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Dieckol and Diphlorethohydroxycarmalol isolated from Ecklonia cava and Ishige okamurae promote vasodilation in the co-culture system and zebrafish model

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Abstract
It is well known that the imbalance between vasoconstriction and vasodilation is deeply related to cardiovascular diseases, especially hypertension (HTN)[1, 2]. Vascular endothelial cells (EC) and vascular smooth muscle cells (VSMC) are the essential components of a typical vessel wall. These two types of cells would cooperate to managing the contraction and relaxation to balance the vascular tone under different physiological conditions. [3-5]. It has been reported that the main problems of HTN are associated with vascular changes characterized by endothelial dysfunction and increased vascular contraction [6, 7]. Based on these viewpoints, the main concepts to reduce blood pressure (BP) could be considered to generate vasodilating factors in EC and decrease the contraction effect in VSMC. Further, resulting in enlarging the vessel diameter and increasing the blood flow rate.
Ecklonia cava (E. cava) and Ishige okamurae (IO) are famous for the different biological activities, including antioxidant, anti-inflammatory, attenuation of endothelial cell dysfunction, and anti-hypertension, in numerous studies [8-11]. Son et al. have indicated that E. cava ethanol extract (ECE) significantly alleviates BP in a mouse model of HTN. Furthermore, dieckol (DK), a polyphenolic compound present in ECE, has been suggested as one of the bioactive components responsible for the potential ACE inhibitory activity [12, 13]. Notably, IO ethanol extract (IOE) and its bioactive substances, diphlorethohydroxycarmalol (DPHC), have shown the remarkable ability to regulate endothelial-dependent vasodilation [14]. However, the molecular signaling pathways were rarely mentioned. Therefore, in the present study, we aim to investigate the vasodilative effect of ECE, DK, IOE, and DPHC and also its molecular signaling pathway in vitro and in vivo.
The endothelial-dependent vasodilation mechanisms are indicated closely related to nitric oxide (NO), a principal regulator of endothelial vasodilator produced from L-arginine by endothelial nitric oxide synthase (eNOS) in the presence of oxygen and the cofactors calcium ([Ca2+]) and calmodulin (CaM) [15]. A previous study has indicated that genetically deficient eNOS mice are hypertensive, with lower circulating NO levels, thus indicating the critical role of eNOS and NO in Cardiovascular disease [16, 17]. Furthermore, the [Ca2+] in the endoplasmic reticulum (ER) ([Ca2+]ER) has long been proposed as a critical factor in regulating eNOS activity, resulting in vasodilation [17]. [Ca2+] homeostasis is affected by acetylcholine (Ach) or vascular endothelial growth factor (VEGF), which plays a major role in regulating vasodilation by increasing the [Ca2+] levels presented in the cytosol ([Ca2+]cytol) via calcium influx, further promoting the expression of downstream proteins such as eNOS [18, 19]. Additionally, the increased expression of phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway directly phosphorylates eNOS, thereby increasing its binding to intracellular CaM, activating eNOS, and promoting NO release [18, 20]. Hence, the increase in NO production effectively dilates the vascular tone to enhance vasodilation. Thus, in the first part, we investigated the endothelial vasodilation effect of ECE, DK, IOE, and DPHC. We observed that all the samples could successfully be generated NO by increasing [Ca2+]ER and [Ca2+]cytol levels and the PI3K/Akt/eNOS expression. Thus, we suggested that ECE, DK, IOE, and DPHC, these natural marine resources, can efficiently enhance endothelial-dependent vasodilation.
VSMCs contractile mechanism and [Ca2+] handling are another major controlling point to achieve vasodilation. An increase in free intracellular [Ca2+] can result from either increased influx of [Ca2+] from the extracellular space or the increased [Ca2+] release from sarcoplasmic reticulum (SR) stores ([Ca2+]SR) in VSMC. Moreover, the free [Ca2+] binds to a special protein, calmodulin (CaM), and the [Ca2+]-CaM complex would further activate the phosphorylation of myosin light chains kinase (MLCK), a kinase enable to interact with myosin light chain (MLC) and actin then lead to contraction [21]. Therefore, the VSMC relaxation occurs only when there is reduced phosphorylation of MLC. In the second part of the study, we aim to verify the relaxation effect of ECE, DK, IOE, and DPHC in VSMC; we have used the EC-VSMC coculture system to mimic the composition of the human vessel and studied its interaction. Based on our results, we found that the NO stimulated by samples in EC would reduce the [Ca2+]cytol and also the decreased expressions of CaM and p-MLC/MLC ratio in VSMC. In particular, the results demonstrated that DK has a high potential to be the vasodilator.
After knowing that DK and DPHC effectively promote the vasodilator NO in EC then further resulting in vasorelaxation in VSMC in vivo experiments. In the third part, we targeted to confirm the vasodilation property in vitro model. The zebrafish model has previously revealed several essential insights into vascular structure development and helped verify underlying molecular mechanisms [22]. In order to confirm all the results of in vivo, the wild-type zebrafish (Danio rerio) and Tg(flk: EGFP) transgenic zebrafish were employed for further experiments. The previous study has shown that contractile marker would increase the expression within the period between 4 dpf and 6 dpf [23]. Therefore, we made up a vasocontraction model by exposing the zebrafish to the classic vasoconstricting agent, Phenylephrine hydrochloride (PE), to determine the vasoactivity at 5 dpf. Further, to evaluate the vasodilation effect of DK and DPHC by measuring the associated cardiovascular parameters such as blood flow (nL/s), linear velocity (μM/s), vessel diameter (μM), and arterial pulse (beats per min). As expected, DK and DPHC treatments effectively promoted vasodilation by increasing the diameter of the dorsal aorta, further regulating blood flow velocity and arterial pulse in the zebrafish model.
In summary, we suggested that the DK and DPHC, which were isolated from Ecklonia cava and Ishige okamurae, exerted vasodilatory property by regulating the calcium signaling and activating the PI3K/Akt/eNOS in EC and further relaxing the VSMC by decreasing the CaM and p-MLC expressions. The vasodilative results were performed in the zebrafish model by showing the increased dorsal aorta diameter, further regulating blood flow velocity and arterial pulse. Based on the present study results, DK and DPHC have a high potential to be developed into a vasodilator for further HTN treatment.
Author(s)
Lu, Yu An
Issued Date
2021
Awarded Date
2021. 8
Type
Dissertation
URI
https://dcoll.jejunu.ac.kr/common/orgView/000000010178
Alternative Author(s)
육유안
Affiliation
제주대학교 대학원
Department
대학원 해양생명과학과
Advisor
Jeon, Yuu Jin
Table Of Contents
Part Ⅰ 1
Bioactive compounds isolated from Ecklonia cava and Ishige okamurae promote vasodilation in endothelial via calcium signalling and PI3K/Akt/eNOS pathway 1
1.1. Introduction 2
1.2. Material and methods 4
1.2.1. Chemicals and reagents 4
1.2.2. Extraction and isolation of DK 4
1.2.3. Extraction and isolation of DPHC 5
1.2.4. EA.hy926 cells culture and cytotoxicity analysis 5
1.2.5. Evaluation of the intracellular NO production 6
1.2.6. Measurement of the intracellular H2S levels 6
1.2.7. Quantification of the cytosolic calcium levels 6
1.2.8. Western blot analysis 7
1.2.9. Statistical analysis 8
1.3. Results 9
1.3.1. Evaluation of cytotoxicity and intracellular NO concentrations under the ECE, DK, IOE, and DPHC treatments 9
1.3.2. Intracellular H2S levels induced by DK and DPHC treatments 14
1.3.3. ECE, DK, IOE, DPHC promoted phosphorylation of the PI3K/Akt/eNOS pathway in E.A.hy926 cells 16
1.3.4. Regulatory effects of ECE, IOE, DK, and DPHC on the [Ca2+]cytol levels 19
1.3.5. Computational prediction of the AchR/VEGFR2 and docking stimulation with DK/DPHC 22
1.3.6. DK and DPHC modulated [Ca2+]cytol levels by activating AchR, VEGFR2, and VDCC 26
1.4. Discussion 30
1.5. Conclusion 33
Part Ⅱ 34
Bioactive compounds isolated from Ecklonia cava and Ishige okamurae promote vasodilation in endothelial-smooth muscle cell co-culture system via down-regulation of CaM and p-MLC expression 34
2.1. Introduction 35
2.2. Material and methods 37
2.2.1. Chemicals and reagents 37
2.2.2. Human coronary artery endothelial cells (HCAECs) monoculture 37
2.2.3. Measurement of cytotoxicity and NO production in HCAEC 37
2.2.4. Evaluation of intracellular H2S levels 38
2.2.5. Quantitative of cytosolic calcium levels in HCAEC 39
2.2.6. Human coronary artery smooth muscle cells (HCASMCs) monoculture 40
2.2.7. Measurement of cytotoxicity and intracellular NO production in HCASMC 40
2.2.8. The protocol for conditional medium preparation 40
2.2.9. The protocol of conditional medium transferred from HCAEC to HCASMC 42
2.2.10. Western blotting analysis 43
2.2.11. The methodology of the HCAEC-HCASMC co-culture model 44
2.2.12. Quantitative of CaM and p-MLC expression in HCASMC by immunofluorescence 45
2.2.13. Statistical analysis 46
2.3. Results 47
2.3.1. Measurement of cell viability and intracellular NO production in HCAEC 47
2.3.2. The levels of intracellular H2S induced by DK and DPHC in HCAEC 50
2.3.3. Evaluation of [Ca2+]cytol under the IOE. DPHC, ECE, and DK treatments in HCAEC 52
2.3.4. Measurement of cytotoxicity under the IOE, DPHC, ECE, and DK treatments in HCASMC 54
2.3.5. Measurement of cell viability and NO production of HCASMC exposured under conditional medium 56
2.3.6. DK and DPHC suppressed the contractile effect by down-regulating the CaM and p-MLC expression in HCASMC 59
2.3.7. Detection of CaM and p-MLC expression by immunofluorescence staining in the co-culture system 62
2.3.8. DK and DPHC down-regulated the expression of CaM and p-MLC in the co-culture system 67
2.4. Discussion 70
2.5. Conclusions 73
Part Ⅲ 74
Bioactive compounds isolated from Ecklonia cava and Ishige okamurae promote vasodilation in the zebrafish model 74
3.1. Introduction 75
3.2. Material and methods 77
3.2.1. Zebrafish husbandry and fish strains 77
3.2.2. Toxicity of DK and DPHC in zebrafish embryos 77
3.2.3. Toxicity of vasoconstriction drug - phenylephrine in zebrafish embryos 78
3.2.4. The vasoconstrictive zebrafish model set up 79
3.2.5. Assessment of whole-body fluorescence intensity in the Tg(flk: EGFP) transgenic zebrafish 79
3.2.6. Evaluation of associated cardiovascular parameters in the zebrafish model 80
3.2.7. Statistical analysis 81
3.3. Results 82
3.3.1. Toxicity of DK and DPHC in zebrafish embryos 82
3.3.2. The vasoconstractive zebrafish model set up by using vasoconstrictor 84
3.3.3. Assessment of whole-body fluorescence intensity in the Tg(flk: EGFP) transgenic zebrafish 86
3.3.4. Evaluation of associated cardiovascular parameters in the zebrafish model 88
3.4. Discussion 94
3.5. Conclusions 96
Acknolegements 97
References 98
Degree
Doctor
Publisher
제주대학교 대학원
Citation
Lu, Yu An. (2021). Dieckol and Diphlorethohydroxycarmalol isolated from Ecklonia cava and Ishige okamurae promote vasodilation in the co-culture system and zebrafish model
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General Graduate School > Marine Life Sciences
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