卡路里限制對高脂飲食母鼠血管功能失調與組織氧化壓力之影響

Effects of calorie restriction on vascular dysfunction and tissue oxidative stress in high-fat diet treated female rats

江珮瑜(Pei-Yu Chiang)

指導教授 ： 顏嘉宏

國立屏東科技大學/農學院/生物科技研究所/碩士(2016年)

摘要

現代人高熱量且不均衡的飲食型態造成嚴重的肥胖人口問題，根據非傳染性疾病風險協作組織(NCD-RisC)統計，1975年至2014年全球肥胖人口比例由9.6%增加到25.7%而且女性占了14.9%，加上先天上脂肪的儲存與代謝的差異，使女性肥胖的問題較男性嚴重。肥胖是許多疾病的危險因子之一，會造成血管功能病變，且本實驗室先前研究顯示肥胖引起的組織氧化壓力在不同血管床上存在著差異性。隨著肥胖人口的增加，社會及醫療體系的負擔也與日俱增，因此預防肥胖發生與改善肥胖所帶來的危害是重要的課題，而解決之道就是運動與生活型態的改變尤其是飲食型態，許多研究已證實適度減少卡路里的攝取有助於體重管理及降低自由基和氧化壓力，進而延緩老化甚至延長生命週期。因此本研究欲探討高脂飲食對於不同主動脈血管床造成的功能性失調是否具有敏感性的差異，以及卡路里限制(calorie restriction, CR)是否可以藉由減少氧化壓力與增加一氧化氮生物利用率，進而改善受損的血管功能。
實驗將雌性Wistar大鼠分成3組：一般飲食組(normal diet, ND)、高脂飲食組(high-fat diet, HFD)與高脂飲食卡路里限制組(high-fat diet with calorie restriction, HFD+CR)，以高脂飲食誘導母鼠當體重高於ND組30%即為肥胖，且持續10週，再將肥胖鼠隨機分為HFD組與HFD+CR組，HFD組持續攝取高脂飲食直到犧牲，而HFD+CR組則是改由攝取一般飼
料並且進行40%的卡路里限制，直到體重下降至與ND組相近且維持10週。實驗期間定期抽血檢測血清中三酸甘油脂(TG)與總膽固醇(TC)濃度，於實驗終點，將大鼠犧牲分別取下胸主動脈與腹主動脈進行血管功能分析，以氯化鉀(potassium chloride, KCl；L-type voltage-gated calcium channel agonist)和苯腎上腺素(phenylephrine, PE；α1-adrenergic receptor agonist)評估血管平滑肌的收縮反應及使用乙醯膽鹼(acetylcholine, ACh；NO-cyclic GMP 路徑stimulator)與硝普納(sodium nitroprusside, SNP；NO donor)評估血管放鬆反應，也利用一氧化氮合成酶(eNOS)抑制劑(Nitro-L-arginine methyl ester, L-NAME)評估內皮層基礎一氧化氮(basal nitric oxide, bNO)釋出量。同時也以lucigenin進行化學冷光分析各種組織中活性氧物質(reactive oxygen species, ROS)產量及間接評估組織中基礎ROS(bROS)釋放量與NADPH氧化酶的活性。
實驗結果顯示：(1)HFD組體重為537.7±20.4g顯著高於ND組356.9±5.2g，而HFD+CR組體重則下降為316.8±2g顯著低於HFD組；(2)在BMI方面，HFD組0.97±0.02 g/cm2顯著高於ND組0.73±0.02 g/cm2，而HFD+CR組則是0.59±0.01 g/cm2顯著低於HFD組；(3)HFD 組TG濃度為165.4±41.7 mg/dL顯著高於ND組51.2±6.2 mg/dL，而HFD+CR組則下降為51.7±7.7 mg/dL；(4)TC濃度方面HFD組(67.4±3.5 mg/dL)與ND組(69.3±4.4 mg/dL)相近，HFD+CR組(68.2±3.3 mg/dL)與HFD組也無差異；(5)在胸主動脈KCl引起的血管收縮反應上，HFD組血管收縮反應與ND組相似，而HFD+CR組與HFD組收縮反應類似；在腹主動脈HFD組血管收縮反應與ND組相似，但HFD+CR組在30mM~90Mm各個濃度收縮反應顯著高於HFD組；(6)在胸主動脈PE引起的收縮反應中，HFD組在3×10-8M~10-5M各濃度下的收縮反應都顯著地低於ND組，HFD+CR組則是在3×10-8M~10-5M各濃度下的收縮反應都顯著地高於HFD組；在腹主動脈HFD組收縮反應相較於ND組有減弱的趨勢但無統計差異，而HFD+CR組的收縮反應在3×10-8M~10-5M各濃度下都顯著地高於HFD組；(7)在胸主動脈ACh引起的血管內皮依賴性放鬆反應中，HFD組在3×10-8M~3×10-5M各個濃度間的放鬆反應都顯著低於ND組，而HFD+CR組的放鬆反應與HFD組相似；在腹主動脈上HFD組在10-7M~3×10-5M各個濃度間的放鬆反應都顯著低於ND組，而HFD+CR組的放鬆反應與HFD組相似；(8)在胸主動脈SNP引起的非內皮依賴性放鬆反應，HFD組在各個濃度下的放鬆反應都與ND組相似，而HFD+CR組的放鬆曲線與HFD組類似；在腹主動脈方面HFD組的放鬆反應與ND組相似，兩組間無差異，而HFD+CR組與HFD組之間也無統計差異；(9)在胸主動脈bNO釋放量上，HFD組釋放量相較於ND組有增加的趨勢，但無統計差異，而HFD+CR組bNO釋放量相較於HFD組顯著的減少；在腹主動脈bNO釋放量分面，HFD組與ND組間無差異，HFD+CR組釋放量相較於HFD組明顯的減少；(10)在胸主動脈血管平滑肌層，HFD組厚度為231.30±7.29μm顯著地高於ND組的厚度204.00±8.06μm，而HFD+CR組血厚度為200.0±7.07μm顯著低於HFD組；在腹主動脈HFD組血管平滑肌層厚度為175.00±3.99μm顯著高於ND組156.30±7.30μm，而HFD+CR組為170.00±9.05μm與HFD組相似；(11)組織bROS釋出量方面，HFD組胸主動脈、腹主動脈、脂肪組織和小腸組織與ND組間並無差異，但HFD+CR組胸主動脈與腹主動脈bROS產量都顯著高於HFD組；(12)NADPH氧化酶活性方面，HFD組胸主動脈與腹主動脈都與ND組相似，但脂肪組織與小腸組織NADPH氧化酶活性都顯著高於ND組，而卡路里限制後HFD+CR組胸主動脈、脂肪組織和小腸中NADPH氧化酶活行都顯著低於HFD組，但腹主動脈卻顯著高於HFD組。
由上述結果可知高脂飲食造成血管功能障礙，在胸主動脈上造成血管平滑肌α1-adrenergic receptor功能失調、內皮依賴性放鬆功能障礙以及血管平滑肌層增生，在腹主動脈上則是導致內皮依賴性放鬆功能障礙以及血管平滑肌層增生，但透過卡路里限制反逆了胸主動脈因高脂飲食造成血管平滑肌α1-adrenergic receptor功能失調以及血管平滑肌層增生的現象，而腹主動脈血管的損傷則是沒有被改變。卡路里限制也改變了脂肪與小腸組織因高脂飲食而導致NADPH氧化酶活性上升的情形。因此我們建議心血管疾病之高危險群的肥胖患者應定期追蹤胸主動脈血管功能以利病變被早期發現，能盡早治療並搭配攝食卡路里的控管可以有助於改善血管功能失調。

關鍵字

高脂飲食 ； 肥胖 ； 主動脈血管功能失調 ； 卡路里限制

並列摘要

High-fat and imbalanced diets have led to a significant increase in obesity in modern populations. Statistics compiled by the Noncommunicable Diseases Risk Factor Collaboration (NCD-RisC) indicate that the proportion of obese individuals in the global population has increased from 9.6% in 1975 to 25.7% in 2014, among which 14.9% are female. Due to inherent differences in fat storage and metabolism, the issue of obesity is much more severe in women than in men. Obesity is a risk factor of a number of diseases and leads to vascular diseases. A previous study in our laboratory linked obesity to differences in oxidative stress in different vascular beds. The growing obese population is also increasing the burden on society and medical care systems. Thus, preventing obesity and reducing the risks that obesity incurs are crucial issues, the solutions to which include exercise and changing one’s lifestyle, particularly one’s dietary habits. Many studies have proven that moderately reducing calorie intake facilitates weight management and reduces free radicals as well as oxidative stress, which in turn slow down aging and even extends life spans. In view of the above, this study investigated whether differences in sensitivity exist in the functional disorders of different aortic vascular beds
caused by high-fat diets and whether calorie restriction (CR) can improve the functions of damaged blood vessels via oxidative stress reduction.
In this study, we used 6-week-old female Wistar rats and randomly divided them into three groups: normal diet group (ND group), high-fat diet group (HFD group), and high-fat diet with calorie restriction group (HFD+CR group). High-fat diet induced body weight percentage in HFD related groups. When high-fat diet rat body weight higher 30% than ND group defined obese rat models. Maintain 10 weeks. Obese rats were randomly divided into HFD group and HFD+CR group. HFD group remain treated with high-fat diet. HFD+CR group was treated with normal diet and 40% calorie restriction. Until reduced the weight. And close ND group. Maintain 10 weeks. We also collect blood to further diagnose the level of triglyceride (TG) and total cholesterol (TC). At the end of study, we sacrificed the rats to isolate the thoracic aorta and abdominal aorta for vascular function assay. Potassium chloride (KCl; L-type voltage-gated calcium channel agonist) and phenylephrine (PE; α1-adrenergic receptor agonist) are used to evaluate the vascular smooth muscle contractile response whereas acetylcholine (ACh; NO-cyclic GMP pass way stimulator) and sodium nitroprusside (SNP; NO donor) are used to evaluate vasorelaxation reaction. Nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor is used to evaluate the endothelium-dependent basal NO release. At the same time, we used lucigenin-based chemiluminescence assay to determine various tissues basal reactive oxygen species (bROS) and the NADPH-related oxidase activity.
Experimental results show that：(1)HFD group have an average body weight of 537.7±20.4g, which was significantly higher than ND group 356.9±5.2g. The body weight of HFD+CR group reduced to 316.8±2g, which was significantly lower than HFD group.(2) The BMI of HFD group was 0.97±0.02 g/cm2, significantly higher than ND group 0.73±0.02 g/cm2, and the BMI of HFD+CR group is 0.59±0.01 g/cm2 , which was significantly lower than HFD group. (3)The TG level of HFD group (165.4±41.7 mg/dL) was significantly higher than ND group (51.2±6.2 mg/dL), and HFD group (51.7±7.7 mg/dL) was significantly reduce than HFD group. (4) But, the concentration of TC was similar between HFD (67.4±3.5 mg/dL) and ND (69.3±4.4 mg/dL) groups, and also similar with HFD+CR (68.2±3.3 mg/dL). (5)The KCl-mediated vasocontraction of thoracic aorta in HFD group and HFD+CR group was similar to ND group, and HFD+CR group was also similar with HFD group; The vasocontraction of abdominal aorta in HFD group was similar to ND group whereas HFD+CR group was significantly higher than HFD group in 30mM~90Mm. (6) The PE-mediated vasocontraction of thoracic aorta in HFD group was significantly lower than ND group in concentration of 3×10-8M~10-5M, whereas HFD+CR group was significantly higher than HFD group in concentration of 3×10-8M~10-5M; The vasocontraction of abdominal aorta in HFD group is similar to ND group, but HFD+CR group was significantly higher than HFD group in 3×10-8M~10-5M.
(7) The ACh-mediated endothelium-dependent vasorelaxation of thoracic aorta in HFD group was significantly lower than ND group in concentration of 3×10-8M~10-5M, whereas HFD+CR group was similar to HFD group; abdominal aorta vasodilation of HFD group was significantly lower than ND group in 10-7M~10-5M, HFD+CR group was similar with HFD group. (8) The SNP-mediated endothelium-independent vasorelaxation of thoracic aorta in HFD group was similar to ND group, whereas HFD+CR group was similar to HFD group; this phenomenon was also observed in abdominal aorta between HFD group and ND group, HFD+CR group and HFD group. (9) The bNO released by thoracic aorta in HFD group was similar to ND group, but HFD+CR group was significantly lower with compared to HFD group; bNO released by abdominal aorta in HFD group was similar to ND group, but HFD+CR group was significantly lower than HFD group. (10) The smooth muscle of thoracic aorta vascular in HFD group was 231.30±7.29μm, which was significantly higher than ND group 204.00±8.06μm, whereas HFD+CR group 200.0±7.07μm was significantly lower than HFD group. Abdominal aorta vascular wall of HFD group was 175.00±3.99μm, which was significantly higher than ND group 156.30±7.30μm. However, HFD+CR group 170.00±9.05μm was similar to HFD group. (11) The bROS of thoracic aorta, abdominal aorta, adipose tissue and small intestine released in HFD group were similar with ND group. However, in HFD+CR group, the bROS released by thoracic aorta and abdominal aorta were significantly higher than HFD group. (12) The NADPH-related oxidase activity in thoracic aorta and abdominal aorta was similar in both HFD group and ND group. The activity of adipose tissue and small intestine in HFD group was significantly higher than ND group; HFD+CR group thoracic aorta, adipose and small intestine were significantly lower than HFD group, but abdominal aorta was higher than HFD group. The aforementioned results revealed that a high-fat diet caused vascular dysfunctions in the thoracic aorta (e.g., dysfunction of the α1-adrenergic receptor in the vascular smooth muscle, dysfunction of endothelium-dependent relaxation, and vascular smooth muscle proliferation) and in the abdominal aorta (e.g., dysfunction of endothelium-dependent relaxation and vascular smooth muscle proliferation). However, a caloric restriction approach was administer to successfully inverse the phenomena regarding the dysfunction of the α1-adrenergic receptor in the vascular smooth muscle as well as vascular smooth muscle proliferation, both of which were caused by a high-fat diet; nevertheless, damages in the abdominal aorta were not reversed. The caloric restriction also altered the increased NADPH oxidase activity in fat and intestinal tissues. Therefore, we recommend that obese patients in the high-risk group for cardiovascular disease receive regular follow-up examinations of the vascular function in their thoracic aorta.

並列關鍵字

high-fat diet ； obesity ； aortic vascular dysfunction ； calorie restriction

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