The flavonoid quercetin reverses pulmonary hypertension in rats

Abstract

Quercetin is a dietary flavonoid which exerts vasodilator, antiplatelet and antiproliferative effects and reduces blood pressure, oxidative status and end-organ damage in humans and animal models of systemic hypertension. We hypothesized that oral quercetin treatment might be protective in a rat model of pulmonary arterial hypertension. Three weeks after injection of monocrotaline, quercetin (10 mg/kg/d per os) or vehicle was administered for 10 days to adult Wistar rats. Quercetin significantly reduced mortality. In surviving animals, quercetin decreased pulmonary arterial pressure, right ventricular hypertrophy and muscularization of small pulmonary arteries. Classic biomarkers of pulmonary arterial hypertension such as the downregulated expression of lung BMPR2, Kv1.5, Kv2.1, upregulated survivin, endothelial dysfunction and hyperresponsiveness to 5-HT were unaffected by quercetin. Quercetin significantly restored the decrease in Kv currents, the upregulation of 5-HT2A receptors and reduced the Akt and S6 phosphorylation. In vitro, quercetin induced pulmonary artery vasodilator effects, inhibited pulmonary artery smooth muscle cell proliferation and induced apoptosis. In conclusion, quercetin is partially protective in this rat model of PAH. It delayed mortality by lowering PAP, RVH and vascular remodeling. Quercetin exerted effective vasodilator effects in isolated PA, inhibited cell proliferation and induced apoptosis in PASMCs. These effects were associated with decreased 5-HT2A receptor expression and Akt and S6 phosphorylation and partially restored Kv currents. Therefore, quercetin could be useful in the treatment of PAH.

Figure 1. Quercetin increases survival.

(A) Study protocol. Numbers in parenthesis indicate the number of rats which started and finished each period. The numbers at the far right indicate the number of rats in which pulmonary arterial pressure could be recorded; the missing animals in the monocrotaline (MCT) group died during the anesthesia or surgery. (B) Kaplan-Meier analysis of survival in rats treated with monocrotaline and monocrotaline plus quercetin (Quer). * indicates P<0.05.

Figure 2. Quercetin reduces pulmonary artery pressure.

Heart rate (A), right systolic (B) and diastolic (C) ventricular pressure, mean (D), systolic (E) and diastolic (F) pulmonary arterial pressure. Panels G, H, I show pulmonary artery pressure recordings in the control (Ctrl), monocrotaline (MCT) and monocrotaline plus quercetin (Quer) group, respectively. Results are means ± SEM of 4–9 animals, * indicates P<0.05 versus MCT and #P<0.05, ## P<0.01 versus Ctrl.

Figure 3. Quercetin reduces right ventricular hypertrophy and vascular remodeling.

(A) Fulton index [RV/(LV+S) ratio]. (B) Right ventricular weight relative to body weight (RV/BW). (C) Representative images of lung histology. (D) Percentage of muscular, partially muscular and non-muscular arteries in different groups. For panels A and B each column represents the mean ± SEM of 7–9 animals. ** For panel D between 26 and 110 arteries (25–100 µm) were analyzed in cross-sections of lungs (stained with haematoxylin and eosin) from at least four animals in each group. ** indicates P<0.01 versus monocrotaline (MCT) and ## indicates P<0.01 versus control (Ctrl).

Figure 4. Quercetin prevents the inhibition of K_V currents.

(A) Cell capacitance (Cm). (B) Current–voltage relationships measured at the end of the pulse. (C) Membrane potential (Em). * indicates P<0.05 versus monocrotaline (MCT). # and ## indicate P<0.05 and P<0.01 versus control (Ctrl). Each column or symbol represents the mean value ± SEM (n = 4–6).

Figure 5. Changes in lung expression of (A) BMPRII, (B) Survivin, (C) 5HT_2A, (D) iNOS, (E) Kv1.5, (F) Kv2.1, (G) Kv7.1 and (H) Kv7.5 mRNA by RT-PCR.

Results are means ± SEM of 4–8 animals normalized by the expression of β-actin. * indicates P<0.05 versus monocrotaline (MCT), # and ## P<0.05 and P<0.01 versus control (Ctrl).

Figure 6. Changes in the expression of survivin at the level of protein.

Lung homogenates were analyzed by Western blot. Results are means ± SEM of 5–8 animals. Protein levels were normalized by the expression of β-actin.

Figure 7. Quercetin (Quer) does not prevent vascular dysfunction.

(A) Relaxant effects of pulmonary arteries to the endothelium-dependent vasodilator acetylcholine in pulmonary arteries stimulated with phenylephrine. (B) Expression of eNOS mRNA in pulmonary arteries homogenates analyzed by RT-PCR. (C) Contractile responses to 5-HT in pulmonary arteries expressed as a percent of a previous response to KCl. Results are means ± SEM of 4–9 experiments. Results are means ± SEM of 4–8 animals normalized by the expression of β-actin. * indicates P<0.05 versus monocrotaline (MCT), # and ## P<0.05 and P<0.01 versus control (Ctrl).

Figure 8. Lack of changes in the activation of MAPKs: ERK1/2 and p38MAPK.

Lung homogenates were analyzed by Western blot and probed with the anti-phospho MAPK or the anti-total MAPK antibodies. Results expressed as phosphorylated forms normalized by the total protein, are means ± SEM of 5–8 animals.

Figure 9. Changes in the PI3K/Akt/mTOR/S6 signaling pathway.

Lung homogenates were analyzed by Western blot and probed with the anti-phospho Akt, the anti-total Akt or anti-β actin (A) or antiphospho S6 ribosomal protein (serines 240, 244) or total S6 (B) antibodies. Results expressed as phosphorylated forms normalized by the total protein, are means ± SEM of 5–8 animals. * indicates P<0.05 versus monocrotaline (MCT), #, ## indicate P<0.05, and P<0.0001 versus control (Ctrl).

Figure 10. Quercetin decreases PASMC and fibroblast proliferation.

(A) PASMC and (B) fibroblasts were isolated from monocrotaline-treated rats and grown in culture. Viable cells were estimated by the MTT test exposed to quercetin in culture for 24 or 48 h. (C) PASMC proliferation was analyzed by the BrdU incorporation after 24 h of treatment with increasing concentrations of quercetin. Results are means ± SEM of 3–4 experiments performed in triplicate. ** indicates P<0.01 versus vehicle (DMSO).

Figure 11. Quercetin decreases proliferation (A) and induces apoptosis (B) in cultured PASMC from control rats.

(A) PASMC proliferation was analyzed by the BrdU incorporation after 24 h of treatment with increasing concentrations of quercetin. Results are means ± SEM of 4–5 experiments. (B) Photographs of PASMC after 48 h treatment with quercetin in culture stained with the membrane permeable blue dye Hoescht 33258 (left, viable and non-viable cells) and with the membrane impermeable red dye propidium iodide (right, non-viable cells). (C) Magnified photographs of cells treated with 10 µM quercetin showing three apoptotic cells (top and middle cells) with chromatin condensation and nuclear fragmentation and two healthy cells (bottom).