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Protective Effect of Hsian-tsao Against tert-butyl hydroperoxide induced acute liver damage and oxidative damage of HepG2 cells
|作者:||陳嬿如||關鍵字:||仙草;oxidative damage;咖啡酸;t-BHP;抗氧化性;活性氧與自由基;人類肝癌細胞株;氧化傷害;caffeic acid;t-BHP;HepG2 cell;Hsian-tsao;Mesona procumbens Hemsl||出版社:||食品科學系||摘要:||
The aims of this study were to evaluate the protective effect of Hsian-tsao (Mesona procumbens Hemsl.). against tert-butyl hydroperoxide (t-BHP)-induced acute liver damage in rats and oxidative damage of HepG2. Chapter 2 focuses on the investigation of the water extracts of Hsian-tsao (WEHT) on t-BHP-induced acute hepatotoxicity and nephrotoxicity in rats. The results showed that the oral pretreatment of WEHT (0.1, 0.5 and 1.0 g/kg ) or caffeic acid (CA) (0.1 g/kg) before t-BHP ( 0.2nmmol/kg ) treated significantly lowered the serum levels of the hepatic enzyme markers (alanine, aspartate aminotransferase and lactate dehydrogenase) and reduced oxidative stress in liver by the evaluation of malondialdehyde (MDA), glutathione (GSH), total thiols groups (TSH), 8-hydroxy-2-deoxy-guanosine (8-OH-dG), glutathione peroxidase and glutathione reductase. The histopathological evaluation of the rat livers showed that WEHT and caffeic acid reduced the incidence of liver lesions including cloudy swelling, pyknosis, ballooning change, cytolysis, and leucocytes infiltration induced by t-BHP in rats. The kidney pathological histology showed that WEHT and caffeic acid reduced the incidence of germinal vesicle breakdown in proximal convoluted. Based on the results of this study, we speculate that Mesona procumbens may play a preventive role on t-BHP-induced acute hepatotoxicity and nephrotoxicity in living systems.
In order to study the protective mechanisms, various concentrations of WEHT and CA were pre-incubated with HepG2 cells to investigate their inhibitory effect on oxidative damage in cells induced by t-BHP (Chapter 3). The addition of t-BHP to culture medium resulted in enhancing lipid peroxidation as measured by the production of malondialdehyde (MDA) and enhancing cytotoxicity as detected by trypan blue and LDH leakage. When WEHT (100-500 g/ml) were incubated with HepG2 cells for 6 h and then treated with 2.0 mM t-BHP for 40 min, it caused the increase of cell viability and the decreases of LDH leakage, reactive oxygen species (ROS), cell surface blebs formation, lipid peroxidation and GSH oxidation (p<0.05). The activities of glutathion peroxidase and glutathione reductase in cells induced by t-BHP were also significantly decreased by WEHT, whereas the activity of glutathione S-transferase didn't change. Caffeic acid is the highest content of phenolic compounds in extracts of Hsian-tsao. When the caffeic acid (10-50 g/ml) was incubated with HepG2 for 6 h and then treated with 2.0 mM t-BHP for 40 min, the LDH leakage, ROS formation, lipid peroxidation and GSH oxidation in HepG2 cells were significantly (p<0.05) decreased with a dose-dependent manner. The results suggest that (1) the protective action of WEHT on oxidative damage in HepG2 cells may via preventing lipid peroxidation, up-regulating GSH-dependent enzymes and maintaining the membrane integrity to against t-BHP induced cytotoxic and oxidative damage, (2) caffeic acid was a major antioxidant compound for the scavenging free radical, inhibiting biomembrane damage, and the decreasing of intracellular reactive oxygen species in HepG2 cells.
Since the data concerning the effects of WEHT on apoptosis in cells is not available, we assumed that WEHT might have preventive effect on apoptosis in cells due to its free radical scavenging capacity and antioxidative properties. Chapter 4 focuses on the study of the potentially protective effect of WEHT on t-BHP induced apoptosis in HepG2 cells. Results showed that HepG2 cells treated with 0.5 mM t-BHP for 12 h resulted in enhancing cytotoxicity and LDH leakage. Based on the results of ROS formation, DNA fragmentation analysis, caspase3 activity and PARP expression, showed that t-BHP induced ROS, DNA fragmentation and down regulation of caspase 3 activity to cleavage PARP, we suggest that t-BHP triggers apoptosis through a mitochondria-denpendent pathway.
When cells were growth for 12 h in the presence of t-BHP and WEHT (100-500 g/ml), the cell injury was inhibited by increasing cell viability, diminishing ROS and DNA ladder, reducing caspase 3 activity and PARP cleavage. In DNA fragmentation and ROS analysis, caffeic acid co-incubated with t-BHP exhibited does dependent activity to reduce DNA ladder and ROS formation in cell.
In conclusion, WEHT exhibited potent protective effects against t-BHP induced liver injury in rat and cell necrosis and apoptosis in HepG2 cells. These results might be resulted in their ability of quenching free radicals, and caffeic acid might be the active ingredient in the WEHT. Therefore, we suggest that daily consumption of WEHT might be beneficial for lowering the possible oxidative damage in living system.
本研究主要是探討仙草對tert-butyl hydroperoxide (t-BHP) 誘發大鼠急性肝損傷與人類肝癌細胞 (HepG2) 氧化傷害之保護效果。第二章以動物模式系統探討仙草水萃取物 (WEHT) 對t-BHP誘發之大鼠急性肝、腎損傷是否具有保護效力。結果顯示，管餵仙草水萃取物 (0.1, 0.5, 1.0 g/kg b.w.) 或咖啡酸 (0.1 g/kg b.w.)，能顯著降低t-BHP (i.p. 0.2 mmol/kg b.w.) 所誘導之肝細胞內酵素 (AST、ALT、LDH) 滲漏，緩和肝組織中GSH/GSSG、TSH (Total thiols groups)、GSH-Rd含量及活性下降與GSH-Px、MDA、8-OHdG上升情形。在肝組織病理切片中發現，仙草水萃取物及咖啡酸能降低t-BHP對大鼠引發之白血球浸潤 (leucocytes infiltration)、肝臟變性及壞死病變，包括：濁腫 (cloudy swelling)、氣泡樣變性 (ballooning change)、核濃縮 (pyknosis) 與細胞質溶解 (cytolysis) 等現象；在腎組織病理切片中發現，仙草水萃取物及咖啡酸能降低t-BHP對大鼠引發之腎臟近曲小管損傷。由上述結果顯示，仙草具減緩肝細胞內酵素滲漏，維持肝組織之GSH/GSSG恆定比率、TSH含量及GSH酵素活性等功效，且以高劑量組 (1 g/kg) 效力最佳。證實仙草應具有直接清除自由基，預防活體內氧化傷害，達到保肝護腎之功能。
為了延續仙草水萃取物及咖啡酸於生物體結果，以細胞模式印證其作用機制，本研究乃利用HepG2為實驗細胞株，探討不同濃度仙草水萃取物及咖啡酸與HepG2預培養後，對t-BHP誘發細胞之氧化損傷是否具有抑制效果 (第三章)。結果顯示：與各濃度仙草水萃物超過6小時培養之HepG2，對2.0 mM t-BHP誘導之傷害，具有提升細胞存活率、減緩lactate dehydrogenase (LDH) 滲漏與胞內活性氧(reactive oxygen species, ROS) 生成能力、並具舒緩細胞因氧化壓力而引起之細胞膜起泡 (membrane blebbing) 、脂質過氧化與GSH氧化現象，且有效降低 t-BHP誘發胞內Glutathione peroxidase活性表現，進而降低Glutathione reducatase活性被誘發機會。而以咖啡酸 (10-50 g/ml) 與HepG2預培養 6小時，則對2.0 mM t-BHP 誘發之細胞LDH滲漏、ROS生成、脂質過氧化及GSH氧化現象，呈現劑量抑制效應 (p<0.05)。因此在人類肝癌細胞株 (HepG2) 模式系統中，仙草水萃取物可抑制t-BHP誘導之HepG2 細胞毒性，對t-BHP誘發之急性氧化傷害 (細胞壞死) 具保護功能，而其中富含之多酚類化物－咖啡酸，在抑制細胞氧氧傷害上扮演重要角色。
因過去學者在仙草研究中尚未探討細胞凋亡領域，仙草是否能憑藉其清除活性氧之抗氧化能力，進而抑制t-BHP誘發細胞凋亡是值得探討的議題，所以第四章設計以t-BHP誘導HepG2 細胞凋亡，評估仙草水萃取物對HepG2的保護作用。結果顯示，HepG2以0.5 mM t-BHP處理12小時會造成細胞存活率下降、胞內LDH酵素滲漏至胞外，並藉由誘發胞內活性氧ROS升高而造成細胞毒性與氧化壓力。在DNA 凝膠電泳與caspase 3酵素活性及PARP蛋白質表現量等細胞凋亡指標實驗顯示，t-BHP所造成的細胞毒性反應，大部份是以細胞凋亡為主，而其凋亡機轉是為Mitochondria-dependent pathway。若以仙草水萃取物 (100-500 g/ml) 與t-BHP共同添加，則可降低細胞傷害：提升細胞存活率、減緩DNA ladder、降低細胞內caspase 3活性上升與PARP分解情形，達到減少DNA損傷、降低細胞毒性而減少細胞走向凋亡。同時仙草中主要抗氧化性酚類化合物－咖啡酸在DNA 凝膠電泳與胞內活性氧 (ROS) 測定中，具有減緩DNA ladder與ROS生成能力，並呈現濃度效應。
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