Ecklonia cava Extract and Its Derivative Dieckol Promote Vasodilation by Modulating Calcium Signaling and PI3K/AKT/eNOS Pathway in In Vitro and In Vivo Models

Abstract

Nitric oxide (NO), an endothelial-derived relaxing factor synthesized by endothelial nitric oxide synthase (eNOS) in endothelial cells, enhances vasodilation by modulating vascular tone. The calcium concentration critically influences eNOS activation in endothelial cells. Thus, modulation of calcium-dependent signaling pathways may be a potential therapeutic strategy to enhance vasodilation. Marine algae reportedly possess protective effects against cardiovascular disorders, including hypertension and vascular dysfunction; however, the underlying molecular signaling pathways remain elusive. In the present study, we extracted and isolated dieckol from Ecklonia cava and investigated calcium transit-enhanced vasodilation. Calcium modulation via the well-known M3 muscarinic acetylcholine receptor (AchM3R), which is linked to NO formation, was investigated and the vasodilatory effect of dieckol was verified. Our results indicated that dieckol effectively promoted NO generation via the PI3K/Akt/eNOS axis and calcium transients influenced by AchM3R. We also treated Tg(flk: EGFP) transgenic zebrafish with dieckol to assess its vasodilatory effect. Dieckol promoted vasodilation by enlarging the dorsal aorta diameter, thus regulating blood flow velocity. In conclusion, our findings suggest that dieckol modulates calcium transit through AchM3R, increases endothelial-dependent NO production, and efficiently enhances vasodilation. Thus, E. cava and its derivative, dieckol, can be considered as potential natural vasodilators.

Keywords: Ecklonia cava; M3 muscarinic acetylcholine receptor; NO production; calcium transit; dieckol; endothelial cell; vasodilation; zebrafish.

Figure 1

Cell viability analysis using EA.hy926 cells treated with (A) ECE (0, 3, 10, 30, and 100 µg/mL) and (B) DK (0, 4, 13, 40, and 134 µM). Intracellular NO production by (C) ECE and (D) DK. Results are expressed as the mean ± standard deviation (S.D.) of three independent experiments. * p < 0.05, *** p < 0.001 significantly different compared with the control group. EC, E. cava extract; DK, Dieckol; NO, nitric oxide; ns, not significant; -: no sample treatment (control)

Figure 2

Evaluation of vasodilation-related protein expression. Examples of Western blot analysis, (A,E). The protein expressions of ECE treatment on (B) phosphorylated-PI3K (p-PI3K), (C) p-Akt, and (D) p-eNOS; the protein expressions of (F) p-PI3K, (G) p-Akt, and (H) p-eNOS following DK treatment. The protein bands were ultimately developed and photographed with the FUSION Solo Vilber Lourmat system. Quantitative data were analyzed using ImageJ software. Results are expressed as the mean ± standard deviation (S.D.) of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 significantly different compared with the control group. EC, E.Cava extract; DK, Dieckol; PI3K, phosphoinositide 3-kinases; eNOS, endothelial nitric oxide synthase; -: no sample treatment (control)

Figure 3

Quantification of [Ca²⁺]_cytol levels following treatment with different ECE and DK concentable 926. cells. In the absence of [Ca²⁺]_cytol using the Fluo-4 calcium indicator, (A) traces and (B) box plot representation of [Ca²⁺]_cytol levels under ECE treatments (3, 10, 30, and 100 µg/mL); The absence of [Ca²⁺]_cytol levels in cell (C) traces and (D) box plot representation of [Ca²⁺]_cytol levels in response to the addition of DK (0, 4, 13, 40, and 134 µM). Results are expressed as the mean ± standard deviation (S.D.) of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 significantly different compared with the control group. EC, E.Cava extract; DK, Dieckol; ns, not significant; AUC, area under the curve; [Ca²⁺]_cytol, calcium level in the cytosol.

Figure 4

Computational prediction of the AchM3R and docking stimulation with DK. (A) 3D diagram and (B) 2D diagram of AchM3R and DK complex. (C) Results of the docking experiments with DK with AchM3R. DK, Dieckol; AchM3R, muscarinic M3-receptor.

Figure 5

Influence of a specific antagonist on [Ca²⁺]_cytol levels in EA.hy926 cells treated with DK. (A) Traces and (B) box plots indicating [Ca²⁺]_cytol levels in response to treatment with DK (134 μM) and antagonist. (C) Effect of DK on NO production in EA.hy926 cells pretreated with atropine. The NO levels were detected by adding 10 μM of 4 amino-5-methylamino-2′, 7′-difluorescein diacetate (DAF-FM-DA). Results are expressed as the mean ± standard deviation (SD) of three independent experiments, *** p < 0.001 significantly different compared with the control group; ### p < 0.01 significantly different compared with DK only group. DK, Dieckol; ns, not significant; AUC, area under the curve; [Ca²⁺]_cytol, calcium level in the cytosol; NO, nitric oxide; -: no sample treatment (control)

Figure 6

The survival rate of the zebrafish embryos under DK treatment. The test was based on the exposure of newly fertilized zebrafish eggs to 4, 13, 40, and 134 µM of DK for up to 120 h (n = 15 per treatment, three replicates). Embryos were observed at each time point under the stereomicroscope (magnification used in the stereomicroscope for observations was 4×). DK, Dieckol.

Figure 7

Cardiovascular parameters measured at the dorsal aorta (DA) of 3 dpf Tg(flk:EGFP) transgenic zebrafish. (A1) Images of the DA captured by fluorescence microscope (20×) (a–e) Vasodilation observed by treatment with DK. (a) PBS (Control); (b) 4 μM of DK; (c) 13 μM of DK; (d) 40 μM of DK; (e) 134 μM of DK. (A2) Evaluation of DA vessel diameter. (B) Arterial pulse (beats per minute); (C) Mean blood flow velocity (μM/s); (D) Blood flow (nL/s) were measured from n = 6 larvae per group. The yellow box represented the area used for evaluation. Results are expressed as the mean ± standard deviation (S.D.). * p < 0.05, ** p < 0.01. ^## p < 0.01. *** p < 0.001 significantly different compared with the control group. dpf, days post-fertilization; ns, not significant; -: no sample treatment (control)

Figure 8

A schematic represented the potential endothelial-dependent vasodilation mechanisms of DK. DK possess potential vasodilatory effects by upregulating the expression of PI3K/Akt/eNOS and enhancing the [Ca²⁺]_cytol transit via AchM3R, resulting in NO formation in endothelial cells.