Quercetin reduces inflammatory responses in LPS-stimulated cardiomyoblasts

Abstract

Flavonoids possess several biological and pharmacological activities. Quercetin (Q), a naturally occurring flavonoid, has been shown to downregulate inflammatory responses and provide cardioprotection. However, the mechanisms behind the anti-inflammatory properties of Q in cardiac cells are poorly understood. In inflammation, nitric oxide (NO) acts as a proinflammatory mediator and is synthesized by inducible nitric oxide synthase (iNOS) in response to pro-inflammatory agents such as lipopolysaccharide (LPS), a causative agent in myocardial depression during sepsis. In the present study, we evaluated the protective effect of Q on rat cardiac dysfunction during sepsis induced by LPS. Pretreatment of H9c2 cardiomyoblasts with Q inhibited LPS-induced iNOS expression and NO production and counteracted oxidative stress caused by the unregulated NO production that leads to the generation of peroxynitrite and other reactive nitrogen species. In addition, Q pretreatment significantly counteracted apoptosis cell death as measured by immunoblotting of the cleaved caspase 3 and caspase 3 activity. Q also inhibited the LPS-induced phosphorylation of the stress-activated protein kinases (JNK/SAPK) and p38 MAP kinase that are involved in the inhibition of cell growth as well as the induction of apoptosis. In conclusion, these results suggest that Q might serve as a valuable protective agent in cardiovascular inflammatory diseases.

Figure 1

Effect of Q on iNOS expression in LPS-stimulated H9c2 cells. Cells were pretreated with 30 μM Q for 2 hours before stimulation with 10 μg/mL LPS. After the indicated time points, cells were harvested and lysed. Crude homogenates (20 μg) were immunoblotted with an antibody that detects endogenous levels of iNOS (NOS2). Equal loading was verified with an anti-β-actin antibody. Densitometric analysis of the protein bands was performed using Bio-Rad Quantity One 1-D Analysis software. Each column represents the mean ± SD of three independent experiments. Data were analyzed by one-way ANOVA followed by Bonferroni's test. *P < 0.05 compared to Control; °P < 0.05 compared to the corresponding LPS exposure times.

Figure 2

Effect of Q on NO production in LPS-stimulated H9c2 cells. Cells were pretreated with 30 μM Q for 2 hours before stimulation with 10 μg/mL LPS for 24 hours. After the treatment, the culture medium was collected for NO assay. The concentration of NO was determined by the Griess reagent using NaNO₂ as standard. Each column represents the mean ± SD of three independent experiments. Data were analyzed by one-way ANOVA followed by Bonferroni's test. *P < 0.05 compared to Control; °P < 0.05 compared to LPS.

Figure 3

Effect of Q on intracellular ROS production in LPS-stimulated H9c2 cells. Cells were pretreated with 30 μM Q for 2 hours before stimulation with 10 μg/mL LPS. After 24 h intracellular ROS were measured using the peroxide-sensitive fluorescent probe DCHF-DA. Each column represents the mean ± SD of three independent experiments. Data were analyzed by one-way ANOVA followed by Bonferroni's test. *P < 0.05 compared to Control; °P < 0.05 compared to LPS.

Figure 4

Effect of Q on cleaved caspase 3 protein expression in LPS-stimulated H9c2 cells. Cells were pretreated with 30 μM Q for 2 hours before stimulation with 10 μg/mL LPS. At the indicated time points, cells were harvested and lysed. Crude homogenates (20 μg) were immunoblotted with an antibody that detects endogenous levels of cleaved caspase 3. Equal loading was verified with an anti-β-actin antibody. Densitometric analysis of the protein bands was performed using Bio-Rad Quantity One 1-D Analysis software. Each column represents the mean ± SD of three independent experiments. Data were analyzed by one-way ANOVA followed by Bonferroni's test. *P < 0.05 compared to Control; °P < 0.05 compared to the corresponding LPS exposure times.

Figure 5

Effect of Q on caspase 3 activity in LPS-stimulated H9c2 cells. Cells were pretreated with 30 μM Q for 2 hours before stimulation with 10 μg/mL LPS. At the indicated time points, caspase 3 activity was measured spectrofluorimetrically in cell lysates as reported in Section 2. Each column represents the mean ± SD of three independent experiments. Data were analyzed by one-way ANOVA followed by Bonferroni's test. *P < 0.05 compared to Control; °P < 0.05 compared to the corresponding LPS exposure times.

Figure 6

Effect of Q on p38 MAPK activation in LPS-stimulated H9c2 cells. Cells were pretreated with 30 μM Q for 2 hours before stimulation with 10 μg/mL LPS. At the indicated time points, cells were harvested and lysed. Crude homogenates (20 μg) were immunoblotted with anti-phospho-p38 and anti-p38 antibodies. Equal loading was verified with an anti-β-actin antibody. Densitometric analysis of the protein bands was performed using Bio-Rad Quantity One 1-D Analysis software. Each column represents the mean ± SD of three independent experiments. Data were analyzed by one-way ANOVA followed by Bonferroni's test. *P < 0.05 compared to Control; °P < 0.05 compared to the corresponding LPS exposure time.

Figure 7

Effect of Q on JNK activation in LPS-stimulated H9c2 cells. Cells were pretreated with 30 μM Q for 2 hours before stimulation with 10 μg/mL LPS. At the indicated time points, cells were harvested and lysed. Crude homogenates (20 μg) were immunoblotted with anti-phospho-JNK and anti-JNK antibodies. Equal loading was verified with an anti-β-actin antibody. Densitometric analysis of the protein bands was performed using Bio-Rad Quantity One 1-D Analysis software. Each column represents the mean ± SD of three independent experiments. Data were analyzed by one-way ANOVA followed by Bonferroni's test. *P < 0.05 compared to Control; °P < 0.05 compared to the corresponding LPS exposure time.