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|標題:||(一) 奈米鈣化合物對大鼠生物利用率之探討(二) β-類澱粉蛋白干擾神經細胞葡萄糖之利用效應及橙皮素與橙皮&;#33527;之保護作用
1. Bioavailability of nano calcium in Sprague-Dawley rats2. Impairment of glucose transport by amyloid β-protein in neuron cells: Protective effects of hesperetin and hesperidin
|關鍵字:||奈米鈣;nano-calcium;血鈣;生物利用率;碳酸鈣;氯化鈣;阿茲海默症;β-類澱粉蛋白 (Aβ1-42);胰島素阻抗;糖尿病;葡萄糖吸收;plasma calcium;bioavailability;calcium carbonate (CaCO3);calcium chloride (CaCl2);Alzheimer's disease (AD);amyloid β-protein (Aβ1-42);insulin resistance;diabetes;glucose uptake||出版社:||食品暨應用生物科技學系所||摘要:||
本論文區分為兩大部份，第一部份乃探討奈米鈣化合物對大鼠生物利用率之影響，藉以釐清鈣質之生物利用率可否因奈米化處理後而提升。第二部份以細胞培養模式探討β-類澱粉蛋白 (amyloid β-protein, Aβ1-42) 對神經元細胞葡萄糖吸收之影響與介入橙皮素和橙皮&;#33527;之保護作用以及相關之分子機轉。
(一) 奈米科技 (nanotechnology) 於食品之應用日趨廣泛與多元，唯市面上多數奈米產品是將奈米物質以食品添加物形式與半成品混和製成，以此宣稱奈米化能提升產品吸收效果及生物利用率 (bioavailability)。然而生物利用率可否因其粒徑縮小而有所提升，目前仍具爭議且未有充足的文獻可循。本研究目的在探討市售之奈米鈣化合物與水溶性氯化鈣於大鼠體內鈣質生物利用率之差異。實驗設計給予 Sprague Dawley 大鼠奈米碳酸鈣、碳酸鈣和氯化鈣之鈣化合物 (100 mg Ca/kg of b.w.)，利用原子吸收光譜儀 (Atomic Absorption Spectrophotometer, AAS) 分析其於七小時內之血鈣濃度變化，以及給予奈米碳酸鈣、碳酸鈣後 24 小時內之尿鈣含量及糞便鈣含量之差異，並以電子顯微鏡與奈米粒子分析儀觀察樣品粒子之形態及粒徑分布情況。結果發現，奈米碳酸鈣的粒徑範圍廣 (100–1000 nm) 且有聚集與無法均勻分散於水溶液中之現象。在血鈣值方面，氯化鈣組其血鈣濃度上升幅度較其他兩種鈣化合物樣品為高，原因可能與其較佳之溶解性有關。碳酸鈣組之血鈣濃度 Tmax 為 60 min，較給樣前提高 4.4%。而給予奈米碳酸鈣之大鼠，其 Tmax 為 180 min，較攝取前增加 5.6% (p<0.05)，且與碳酸鈣組相比可維持較長時間之高血鈣濃度。於採樣時間內，給予 SD 大鼠碳酸鈣之尿鈣含量顯著地高於奈米碳酸鈣組 (p<0.05)，而糞便鈣含量則無明顯之差異。綜合上述，奈米化可增加鈣質於生理之滯留時間，鈣化合物溶解性為決定其生物利用率之關鍵，而奈米化與否則對生物利用率之影響較為有限，其原因可能與奈米粒子聚集而削弱表面積效應有關，有待進一步探討。
(二) 臨床研究顯示，糖尿病患者有較常人高之阿茲海默症 (Alzheimer’s disease, AD) 罹患率與急劇之 AD 病程發展，此現象可能與 β-類澱粉蛋白 (β-amyloid protein, Aβ) 誘發神經元細胞產生葡萄糖代謝異常有關。本研究利用 Neuro-2A 神經元細胞，探討 Aβ1-42 對該細胞葡萄糖吸收之影響及相關分子機轉，並介入柑橘屬橙皮素 (hesperetin) 與橙皮&;#33527; (hesperidin) 類黃酮化合物以評估其對神經細胞之保護效應。結果顯示，Aβ1-42 於 100–500 nM 作用劑量下，可顯著降低 Neuro-2A 神經元細胞之葡萄糖吸收；在胰島素訊息調控機轉方面，Aβ1-42 (500 nM) 可抑制胰島素膜受器之基因表現，與 protein kinase B (Akt) 蛋白之磷酸化，並降低葡萄糖運輸蛋白 (Glucose transporter proteins, GLUTs) GLUT3 和 GLUT4 之蛋白質表現量，但對於轉錄因子 Hypoxia-inducible factor 1α (HIF-1α) 則無顯著影響。此外，預先介入 hesperetin 和 hesperidin可顯著改善 Aβ1-42 對於神經元細胞葡萄糖吸收利用之干擾，並維持上述與胰島素訊息傳遞相關之基因與蛋白質之正常表現。綜合上述結果，柑橘屬類黃酮化合物可抑制 Aβ1-42 所誘導之神經元細胞胰島素訊息傳遞路徑與葡萄糖運輸蛋白 GLUT3 和 GLUT4 表現量之異常，進而改善其葡萄糖之吸收作用，因此推測可能具有延緩 AD 發展之保健功效。
This thesis includes two topics. The first topic was to study the bioavailability and uptake of nano-calcium in Sprague Dawley rats. The second topic was to study the protective effect of citrus flavonoids, hesperetin and hesperidin, against amyloid β-protein (Aβ1-42) induced damage and influencing glucose uptake in neuronal cells.
Chapter 1 was aimed to investigate the bioavailability of nano-calcium in Sprague-Dawley (SD) rats. The application of nanotechnology in food industry has become widely diverse. Numerous commercial nano-food products have been made by blending nanomaterials as food additives with half-finished goods. Nanosized ingredients usually be claimed to be able to enhance absorption and bioavailability in human body. Until now, the literature data concerning the effect of decreasing the particle size of bioactive compound in improving its bioavailability are limited. In this study, we investigated the difference of bioavailability of calcium between nano-calcium powder and calcium compounds. Sprague Dawley (SD) rats were orally administered with 100 mg Ca/kg of body weight of nanosized calcium carbonate, calcium carbonate or calcium chloride. Sequence blood samples were taken periodically up to 7 h, then the urine and fecal samples were collected after administration. The calcium contents in plasma, urine and fecal were analyzed using atomic absorption spectrophotometer (AAS). The characteristics of nano-particles were determined by tabletop electron microscope and dynamic laser particle size analyzer. Results showed that nano-calcium compounds with a wide range in particle size (100-1000 nm) tended to agglomerate and did not disperse into a nanoparticulate dispersion. The plasma calcium content of rats administered with calcium chloride was significantly higher than those of nanosized calcium carbonate and calcium carbonate groups because of its high solubility. The plasma calcium in rats administered with calcium carbonate reached the maximum concentration (Cmax), and was elevated by 4.4% (p<0.05) after 60 min (Tmax), whereas those with nano-calcium carbonate reached the Cmax after 180 min (increased by 5.6%, p<0.05). Nano-calcium carbonate group maintained a longer period of high blood calcium level than that of carbonate group. The urinary calcium of calcium carbonate group was significantly (p <0.05) higher than that of nano-calcium carbonate group; however, there was no significant difference in fecal calcium content. These results indicated that nanonizaton increased the retention time of calcium in rats. The solubility of calcium compounds was a determinant of calcium bioavailability. However, further studies were needed to explore the effect of nanonization on bioavailability from poorly soluble calcium compound due to agglomeration and the reduction of surface area.
Chapter 2 was aimed to investigate the protective effects of hesperetin and hesperidin against Aβ1-42-induced damage in neuronal cells and involved molecular mechanism. Clinical and experimental evidence have suggested that diabetics faced a higher risk of developing Alzheimer's disease (AD) and had a more rapid symptom development than nondiabetics. This phenomenon might be related to the abnormalities of basal glucose utilization in neuron cells caused by Aβ1-42. In this study, we investigated the effect of Aβ1-42 on glucose uptake in Neuro-2A cells and involved molecular mechanism. We also evaluated the neuroprotection of hesperetin and hesperidin which were the major citrus flavonoids against Aβ1-42-induced damage in Neuro-2A cells. Results showed that Aβ1-42 (100-500 nM) significantly inhibited the insulin stimulated glucose uptake in Neuro-2A cells. The possible mechanism underlying this action was that Aβ1-42 treated-neuron caused a decrease in the gene expression of insulin receptor and protein kinase B (Akt) activation in insulin signaling pathway. Additionally, the protein expressions of GLUT3 and GLUT4 were attenuated in Aβ1-42-treated cells which might be associated with the deficiency of glucose uptake. The expression of hypoxia-induced factor-1α (HIF-1α) and GLUTs genes had no change in Aβ1-42-treated neurons. Treatments with hesperetin or hesperidin could significantly improve the interference of glucose absorption in neuronal cells and inhibit the damage effects on the above gene and protein expressions in insulin signaling cascade caused by Aβ1-42. Therefore, the present study demonstrated that Aβ1-42 impaired the basal glucose uptake of neuronal cells via downregulating the expression of GLUT3 and insulin signaling cascade. Supplementation of citrus flavonoid compounds (hesperetin and hesperidin) could protect neuronal cells against Aβ1-42-induced damage. Results suggest that dietary citrus flavonoids might have potential to delay AD progression.
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