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研究生:KUNANYA,MASODSAI
研究生(外文):KUNANYA,MASODSAI
論文名稱:老化高血壓之心血管病變與運動和原兒茶酸介入之影響
論文名稱(外文):Aging-related Cardiovascular Dysfunction in Hypertension and its Reversal through Exercise and Protocatechuic Acid
指導教授:楊艾倫楊艾倫引用關係
指導教授(外文):Yang, Ai-Lun
口試日期:2019-01-08
學位類別:博士
校院名稱:臺北市立大學
系所名稱:運動科學研究所
學門:民生學門
學類:運動科技學類
論文種類:學術論文
論文出版年:2019
畢業學年度:107
語文別:英文
論文頁數:121
中文關鍵詞:老化高血壓內皮功能一氧化氮胰島素阻抗氧化壓力發炎有氧運動多酚原兒茶酸
外文關鍵詞:AgingHypertensionEndothelial functionNitric oxideInsulin resistanceOxidative stressInflammationAerobic exercisePolyphenolProtocatechuic acid
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  • 點閱點閱:118
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背景和目的: 根據流行病學研究,高血壓的發生率可能無法避免隨著年齡增加,高血壓與老化兩者皆對心血管病變有顯著的影響,特別是內皮功能障礙。規律的運動已被證明是非藥物性預防與治療高血壓的方式。此外,天然多酚化合物,如原兒茶酸,被認為具有促進健康的效果,包括心血管保護作用。在本研究中,我們欲探討是否老化對高血壓之內皮功能障礙有加成效應。此外,我們也進一步探討運動或原兒茶酸介入對老化高血壓之內皮功能障礙的影響。方法: 本研究使用雄性自發性高血壓大鼠(spontaneously hypertensive rats, SHR)和正常血壓大鼠(Wistar-Kyoto, WKY),隨機分為四組:24週齡正常血壓組(24wk-WKY)、48週齡正常血壓組(48wk-WKY)、24週齡高血壓組(24wk-SHR)與48週齡高血壓組(48wk-SHR)。此外,只有48週齡高血壓組另接受運動或原兒茶酸介入(分別為48wk-SHR-Ex和48wk-SHR-PCA),須完成12週中等強度跑步機介入(21公尺/分鐘,60分鐘/天,5天/週)或每日於飲用水中口服原兒茶酸(200毫克/公斤/天)。在所有動物達到其年齡並完成介入後,使用組織浴系統評估其內皮功能,即血管反應性,且分析各組一氧化氮含量、氧化與抗氧化特性、發炎反應、及胰島素阻抗等,並進行比較。此外,利用西方墨點法檢測主動脈蛋白表現量。結果: 我們發現,不同品系(高血壓)會有血壓顯著增加的情形,而非老化造成(P<0.05)。由胰島素和類胰島素生長因子-1誘導之內皮依賴性血管舒張反應於SHR兩個週齡中均顯著降低(P<0.05),而48wk-SHR組比24wk-SHR組更為嚴重(P <0.05)。然而,這些血管舒張反應於48wk-SHR-Ex和48wk-SHR-PCA組中皆有顯著提升(P <0.05)。相似的情形也發生於一氧化氮濃度、氧化與抗氧化特性、發炎反應和胰島素阻抗等。而於磷脂酸肌醇3-酶(PI3K)或一氧化氮合成酶(NOS)抑制後,胰島素和類胰島素生長因子-1誘導之血管舒張反應皆無顯著差異。此外,與同週齡的正常血壓組相比,兩個週齡的高血壓組中胰島素受體,類胰島素生長因子-1受體,蛋白激酶B(Akt)和內皮型一氧化氮合成酶(eNOS)的蛋白表現量皆顯著下降(P <0.05)。然而,於運動或原兒茶酸介入後皆有顯著改善(P <0.05)。結論: 本研究證實老化會使高血壓之內皮功能障礙更為嚴重,然而,經12週運動或原兒茶酸介入可顯著改善老化高血壓之內皮功能障礙,可能經由胰島素/類胰島素生長因子-1-PI3K-NOS-NO途徑之影響。基於這些發現,我們建議運動或原兒茶酸介入可作為預防或治療老化高血壓之非藥物性方式。
Background and purpose: Based on the epidemiological studies, the prevalence of hypertension may unavoidably increase with advancing age. Both of hypertension and aging have remarkable effects on cardiovascular disorders, particularly endothelial dysfunction. Regular exercise has been proved as the non-pharmacological strategy in prevention and treatment for hypertension. In addition, natural polyphenolic compounds, such as protocatechuic acid (PCA), have been reviewed for various actions in health promotion, including cardiovascular-protective effects. In the present study, we investigated whether aging additively affected endothelial dysfunction in hypertension. Moreover, we explored the effects of exercise intervention or PCA administration on endothelial dysfunction in aging hypertension. Methods: Male spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats were randomly divided into four groups: the 24-week-old WKY (24wk-WKY), 48-week-old WKY (48wk-WKY), 24-week-old SHR (24wk-SHR), and 48-week-old SHR (48wk-SHR) groups. Also, only the 48-week-old SHR group received either exercise intervention or PCA administration (48wk-SHR-Ex, 48wk-SHR-PCA, respectively). They completed the 12 weeks of either moderate-intensity treadmill running (21 m/min for 60 min/days, 5 days/week) or daily oral PCA administration (200 mg/kg/day) in drinking water. After all animals reached their age and completed interventions, endothelial function in term of vascular reactivity was evaluated by using the organ bath system. Also, the production of nitric oxide (NO), oxidant and antioxidant properties, inflammatory profiles, and insulin resistance were determined and compared among groups. Western immunoblot analysis was performed to determine the aortic protein expression. Results: Our results showed that blood pressure was significantly increased with the stain (SHR) but not with aging (P<0.05). The endothelium-dependent vasorelaxation induced by insulin and insulin-like growth factor-1 (IGF-1) was significantly decreased in both ages of SHRs (P<0.05), and it was significantly more severe in the 48wk-SHR group compared with the 24wk-SHR group (P<0.05). However, this vasorelaxation was significantly restored in 48wk-SHR-Ex and 48wk-SHR-PCA groups (P<0.05). Similar trends were also found in NO production, oxidant and antioxidant properties, inflammatory profiles, and insulin resistance. There were no significant differences in insulin- and IGF-1-induced vasorelaxation among groups after the pre-incubation of phosphatidylinositol 3-kinase (PI3K) or nitric oxide synthase (NOS) inhibitors, respectively. Moreover, the protein levels of insulin receptors, IGF-1 receptors, protein kinase B (Akt), and endothelial nitric oxide synthase (eNOS) were significantly declined in both ages of SHRs compared with the age-matched WKYs (P<0.05). However, they were significantly improved after exercise intervention or PCA administration (P<0.05). Conclusion: This study suggested that aging could additively induce severe endothelial dysfunction in hypertension, however, the 12 weeks of exercise intervention or PCA administration remarkably ameliorated endothelial dysfunction in aging hypertension partly through insulin/IGF-1-PI3K-NOS-NO pathways. Based on these findings, exercise intervention or PCA administration could be suggested as the non-pharmacological strategy for the prevention and/or treatment for aging hypertension.
CONTENTS
Page
ACKNOWLEDGEMENTS i
ABSTRACT ii
CONTENTS v
LIST OF TABLES vi
LIST OF FIGURES vii
LIST OF ABBREVIATIONS xii
CHAPTER I INTRODUCTION 1
CHAPTER II LITERATURE REVIEWS 8
CHAPTER III MATERIALS AND METHODS 32
CHAPTER IV RESULTS 38
4.1 First study 38
4.2 Second study 42
CHAPTER V DISCUSSION 46
5.1 First study 46
5.2 Second study 55
CHAPTER VI CONCLUSION 64
REFERENCES 65
TABLES AND FIGURES 90
First study 90
Second study 103
BIOGRAPHY 116

LIST OF TABLES

Page
Table 1 General characteristics (first study) 90
Table 2 Insulin resistance (first study) 90
Table 3 General characteristics (second study) 103
Table 4 Insulin resistance (second study) 103

LIST OF FIGURES

Page
Figure 1 Chemical Structure of protocatechuic acid (PCA) 28
Figure 2 Vasorelaxant responses of insulin (310-8 to 310-6 M) at cumulative concentration-response curves for endothelium-intact (A) and endothelium-denuded (B) aortic rings in 24-week-old (24wk) and 48-week-old (48wk) Wistar-Kyoto rats (WKY), and 24wk- and 48wk-spontaneously hypertensive rats (SHR). 90
Figure 3 Vasorelaxant responses of insulin-like growth factor-1 (IGF-1) (10-9 to 10-7 M) at cumulative concentration-response curves for endothelium-intact (A) and endothelium-denuded (B) aortic rings in 24-week-old (24wk) and 48-week-old (48wk) Wistar-Kyoto rats (WKY), and 24wk- and 48wk-spontaneously hypertensive rats (SHR). 91
Figure 4 Vasorelaxant responses of insulin (A, 10-6 M) and insulin-like growth factor-1 (IGF-1) (B, 310-8 M) in the presence of Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) (10-6 M) and Wortmannin (310-7 M) in aortic rings of 24-week-old (24wk) and 48-week-old (48wk) Wistar-Kyoto rats (WKY), and 24wk- and 48wk-spontaneously hypertensive rats (SHR). 92
Figure 5 Vasorelaxant responses of sodium nitroprusside (SNP) (310-10 to 310-8 M) at cumulative concentration-response curves for aortic rings in 24-week-old (24wk) and 48-week-old (48wk) Wistar-Kyoto rats (WKY), and 24wk- and 48wk-spontaneously hypertensive rats (SHR). 93
Figure 6 Serum Nitrate/Nitrite concentration in 24-week-old (24wk) and 48-week-old (48wk) Wistar-Kyoto rats (WKY), and 24wk- and 48wk-spontaneously hypertensive rats (SHR). 94

LIST OF FIGURES (CONT.)

Page
Figure 7 Serum malondialdehyde (MDA, A) concentration and superoxide dismutase (SOD, B) activity in 24-week-old (24wk) and 48-week-old (48wk) Wistar-Kyoto rats (WKY), and 24wk- and 48wk-spontaneously hypertensive rats (SHR). 95
Figure 8 Serum catalase activity(A) and antioxidant capacity (D) in 24-week-old (24wk) and 48-week-old (48wk) Wistar-Kyoto rats (WKY), and 24wk- and 48wk-spontaneously hypertensive rats (SHR). 96
Figure 9 Serum interleukin (IL)- 1beta (β) (A) and tumor necrosis factor-alpha (TNF-α, B) concentration in 24-week-old (24wk) and 48-week-old (48wk) Wistar-Kyoto rats (WKY), and 24wk- and 48wk-spontaneously hypertensive rats (SHR). 97
Figure 10 Representative immunoblots of insulin receptor (Insulin-R) protein extracted from aortas and relative protein quantification of Insulin-R normalized by the corresponding actin (B) in 24-week-old (24wk) and 48-week-old (48wk) Wistar-Kyoto rats (WKY), and 24wk- and 48wk-spontaneously hypertensive rats (SHR) 99
Figure 11 Representative immunoblots of insulin-liked growth factor 1 receptor (IGF-1R) protein extracted from aortas (A) and relative protein quantification of IGF-1R normalized by the corresponding actin (B) in 24-week-old (24wk) and 48-week-old (48wk) Wistar-Kyoto rats (WKY), and 24wk- and 48wk-spontaneously hypertensive rats (SHR) 100
Figure 12 Representative immunoblots of phospho-protein kinase B (p-Akt) protein extracted from aortas (A) and relative protein quantification of p-Akt normalized by the corresponding Akt (B) in 24-week-old (24wk) and 48-week-old (48wk) Wistar-Kyoto rats (WKY), and 24wk- and 48wk-spontaneously hypertensive rats (SHR) 101

LIST OF FIGURES (CONT.)

Page
Figure 13 Representative immunoblots of phospho-endothelial nitric oxide synthase (p-eNOS) protein extracted from aortas (A) and relative protein quantification of p-eNOS normalized by the corresponding eNOS (B) in 24-week-old (24wk) and 48-week-old (48wk) Wistar-Kyoto rats (WKY), and 24wk- and 48wk-spontaneously hypertensive rats (SHR) 102
Figure 14 Vasorelaxant responses of insulin (3x10-8 to 3x10-6 M) at cumulative concentration-response curves for endothelium-intact (A) and endothelium-denuded (B) aortic rings in 48-week-old Wistar-Kyoto rats (48wk-WKY), spontaneously hypertensive rats (48wk-SHR), SHR with exercise (48wk-SHR-Ex), and SHR with protocatechuic acid (48wk-SHR-PCA). 104
Figure 15 Vasorelaxant responses of insulin-like growth factor-1 (IGF-1) (10-9 to 10-7 M) at cumulative concentration-response curves for endothelium-intact (A) and endothelium-denuded (B) aortic rings in 48-week-old Wistar-Kyoto rats (48wk-WKY), spontaneously hypertensive rats (48wk-SHR), SHR with exercise (48wk-SHR-Ex), and SHR with protocatechuic acid (48wk-SHR-PCA). 105
Figure 16 Vasorelaxant responses of insulin (A, 10-6 M) and insulin-like growth factor-1 (IGF-1) (B, 3x10-8 M) in the presence of Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) (10-6 M) and Wortmannin (3x10-7 M) in aortic rings of 48-week-old Wistar-Kyoto rats (48wk-WKY), spontaneously hypertensive rats (48wk-SHR), SHR with exercise (48wk-SHR-Ex), and SHR with protocatechuic acid (48wk-SHR-PCA). 106


LIST OF FIGURES (CONT.)

Page
Figure 17 Vasorelaxant responses of sodium nitroprusside (SNP) (3x10-10 to 310-8 M) at cumulative concentration-response curves for aortic rings in 48-week-old Wistar-Kyoto rats (48wk-WKY), spontaneously hypertensive rats (48wk-SHR), SHR with exercise (48wk-SHR-Ex), and SHR with protocatechuic acid (48wk-SHR-PCA). 107
Figure 18 Serum Nitrate/Nitrite concentration in 48-week-old Wistar-Kyoto rats (48wk-WKY), spontaneously hypertensive rats (48wk-SHR), SHR with exercise (48wk-SHR-Ex), and SHR with protocatechuic acid (48wk-SHR-PCA). 108
Figure 19 Serum malondialdehyde (MDA, A) concentration and superoxide dismutase (SOD, B) activity in 48-week-old Wistar-Kyoto rats (48wk-WKY), spontaneously hypertensive rats (48wk-SHR), SHR with exercise (48wk-SHR-Ex), and SHR with protocatechuic acid (48wk-SHR-PCA). 109
Figure 20 Serum catalase activity (A) and antioxidant capacity (B) in 48-week-old Wistar-Kyoto rats (48wk-WKY), spontaneously hypertensive rats (48wk-SHR), SHR with exercise (48wk-SHR-Ex), and SHR with protocatechuic acid (48wk-SHR-PCA). 110
Figure 21 Serum interleukin (IL)- 1 beta (β) (A) and tumor necrosis factor-alpha (TNF-α, B) concentration in 48-week-old Wistar-Kyoto rats (48wk-WKY), spontaneously hypertensive rats (48wk-SHR), SHR with exercise (48wk-SHR-Ex), and SHR with protocatechuic acid (48wk-SHR-PCA). 111




LIST OF FIGURES (CONT.)

Page
Figure 22 Representative immunoblots of insulin receptor (Insulin-R) protein extracted from aortas (A) and relative protein quantification of Insulin-R normalized by the corresponding actin (B) in 48-week-old Wistar-Kyoto rats (48wk-WKY), spontaneously hypertensive rats (48wk-SHR), SHR with exercise (48wk-SHR-Ex), and SHR with protocatechuic acid (48wk-SHR-PCA) 112
Figure 23 Representative immunoblots of insulin-liked growth factor 1 receptor (IGF-1R) protein extracted from aortas (A) and relative protein quantification of IGF-1R normalized by the corresponding actin (B) in 48-week-old Wistar-Kyoto rats (48wk-WKY), spontaneously hypertensive rats (48wk-SHR), SHR with exercise (48wk-SHR-Ex), and SHR with protocatechuic acid (48wk-SHR-PCA) 113
Figure 24 Representative immunoblots of phospho-protein kinase B (p-Akt) protein extracted from aortas (A) and relative protein quantification of p-Akt normalized by the corresponding Akt (B) in 48-week-old Wistar-Kyoto rats (48wk-WKY), spontaneously hypertensive rats (48wk-SHR), SHR with exercise (48wk-SHR-Ex), and SHR with protocatechuic acid (48wk-SHR-PCA) 114
Figure 25 Representative immunoblots of phospho-endothelial nitric oxide synthase (p-eNOS) protein extracted from aortas (A) and relative protein quantification of p-eNOS normalized by the corresponding eNOS (B) in 48-week-old Wistar-Kyoto rats (48wk-WKY), spontaneously hypertensive rats (48wk-SHR), SHR with exercise (48wk-SHR-Ex), and SHR with protocatechuic acid (48wk-SHR-PCA) 115
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