薑黃素或/和柴胡皂素a對四氯化碳誘發老鼠肝損傷之抑制效果及其機制探討

• Main Author: 吳淑如
• Other Authors: 藥學系(博士班)
• Language: Chinese; English
• Published: 2010
• Subjects: 薑黃素 柴胡皂素a 抗氧化 發炎反應 纖維化; Indian; Moreno; Andreas; Crawford; Wilkins; Lowry; Randall; Levy; Sirius; Fang; The Portal; Ulmer; Maher; Rivera; Bello; Chiang; Prieto; Leet; Segovia; Larrea; Valla; Espinoza; Huet; Terranova
• Online Access: http://libir.tmu.edu.tw/handle/987654321/36339

(一)論文目次 目錄 摘要 Abstract 目錄 圖目次 表目次 第一章 緒論 第二章 文獻回顧 第一節 肝臟結構與細胞組成 第二節 四氯化碳誘發肝傷害之模式 第三節 肝纖維化之形成機轉 第四節 氧化壓力與肝損傷 第五節 肝損傷與相關細胞激素的表現 一、 腫瘤壞子因子-α 二、 介質素-1β 三、 介質素-6 四、 介質素-10 五、 轉化生長因子-β1 第六節 薑黃素之生理功能及相關研究 一、薑黃素簡介 二、薑黃素之藥理功能 第七節 柴胡皂素a之生理功能及相關研究 一、柴胡皂a素簡介 二、柴胡皂a之藥理功能 第三章 實驗材料與方法 第一節 薑黃素和柴胡皂素a劑量的決定 第二節 實驗動物 第三節 實驗設計 第四章 測量項目 第一節 病理組織切片分析 第二節 免疫組織染色分析 第三節 肝功能指標測定 一、 血漿ALT活性 二、 血漿AST活性 第四節 血漿脂質濃度的測定 一、血漿中總膽固醇(total cholesterol; TC)濃度 二、血漿中三酸甘油酯(triglycerides; TG)濃度 第五節 脂肪變性指標 一、肝臟中總膽固醇濃度 二、肝臟中甘油酯濃度 第六節 肝臟抗氧化能力指標 一、肝臟抗氧化酵素活性 二、肝臟抗氧化物質濃度 三、肝臟脂質過氧化物濃度 四、肝臟總抗氧化狀態 第七節 肝臟發炎反應指標測定 一、肝臟腫瘤壞死細胞-α濃度 二、介質素-1β濃度 三、介質素-6濃度 四、介質素-10濃度 第八節 肝臟纖維化指標測定 一、肝臟轉化生長因子-β1濃度 二、肝臟膠原蛋白前驅物含量 第九節 統計方法 第五章 結果 第一節 薑黃素和柴胡皂素a劑量的決定 第二節 老鼠體重、攝食量、絕對與相對肝臟重量 第三節 肝臟組織病理切片 一、Hematoxylin-eosin (H&E) 染色 二、Masson trichrome 與 silver染色 第四節 免疫組織染色法 一、 肝臟組織切片Sirius red染色 二、 肝臟組織NF-κB免疫染色 第五節 血漿肝功能指標 第六節 脂質代謝 一、血漿脂質濃度 二、肝臟脂質濃度 第七節 肝臟抗氧化能力 一、抗氧化酵素活性 二、抗氧化物質濃度 三、脂質過氧化物濃度 四、總抗氧化狀態 第八節 肝發炎反應指標 一、TNF-α之含量 二、IL-1β之含量 三、IL-6之含量 四、IL-10之含量 第九節 肝纖維化指標 一、肝臟TGF-β1濃度 二、肝臟膠原蛋白前驅物hydroxyproline濃度 第六章 討論 第一節 薑黃素、柴胡皂素a 的劑量 第二節 體重與肝臟相對重量 第三節 免疫組織染色法 第四節 肝功能指數 第五節 脂質代謝 第六節 肝臟抗氧化能力 第七節 肝臟發炎反應與纖維化情形 第八節 探討薑黃素與柴胡皂素a減輕肝傷害之可能機制 第七章 總結 第八章 參考文獻 圖目次 圖一 肝小葉的構造 圖二 肝細胞的構造 圖三 肝臟受損時肝細胞的變化 圖四 四氯化碳代謝誘發肝傷害 圖五 肝纖維化過程 圖六 氧化壓力與肝纖維化之機制 圖七 肝纖維化過程與相關細胞激素的表現 圖八 薑黃素構造圖 圖九 柴胡皂素 a 結構式 圖十 實驗流程簡圖 圖十一 薑黃素和柴胡皂素a 對老鼠正常肝臟細胞 超氧歧化酶活性之影響 圖十二 薑黃素和柴胡皂素a 對老鼠正常肝臟細胞 過氧化氫酶活性之影響 圖十三 薑黃素和柴胡皂素a 對老鼠正常肝臟 脂質過氧化物MDA濃度之影響 圖十四 病理切片(H&E染色) 圖十五 病理切片(Masson染色) 圖十六 病理切片(silver染色) 圖十七 薑黃素或/和柴胡皂素a 對各組老鼠肝臟 組織病理切片H&E染色積分圖 圖十八 薑黃素或/和柴胡皂素a 對各組老鼠肝臟組織 病理切片Masson trichrome與silver染色積分圖 圖十九 免疫組織染色(Sirius red染色) 圖二十 免疫組織染色(NF-κB染色) 圖二十一 薑黃素或/和柴胡皂素a 對各組老鼠肝臟 組織Sirius red染色積分圖 圖二十二 薑黃素或/和柴胡皂素a 對各組老鼠肝臟 組織NF-κB染色積分圖 圖二十三 薑黃素或/和柴胡皂素a 對各組老鼠血漿 (A)ALT與(B)AST活性(IU/L)之影響 圖二十四 薑黃素或/和柴胡皂素a 對各組老鼠血漿 (A)總膽固醇與(B)三酸甘油酯濃度之影響 圖二十五 薑黃素或/和柴胡皂素a 對各組老鼠肝臟 中總膽固醇與三酸甘油酯濃度之影響 圖二十六 薑黃素或/和柴胡皂素a 對各組老鼠肝臟 (A)SOD與(B)CAT活性之影響 圖二十七 薑黃素或/和柴胡皂素a 對各組老鼠肝臟 麩胱甘肽氧化還原狀態之影響 圖二十八 薑黃素或/和柴胡皂素a對各組老鼠肝臟中 脂質過氧化物malondialdehyde濃度之影響 圖二十九 薑黃素或/和柴胡皂素a對各組老鼠肝臟 總抗氧化狀態之影響 圖三十 老鼠肝臟中TNF-α之含量(μg/mg protein) 圖三十一 老鼠肝臟中IL-1β之含量(μg/mg protein) 圖三十二 老鼠肝臟中IL-6之含量(μg/mg protein) 圖三十三 老鼠肝臟中IL-10之含量(μg/mg protein) 圖三十四 老鼠肝臟中TGF-β1之含量(μg/mg protein) 圖三十五 老鼠肝臟中hydroxyproline之含量(μg/mg protein) 圖三十六 抗氧化劑清除自由基 圖三十七 活性氧及TNF-α活化NF-κB，增加促發炎基因轉錄 圖三十八 薑黃素阻斷肝星狀細胞PDGF和EGF 途徑、 活化PPAR-γ，降低HSC增殖 表目次 表 一 民國九十七年台灣地區十大死因排名 表 二 民國九十七年台灣地區癌症十大死因排名 表 三 老鼠飼料中薑黃素與柴胡皂素a 之含量 表 四 實驗期間各組老鼠之體重 表 五 各組老鼠之體重增加、肝臟重量、絕對與相對肝臟重量 (二)參考文獻 Aggarwal BB, Sung B. 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Zhonghua Gan Zang Bing Za Zhi 2006;14:449–452. 王錫崗、于家城、林嘉志、施科念、高美眉、張林松、陳瑩玲、陳聰文、黃慧真、溫小娟、廖美華、蔡宜容。人體生理學。台北縣；新文京開發出版股份有限公司。2004；pp.603-609。 尤紅、王寶恩。介導的肝臟實質細胞和非實質細胞的凋亡。首都醫科大學學報 2000；21：362-364。 行政院衞生署(2008) http://www.doh.gov.tw/statistic/index.htm。 張志軍。日本對小柴胡湯的研究。中醫雜誌1993；4：36。 李舒敏。小柴胡湯在內科臨床上的應用概況。廣西中醫學院學報 2002；17：52-54。 張莉娟、李斌、黃月紅、陳治新、王小眾 (2001a) 轉化因子β1和腫瘤壞死子α在門靜脈高血壓發病中的意義。福建醫科大學學報，35:142-143。 薑黃素屬於多酚類，柴胡皂素a屬於皂素類；薑黃素與柴胡皂素a都被研究指出具有抗氧化活性。本研究使用兩種單離純化的植物化學物質薑黃素與柴胡皂素a，以單方或複方的形式更進一步瞭解其複方對四氯化碳誘發老鼠肝損傷之影響。將老鼠隨機分成五組：控制組(正常飲食＋腹腔注射橄欖油)、CCl4組(正常飲食＋腹腔注射CCl4)、薑黃素組 (添加0.005 % 薑黃素於飼料＋腹腔注射CCl4)、柴胡皂素組(添加0.004 % 柴胡皂素a於飼料＋腹腔注射CCl4)、複方組(添加0.005 %薑黃素＋0.004 %柴胡皂素a於飼料＋腹腔注射CCl4)。薑黃素萃取物中薑黃素的純度是95 %，柴胡皂素萃取物含有98 % 以上的柴胡皂素a，分別以0.005 % 及0.004 % (w/w) 之比例添加於飼料中。於注射CCl4前一週先給予薑黃素或/和柴胡皂素a，之後每週腹腔注射一次CCl4，CCl4的注射劑量為每公斤體重0.75 mL之40 % 的CCl4；控制組則注射同等劑量之橄欖油，實驗期共八週。實驗結束後進行犧牲並採集血液樣本及肝組織，進行肝臟病理組織切片和分別利用Sirius Red染色法和核因子-κB (nuclear factor-κB;NF-κB) 免疫組織染色法，觀察肝小葉中央靜脈區膠原蛋白堆積及肝細胞壞死情形，血漿中肝傷害指標酵素、脂質代謝、肝臟抗氧化狀態、肝發炎反應指標及肝纖維化指標之分析測定。病理切片顯示，薑黃素與柴胡皂素a可以降低肝門靜脈脂肪變性與肝細胞壞死情形，其效果又以複方組較為顯著 (P < 0.05)；而肝纖維化和膠原蛋白堆積情形，柴胡皂素組和複方組顯注降低肝臟纖維化情形 (P < 0.05)。肝臟Sirius Red染色法顯示，薑黃素和柴胡皂素a可以降低膠原蛋白纖維架橋增生的情形 (P < 0.05)。然而，複方組膠原蛋白纖維架橋增生的情形與四氯化碳組之間均未達統計差異。肝臟NF-κB免疫組織染色法顯示，薑黃素組、柴胡皂素組、複方組之NF-κB表現均顯著低於四氯化碳組(P < 0.05)。實驗期間，給予薑黃素及柴胡皂素a能顯著降低因肝損傷而引起血漿中丙胺酸轉胺酶 (alanine aminotransferase; ALT) 及天門冬胺酸轉胺酶 (aspartate aminotransferase; AST) 之活性上升的情形 (P < 0.05)，且可顯著降低血漿與肝臟中膽固醇及三酸甘油酯 (P < 0.05)含量。在肝臟抗氧化能力方面，薑黃素組、柴胡皂素組和複方組都顯著提升肝臟的總抗氧化狀態 (total antioxidant status)，降低肝臟脂質過氧化物 (malondialdehyde; MDA) 濃度；於複方組肝臟中超氧歧化酶 (superoxide dismutase; SOD) 活性和肝臟抗氧化物質(total glutathionine)的濃度都顯著較四氯化碳組高 (P < 0.05)。在發炎反應指標方面，薑黃素與柴胡皂素a可降低肝臟中促發炎細胞激素，腫瘤壞死因子-α(tumor necrosis factor-α; TNF-α)、介質素-1β(interleukin-1β; IL-1β)和介質素-6 (interleukin-6; IL-6) 之含量；而提升肝臟中抗發炎細胞激素，介質素-10 ( interleukin-10; IL-10) 之含量 (P < 0.05)。肝纖維化指標方面，薑黃素及柴胡皂素a均能顯著抑制肝臟中膠原蛋白前驅物-羥基脯胺酸 (hydroxyproline) 含量及抑制導致肝臟纖維化之轉化生長因子-β1 (transforming growth factor-β1; TGF-β1) 之含量 (P < 0.05)，具有抑制肝纖維化的功能。因此，給予薑黃素或/和柴胡皂素a可藉由降低肝小葉中央靜脈區膠原蛋白堆積及肝細胞壞死情形、減輕肝臟脂質堆積、減少肝臟脂質過氧化和提升抗氧化能力、降低發炎反應，達到減輕肝損傷和肝纖維化之功能。然而，薑黃素與柴胡皂素a複方在肝臟抗發炎和抗纖維化反應，並無顯著協同效果。 Curcumin and saikosaponin a, as the antioxidants, are bioactive phytochemicals of turmeric and Bupleurum. The combined effect of curcumin and saikosaponin a on hepatoprotection against CCl4-induced liver injury has not been studied yet. Therefore, we used pure phytochemical compounds of curcumin and saikosaponin a to determine their hepatoprotective effects on CCl4-induced liver injury. This study investigated the effects of supplementation with curcumin and/or saikosaponin a on carbon tetrachloride (CCl4)-induced liver injury in rats. Male Sprague-Dawley rats were randomly divided into 5 groups: control, CCl4, CCl4+curcumin (0.005%; CU), CCl4+ saikosaponin a (0.004%; SS), and CCl4+curcumin+saikosaponin a (0.005%+0.004%; CU+SS) groups. CCl4 (40% in olive oil) was injected intraperitoneally at a dose of 0.75 mL/kg body weight once a week. Curcumin and/or saikosaponin a was administered orally 1 week before CCl4 injection for 8 weeks. Rats were intraperitoneally injected with an equivalent dosage of olive oil as the control group. After 8-week treatments with curcumin and/or saikosaponin a, plasma and liver were collected for biochemical and pathological analyses. The pathological results showed that curcumin and saikosaponon a reduced fatty change and hepatic necrosis in the portal veins, and the effect of the CU+SS group was more obvious (P <0.05). Liver fibrosis was ameliorated in the SS and CU+SS groups. Hstopathological results showed hepatic collagen deposition was significantly reduced in the CU and SS groups, and activated nuclear factor-?羠 expression induced by CCl4 in the liver was significantly inhibited by curcumin and/or saikosaponin a. After 8 weeks, supplementation with curcumin and/or saikosaponin a significantly decreased plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, as well as plasma and hepatic cholesterol and triglyceride levels. Supplementation with curcumin and/or saikosaponin a significantly improved hepatic antioxidant status and suppressed malondialdehyde formation. The CU+SS group showed increases in hepatic superoxide dismutase activity and total glutathione level compared with the CCl4 group. Hepatic proinflammatory cytokines tumor necrosis factor-?? (TNF-α), interleukin-1?? (IL-1β), and interleukin-6 (IL-6) were significantly decreased, and the anti-inflammatory cytokine interleukin-10 (IL-10) was significantly increased by supplementation with curcumin and/or saikosaponin a. Additionally, curcumin and/or saikosaponin a significantly decreased hepatic transforming growth factor-??1 (TGF-??1) and hydroxyproline levels. Therefore, supplementation with curcumin and/or saikosaponin a protects against CCl4 –induced liver injury and fibrosis by attenuating hepatic lipids and lipid peroxidation, enhancing antioxidant defense, and suppressing inflammation and fibrogenesis. However, Curcumin and saikosaponin a had no additive effects on hepatoprotection.