| Summary: | 博士 === 國立中興大學 === 食品暨應用生物科技學系所 === 101 === 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.
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