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. 2021 Mar 5;10(6):e018097. doi: 10.1161/JAHA.120.018097

Figure 7. AdipoRon inhibits the emergence of a systemic inflammatory response syndrome–associated inflammatory phenotype in cardiac fibroblasts.

Figure 7

Cardiac fibroblasts were preincubated with AdipoRon (concentration range 40–100 µmol/L) or vehicle (dimethyl sulfoxide) for 2 hours before stimulation with lipopolysaccharide (1 mg/mL) for 1 hour. A, Concentration of TNF‐α (tumor necrosis factor α) in culture supernatants, (B) phosphorylation status of AMP‐activated protein kinase (AMPK; molecular weight: 62 kDa) in cell lysates and (C) content of lactate dehydrogenase (LDH) in culture supernatants was quantified by ELISA (n=5), immunoblot (n=2), and activity assay (n=5), respectively. Cardiac fibroblasts were preincubated with AdipoRon (80 µmol/L) or vehicle for 2 hours before stimulation with TNF‐α (10 ng/mL) for 1 hour. mRNA expression of (D) the chemokine CCL2 (C‐C chemokine ligand 2), (E) the enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox2 subunit), and (F) the cytokine IL (interleukin) 10 was measured relative to 18S ribosomal RNA (rRNA) by quantitative real‐time polymerase chain reaction (n=6). G, Phosphorylation of AMPK in cell lysates was analyzed by immunoblot (n=5). Upper panel: representative pictures of the resulting phosphorylated AMPK (P‐AMPK; molecular weight: 62 kDa) and AMPK (molecular weight: 62 kDa) band patterns. Lower panel: column bars indicate quantified P‐AMPK/AMPK expression ratios. Results are presented as mean+SEM. Differences of marker expression levels between experimental groups were analyzed statistically by performing the indicated pairwise comparisons.