Potentilla reptans L. postconditioning protects reperfusion injury via the RISK/SAFE pathways in an isolated rat heart

Abstract

Background: Our previous study indicated that Potentilla reptans root has a preconditioning effect by its antioxidant and anti-apoptotic effects in an isolated rat heart ischemia/reperfusion (IR) model. In the present study, we investigated the post-conditioning cardio-protective effects of Potentilla reptans and its active substances.

Methods: The ethyl acetate fraction of P. reptans root (Et) was subjected to an IR model under 30 min of ischemia and 100 min of reperfusion. To investigate the postconditioning effect, Et was perfused for 15 min at the early phase of reperfusion. RISK/SAFE pathway inhibitors, 5HD and L-NAME, were applied individually 10 min before the ischemia, either alone or in combination with Et during the early reperfusion phase. The hemodynamic factors and ventricular arrhythmia were calculated during the reperfusion. Oxidative stress, apoptosis markers, GSK-3β and SGK1 proteins were assessed at the end of experiments.

Results: Et postconditioning (Etpost) significantly reduced the infarct size, arrhythmia score, ventricular fibrillation incidence, and enhanced the hemodynamic parameters by decreasing the MDA level and increasing expression of Nrf2, SOD and CAT activities. Meanwhile, Etpost increased the BCl-2/BAX ratio and decreased Caspase-3 expression. The cardioprotective effect of Etpost was abrogated by L-NAME, Wortmannin (a PI3K/Akt inhibitor), and AG490 (a JAK/STAT3 inhibitor). Finally, Etpost reduced the expression of GSK-3β and SGK1 proteins pertaining to the IR group.

Conclusion: P. reptans reveals the post-conditioning effects via the Nrf2 pathway, NO release, and the RISK/SAFE pathway. Also, Etpost decreased apoptotic indexes by inhibiting GSK-3β and SGK1 expressions. Hence, our data suggest that Etpost can be a suitable natural candidate to protect cardiomyocytes during reperfusion injury.

Keywords: Anti-apoptotic; Antioxidant; GSK-3β; Ischemia/reperfusion; NO; Postconditioning; Potentilla reptans root; SGK1.

Fig. 1

Experimental protocols. All experimental groups were first perfused for 30 mins on the Langendorff apparatus to allow the isolated hearts to stabilize. The hearts were then divided into different groups. All groups were subjected to 30 mins of regional ischemia followed by 100 mins of reperfusion. IR; Ischemia-reperfusion, IPOST; Ischemic postconditioning (3 × 10 s ischemia and reperfusion at the onset of reperfusion period), Etpost; ethyl acetate fraction from Potentilla reptans root (2 μg/ml) was applied at the onset of reperfusion, L-NAME; NO inhibitor (50 μM), Wort; Wortmannin a PI3K/AKT inhibitor (400 nM), PD; PD98059 (2′-Amino-3′-methoxyflavone) an ERK1/2 inhibitor (400 nM), AG; tyrphostin (AG490, 400 nM) a JAK/STAT3 inhibitor, HD; 5-hydroxy decanoate (5HD,1 μM) a mitoKATP channel blocker

Fig. 2

Cardioprotective effect of Etpost from Potentilla reptans root. Representative photographs of TTC (2,3,5-triphenyl-tetrazolium chloride) stained rat heart sections and statistical data of myocardial infarct size (IS/AAR%) and area at risk (AAR/LV%) in the isolated perfused rat hearts subjected to IR, IPOST, Etpost (2 μg/ml), IPOST + inhibitors, Etpost + inhibitors. Data are presented as means ± SEM and expressed in percentages. *P < 0.05 vs. IR; ^& P < 0.05 vs. IPOST; ^# P < 0.05 vs. Etpost (2 μg/ml); ^$ P < 0.05 vs. IPOST+ inhibitor. (LV: Left ventricular, Etpost: ethyl acetate fraction of P. reptans root at postconditioning mechanism)

Fig. 3

Antioxidant effect of Etpost (2 μg/ml) on the isolated rat heart tissue. Antioxidant effect of Etpost (2 μg/ml) on the isolated rat heart tissue. The effect of IR, L-NAME, Etpost (2 μg/ml), and Etpost (2 μg/ml) + L-NAME on oxidative stress markers. A The expression of Nrf2 mRNA, B malondialdehyde (MDA), C) catalase (CAT), and D) superoxide dismutase (SOD). Data are presented as means ± SEM. ^*P < 0.05 vs. IR, ^$P < 0.05 vs. IPOST+L-NAME and ^#P < 0.05 vs. Etpost (2 μg/ml)

Fig. 4

Anti-apoptosis effect of Etpost (2 μg/ml) on the isolated sections of rat heart tissue. Anti-apoptosis effect of ethyl acetate fraction from P. reptans on the isolated sections of rat heart tissue. The effect of IR, L-NAME, Etpost (2 μg/ml) and Etpost (2 μg/ml) + L-NAME on apoptotic markers. A Expression of BAX and BCl-2 proteins after nuclei staining with DAPI and obtained from fluorescence microscope (observed under 400 magnifications). B Expression of the levels of caspase-3 protein. C Representative TUNEL stained and captured by fluorescence microscopy of rat heart sections with IR and Etpost (2 μg/ml) groups. The green nuclear structures represent apoptotic cells. The nuclei are stained by PI. The data were expressed as mean ± SEM. *P < 0.05 vs. IR group. Etpost: ethyl acetate fraction of P. reptans root

Fig. 5

Western blot analysis. A Western blot analysis of BCl-2, BAX and levels of cleaved caspase-3 expression in heart tissue of Etpost (2 μg/ml) adjusted relative to GAPDH and IR. B Western blot analysis of GSK-3β and SGK1 expression in the heart tissue of Etpost (2 μg/ml) adjusted relative to GAPDH and IR. GAPDH was used as a loading control. The data were expressed as mean ± SEM. *P < 0.05 vs. IR group. Etpost: ethyl acetate fraction of P. reptans root

Fig. 6

Western blot analysis. A Western blot analysis shows the relative intensity of AKT (Tyr473) phosphorylation in heart tissue of IR, Etpost (2 μg/ml) and Etpost+Wort groups that were adjusted relative to GAPDH. B Western blot analysis illustrates the relative intensity of ERK1/2 (Thr183/185) phosphorylation in heart tissue of IR, Etpost (2 μg/ml) and Etpost+PD groups that were adjusted relative to GAPDH. C Western blot analysis displays the relative intensity of STAT3 (Tyr705) phosphorylation in heart tissue of IR, Etpost (2 μg/ml) and Etpost+AG groups that were adjusted relative to GAPDH. The data were expressed as mean ± SEM. ^*P < 0.05 vs. IR group and ^#P < 0.05 vs. Etpost (2 μg/ml). Etpost: ethyl acetate fraction of P. reptans root; Wort: Wortmanin (PI3K/Akt inhibitor); PD: PD98059 (ERK inhibitor); AG: AG490 (JAK/STAT3 inhibitor)

Fig. 7

Effect of Etpost (2 μg/ml) on time course recovery of post-ischemic changes of hemodynamic parameters, Arrhythmia score and VF incidence in the isolated rat heart. A The effect of Etpost (2 μg/ml) and Etpost+L-NAME on LVDP, data are presented as % of per-ischemia (PS) values. ^* P < 0.05 vs. IR and ^# P < 0.05 vs. Etpost (2 μg/ml); B The effect of Etpost (2 μg/ml) and Etpost+L-NAME on Post-ischemic changes of rate pressure product (RPP) in the isolated rat heart. Data are presented as % of pre-ischemia (PS) values. ^* P < 0.05 vs. IR and ^# P < 0.05 vs. Etpost (2 μg/ml). C Arrhythmia scores calculation of the reperfusion period in the isolated rat heart. The effect of IR, IPOST, IPOST+ L-NAME, Etpost (2 μg/ml), and Etpost (2 μg/ml) + L-NAME on the arrhythmia score. Scale range is (0 to 7). ^* P < 0.05 vs. IR, ^@ P < 0.05 vs. IPOST, and ^# P < 0.05 vs. Etpost; D The incidence of ventricular fibrillation (VF) calculation of the reperfusion period in the isolated rat heart. The effect of IR, IPOST, IPOST+ L-NAME, Etpost (2 μg/ml), and Etpost (2 μg/ml) + L-NAME on the incidence of VF. Data are presented as the percentage of incidence. ^* P < 0.05 vs. IR, ^@ P < 0.05 vs. IPOST and ^# P < 0.05 vs. Etpost (2 μg/ml)