The cardioprotective effects of secoisolariciresinol diglucoside (flaxseed lignan) against cafeteria diet-induced cardiac fibrosis and vascular injury in rats: an insight into apelin/AMPK/FOXO3a signaling pathways

Abstract

Introduction: Fast food is a major risk factor for atherosclerosis, a leading cause of morbidity and mortality in the Western world. Apelin, the endogenous adipokine, can protect against cardiovascular disease via activating its receptor, APJ. Concurrently, secoisolariciresinol diglucoside (SDG), a flaxseed lignan extract (FLE), showed a therapeutic impact on atherosclerosis. The current study aimed to examine the effect of SDG on cafeteria diet (CAFD)-induced vascular injury and cardiac fibrosis via tracking the involvement of the apelin/APJ pathway. Methods: Thirty male rats were allocated into control, FLE-, CAFD-, CAFD/FLE-, and CAFD/FLE/F13A-treated rats, where F13A is an APJ blocker. All treatments lasted for 12 weeks. Results and discussion: The CAFD-induced cardiovascular injury was evidenced by histological distortions, dyslipidemia, elevated atherogenic indices, cardiac troponin I, collagen percentage, glycogen content, and apoptotic markers. CAFD increased both the gene and protein expression levels of cardiac APJ, apelin, and FOXO3a, in addition to increasing endothelin-1, VCAM1, and plasminogen activator inhibitor-1 serum levels and upregulating cardiac MMP-9 gene expression. Moreover, CAFD reduced serum paraoxonase 1 and nitric oxide levels, cardiac AMPK, and nuclear Nrf2 expression. FLE attenuated CAFD-induced cardiovascular injury. Such effect was reduced in rats receiving the APJ blocker, implicating the involvement of apelin/APJ in FLE protective mechanisms. Conclusion: FLE supplementation abrogated CAFD-induced cardiac injury and endothelial dysfunction in an apelin/APJ-dependent manner.

Keywords: apelin; cafeteria diet; cardiac fibrosis; dyslipidemia; flaxseed; secoisolariciresinol diglucoside; vascular injury.

FIGURE 1

The mean change of the (A) body weight, (B) food intake, and (C) caloric intake per week in the control, cafeteria diet (CAFD)-, flaxseed lignan extract (FLE)-, and FLE/F13A-treated rat groups over 12 weeks. F13A is an apelin-13 receptor blocker. Values are presented as the mean ± SD (n = 6 per group; repeated measures ANOVA followed by Tukey’s multiple comparison test; ^* p < 0.05, ^**** p < 0.0001).

FIGURE 2

Effect of flaxseed lignan extract (FLE) on serum (A) malondialdehyde (MDA), (B) reduced glutathione (GSH), (C) interleukin-6 (IL-6), (D) tumor necrosis factor-alpha (TNF-α), and (E) C-reactive protein (CRP), as well as the cardiac tissue levels of (F) TNF-α, (G) interferon-gamma (IFN-γ), and (H) IL-10 of the cafeteria diet (CAFD)-fed rats. F13A is an apelin-13 receptor blocker. Values are presented as the mean ± SD (n = 6 per group; one-way ANOVA followed by Tukey’s multiple comparison test; ^* p < 0.05, ^** p < 0.01, ^**** p < 0.0001).

FIGURE 3

Effect of flaxseed lignan extract (FLE) on the serum levels of (A) nitric oxide (NO), (B) cardiac troponin I (cTnI), (C) paraoxonase-1 (PON1), (D) plasminogen activator inhibitor-1 (PAI-1), (E) vascular cell adhesion molecule 1 (VCAM1), and (F) endothelin-1 (ET-1), as well as the (G) Bcl-2-associated X protein (Bax) levels, and (H) caspase-3 activity in the cardiac tissue of the cafeteria diet (CAFD)-fed rats. F13A is an apelin-13 receptor blocker. Values are presented as the mean ± SD (n = 6 per group; one-way ANOVA followed by Tukey’s multiple comparison test; ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001).

FIGURE 4

Effect of flaxseed lignan extract (FLE) on cardiac mRNA expression of (A) apelin-13, (B) apelin receptor (APJ), (C) nuclear factor erythroid 2-related factor 2 (Nrf2), (D) matrix metalloproteinase-9 (MMP-9), and (E) forkhead box O3 a (FOXO3a) in the cafeteria diet (CAFD)-fed rats. F13A is an apelin-13 receptor blocker. Values are presented as the mean ± SD (n = 6 per group; one-way ANOVA followed by Tukey’s multiple comparison test; ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001).

FIGURE 5

Relative protein expression levels and representative Western blot of (A) apelin-13, (B) apelin receptor (APJ), (C) phosphorylated AMP-activated protein kinase (p-AMPK) (Thr172), (D) phosphorylated forkhead box O3 a (p-FOXO3a) (Ser574), (E) cytosolic nuclear factor erythroid 2-related factor 2 (Nrf2), and (F) nuclear Nrf2 in the cardiac tissues of CAFD-fed rats treated with flaxseed lignan extract (FLE). Values are presented as the mean ± SD (n = 6 per group; one-way ANOVA followed by Tukey’s multiple comparison test; ^* p < 0.05, ^** p < 0.01, ^**** p < 0.0001).

FIGURE 6

(A,D) The control and flaxseed lignan extract (FLE)-treated groups show irregularly arranged and anastomosing myocytes covered by perichondrium. (B,E) Longitudinal section (LS) of anastomosing muscle fibers with apparent striation and intercalated discs (Blue arrow), and central vesicular nuclei (Black arrow). (C,F) Transverse section (TS) of cardiac muscles having central vesicular nuclei and fibroblast nuclei (Circles). (G–I) The cafeteria diet (CAFD)-treated group shows thickened pericardium, with sub-pericardial hemorrhage and fat cells deposition (F) (LS & TS), separated cardiac myocytes with some destructed fibers (Yellow arrow) and degenerated areas (Arrowhead), fat cell infiltration (F), cellular infiltration (Star), and hemorrhage (C). (J–L) The CAFD/FLE-treated group shows pericardium and cardiac muscles (LS & TS) with less congestion (C), more cellular infiltrations (Star), and some destructed myocytes (Yellow arrow). (M–O) The CAFD/FLE/F13A-treated group shows irregularly arranged and widely separated muscle fibers (LS & TS), less hemorrhage (C), cellular infiltration (Star), as well as destructed (Yellow arrow) and degenerated myocytes (Arrowhead), where F13A is an apelin-13 receptor blocker. (H&E stain; X100, X200).

FIGURE 7

Photomicrographs of (A) Masson’s trichrome- and (B) Periodic acid–Schiff (PAS)-stained sections of cardiac muscles, (C) collagen area percentage, and (D) optical density (O.D.) of PAS reaction in rat groups. Masson’s trichrome and PAS; X200. Black arrows: collagen fibers. Blue arrows: positive reaction to the PAS stain. Data are presented as the mean ± SD (n = 6 per group; one-way ANOVA followed by Tukey’s multiple comparison test; ^*** p < 0.001, ^**** p < 0.0001). CAFD, cafeteria diet; FLE, flaxseed lignan extract, F13A; an apelin-13 receptor blocker.