| Summary: | 博士 === 中國醫藥大學 === 中國藥學研究所博士班 === 95 === We investigated the efficacy of 7 kinds of natural products on chemical-induced liver fibrosis in animal model. The purpose of this study was to investigate the hepatoprotective effects of olive oil, palm oil and corn oil, Anoectochilus formosanus effective fraction (AFEF), ethanolic extract of the fruit of Hovenia dulcis (EHD) and fermented substance from Aspergillus phoenicis (FSAP) or Saccharomyces cerevisiae (FSSC), via CCl4-induced chronic liver damage animal model. One discovery has been made: corn oil enhanced the hepatic lipid peroxidation activity, but did not affect the liver fibrosis. Subsequently, we further reaearched into the role of lipid peroxidation in liver fibrosis pathway by in vivo and in vitro study.
The results of plasma biochemistry data and hepatic hydroxyproline demonstrated that these natural medicines decreased liver fibrosis. The pathohistolgical and fibrogenic mRNA expression also confirmed these results. RT-PCR analysis showed that CCl4 increased hepatic mRNA expressions of CD14, Toll-like receptor 4, NADPH oxidase, nuclear factor-kappa beta, collagen (??1)(I), collagen (??1)(III), transforming growth factor ??1, lipopolysaccharide binding protein. These mRNA expressions could be decreased by olive oil treatment. In addition, Western blot analysis also supported these results. Olive oil treatment decreased hepatic levels of malondialdehyde and hydroxyproline induced by CCl4. Histological evaluations showed that olive oil could attenuate liver fibrosis, necrosis and expression of smooth muscle ??-actin induced by CCl4. In this study, since non-virgin olive oil was used in this experiment, it is speculated that fatty acid of olive oil significantly reduced CCl4-induced hepatic fibrosis in rats.
Palm oil might tend to increase hepatic and plasma triglyceride indefinitely, but could not influence liver fibrosis in CCl4-induced liver injury model.
Corn oil did not affect the enhanced plasma ALT and AST activity and hypoalbuminemia caused by CCl4 administration. Corn oil did not affect the enhanced hepatic hydorxyproline and MDA caused by CCl4 administration. Histology examination with Sirius red stain also confirmed that corn oil did not affect the CCl4-induced liver fibrosis. Corn oil treatment markedly enhanced the hepatic MDA and 8-iso-PGF2?? concentrations induced by CCl4, but not the hepatic 15-keto-dihydro-PGF2?? (15-keto-PGF2??) content. Indeed, quantitative real-time RT-PCR analysis showed that hepatic GPAT (glycerol-3-phosphate acyltransferase) and UCP-2 (uncoupling protein 2) mRNA expression was increased by CCl4 treatment, but not CD36. In this study, corn oil markedly increased hepatic GPAT and CD36 expression, whereas hepatic UCP-2 expression was not affected. In this study, the amount of enzymatic lipid peroxidation product and inflammatory states induced by CCl4 were not affected by corn oil treatment. Corn oil enriches polyunsaturated fatty acids through non-enzymatic pathways to increase LPO products that cannot enhance liver fibrosis induced by CCl4.
We investigated the relationship of lipid peroxidation and hepatic stellate cell activation in vivo model. The Wistar rats were orally administered with CCl4 and / or corn oil and killed in week 2, 4, 8. Corn oil did not affect the enhanced liver fibrosis in hepatic hydorxyproline. Histology examination also confirmed these results. Corn oil enhanced the free radical lipid peroxidation product (MDA and 8-iso-PGF2α), not enzymatic product (15-keto-PGF2??). Therefore, CCl4 enhanced the hepatic hydroxyprline of fibrosis marker until week 2. Histology examination also confirmed these liver fibrosis results in CCl4 model. CCl4 enhanced the free radical lipid peroxidation product (MDA, 8-iso-PGF2α) and enzymatic product (15-keto-PGF2??).
In vitro model, the treatment of lipid peroxidation product (MDA、8-iso-PGF2α、15-keto-PGF2α) did not directly induce hepatic stellate cell activation by the confocal and western blot analysis of α-SMA. These cells were activated by fibrogenic cytokines such as TGF-ß1. Therefore, the high dose of lipid peroxidation product combined with TGF-ß1-treated hepatic stellate cell has a bearing on the cause of hepatic stellate cell apoptosis and cells activation was not consequent. So, the product of lipid peroxidation did not directly induce hepatic stellate cell activation.
In this study, we initialized the hepatoprotective effect of Anoectochilus formosanus effective fraction (AFEF) and content kinsenoside 180 mg/g. Plasma GPT, hepatic levels of hydroxyproline and malondialdehyde were significantly lower in mice treated with AFEF compared to those treated with CCl4 only. Liver pathology in the AFEF-treated mice was also improved. RT-PCR analysis showed that AFEF treatment increased the expression of methionine adenosyltransferase 1A, and decreased the expression of collagen(??1)(I) and transforming growth factor-??1. These results clearly demonstrated that the AFEF reduced the hepatic damage induced by CCl4 in mice.
In the ethanolic extract of the fruit of Hovenia dulcis (EHD) results, the Plasma activities of GPT and GOT, and hepatic levels of malondialdehyde were significantly lowered in mice treated with EHD as compared to mice treated with CCl4 only. Histological evaluation showed that EHD could attenuate the liver fibrosis and necrosis caused by CCl4. RT-qPCR analysis also showed that EHD treatment decreased hepatic collagen (??1)(I) and collagen (??1)(III) mRNA expressions. Chronic CCl4 treatment caused liver injuries in mice, characterized by an increase in hepatic methionine adenosyltransferase (MAT)2A gene expression, and decreased MAT1A gene expression. EHD significantly reduced the changes in MAT gene expression due to chronic CCl4 treatment. These results clearly demonstrated the EHD could reduce hepatic injuries in mice induced by CCl4.
In fermented substance from Aspergillus phoenicis (FSAP) or Saccharomyces cerevisiae (FSSC) results, the plasma ALT and AST, spleen weight, and hepatic levels of lipid peroxidation and hydroxyproline were significantly lower in the rats treated with FSAP or FSSC as compared to CCl4 only. Liver pathology in the FSAP or FSSC - treated rats was also improved. mRNA expression analysis showed that FSAP or FSSC treatment increased the expression of matrix metalloproteinase 13 and decreased the expression of methionine adenosyltransferase 2A, collagen (??1)(I), collagen (??1)(III), transforming growth factor-??1, and tissue inhibitor of metalloproteinase 1. These results clearly indicate that FSAP or FSSC partially reduced the liver fibrosis in rats induced by CCl4.
The effect of Saccharomyces cerevisiae (FSSC) in mice induced acute liver injury by acteaminophen and bromobenzene, the plasma ALT and AST was decreased, and hepatic levels of glutathione and activity of superoxide dismutase (SOD)、catalase、GSH-peroxidase (GSH-Px)、GSH-reducdase (GSH-Rd)、GSH-transferase (GSH-T) were significantly increased in the mice treated with FSSC as compared to acteaminophen and bromobenzene only. These results clearly indicate that FSSC partially reduced the liver injury in mice induced by acteaminophen and bromobenzene.
In summary, (1) the five kinds of natural drugs, including the AFEF, EHD, FSAP, FSSC and olive oil, have hepatoprotective effect on liver fibrosis in animal model; (2) the product of lipid peroxidation did not directly induce hepatic stellate cell activation, and also not enhance the TGF-β1 induced hepatic stellate cell activation.
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