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標題: 仙草改善化學性肝損傷及其活性成分齊墩果酸與熊果酸改善非酒精性脂肪肝疾病之功效與作用機轉
Assessments of Hsian-tsao (Mesona procumbens Hemsl.) and its active compounds, olenaolic acid and ursolic acid, on improvements of chemical-induced liver injury and nonalcoholic fatty liver disease (NFALD)
作者: 徐明煥
Shyu, Ming-Huan
關鍵字: 仙草;Hsian-tsao (Mesona procumbens Heml.);齊墩果酸;熊果酸;肝纖維化;肝癌細胞;凋亡;游離脂肪酸;非酒精性脂肪肝臟疾病;oleanolic acid;ursolic acid;hepatocellular carcinoma cells;apoptosis;free fatty acid and nonalcoholic fatty liver disease (NAFLD)
出版社: 食品暨應用生物科技學系所
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齊墩果酸 (oleanolic acid, OA) 與熊果酸 (ursolic acid, UA) 為五環三萜化合物,存在於許多中藥草藥植物中,例如:仙草 (Mesona procumbens Hemsl.)。目前已知具有抗氧化、抗發炎、保肝、降血糖及誘導癌細胞凋亡等功效。本研究將進一步評估齊墩果酸及熊果酸於保護不同危險因子誘導肝臟疾病之功效。

仙草已證實可減緩 t-BHP (tert-butyl hydroperoxide) 誘導急性肝損傷是透過提升 SD (Sprague Dawley) 大鼠體內抗氧化能力的作用。本研究探討仙草萃取物 (extracts of Hsian-tsao, EHT) 及活性成分 (齊墩果酸及熊果酸) 對慢性肝損傷及纖維化之保護效應。結果顯示,餵食 EHT 可降低因四氯化碳 (carbon tetrachloride, CCl4) 傷害所造成 SD 大鼠中肝臟腫大之現象;血清生化值分析也顯示餵食EHT 可以降低因 CCl4 引發大鼠之 aspartate aminotransferase (AST) 及 alanine aminotranferease (ALT) 活性上升 (p < 0.05)。由病理切片結果亦證實EHT 可有效減輕因 CCl4 誘發大鼠肝纖維化現象。從以上結果得知 EHT 的確可減緩 CCl4 誘導慢性肝損傷及肝纖維化。此外,結果也發現,餵食 EHT可顯著提升肝臟中總抗氧化能力 (total antioxidant capacity, TEAC) 及降低肝中脂質過氧化物 (malondialdenhyde, MDA) 含量 (p < 0.05)。抗氧化酵素分析方面,餵食 EHT 對大鼠體內 glutathione peroxidase (GPx)、glutathione S-transfertase (GST) 與catalase 之活性及 total glutathione (GSH) 的含量亦具提升之作用 (p < 0.05),證實 EHT 可提升抗氧化酵素,降低脂質過氧化來提升肝臟總抗氧化力。蛋白質表現量方面,EHT 也可顯著降低發炎指標 inducible nitric oxide synthase (iNOS) 及 cyclooxygenase-2 (COX-2) 的表現。此外,EHT 確實可以減少肝纖維化指標 smooth muscle α-actin (α-SMA) 蛋白質及 matrix metalloproteinase (MMP)-2 及 MMP-9 活性之表現,表示仙草萃取物的確可減緩肝纖維化之效用。另外,在細胞實驗的結果發現齊墩果酸及熊果酸可藉降低 α-SMA蛋白質及 MMP-2 活性表現來抑制 phorbol 12-myristate 13-acetate (PMA) 誘導大鼠肝星狀 HSC-t6 細胞的活化。以上結果顯示齊墩果酸及熊果酸可能為仙草萃取物中抗肝纖維化的活性成分。

齊墩果酸及熊果酸存於許多中草藥植物中,且具有保肝、抗發炎及抗癌之能力。本研究探討齊墩果酸及熊果酸誘發人類肝癌 HuH 7 細胞凋亡之效果,並且分析其相關機制。結果發現,齊墩果酸及熊果酸可抑制 HuH 7 細胞生長,且 IC50 濃度分別為 100 及 75 μM。經由細胞週期分析發現,齊墩果酸及熊果酸可增加 HuH 7 細胞之 sub-G1 pahse 的表現,證實齊墩果酸及熊果酸可透過誘導 HuH 7 細胞凋亡的方式來抑制 HuH 7 細胞生長。其中,齊墩果酸及熊果酸會造成 HuH 7 細胞粒線體膜電位的喪失,並發現與 Bcl-2 家族失衡有關。此外,結果發現齊墩果酸及熊果酸也會促使粒線體中的 cytochrome c 釋放到細胞質中,並活化下游蛋白 caspase-9 及 caspase-3 的表現,接著造成 poly (ADP-ribose) polymerase (PARP) 的裂解。另外,齊墩果酸及熊果酸透過降低 HuH 7 細胞中 nuclear factor-κB (NF-κB) 的活性,進而抑制 X-linked inhibitor of apoptotic protein (XIAP) 的基因表現。從以上結果得知,齊墩果酸及熊果酸可透過粒線體路徑及調控 NF-κB 及其下游的表現來誘導 HuH 7 細胞凋亡。

過去研究發現當體內造成代謝性胰島素阻抗時,由於會促使脂肪組織中的三酸甘油酯大量代謝,進而造成體內游離脂肪酸增加,過多的游離脂肪酸會轉運回肝臟中進行脂肪合成,造成非酒精性脂肪肝疾病。其中主要以棕櫚酸 (palmitic acid, PA) 及油酸 (oleic acid, OLA) 為主。利用游離脂肪酸混合物誘導 HepG2 細胞來建立非酒精性脂肪肝疾病之細胞模式。結果發現1 mM 的棕櫚酸及游離脂肪酸混合物可以顯著抑制細胞生長,但油酸則不會影響。此外,當細胞只處理棕櫚酸時,的確會造成 HepG2 細胞中 caspase-3 的活性、cathepsin B的活性及細胞內 Ca2+ 的濃度顯著增加,也會增加內質網壓力蛋白 78-kDa glucose-regulated protein (GRP78) 及 C/EBP homologous protein (CHOP) 的基因表現。但油酸及游離脂肪酸混合物則只有輕微的增加,顯示棕櫚酸為造成細胞損傷的主要游離脂肪酸。在氧化壓力結果也發現棕櫚酸、油酸及其游離脂肪酸混合物都會造成細胞內 reactive oxygen species (ROS) 的生成,顯示氧化壓力可能為脂肪酸誘導肝損傷的原因。此外,結果也發現棕櫚酸、油酸及其游離脂肪酸混合物也可增加發炎相關基因 tumor necrosis factor-α (TNF-α) 及 interleukin-1 β (IL-1β) 之基因表現。從 Oil Red O-stained material (OROSM) 的結果顯示油酸為造成 HepG2 細胞中脂肪滴形成的主要游離脂肪酸,進而促使游離脂肪酸混合物也會大量增加脂肪滴的生成,而棕櫚酸只有輕微的脂肪滴累積。然而在脂肪代謝相關基因表現發現棕櫚酸、油酸及其游離脂肪酸混合物皆可以增加 stearoyl-CoA desaturase 1 (SCD-1)、fatty acid synthase (FAS)、acetyl-CoA carboxylase α (ACCα)、HMG-CoA reductas (HMGCoAR)、low-density lipoprotein receptor (LDLR)、sterol regulatory element binding proteins 1 (SREBP-1) 及 peroxisome proliferator-activated receptor γ (PPARγ) 的基因表現,也可以輕微增加 carnitine palmitoyltransferase 1 (CPT-1),但對 uncoupling protein 2 (UCP2) 及 peroxisome proliferator-activated receptor α (PPARα) 則無影響。因此游離脂肪酸混合物誘導 HepG2 細胞可做為非酒精性脂肪肝疾病之細胞模式。

先前結果已證實棕櫚酸及油酸為形成非酒精性脂肪肝疾病之游離脂肪酸,也發現其游離脂肪酸混合物 (free fatty acid mixture, FFA mixture, PA : OLA = 1 : 2) 可使 HepG2 細胞中會產生胞器受損及脂肪滴的生成,可做為非酒精性脂肪肝疾病的細胞模式。最後進一步評估齊墩果酸及熊果酸在游離脂肪混合物誘導 HepG2 細胞形成非酒精脂肪肝疾病中是否具有保護肝臟之良好功效。結果發現齊墩果酸及熊果酸的確可以降低因游離脂肪酸誘導的 ROS 生成,主要發現是齊墩果酸及熊果酸皆可提升細胞內 glutathione peroxidase 1 (GPx1)、γ-glutamylcysteine synthetase (γGCS)、CuZn superoxide dismutase (CuZnSOD)、Mn superoxide dismutase (MnSOD) 及 catalase 的基因表現來減少 ROS 的生成。發現齊墩果酸及熊果酸也可降低細胞內 Ca2+ 的濃度及內質網壓力相關蛋白 GRP78 與 CHOP 的基因表現及細胞內 caspase-3 的活性及 cathepsin B 的活性,來證實齊敦果酸及熊果酸可透過降低 ROS 生成及提升抗氧化酵素來減少游離脂肪酸對 HepG2 細胞中粒線體、溶酶體及內質網等胞器的傷害。此外,齊墩果酸及熊果酸也被證實可減少因游離脂肪酸誘導 HepG2 細胞中 TNF-α及 IL-1β基因表現。從OROSM的結果發現齊墩果酸及熊果酸可以減少游離脂肪酸物於 HepG2 細胞中所生成的脂肪滴,主要是減少脂肪合成相關酵素 SCD-1、FAS、ACCα、HMGCOAR、LDLR、SREBP-1 及 PPARγ 的基因表現,並且增加脂肪代謝相關酵素 CPT-1、UCP2 及 PPARα 的基因表現。以上結果顯示齊墩果酸及熊果酸可降低細胞內脂肪滴的生成是透過降低脂肪及膽固醇合成相關酵素及提升脂肪酸氧化相關酵素所致。綜合上述,齊墩果酸及熊果酸的確可以降低氧化壓力所造成胞器的損傷及細胞內脂肪滴的生成,來減緩游離脂肪酸誘導 HepG2 細胞形成非酒精性脂肪肝疾病的現象。


Oleanolic acid (OA) and ursolic acid (UA) are triterpenoids in chinese herbal medicine plants (e.g. Mesona procumbens Hemsl, Hsian-tsao) and have beneficial effects on antioxidant capacity, anti-inflammation, hepatoprotection and induction of cancer cells apoptosis. The aim of study was to evaluate the potential protective effects of OA and UA on different risk factor-induced liver diseases.

The previously study indicated that water extract of Hsian-tsao could decrease acute liver damage in SD rats induced by t-BHP (tert-butyl hydroperoxide) through enhancing antioxidant capacity in rats. First, the protective effects of EHT (extracts of Hsian-tsao) and its active compounds (OA and UA) on chronic liver damage and fibrosis in vivo and in vitro were evaluated. The results showed that EHT decreased the relative liver weight in carbon tetrachloride (CCl4)-treated SD rats. Serum aspartate aminotransferase (AST) and alanine aminotranferease (ALT) levels in rats with EHT treatment were significantly lower than that in rats by CCl4-induced only (p < 0.05). Histological examination expressed EHT had significantly protective against liver fibrosis (p < 0.05). These data showed that EHT could decrease liver fibrosis in rats induced by CCl4. When rats fed EHT could also significantly increase total antioxidant capacity (TEAC) and decease malondialdenhyde (MDA) in liver than only CCl4-induced rats (p < 0.05). In antioxidant enzymes, orally treated EHT raised glutathione peroxidase (GPx), glutathione S-transfertase (GST), catalase activity and total glutathione (GSH) content (p < 0.05). These data showed that EHT could increase the antioxidant enzymes and decrease lipid peroxidation to enhance total antioxidant capacity in CCl4-induced rats. EHT also diminished the protein expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and decreased the protein level smooth muscle α-actin (α-SMA) and the activities of matrix metalloproteinase (MMP)-2 and -9 in CCl4-induced rats. These data showed that EHT could inhibit inflammation and liver fibrosis in CCl4-induced rats. Rat hepatic stellate HSC-t6 cells activation induced by phorbol-12-myristate-13-acetate (PMA) was uesd to study the anti-fibrotic effects of OA and UA in vitro. Treating these cells with OA or UA caused a decrease in the protein level of α-SMA and the activity of MMP-2. These data suggested that OA and UA might be the anti-fibrotic compounds in Hsian-tsao.

OA and UA are commonly found in plants and herbs, and have been reported to prossess hepatoprotective, anti-inflammatory and anticancer activitires. The effects of OA and UA on induction of apoptosis in human hepatocellular carcinoma HuH 7 cells and the related mechanisms were investigated. The results demonstrated that OA and UA could inhibit the growth of HuH 7 cells with IC50 values of 100 and 75 μM, respectively. Cell cycle analysis using flow cytometry indicated that OA and UA progressively increased the fraction of HuH 7 cells in sub-G1 phase in dose-dependent manner. These data showed that OA and UA could induce HuH 7 cell apoptosis to inhibit HuH 7 cell growth. Treatment with OA or UA induced a dramatic loss of the mitochondria membrane potential and interfered with the ratio of expression levels of pro- and antiapoptotic Bcl-2 family members in HuH 7 cells. OA and UA-induced apoptosis involved the release of mitochondria cytochrome c into the cytosol and subsequently induced the activation of caspase-9 and caspase-3, followed by cleavage of poly (ADP-ribose) polymerase (PARP). Moreover, HuH 7 cells treated with OA and UA also suppressed the activity of nuclear factor-κB (NF-κB) to modulate the mRNA expression of X-linked inhibitor of apoptotic protein (XIAP) as compared with untreated cells. These results demonstrated that OA and UA induce HuH 7 cells apoptosis through a mitochondria-mediated pathway and regulation of the activity of NF-κB and the mRNA expression of its downstream protein, XIAP.

Insulin resistance could promote triglyceride (TG) lipolysis in the adipose tissue to release a number of free fatty acids into blood, and fre fatty acids could transfer into liver. But excess free fatty acids in liver could promote a large amount of TG synthesis to cause nonalcoholic fatty liverdisease (NAFLD). Palmitic acid (PA) and Oleic acid (OLA) are the main type of free fatty acids in human blood, and the ratio of saturated fatty acid and unsaturated fatty acid is 1 : 2. We established the fatty acid mixture (FFA mixture, PA : OA = 1 : 2)-induced nonalcoholic fatty liver disease in HepG2 cells as the in vitro model for the study of NAFLD. The results showed that 1 mM of PA or FFA mixture could significantly inhibit HepG2 cells growth (p < 0.05), but OLA was no cytotoxicity. In addition, the results showed that PA could increase the intracellular Ca2+ concentration and the gene expressions of endoplasmic reticulum (ER) stress related proteins, 78-kDa glucose-regulated protein (GRP78) and C/EBP homologous protein (CHOP), and the activities of cathepsin B and caspase-3. Moverover, OLA and FFA mixture could slightly increase organelles damage. These results suggested that PA was the main type of free fatty acid to induce organelles damage. PA, OLA and FFA mixture also increased intracellular reactive oxygen species (ROS) production in HepG2 cells, suggesting ROS plays a role in free fatty acid induced liver damage. Besides, PA, OLA and FFA mixture could increase the gene expressions of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). Oil Red O-stained material (OROSM) was used to investigate the effects of free fatty acids on fat-droplet formation in HepG2 cells. The results showed that OLA and FFA mixture significantly increased fat-droplet formation in HepG2 cells (p < 0.05), but PA could slightly increase fat-droplet formation. The data suggested that OLA was the main type of free fatty acid to increasing triglyceride (TG) synthesis in HepG2 cells. In addition, The results showed that PA, OLA and FFA mixture cloud increase the gene expressions of stearoyl-CoA desaturase 1 (SCD-1), fatty acid synthase (FAS), acetyl-CoA carboxylase α (ACCα), HMG-CoA reductase (HMGCoAR), low-density lipoprotein receptor (LDLR), sterol regulatory element binding proteins-1 (SREBP-1) and peroxisome proliferator-activated receptor γ (PPARγ), and these fatty acids also slightly increased the gene expression of carnitine palmitoyltransferase 1 (CPT-1), but free fatty acids could not influence the gene expressions of uncoupling protein 2 (UCP2) and peroxisome proliferator-activated receptor α (PPARα). The data suggested that FFA mixture-induced HepG2 cells could be the in vitro model for the study of NAFLD.

Previously study showed that palmitic acid (PA) and oleic acid (OLA) are the main type of free fatty acids to promote the formation of nonalcoholic fatty liver disease, and free fatty acid mixture (FFA mixture, PA : OLA = 1 : 2) could induce organelles damage and lipid-droplet formation to promote the formation of nonalcoholic fatty liver disease (NAFLD) in HepG2 cells. The potential protective effects of OA and UA on NAFLD in FFA mixture-induced HepG2 cells were further determined. The results showed that OA and UA could decrease ROS production, and increase the gene expressions of glutathione peroxidase 1 (GPx1), γ-glutamylcysteine synthetase (γGCS), CuZn superoxide dismutase (CuZnSOD), Mn superoxide dismutase (MnSOD) and catalase in FFA mixture-induced HepG2 cells. Besides, OA and UA could decrease the intracellular Ca2+ concentration, the gene expressions of GRP78 and CHOP, and the activities of caspase-3 and cathepsin B in FFA mixture-induced HepG2 cells. These data suggested that OA and UA could decrease ER stress, lysosome and mitochondria damage through decreasing ROS production and increasing the gene expressions of antioxidant enzymes in HepG2 cells induced by FFA mixture. Besides, OA and UA could decrease the gene expressions of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in FFA mixture-induced HepG2 cells. Moreover, OA and UA could decrease fat-droplet formation in FFA mixture-induced HepG2 cells. These results showed that OA and UA could decrease the expressions of SCD-1, FAS, ACCα, HMGCoAR, LDLR, SREBP-1 and PPARγ, and increase the gene expressions of CPT-1, UCP2 and PPARα in FFA-mixture induced HepG2 cells. These data suggested that OA and UA could decrease fat-droplet formation by decreasing fat and cholesterol synthesis-related gene expression and increasing fatty acid oxidation-related gene expression in HepG2 cells induced by FFA mixture.

In conclusion, the potential effects of OA and UA could be inhibition of liver fibrogenesis, depletion of NAFLD induced by FFA and induction of hepatoma cell apoptosis to protect against liver diseases.
其他識別: U0005-2608201316542800
Appears in Collections:食品暨應用生物科技學系

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