Luteolin, quercetin, genistein and quercetagetin inhibit the effects of lipopolysaccharide obtained from Porphyromonas gingivalis in H9c2 cardiomyoblasts

Fig. 1. The chemical structures of quercetin, quercetagetin, genistein and luteolin. The central structure is that of a flavone. It has two phenyl rings (A and B) joined by a pyran ring (C). The boxes frame the substituents of their structure with respect to the flavonoid quercetin

Fig. 2. Flavonoid effect on cell viability. Cell viability in the presence of genistein (a), luteolin (b), quercetin (c) and quercetagetin (d) was evaluated using the MTT assay. H9c2 cells were treated with flavonoid (10 nM −10 μM) for 24, 48 and 72 h. Quercetagetin reduced cell viability at the highest concentration at 24 and 48 h without significant difference. No other flavonoid modified cell viability. The results are expressed as means ± SEM from at least three independent assays

Fig. 3. Lipopolysaccharide stimulation induces phosphorylation of ERK 1/2, p38, JNK in cardiomyoblast. H9c2 cells were incubated with LPS (1 μg/ml) for the indicated periods of time or with the indicated concentrations of LPS for 15 or 30 min. Cell lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a PVDF membrane, and immunoblotted with anti-phosphorylated-ERK 1/2 (a), −p38 (b) or -JNK antibodies (c). The membrane was then stripped and incubated with anti-tubulin antibodies. The data are representative of three independent experiments. The means ± SEM were obtained by densitometry. *p < 0.05 vs. untreated group

Fig. 4. The effect of LPS on ERK 1/2, p38 and JNK phosphorylation in time- and dose-dependent response. H9c2 cells were placed in 6-well plates. Cells were stimulated with LPS (1 μg/ml) for the times and doses indicated in the figure. Cell lysates were fractionated via SDS-PAGE and analyzed via western Blotting using an antibody against phospho-ERK 1/2 (a), −p38 (b) or -JNK (c). Membranes were then stripped and reprobed using an antibody against each kinase as a control for equal loading. The data are representative of one of three independent experiments performed with similar results. Phosphorylation was quantified via densitometry with LabWorks 4.0 (Upland, CA, USA) commercial software. The results are expressed as means ± S.D. *p < 0.05 vs. untreated group

Fig. 5. The effect of LPS on IkB phophorylation and degradation and flavonoids on IkB degradation. The time course of IκBα (a) and IκBβ degradation (b) and IκBα phosphorylation (c). Cells were pre-treated with flavonoids (10 μM) for 30 min and later exposed to LPS (1 μg/ml) for 15 min, to then be lysed. d – H9c2 cells were incubated with flavonoids (10 μM) for 30 min, then stimulated with LPS (1 μg/ml) for 60 min. Whole cell extract was processed via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and the membranes were blotted with IκB-specific phosphoantibodies. The results are representative of three separate experiments. Densitometric analysis represents the means ± SEM. *p < 0.05 vs. untreated basal value

Fig. 6. Effect of LPS and flavonoids on COX-2 expression. a – H9c2 cells were incubated with LPS at different doses. b – H9c2 cells were pretreated with flavonoids (10 μM) for 1 h and then incubated with LPS (1 μg/ml) for 12 h. c – H9c2 cells were pretreated with different inhibitors and then treated with LPS (1 μg/ml). The lysates of the cells were processed via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and the membranes were blotted with COX-2-specific antibodies. The results are representative of three separate experiments. Tubulin was used as an internal control for western blot analysis. Statistical significance was determined with a two-way ANOVA test. *p < 0.05 vs. untreated group