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研究生:林湘婷
論文名稱:微脂體蝦紅素對脂多醣誘導引起肝與腎損傷之保護作用
論文名稱(外文):The Protective Effects of Liposome-Encapsulated Astaxanthin on Lipopolysaccharide-Induced Liver and Kidney Damages
指導教授:喬長誠 教授邱駿紘 助理教授
學位類別:碩士
校院名稱:弘光科技大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
畢業學年度:100
語文別:中文
論文頁數:97
中文關鍵詞:蝦紅素微脂體脂多醣抗發炎抗氧化
外文關鍵詞:AstaxanthinLiposomeLipopolysaccharideAnti-inflammationAnti-oxidation
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本研究的目標在於發展出針對改善脂多醣誘導肝及腎臟組織損傷的微脂體蝦紅素傳輸系統。我們假設蝦紅素包覆於微脂體中可以促進其對脂多醣作用於實驗動物的治療效用。在微脂體蝦紅素製備中,蛋黃磷脂質與蝦紅素經高壓均質化後形成直徑為240 ±58 nm球狀粒子,其包埋率為58.06±8.0%。由表面電位和場發射掃瞄式電子顯微鏡,顯示所製備的微脂體蝦紅素,在溶液中有較好的穩定性及分散性。在動物研究中,雄性SD大鼠以口服方式連續7天給予生理食鹽水、N-acetyl cysteine (NAC 200 mg/kg B.W.)、蝦紅素( 10 mg/kg B.W.)或蝦紅素微脂體(2,5,10 mg/kg B.W.),於第七天以腹腔注射方式施打脂多醣 (5 mg/kg B.W.),12小時後犧牲實驗動物進行分析。肝臟和腎臟損傷藉由測量血清中麩草酸轉胺基酶(GOT)、麩丙酮轉胺基酶 (GPT)、尿素氮和肌酸酐的濃度來判斷。脂多醣活化發炎反應乃藉由分析血清中一氧化氮、腫瘤壞死因子(TNF-α)和介白素-6的濃度及誘導型一氧化氮合成酶蛋白質表現量來評估。組織中脂質過氧化產物 (丙二醛) 的含量和抗氧化酵素活性(超氧歧化酶、過氧化氫酶和麩胱苷肽過氧化酶)被用來分析氧化壓力機制參予的程度。
結果發現,當與控制組比較,LPS誘導組中發炎反應調控因子濃度的上升(如一氧化氮濃度增加5.7倍、腫瘤壞死因子濃度增加4.5倍和介白質-6濃度增加2.8倍)、脂質過氧化產物含量的增加(肝臟及腎臟丙二醛濃度分別增加3.2倍和2.5倍)、抗氧化酵素活性的降低(組織中超氧歧化酶活性下降1.6-1.8倍、組織中過氧化氫酶活性下降2.4-2.8倍和組織中麩胱苷肽過氧化酶活性下降1.2倍),及嚴重性的組織損傷,都顯示僅以脂多醣處理的動物會造成組織傷害。以抗氧化物 (NAC、蝦紅素及蝦紅素微脂體)前處理的動物,對脂多醣的挑戰都有明顯的保護作用。在給予劑量為10 mg/kg B.W. 的蝦紅素微脂體在減緩脂多醣所造成組織傷害的效果上,顯著地比NAC (200 mg/kg B.W.) 或游離型蝦紅素 (10 mg/kg B.W.) 效果更佳,包括一氧化氮濃度降低 (74.2 v.s.55.0 v.s. 56.0%)、腫瘤壞死因子濃度濃度降低(74.4% v.s. 56.8% v.s. 56.1%) 和介白素-6濃度降低 (55.2 v.s. 42.5 v.s. 27.8%)、肝臟丙二醛濃度下降(62.6 v.s. 55.5 v.s. 53.8%)與腎臟丙二醛濃度下降(55.4 v.s. 35.1 v.s. 31.7%)。此外,蝦紅素微脂體(10 mg/kg B.W.)增加抗氧化酵素活性的能力優於NAC或游離型蝦紅素,而抑制誘導型一氧化氮合成酶蛋白質表現上也比NAC或游離型蝦紅素效果好。結果顯示,蝦紅素微脂體的製備,明顯較游離型蝦紅素有效防止脂多醣誘導組織的損傷,並以透過抗發炎及抗氧化之機制,提供有效的保護作用。

In the present study, we aimed at developing liposomal delivery system for Astaxanthin (Ast) targeting in ameliorating lipopolysaccharide (LPS)-induced hepatic and renal damages. We hypothesized that encapsulation of Ast in liposomes might promote the therapeutic potential against experimental animals challenged with LPS. For the preparation of liposomal encapsulated Ast, the egg yolk phospholipids (EPC) and Ast were homogenized with high pressure homogenizer to form spherical particles with diameter of 240 ± 58 nm. The encapsulation efficacy is calculated as 58.06 ± 8.0%. From the zeta potential and field-emission electron scanning microscopy (FE-SEM) analysis, it was suggested that the prepared liposomal Ast could be formed as a stable dispersion in solution. During the in vivo experiment, male Sprague-Dawley rats were pretreated orally with saline, N-acetyl cysteins (NAC200 mg/kg B.W.), Astaxanthin (10 mg/kg B.W.) or liposomal Ast (2, 5 and 10 mg/kg B.W.) for 7 consecutive days. LPS (5 mg/kg B.W.) was introduced intraperitoneally at the 7th day and animals were sacrificed 12 h post-LPS challenge for analysis. Hepatic and renal damages were evaluated by measuring GOT (Glutamic oxaloacetic transaminase), GPT( Glutamic pyruvic transaminase), BUN(Blood urea nitrogen) and CRE (Creatinine) in serum. LPS-induced activation of the inflammatory response was evaluated by measuring the levels of serum NO, TNF-α and IL-6 as well as iNOS protein expression. The tissue levels of lipid peroxidation product(malondialdehyde) and the activities of antioxidant enzymes (SOD, CAT and GSH-Px) were used to assess the extent of involvement of oxidative stress mechanisms.
The challenge of animals with LPS resulted significantly in tissue damages, as evidenced by the activation of inflammatory responses (for example; NO, TNF-α and IL-6 increased 5.7, 4.5 and 2.8 fold, respectively); increase in the level of lipid peroxidation product (hepatic and renal MDA increased 3.2 and 2.5 fold, respectively); decrease in the activities of antioxidant enzymes (tissue SOD activities reduced 1.6-1.8 fold, CAT activities reduced 2.4-2.8 fold and GSH-Px activities reduced 1.2 fold) and enhanced histological injuries as shown in histology, as compared to the control group. Pretreatment of animals with antioxidants (NAC, Ast or liposomal Ast) resulted in the significant protection against LPS challenges. Moreover, liposomal Ast (10 mg/kg B.W.) was demonstrated to be more effective than NAC (200 mg/kg B.W.) or free Ast (10 mg/kg B.W.) in attenuating the LPS-induced tissue injuries (for example; NO decreased as 74.2% v.s. 55.0% v.s. 56.0%; TNF-α decreased as 74.4% v.s.56.8% v.s. 56.1% and IL-6 decreased as 55.2% v.s. 42.5% v.s. 27.8%; hepatic MDA reduced as 62.6% v.s. 55.5% v.s.53.8% and renal MDA reduced as 55.4% v.s.35.1% v.s.31.7%, respectively ). In addition, the effects of liposomal Ast (10 mg/kg B.W.) on increasing the activities of antioxidant enzymes and inhibiting iNOS protein expression are better than NAC or free Ast. In conclusion, the results suggested that liposomal preparation of as Ast provided effective protection via anti-inflammatory and antioxidant mechanisms against LPS-induced tissue damages.

目錄…………………………………………………………………………………………I
圖目錄………………………………………………………………………………………IV
表目錄………………………………………………………………………………………V
中文摘要……………………………………………………………………………………VI
英文摘要…………………………………………………………………………………VIII
第一章 序論……………………………………………………………………………1
第二章 文獻回顧………………………………………………………………………3
第一節 蝦紅素(Astaxanthin)………………………………………………………3
第二節 微脂體(Liposome)……………………………………………………………8
第三節 脂多醣(Lipopolysaccharide)…………………………………………12
第四節 一氧化氮(NO)及誘導型一氧化氮合成酶(iNOS)…………………………16
第五節 介白素-6(Interleukin-6; IL-6)與腫瘤壞死因子-α (Tumor necrosisfactor;TNF-α)…………………………………………………………19
第六節 自由基氧化傷害及對生物體的傷害…………………………………………20
第七節 生物體內抗氧化防禦系統……………………………………………………23
第三章 研究目的與實驗架構……………………………………………………………25
第四章 材料與方法………………………………………………………………………27
第一節 實驗試劑與材料………………………………………………………………27
第二節 微脂體蝦紅素之製備…………………………………………………………30
第三節 微脂體蝦紅素之含量分析……………………………………………………30
第四節 粒徑測定………………………………………………………………………32
第五節 表面電位 (Zeta Potential) 之特性分析……………………………32
第六節 微脂體蝦紅素之表面形態及微結構觀察……………………………………32
第七節 動物的來源分組與實驗………………………………………………………33
第八節 實驗動物血液生化值測定……………………………………………………34
第九節 一氧化氮﹙NO﹚濃度分析……………………………………………………34
第十節 酵素免疫分析法………………………………………………………………35
第十一節 總蛋白質含量測定…………………………………………………………35
第十二節 組織脂質過氧化分析………………………………………………………36
第十三節 組織抗氧化酵素活性測試…………………………………………………36
1. 組織均質液之製備…………………………………………………………………36
2. 組織超氧歧化酶 (Superoxide dismutase; SOD)之活性分析…………37
3. 組織過氧化氫酶 (Catalase; CAT) 之活性分析……………………………38
4. 組織麩胱苷肽過氧化酶 (Glutathione peroxidase;GSH-Px)
之活性分析………………………………………………………………………………39
第十四節西方墨點法 (Western blot)……………………………………………40
第十五節組織切片 (Tissue section)……………………………………………42
第十六節統計分析………………………………………………………………………42
第五章結果…………………………………………………………………………………43
第一節 微脂體蝦紅素中蝦紅素含量之分析……………………………………………43
第二節 微脂體蝦紅素粒徑範圍測定……………………………………………………44
第三節 微脂體蝦紅素之表面電位測定…………………………………………………45
第四節 微脂體蝦紅素之表面形態及微結構觀察………………………………………46
第五節 各組大鼠體重變化及組織重量比………………………………………………47
第六節N-Acetylcysteine、游離型蝦紅素以及不同濃度之微脂體蝦紅
素之投予對大鼠血清中麩草酸轉胺基酶(GOT)及麩丙胺酸轉胺基(GPT)的影響…48
第七節 N-Acetylcysteine、游離型蝦紅素以及不同濃度微脂體蝦紅素
之投予對大鼠血清中尿素氮﹙BUN﹚及肌酸酐﹙CRE﹚的影響………………………51
第八節 N-Acetylcysteine、游離型蝦紅素以及不同濃度之微脂體蝦紅
素之投予對大鼠血清中一氧化氮 (NO)生成影響……………………………54
第九節 N-Acetylcysteine、游離型蝦紅素以及微脂微脂體蝦紅素之投
予對大鼠血清中發炎前驅物質生成的影響……………………………………………56
第十節 N-Acetylcysteine、游離型蝦紅素以及微脂體蝦紅素之投予對
大鼠組織中脂質過氧化的影響…………………………………………………………59
第十一節 N-Acetylcysteine、游離型蝦紅素以及微脂體蝦紅素之投予
對大鼠組織中超氧歧化酶 (SOD) 活性的影響………………………………………62
第十二節 N-Acetylcysteine、游離型蝦紅素以及微脂體蝦紅素之投予
對大鼠組織中過氧化氫酶 (CAT) 活性的影響………………………………………65
第十三節 N-Acetylcysteine、游離型蝦紅素以及微脂體蝦紅素之投予
對大鼠組織中麩胱苷肽過氧化酶 (GSH-Px) 活性的影響…………………………68
第十四節 N-Acetylcysteine、游離型蝦紅素以及微脂體蝦紅素之投予
對大鼠組織中誘導型一氧化氮合成酶(iNOS)蛋白質表現的影響…………………70
第十五節 N-Acetylcysteine、游離型蝦紅素以及微脂體蝦紅素對肝臟及腎臟 組織型態的影響…………………………………………………………………………72
第六章討論………………………………………………………………………………76
第七章結論………………………………………………………………………………82
第八章參考文獻…………………………………………………………………………84


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