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標題: | 以體內和體外試驗探討金銀花水萃液對TAA誘導肝損傷的保護功效 Protective effect of Lonicera japonica water extract solution on TAA induced liver lesion in vivo and in vitro |
作者: | Ming-Yuan Chao 趙名媛 |
指導教授: | 陳億乘(Yi-Chen Chen) |
關鍵字: | 抗氧化,抗凋亡,肝纖維化,硫代乙醯胺,金銀花水萃液, anti-oxidation,anti-apoptosis,liver fibrosis,thioacetamide,Lonicera japonica water extract solution, |
出版年 : | 2018 |
學位: | 碩士 |
摘要: | 肝纖維化主要特徵為:星狀細胞活化促使肌纖維母細胞增生,細胞外基質堆積在肝組織之間,同時,慢性炎症及氧化壓力亦會伴隨產生。中草藥金銀花(Lonicera japonica)含有藥用活性之多酚類(如:綠原酸)和黃酮類(如:葉黃酮(亦稱木犀草素))等;綠原酸和黃酮類可減少自由基產生,並降低細胞毒素之損傷。因此,本實驗目的在於探究金銀花水萃液(Lonicera japonica water extract solution, LJWES)減緩硫代乙醯胺(thioacetamide, TAA)造成肝纖維化之功效。首先,在動物試驗中40隻大鼠被隨機分配為4組(每組10隻):(1)控制組(CON):生理食鹽水(腹腔注射)+二次去離子水(口服管灌);(2)硫代乙醯胺處理組(TAA):100 mg TAA/kg B.W. (腹腔注射)+二次去離子水(口服管灌);(3)綠原酸處理組(TAA+CGA):100 mg TAA/kg B.W. (腹腔注射) + 100 mg CGA/kg B.W. (口服管灌),並作為正控制組;(4)金銀花水萃液處理組(TAA+LJWES):100 mg TAA/kg B.W. (腹腔注射) + 2.5 mL LJWES /kg B.W. (口服管灌)。TAA每週注射三次,實驗8週後,TAA使得體重及採食率顯著降低(p<0.05),提升(p<0.05)血清中ALT、ALP及BUN濃度。此外,補充LJWES確實增加(p<0.05) TAA誘導後造成的肝臟抗氧化能力(reduced GSH含量、TEAC含量與SOD、CAT、GPx活性),降低(p<0.05)脂質過氧化(TBARS程度);同時亦減少了膠原蛋白及發炎細胞激素含量(如:IL-6、TGF-β),亦在H&E染色及Sirius red 染色中可觀察到減少了纖維疤痕之產生,而在IHC染色中亦看到α-SMA表現量減少。之後,再從體外試驗探討其可能分子機制,在預處理稀釋1/400×、1/200×、1/100×之LJWES原液濃度(10 g LJ/100 mL DDW)下能夠減少(p<0.05) 75 mM TAA造成之乳酸脫氫酶含量,並增加(p<0.05)其細胞存活率。儘管在TAA損傷後FL83B細胞之發炎及凋亡基因有顯著上升(p<0.05),然而預處理LJWES後,調降了發炎基因(Tnf-α、Tnfr1)及凋亡基因(Bax、Cytochrome c),亦下降(p<0.05)了內源性凋亡蛋白質(P53、CLEAVED-CASPASE3、CASPASE9、CLEAVED-CASPASE9)。綜上所述,LJWES可調降TAA處理後凋亡相關基因之表現量、減少氧化壓力傷害、降低發炎細胞激素之分泌量,減少膠原蛋白堆積,LJWES具有減緩肝纖維化形成之功效。 Liver fibrosis is majorly characterized as a cellular activation of hepatic stellate cells which accelerate myofibroblast growth and deposit large extracellular matrix (ECM) components within the liver. Meanwhile, chronic inflammation and oxidative stress in livers are occurred as well. According to a previous research, the traditional herb medicine, Lonicera japonica, contains plenty of polyphenols (i.e. chlorogenic acid) and flavonoid (i.e. luteolin) which has been characterized as pharmacological effects, ROS scavenger, and cytotoxic-injury waver. Hence, the objectives of this study were to investigate the anti-fibrotic effects of Lonicera japonica water extract solution (LJWES) on thioacetamide (TAA) induced liver lesion in vitro and in vivo. Forty rats were randomly divided into four groups (n=10 per group): (1) CON: Saline (ip) + distilled water (oral gavage); (2) TAA: 100 mg TAA/kg B.W. (ip) + distilled water (oral gavage); (3) TAA+CGA: 100 mg TAA/kg B.W. (ip) + 100 mg chlorogenic acid (CGA)/kg B.W. (oral gavage) as a positive control; (4) TAA+LJWES: 100 mg TAA/kg B.W. (ip) + 2.5 mL LJWES /kg B.W. (oral gavage). TAA was injected three times per week. After 8 weeks of experiment, TAA treatment decreased (p<0.05) body weight and feed efficiency, but increased (p<0.05) serum ALT, ALP, and BUN levels. Although drinking LJWES partially reversed serum liver damage indices, drinking LJWES apparently enhanced (p<0.05) antioxidant capacities (reduced GSH and TEAC levels, and SOD, CAT, and GPx activities) and decreased (p<0.05) lipid peroxidation (TBARS) in livers of TAA-treated rats. Moreover, drinking LJWES decreased (p<0.05) levels of collagen and inflammatory cytokines, i.e. IL-6 and TGF-β. Additionally, drinking LJWES reduced fibrotic scars via observations of H&E and Sirus red stainings in livers of TAA-treated rats while IHC stainings also demonstrated less α-SMA accumulation. Sequentially, via assays of both cell viability and lactate dehydrogenase (LDH) release, 75 mM of TAA was chosen to induce cell lesion (FL83B cell, a normal rat fetal hepatocyte) for further investigations. Results showed that pretreatments of LJWES (1/400×, 1/200×, and 1/100× LJWES) could increase (p<0.05) cell viabilities and decrease (p<0.05) lactate dehydrogenase release in the 75 mM TAA induction. Although both inflammation and apoptosis-related gene expressions in FL83B cells were upregulated (p<0.05) by TAA treatment, the pretreatment of LJWES downregulated (p<0.05) gene expressions of Tnf-α, Tnfr1, Bax, and Cytochrome c. In addition, the pretreatment of LJWES downregulated (p<0.05) protein expressions of P53, CLEAVED-CASPASE3, CASPASE9, and CLEAVED-CASPASE9. Based on current results, LJWES can downregulate the apoptosis-related gene and protein expressions, as well as alleviate oxidative damage, the inflammatory cytokine and collagen deposition in the TAA treatment. Therefore, LJWES owns a preventive effect on the development of oxidative-stress induced hepatic fibrosis. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70063 |
DOI: | 10.6342/NTU201800350 |
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顯示於系所單位: | 動物科學技術學系 |
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