臺灣博碩士論文加值系統

肥胖為已開發中國家常見之文明病之一，其造成高血脂和糖尿病等代謝症候群相關疾病。肥胖可能使體內慢性發炎並提高 ROS 程度，導致精子品質降低、睪丸損傷和性荷爾蒙分泌異常，造成男性不孕。故本研究以牛樟芝子實體探討高脂飲食誘發倉鼠肥胖產生高血脂、慢性發炎和氧化壓力造成男性生殖功能損傷之保護功效。將牛樟芝分別以熱水萃取 (ACW) 和乙醇萃取 (ACE)，顯示出其熱水萃取物具有良好的抗氧化能力。而細胞實驗以 LPS 誘發巨噬細胞發炎反應，顯示 ACW 和 ACE (25-125 g/ml) 可抑制細胞發炎反應和降低 ROS 生成量。以高脂飲食誘發倉鼠肥胖之動物模式牛樟芝子實體 (0.1, 0.2 和 0.5 g/kg．BW) 給予後其結果可降低肥胖倉鼠之三酸甘油酯、膽固醇和血糖，抑制促發炎細胞激素含量和 NO 產生，降低 MDA 含量提高抗氧化酵素活性，並有效提升精子品質。本研究證實牛樟芝子實體可降低高脂飲食引起之肥胖發炎反應，降低肥胖倉鼠睪丸和精子 ROS 的表現，對於調節血脂、抗發炎和抗氧化活性，並且延長餵食時間於低劑量下可降低肥胖之慢性發炎保護肥胖男性生殖功能的傷害。

Obesity has increased drastically in recent years and is currently considered to be a major risk factor for diabetes and dyslipidemia. The development of obesity and excessive fat accumulation leads to the macrophages infiltrate WAT, causing a chronic inflammation as well as oxidative stress. Antrodia cinnamomea (AC) is a medicinal mushroom used for the anti-inflammatory, anti-oxidative and anti-hyperlipidemic. But male obesity induced chronic inflammatory and oxidative stress on sperm and testis damage of this material has not yet been investigated. In this study, the mechanism of wood cultivation AC fruiting bodies extract by hot water (ACW) and ethanol (ACE) (25-125 g/ml) reduced on LPS-induced macrophage cell inflammatory and intracellular ROS content. The protective effects of AC (0.1-0.5 g/kg) against HFD-induced obesity in male Syrian hamsters also been conducted. AC inhibited HFD induced NO, TNF-, IL-6, and MDA production in obesity hamsters’ serum and testicular tissue. Treatment with AC reduced the control level of TG, TC, blood glucose and LDL in the HFD induced obesity group serum. The AC was reversed sperm quality to control levels with HFD groups. The HFD induced decline in the levels of antioxidant enzymes (CAT, SOD, and GPX) and were significantly reversed to control group but HFD induced obesity group serum and testicular tissue were preceded by the administration of AC (0.5 g/kg) has higher levels compared to control. The protective effects of AC suggested to be mediated by their potent antioxidant activities, and provided significant amelioration of oxidative stress parameters.

謝誌 I
名詞縮寫 (Abbreviation) II
摘要 III
Abstract IV
目錄 V
圖目錄 IX
表目錄 XI
附錄 XII
壹、緒論 1
貳、文獻回顧 3
一、男性不孕症 3
(一)不孕症定義 3
1. 內分泌系統 3
2. 睪丸 3
3. 先天性染色體的異常 4
4. 環境 4
5. 疾病 4
6. 生活因素 5
二、精子和氧化壓力 5
(一) 氧化壓力 6
(二) 反應性氧化物和精子功能 6
(三) 氧化壓力對精子粒線體之影響 7
(四) 氧化壓力對精子 DNA 之影響 8
三、抗氧化物和精子之關係 8
(一) 非酵素型防禦系統 8
1. 維生素 C 8
2. Ubiquinol (coenzyme Q) 9
(二) 酵素型防禦系統 9
1. 超氧化物岐化酶 9
2. 觸酶 10
3. 麩胱甘肽過氧化酶 10
四、肥胖與代謝症候群 10
五、男性肥胖和精子之關係 11
六、牛樟芝 (Antrodia cinnomomea) 12
(一)牛樟芝之簡介 13
(二)牛樟芝之生理活性成分 13
1. 多醣體 14
2. 三萜類化合物 15
3. 超氧歧化酶 16
(三) 牛樟芝之生理活性 17
1. 抗氧化活性 17
2. 抗發炎活性 17
3. 抗腫瘤 18
4. 保肝能力 18
5. 預防心血管疾病 18
参、實驗設計 20
一、完整實驗流程 20
二、ACW 和 ACE 萃取流程 21
三、細胞實驗設計 21
四、動物實驗 22
(一) 實驗動物分組 22
(二) 動物實驗設計 22
肆、實驗材料與方法 23
一、材料與藥品 23
(一) 材料來源 23
(二) 細胞株 23
(三) 實驗動物 23
(四) 動物飼料 23
二、藥品 24
三、實驗方法 28
(一) 牛樟芝子實體萃取物之製備 28
1. 牛樟芝子實體水萃取物之製備 28
2. 牛樟芝子實體乙醇萃取物之製備 28
(二) 牛樟芝子實體萃取物之總醣含量測定 28
(三) 牛樟芝子實體萃取物之抗氧化活性分析 29
1. 清除 DPPH 自由基能力之測定 29
2. 還原力之測定 30
3. 螯合亞鐵離子能力之測定 30
(四)牛樟芝子實體萃取物抗發炎之功能評估－細胞實驗 31
1.細胞株解凍 31
2.細胞培養 31
3.細胞存活率測定 (MTT assay) 32
4. NO 釋放測定 (NO release) 33
5.酵素免疫連結法測定細胞激素 IL-6 與 TNF- 33
6. 利用反轉錄聚合酶連鎖反應分析 COX-2 和 iNOS 之表現 35
7.利用流式細胞儀分析細胞內 ROS含量 39
8.利用流式細胞技術分析細胞內粒線體膜電位 ΔΨm 40
(五)牛樟芝子實體對肥胖雄性倉鼠生殖功能之評估-動物實驗 41
1. 動物飼養分組和樣品處理 41
2. 實驗動物分組 42
3. 動物犧牲、採血和臟器摘取 43
4. 倉鼠血液中血脂的測定 44
5. 倉鼠血液中血糖之測定 45
6. 倉鼠血液中胰島素之測定 46
7. 倉鼠血清功能性指標之評估 47
8. 倉鼠精子功能性指標之評估 50
8. 倉鼠睪丸功能性指標之評估 53
9. 統計方法 56
伍、結果與討論 57
一、牛樟芝子實體萃取物之製備 57
二、牛樟芝子實體萃取物中粗多醣之含量 57
三、牛樟芝子實體萃取物之抗氧化活性分析 57
(一) 清除 DPPH 自由基能力之測定 57
(二) 還原力之測定 58
(三) 螯合亞鐵離子能力之測定 58
四、牛樟芝子實體萃取物抗發炎之功能評估－細胞實驗 59
(一) 牛樟芝萃取物對細胞存活率之探討 59
(二) 牛樟芝萃取物對 LPS 誘發細胞產生 NO 之影響 59
(三) 牛樟芝萃取物對 LPS 誘發細胞產生細胞激素之影響 60
(四) 牛樟芝萃取物對 LPS 誘發細胞產生 iNOS 和 COX-2 mRNA 之表現 61
(五) 牛樟芝萃取物對胞內 ROS 產生之影響 62
(六) 牛樟芝萃取物對細胞粒線體膜電位之影響 63
五、牛樟芝子實體對高脂飲食誘導倉鼠男性生殖功能之影響 64
(一) 倉鼠每日飲食攝取量、每日熱量攝取和每週體重之變化 64
(二) 牛樟芝子實體對於高脂飲食誘導倉鼠體重、肝、腎、副睪脂肪和腎週邊脂肪重量之影響 65
(三) 牛樟芝子實體對於高脂飲食誘導倉鼠腹部脂肪含量之影響 66
(四) 牛樟芝子實體對於高脂飲食誘導倉鼠生殖器官之影響 66
(五) 牛樟芝子實體對於高脂飲食誘導倉鼠血脂之影響 66
(六) 牛樟芝子實體對於高脂飲食誘導倉鼠血糖之影響 68
(七) 牛樟芝子實體對於高脂飲食誘導倉鼠血漿、睪丸和精子中發炎物質產生之影響 69
(八) 牛樟芝子實體對於高脂飲食誘導倉鼠血漿、睪丸和精子中脂質過氧化之影響 71
(九) 牛樟芝子實體對於高脂飲食誘導倉鼠精子 ROS 生成之影響 72
(十) 牛樟芝子實體對於高脂飲食誘導倉鼠精子粒線體膜電位之影響 74
(十一) 牛樟芝子實體對於高脂飲食誘導倉鼠精子特性之影響 74
(十二) 牛樟芝子實體對於高脂飲食誘導倉鼠血漿和睪丸組織中抗氧化酵素活性之影響 75
(十三) 牛樟芝子實體對於高脂飲食誘導倉鼠睪固酮含量之影響 76
(十四) 牛樟芝子實體對於高脂飲食誘導倉鼠製精小管型態和生殖細胞厚度之影響 76
(十五) 牛樟芝子實體對於高脂飲食誘導倉鼠脂肪組織型態之影響 77
陸、結論 78
柒、參考文獻 80
捌、圖表 105
附錄 153

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