Luteolin suppresses lipopolysaccharide‑induced cardiomyocyte hypertrophy and autophagy in�vitro

Figures

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  Figure 1.. LTL increases the viability of rat cardiomyocytes treated with LPS. Cells were divided into five groups, including PBS (control group), 10% fetal bovine serum (NC), LPS (100 ng/ml), LTL1 (50 mg/ml) + LPS (100 ng/ml), and LTL2 (100 mg/ml) + LPS (100 ng/ml) for 8 h. (A) The morphology of the cultured rat cardiomyocytes (24 h). (B) Cell viability was detected by the MTT assay. (C) Expression levels of ANP and BNP was determined by reverse transcription-quantitative polymerase chain reaction. *P<0.05 and **P<0.01 vs. NC; +P<0.05 vs. LPS. ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; LC3, microtubule-associated protein 1 light chain 3; LPS, lipopolysaccharide; LTL, luteolin; NC, negative control.

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  Figure 2.. LTL suppresses LPS-induced cardiomyocyte hypertrophy and formation of autophagosomes. (A) The expression levels of α-actinin (a cardiomyocyte specific marker and a hypertrophic marker gene) were measured by immunofluorescence staining in the treated cardiomyocytes; nuclei were stained with DAPI. The Myocyte area (µm2) was calculated by formula (axb)/2 (a: The longest distance through the nucleus; b: The shortest distance through the nucleus) and analyzed by Image Pro-Plus 6.1 software after anti-α-actinin staining (green). (B) The expression levels of LC3 (green) were measured by immunofluorescence assay in the treated cardiomyocytes; nuclei were stained with DAPI. By specific labeling of autophagosome membranes (LC3II) and the cytoplasmic labeling (LC3I) and the amount of LC3 was quantified by densitometry. **P<0.01 and ***P<0.001 vs. NC; +P<0.05 and ++P<0.01 vs. LPS. LC3, microtubule-associated protein 1 light chain 3; LPS, lipopolysaccharide; LTL, luteolin; NC, negative control.

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  Figure 3.. LTL decreases LPS-mediated autophagy- and Wnt signaling pathway-associated gene expression levels. (A) RT-qPCR and (B) western blot assays were performed to detect the LC3 mRNA and LC3I and II protein expression levels, respectively. (C and D) Autophagy-associated gene expression levels were analyzed by RT-qPCR and western blot assays. *P<0.05, **P<0.01 and ***P<0.001 vs. NC; ++P<0.01 and +++P<0.001 vs. LPS. Atg, autophagy-related 4b cysteine peptidase; Bnip, B cell lymphoma l2 interacting protein; LC3, microtubule-associated protein 1 light chain 3; LPS, lipopolysaccharide; LTL, luteolin; NC, negative control; RT-qPCR, reverse transcription-quantitative polymerase chain reaction. LTL decreases LPS-mediated autophagy- and Wnt signaling pathway-associated gene expression levels. (E and F) Wnt signal pathway associated genes expression levels were detected by RT-qPCR and western blot assays. *P<0.05, **P<0.01 and ***P<0.001 vs. NC; ++P<0.01 and +++P<0.001 vs. LPS. Atg, autophagy-related 4b cysteine peptidase; Bnip, B cell lymphoma l2 interacting protein; LC3, microtubule-associated protein 1 light chain 3; LPS, lipopolysaccharide; LTL, luteolin; NC, negative control; RT-qPCR,…

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  Figure 4.. β-Catenin expression is silenced using siRNA in cardiomyocytes. (A) Reverse transcription-quantitative polymerase chain reaction and (B) western blot assays were performed to detect the knockdown efficiency of β-catenin siRNA. (C) Quantification of the western blot analysis from (B). **P<0.01 and ***P<0.001 vs. NC. NC, normal control; siRNA, small interfering RNA.

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  Figure 5.. Silencing of β-catenin inhibits LPS-induced cardiomyocyte hypertrophy and formation of autophagosome. (A) Expression levels of α-actinin (a cardiomyocyte specific marker and a hypertrophic marker gene) were measured by immunofluorescence staining; nuclei were stained with DAPI. The myocyte area was calculated by formula (axb)/2 (a: The longest distance through the nucleus; b: the shortest distance through the nucleus) and analyzed by Image Pro-Plus 6.1 software after anti-α-actinin staining (green). (B) LC3 expression levels were measured by immunofluorescence assay; nuclei were stained with DAPI. By specific labeling of autophagosome membranes (LC3II) and the cytoplasmic labeling (LC3I) and the amount of LC3 was quantified by densitometry. ***P<0.001 vs. NC; +P<0.05 vs. LPS. LC3, microtubule-associated protein 1 light chain 3; LPS, lipopolysaccharide; LTL, luteolin; NC, normal control; siRNA, small interfering RNA.