探討蒟蒻纖維及果寡醣於D-半乳糖注射Balb/c雄鼠心血管功能之保健功效及其相關機制

Healthy effects of konjac fiber and fructo-oligosaccharide on the cardiovascular function in D-galactose-treated Balb/c mice and the related mechanisms

潘彥臻(Yan-Jhen Pan)

指導教授 ： 陳曉鈴

中山醫學大學/健康管理學院/營養學研究所/碩士(2014年)

摘要

目的：本實驗有三個主要的研究目的。第一在探討長期注射D-半乳糖是否成功誘發Balb/c雄性小鼠老化相關的心血管損傷，第二是探討補充蒟蒻纖維、果寡醣及維生素E是否降低D-半乳糖誘發的心血管傷害，以及相關細胞機制。
材料方法：將12週齡大Balb/c雄性小鼠分成6組：控制組 (Control組)、D-半乳糖組 (DG組)、D-半乳糖-蒟蒻纖維組 (DG+K組，5%, w/w)、D-半乳糖-果寡醣組 (DG+FO組，5%, w/w)、D-半乳糖-蒟蒻果寡醣組 (DG+KFO組，各2.5%, w/w)、D-半乳糖-維生素E組 (DG+E組，0.04%, w/w)。Control組給予皮下注射 0.9%生理食鹽水，其他DG組則注射10% 半乳糖生理食鹽水 (1.2 g/Kg BW)，介入49天犧牲。另有自然老化組 (NA組)不接受注射，於56週齡大犧牲。實驗期間NA組、Control組及DG組均給予控制組飼料，其餘組別餵食各實驗飼料。實驗結束前一週測量心電圖。小鼠禁食16小時後，以CO2昏迷犧牲，由下腔靜脈採血收集血液，並收集心臟、主動脈及測量左心室心肌厚度。分析項目包含心臟TUNEL stain、Masson trichrome stain、western blot分析；主動脈Elastin stain、RT-PCR分析及血漿總膽固醇、三酸甘油酯及脂質過氧化物濃度的測定。
結果：結果顯示，DG組及NA組有相似的心血管功能退化情形。在心臟方面，DG組的心跳速率顯著低於Control組，由心電圖顯示DG組之RR區間及QTc皆有延長之情形；另外，NA組及DG組在左心室心肌厚度、心肌細胞凋亡程度及心肌細胞凋亡Bid、Bax、Cytochrome c之蛋白表現量皆顯著高於Control組。補充蒟蒻纖維、果寡醣及維生素E可以降低DG造成的心跳速率異常、降低心肌凋亡程度及凋亡相關蛋白之表現至類似於Control組。在主動脈方面，與Control組比較，NA組及DG組之彈力纖維斷裂多且纖維層鬆弛、平均肌肉層厚度也顯著高於Control組，在LOX基因表現則顯著低於Control組。補充蒟蒻纖維、果寡醣及維生素E可以使血管型態回復至類似Control組，並提升LOX之基因表現量。在血液方面，NA組及DG組顯著增加血漿脂質過氧化產物、總膽固醇及三酸甘油酯的濃度，而在補充蒟蒻纖維、果寡醣及維生素E後，可顯著減少血漿脂質過氧化物、總膽固醇及三酸甘油酯的濃度。
結論：本研究顯示補充蒟蒻纖維、果寡醣及維生素E有助於改善D-半乳糖誘發的心血管損傷，推測可能藉由膳食纖維的啟動抗氧化壓力機制及調節血脂的作用，進而延緩氧化壓力造成的心血管病變、抑制心肌細胞凋亡路徑，並增加血管彈性相關基因表現。

關鍵字

D-半乳糖 ； 老化 ； 蒟蒻纖維 ； 果寡醣 ； 心血管功能 ； 細胞凋亡 ； lysyl oxidse

並列摘要

Objectives：The study has two main goals. The first was to determine whether the long-term subcutaneous (s.c.) injection of D-galactose could induce the aging cardiovascular dysfunction in Balb/c male mice. The second is to determine effects of dietary supplement of konjac glucomannan (KGM), fructo-oligosaccharide (FOS) or vitamin E on the D-galactose-induced dysfunction and the underlying mechanisms.
Materials and Methods： The twelve-week-old male Balb/c mice were divided into control group, D-galactose group (DG group), D-galactose-konjac glucomannan group (DG+K group, 5% w/w), D-galactose-fructo-oligosaccharide group (DG+FO group, 5% w/w), D-galactose-KGM-FOS group (DG+KFO group, 2.5% w/w each), D-galactose-vitamin E group (DG+E group, 0.04% w/w as a antioxidant control). The control group was given 0.9% saline (s.c.) while the other groups were injected with D-galactose (s.c., 1.2 g/kg BW) for 49 days. Another natural aging group (NA group) did not receive any injection and were killed at 56 weeks old. The ECG was determined a week before the end of the experiment. Mice were anaesthetized with CO2 after 18 h fasting and a midline incision was made to collect blood from the inferior vena cava, and to dissect the heart and aorta. Analysis of the project includes ECG, left ventricular thickness, cardiac TUNEL stain, Masson trichrome stain, western blot, aortic elastin stain, RT-PCR, plasma total cholesterol, triglyceride and malondialdehyde.
Result：D-galactose and natural aging groups had similar cardiovascular dysfunction. The heart rate of DG group was significantly lower than that of the control group. Based on the ECG, the RR interval and QTc was greater in the DG than the control group. In addition, the left ventricular thickness, cardiac TUNEL stain level, and the cardic protein levels of Bid, Bax, Cytochrome c was significantly greater in the NA group and DG group than the control group, respectively. Supplementation of KGM, FOS or vitamin E could ameliorate the D-galactose-induced alterations in the heart rate, cardiac TUNEL stain and apoptosis-related proteins. In the aorta, the elastic fiber nicks and the thickness of smooth muscle layer of NA or DG group was significantly greater than those of the control group. The LOX gene expression of the aorta was significantly lower in the NA and DG groups than that of the control group. Supplementation of KGM, FOS or vitamin E could diminish the D-galactose-induced vascular alteration in the morphology and the LOX gene expression. The plasma MDA, total cholesterol, and triglyceride in the NA and DG groups were significantly greater than that in the control group. Supplementation of KGM, FOS or vitamin E effectively decreased these alterations.
Conclusion：Supplementation of KGM, FOS and vitamin E effectively decreased the alterations caused by the D-galactose in the heart and aorta. These effects was likely to be mediated by the antioxidative and lipid-lowering effects of dietary fiber, which subsequently ameliorate the oxidatie stress-related intracellular apoptotic pathway, and increased the related gene expression of elasticity of blood vessels.

並列關鍵字

D-galactose ； aging ； konjec glucomannan ； fructo-oligosaccharide ； cardiovascular function ； apoptosis ； lysyl oxidase

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