Pinocembrin ameliorates post-infarct heart failure through activation of Nrf2/HO-1 signaling pathway

Abstract

Background: Oxidative stress is an important factor involved in the progress of heart failure. The current study was performed to investigate whether pinocembrin was able to ameliorate post-infarct heart failure (PIHF) and the underlying mechanisms.

Methods: Rats were carried out left anterior descending artery ligation to induce myocardial infarction and subsequently raised for 6 weeks to produce chronic heart failure. Then pinocembrin was administrated every other day for 2 weeks. The effects were evaluated by echocardiography, western blot, Masson's staining, biochemical examinations, immunohistochemistry, and fluorescence. In vitro we also cultured H9c2 cardiomyocytes and cardiac myofibroblasts to further testify the mechanisms.

Results: We found that PIHF-induced deteriorations of cardiac functions were significantly ameliorated by administrating pinocembrin. In addition, the pinocembrin treatment also attenuated collagen deposition and augmented vascular endothelial growth factor receptor 2 in infarct border zone along with an attenuated apoptosis, which were related to an amelioration of oxidative stress evidenced by reduction of reactive oxygen species (ROS) in heart tissue and malondialdehyde (MDA) in serum, and increase of superoxide dismutase (SOD). This were accompanied by upregulation of nuclear factor erythroid 2-related factor 2 (Nrf2)/ heme oxygenase-1 (HO-1) pathway. In vitro experiments we found that specific Nrf2 inhibitor significantly reversed the effects resulted from pinocembrin including antioxidant, anti-apoptosis, anti-fibrosis and neovascularization, which further indicated the amelioration of PIHF by pinocembrin was in a Nrf2/HO-1 pathway-dependent manner.

Conclusion: Pinocembrin ameliorated cardiac functions and remodeling resulted from PIHF by ROS scavenging and Nrf2/HO-1 pathway activation which further attenuated collagen fibers deposition and apoptosis, and facilitated angiogenesis.

Keywords: Heart failure; Nrf2/HO-1; Oxidative stress; Pinocembrin.

Fig. 1

The chemical structure of pinocembrin and the animal experimental protocol. A The chemical structure of pinocembrin was illustrated; B the animal experimental protocol where an arrow meant one time of administration of pinocembrin

Fig. 2

The effects exerted by chronic application of pinocembrin on cardiac functions. A Representative echocardiogram images in all groups; B the statistic analysis of parameters of cardiac functions; C the serum BNP concentration and heart weight/tibia length which indicated severity of heart failure and hypertrophy of heart, respectively. †: p < 0.01 when sham group compared with HF group; **: p < 0.01 when HF + Pino group compared with HF group. BNP brain natriuretic peptide, HF heart failure, Pino pinocembrin

Fig. 3

Pinocembrin alleviated post-infarct heart failure-induced ventricular remodeling. A pinocembrin decreased collagen deposition in infarct border zone, evidenced by the Masson staining; B pinocembrin reversed the heart failure-induced rarefaction of vascular endothelial growth factor receptor 2, demonstrating an amelioration of angiogenesis; C western blot results showed that pinocembrin improved apoptosis. N = 3 for quantified analysis of western blot. D Terminal-deoxynucleoitidyl Transferase-Mediated Nick-End Labeling (TUNEL) assay detected the apoptotic cardiomyocytes in infarct border zone or in the approximate region of apex cordis.*: p < 0.05; **: p < 0.01

Fig. 4

Pinocembrin ameliorated post-infarct heart failure-associated oxidative stress. A–C ROS detection, biochemical examinations and western blot results indicated that pinocembrin could scavenge ROS and facilitate antioxidant defense in a Nrf2/HO-1 signaling pathway-dependent manner. *p < 0.05; **p < 0.01. ROS reactive oxygen species, Nrf2 nuclear factor erythroid 2-related factor 2, HO-1 heme oxygenase-1

Fig. 5

In vitro incubation of Nrf2 inhibitor 5 μM ML385 counteract the antioxidant effects of pinocembrin. A, B ROS scavenging and antioxidant defense of pinocembrin were significantly offset by the application of ML385. *p < 0.05; **p < 0.01. ROS reactive oxygen species, Nrf2 nuclear factor erythroid 2-related factor 2

Fig. 6

5 μM ML385 abrogated the salutary effects exerted by pinocembrin on remodeling. A ML385 significantly inhibited Nrf2/HO-1 signaling pathway, subsequently contributing to the exacerbation of key remodeling factors including fibrosis and apoptosis; B the statistic analysis for western blot. N = 4 for quantified analysis for every protein panel. C and D Quantification analysis of collagen I protein level and proliferation of CMFs. *p < 0.05; **p < 0.01. Nrf2 nuclear factor erythroid 2-related factor 2, HO-1 heme oxygenase-1, CMFs cardiac myofibroblasts