Beneficial effects of proanthocyanidins in the cardiac alterations induced by aldosterone in rat heart through mineralocorticoid receptor blockade

Abstract

Aldosterone administration in rats results in several cardiac alterations. Previous studies have demonstrated that proanthocyanidins, phenolic bioactive compounds, have cardioprotective effects. We studied the potential beneficial effects of the proanthocyanidin-rich almond skin extract (PASE) on the cardiac alterations induced by aldosterone-salt treatment, their effects in mineralocorticoid receptor activity and we sought to confirm proanthocyanidins as the specific component of the extract involved in the beneficial cardiac effects. Male Wistar rats received aldosterone (1 mg/Kg/day) +1% NaCl for 3 weeks. Half of the animals in each group were simultaneously treated with either PASE (100 mg/Kg/day) or spironolactone (200 mg/Kg/day). The ability of PASE to act as an antagonist of the mineralocorticoid receptor was examined using a transactivation assay. High performance liquid chromatography was used to identify and to isolate proanthocyanidins. Hypertension and diastolic dysfunction induced by aldosterone were abolished by treatment with PASE. Expression of the aldosterone mediator SGK-1, together with fibrotic, inflammatory and oxidative mediators were increased by aldosterone-salt treatment; these were reduced by PASE. Aldosterone-salt induced transcriptional activity of the mineralocorticoid receptor was reduced by PASE. HPLC confirmed proanthocyanidins as the compound responsible for the beneficial effects of PASE. The effects of PASE were comparable to those seen with the mineralocorticoid antagonist, spironolactone. The observed responses in the aldosterone-salt treated rats together with the antagonism of transactivation at the mineralocorticoid receptor by PASE provides evidence that the beneficial effect of this proanthocyanidin-rich almond skin extract is via as a mineralocorticoid receptor antagonist with proanthocyanidins identified as the compounds responsible for the beneficial effects of PASE.

Figure 1. TGF-β (A), CTGF (B), MMP2 (C), TIMP2 (D) mRNA levels, and MMP2/TIMP2 ratio (E) in control (C), aldosterone-salt-treated animals (ALDO), PASE (PASE), aldosterone-salt plus PASE treated animals (ALDO+PASE), spironolactone treated animals (SPIRO) and aldosterone-salt plus spironolactone (ALDO+SPIRO).

Data are expressed as mean ± SEM derived from 8 animals per group. *p<0.05 vs. C; ^†p<0.05 vs. ALDO.

Figure 2. TNF-α (A), IL-1β (B), p22phox (C) and eNOS (D) mRNA levels in control (C), aldosterone-salt-treated animals (ALDO), PASE (PASE), aldosterone-salt plus PASE treated animals (ALDO+PASE), spironolactone treated animals (SPIRO) and aldosterone-salt plus spironolactone (ALDO+SPIRO).

Data are expressed as mean ± SEM derived from 8 animals per group. *p<0.05 vs. C; ^†p<0.05 vs. ALDO.

Figure 3. SGK-1 mRNA levels in control (C), aldosterone treated animals (ALDO), PASE (PASE), aldosterone plus PASE treated animals (ALDO+PASE), spironolactone treated animals (SPIRO) and aldosterone-salt plus spironolactone (ALDO+SPIRO).

Data are expressed as mean ± SEM derived from 8 animals per group. *p<0.05 vs. CONTROL; ^†p<0.05 vs. ALDO.

Figure 4. HPLC chromatogram and pools representation.

Column: Waters Novapak C18 60Å 4 µm, 30×0.39 cm. Flow rate = 1 ml/min. Buffer A: 2% CH3COOH. Buffer B: 2% CH3COOH, 25% CH3CN. Arrow indicates column strip with 2% CH3COOH, 75% CH3CN. Sample volume loaded = 1 ml. Fractions collected at 1 min intervals. Pools compound of 10 individual fractions; P1∶1–10, P2∶11–20, P3∶21–30. P4∶31–40, P5∶41–50, P6∶51–60, P7∶61–70, P8∶71–80, P9∶81–90 and P10∶91–100.

Figure 5. Effect of PASE on aldosterone-induced human mineralocorticoid receptor (hMR) transcriptional activity.

CV-1 cells transfected with hMR expression and MMTV-LUC reporter plasmids were treated with vehicle (V) or aldosterone 1 nM (A1 nM) in presence of spironolactone 100 nM (S100 nM) or PASE 10 nM, 100 nM or 1 µM (P10 nM, P100 nM or P1 µM). Each data point represents the mean ± SEM derived from 3 independent experiments. *p<0.01 vs V; ^†p<0.05 vs. A1 nM.

Figure 6. Effect of P4 individual fractions on aldosterone-induced human mineralocorticoid receptor (hMR) transcriptional activity.

CV-1 cells transfected with hMR expression and MMTV-LUC reporter plasmids were treated with vehicle (V) or aldosterone 1 nM (A1 nM) in presence of spironolactone 100 nM (S100 nM) or fractions (F) 31 to 40 (1 µM), blank (B) and blank+aldosterone1 nM (B+A1 nM). Each data point represents the mean ± SEM derived from 3 independent experiments. *p<0.01 vs V; ^†p<0.05 vs. A1 nM.