臺灣博碩士論文加值系統

肝臟是人體代謝各類物質的重要器官，當肝臟發生問題、無法進行代謝與解毒等作用時，便產生疾病。中草藥近年在對抗肝病的治療上備受重視，本研究於四氯化碳所誘導之急性與慢性肝損傷動物模式中，觀察中醫常用之肝病治療方劑—柴胡疏肝散變方－對於肝臟解毒系統及抗氧化酵素系統之影響。實驗共分為正常控制組、四氯化碳處理組、柴胡疏肝散變方低劑量中藥組及柴胡疏肝散變方高劑量中藥組；並以管餵方式、每週二次、連續八週給予大鼠50 %四氯化碳（1 ml/Kg），以進行慢性肝損傷誘導。急性肝損傷實驗部分，則在給予柴胡疏肝散變方連續十天後，以管餵方式給予大鼠50 %四氯化碳一次（2 ml/Kg）。除了肝功能血液生化值（AST、ALT）之外，保肝解毒療效之評估指標還包括：脂質過氧化（lipid peroxidation），抗氧化酵素之過氧化氫酶（catalase, CAT）、穀胱甘肽過氧物酶（glutathione peroxidase, GPx）、超氧物歧化酶（superoxide dismutase, SOD），解毒酵素系統中第一階段（phase I）之細胞色素P450（cytochrome P450, CYP450）相關亞型，及第二階段（phase II）穀胱甘肽轉移酶（glutathione S-transferase, GST）。實驗結果發現在慢性肝損傷實驗中，柴胡疏肝散變方高劑量中藥組之AST、ALT低於四氯化碳組（p<0.05），且抗氧化酵素CAT之活性高於四氯化碳組（p<0.05）。GST活性在柴胡疏肝散變方低劑量中藥組高於四氯化碳組（p<0.05），且柴胡疏肝散變方可能會減緩CYP 2E1之降解。而在急性肝損傷實驗中，柴胡疏肝散變方高劑量中藥組之GPx活性明顯高於四氯化碳組（p<0.05）。因此推論柴胡疏肝散變方可藉由增強部分抗氧化酵素之活性及對解毒酵素系統CYP及GST之調節，減低四氯化碳對生物體所造成之傷害，達到保肝之療效。

Liver plays an important role in human metabolism and detoxification. When the metabolic pathways in liver fails, diseases occurr. Traditional Chinese herbal medicine has been a spotlight of research interests in recent years. This study focused on the effect of chi-hu-shu-gan (CHSG) powder, a traditional Chinese herbal medicine commonly used in the treatment of liver diseases by doctors, on hepatic antioxidation system and detoxification system of Sprague-Dawley rats whose chronic or acute liver injury had been induced by carbon tetrachloride (CCl4). Animals were divided into four groups, including normal control group, CCl4-treated only group and two herb-treated groups with CHSG low (1.26 g/Kg) and high dose (6.23 g/Kg). In chronic liver injury model, rats received 50 % CCl4 (1 ml/Kg) orally twice a week for eight weeks while in acute liver injury model after ten days of herbal treatment the rats received a single dose of 50 % CCl4 (2 ml/Kg). The detoxificative and hepatoprotective effects were evaluated by following analyses: (1) lipid peroxidation analysis, (2) antioxidative enzymes assays, including catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), (3) detoxification enzyme analyses, including cytochrome P450s (CYP) of phase I and GST of phase II, (4) activity assays of liver-specific cytosolic enzymes AST, ALT. In chronic liver injury model, the data showed that high dose of CHSG significantly increased AST, ALT and CAT activity and prevented CYP2E1 from degradation as compared with the CCl4 group (p<0.05). Besides, the GST activity of the CHSG low dose group was higher than that of the CCl4 group (p<0.05). In the acute liver injury model, the GPx activity of high dose CHSG group was much higher than that of the CCl4 group (p<0.05). All together, it suggested that the hepatoprotective effect of the modified-CHSG powder was resulted from the enhancement of the activities of some antioxidative enzymes and modulation of detoxification system, which in turn may lower the damage caused by CCl4.

目錄………………………………………………………………………………....I
表次………………………………………………………………………………...V
圖次………………………………………………………………………………..VI
第一章 緒論………………………………………………………………………..1
1.1肝臟之生理功能……………………………………………………………2
1.1.1內分泌功能………………………………………………………….3
1.1.2凝血功能…………………………………………………………….3
1.1.3血漿蛋白質……………………………………………………….…3
1.1.4消化功能…………………………………………………………….3
1.1.5有機物的代謝………………………………………………….……4
1.1.6膽固醇代謝…………………………………………………….……4
1.1.7排除及分解功能…………………………………………………….4
1.2肝臟之解毒酵素系統……………………………………………………....5
1.2.1 Phase I 酵素系統……………………………………………………6
1.2.1.1氧化還原酵素………………………………………………...6
1.2.1.1.1 CYP之介紹……………………………………………6
1.2.1.1.2 CYP之命名……………………………………………7
1.2.1.1.3 CYP在生物體所扮演之角色…………………………8
1.2.1.2水解酵素……………………………………………………...8
1.2.1.2.1 Esterase………………………………………………...8
1.2.1.2.2 Epoxide Hydrolase…………………………………….9
1.2.2 Phase 酵素系統……………………………………………………9
1.2.2.1 Glucuronidation……………………………………………..10
1.2.2.2硫酸化與穀胱甘肽結合反應……………………………….10
1.2.2.3甲基化……………………………………………………….11
1.2.2.4乙基化……………………………………………………….11
1.2.2.5氨基酸結合反應…………………………………………….11
1.3抗氧化防禦系統…………………………………………………………..12
1.3.1超氧物歧化酶（SOD）……………………………………………13
1.3.2過氧化氫酶（CAT）………………………………………………14
1.3.3穀胱甘肽過氧物酶（GPx）……………………………………....14
1.4肝損傷動物模式…………………………………………………………..15
1.5中醫傳統方劑－柴胡疏肝散變方…………………………………….…16
1.6研究動機與目的…………………………………………………………..17
第二章 實驗設計、材料與方法………………………………………………….18
2.1柴胡疏肝散變方的製備…………………………………………………..18
2.1.1. 組成……………………………………………………………….18
2.1.2. 製備方法………………………………………………………….18
2.1.3. 劑量配置………………………………………………………….18
2.2實驗動物…………………………………………………………………..19
2.2.1以四氯化碳誘導大鼠慢性肝損傷動物模式中－柴胡疏肝散變方之保肝解毒療效評估……………………………………………………19
2.2.1.1. 實驗動物分組…………………………………………….19
2.2.1.2. 慢性肝損傷誘導方式…………………………………….19
2.2.1.3. 實驗流程………………………………………………….19
2.2.2以四氯化碳誘導大鼠急性肝損傷動物模式中－柴胡疏肝散變方之保肝解毒療效評估……………………………………………..……..20
2.2.2.1. 實驗動物分組……………………………………………..20
2.2.2.2. 急性肝損傷誘導方式……………………………………..20
2.2.2.3. 實驗流程……………………………………………….….20
2.3實驗樣品之前處理……………………………………………………….21
2.3.1. 血液前處理………………………………………………….…21
2.3.2. 肝臟樣品之前處理及製備…………………………………….21
2.3.3. 蛋白質之定量………………………………………………….21
2.4保肝與解毒療效評估指標………………………………………………..22
2.4.1. 脂質過氧化（lipid peroxidation）之分析…………………….22
2.4.2. Catalase（CAT）活性之測定…………………………………22
2.4.3. Glutathione Peroxidase（GPx）活性之測定……………….…23
2.4.4. Superoxide Dismutase（SOD）活性之測定………………….23
2.4.5. Glutathione S-Transferase（GST）活性之測定………………23
2.4.6. Cytochrome P450（CYP）之定量分析……………………….24
2.4.7. 血液生化值測定……………………………………………….25
2.4.8. 統計分析……………………………………………………….25
第三章 以四氯化碳誘導大鼠慢性肝損傷模式評估柴胡疏肝散變方之保肝解毒療效………………………………………………………………………26
3.1肝功能之評估……………………………………………………………..26
3.2抗氧化能力評估…………………………………………………………..27
3.2.1脂質過氧化程度（MDA）之分析………………………………..27
3.2.2肝臟抗氧化酵素活性之分析……………………………………...28
3.3肝臟解毒能力之評估…………………………………………………….29
3.3.1 GST 酵素活性之分析…………………………………….............30
3.3.2 Cytochrome P450 2E1、3A1、3A2蛋白質表現之分析…………30
第四章 以四氯化碳誘導大鼠急性肝損傷模式評估柴胡疏肝散變方之保肝解毒療效………………………………………………………………………32
4.1肝功能之評估…………………………………………………………….32
4.2抗氧化能力評估………………………………………………………….32
4.2.1脂質過氧化程度（MDA）之分析………………………………..32
4.2.2肝臟抗氧化酵素活性之分析………………………………...........33
4.3肝臟解毒能力之評估……………………………………………………..34
4.3.1GST酵素活性之分析………………………………..……………..34
4.3.2 Cytochrome P450 2E1、3A1、3A2蛋白質表現之分析…………34
第五章 結論………………………………………………………………………36
圖表………………………………………………………………………………...37
附錄………………………………………………………………………………...43
參考文獻…………………………………………………………………………...46

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