyte hypertrophy markers ANP (c) and BNP (d) were determined by RT-qPCR. Expression levels of FABP4 were determined by RT-qPCR (e) and western blotting (f). Data are presented as means ± SD from three independent experiments. *P<0.05, **P<0.01, ***P<0.001 vs. cardiomyocytes in the control group. FABP4, fatty acid-binding protein 4; LPS, lipopolysaccharide; CCK, cell counting kit; LDH, lactate dehydrogenase; ANP, atrial natriuretic peptide; BNP, B-type natriuretic peptide; RT-qPCR, real-time quantitative PCR.

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  Figure 2.. Cultured H9c2 cells were transfected with siFABP4 or siNC for 24 hours and then treated with LPS (10 µg/mL) for a further 24 hours. The transfection efficiency was confirmed by RT-qPCR (a) and western blot analysis (b) after 48 hours. siFABP4 increased the viability (c) and LDH content in culture media (d) of H9c2 cells treated with LPS. Data are presented as means ± SD from three independent experiments. *P<0.05, **P<0.01, ***P<0.001 vs. cardiomyocytes in the control group. ##P<0.01, ###P<0.001 vs. cardiomyocytes with LPS. FABP4, fatty acid-binding protein 4; siFABP4, FABP4-specific small interfering RNA; siNC, negative control siRNA; LPS, lipopolysaccharide; RT-qPCR, real-time quantitative PCR.

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  Figure 3.. siFABP4 inhibited LPS‐induced cardiomyocyte hypertrophy. Cultured H9c2 cardiomyocytes were transfected with siFABP4 or siNC and then treated with LPS (10 µg/mL) for 24 hours. (a) Cardiomyocytes were stained with α-actinin (green) and DAPI (blue). Representative photographs are shown (scale bar: 5 μm). (b) Quantitative analysis of cardiomyocyte surface area from a total of 100 cardiomyocytes per group. ANP (c) and BNP (d) expression was evaluated by RT‐qPCR using GAPDH as the internal control. ***P<0.001 vs. cardiomyocytes in the control group; ##P<0.01, ###P<0.001 vs. cardiomyocytes with LPS. FABP4, fatty acid-binding protein 4; LPS, lipopolysaccharide; siFABP4, FABP4-specific small interfering RNA; siNC, negative control siRNA; DAPI, 4′,6-diamidino-2-phenylindole; ANP, atrial natriuretic peptide; BNP, B-type natriuretic peptide; RT-qPCR, real-time quantitative PCR; GADPH, glyceraldehyde 3-phosphate dehydrogenase.

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  Figure 4.. siFABP4 inhibited LPS‐induced cardiomyocyte apoptosis. (a) Cardiomyocytes were stained with TUNEL (green) and DAPI (blue). Representative photomicrographs are shown (scale bar: 5 μm). (b) Quantification of TUNEL-positive cells. Cell apoptosis was determined by measuring caspase-3 (c) and caspase-9 (d) activities. ***P<0.001 vs. cardiomyocytes in the control group; ##P<0.01, ###P<0.001 vs. cardiomyocytes with LPS. FABP4, fatty acid-binding protein 4; siFABP4, FABP4-specific small interfering RNA; LPS, lipopolysaccharide; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; DAPI, 4′,6-diamidino-2-phenylindole.

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  Figure 5.. siFABP4 inhibited the LPS-induced inflammatory response in cardiomyocytes. ELISA was used to determine the expression of IL-1β (a), IL-6 (b), and TNF-α (c) in the supernatant of cultured H9c2 cells. RT-qPCR was performed to detect the mRNA levels of IL-1β (d), IL-6 (e), and TNF-α (f) in H9c2 cells. ***P<0.001 vs. cardiomyocytes in the control group; ##P<0.01, ###P<0.001 vs. cardiomyocytes with LPS. FABP4, fatty acid-binding protein 4; siFABP4, FABP4-specific small interfering RNA; LPS, lipopolysaccharide; ELISA, enzyme-linked immunosorbent assay; IL, interleukin; TNF, tumor necrosis factor; RT-qPCR, real-time quantitative PCR.

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  Figure 6.. siFABP4 inhibited TLR4 and NF-κB signaling pathways. (a) TLR4 mRNA expression was examined by RT-qPCR. (b) FABP4 was silenced by siRNA. Representative western blot bands of (c) FABP4 and (d) TLR4 are shown. (e) Representative western blot bands of nuclear NF-κB p65 and cytoplasmic I-κBα are shown. The densitometry of cytoplasmic FABP4 (f), NF-κB p65 (g), and I-κBα (i), and nuclear NF-κB p65 (h) are shown. ***P<0.001 vs. cardiomyocytes in the control group; ##P<0.01, ###P<0.001 vs. cardiomyocytes with LPS. FABP4, fatty acid-binding protein 4; siFABP4, FABP4-specific small interfering RNA; LPS, lipopolysaccharide; TLR4, Toll-like receptor 4; NF, nuclear factor; RT-qPCR, real-time quantitative PCR.