| Object: To research the protective effect and possible mechanism of Enteromorpha's Polysacchride(EP) against carbon tetrachloride induced hepatotoxicity in vivo and in vitro.Method:1. Take the enteromorpha which is produced by fujian province as the research object.The EP is extracted by cellulose enzymatic. Then remove protein by neutral protease, remove salts by materialto dialysis bag, remove pigment by DA201 resin.Combinated with water extract-alcohol precipitation to extract the CEP. Then DEAE-52 cellulose will be used to purify and separate the CEP,then the principal components of CEP(EPx) will be getted.Analysis CEP and EPx about polysaccharide content by phenol sulfuric acid method, sulfate radical content by barium sulfate turbidimetry.2. In vitro study, human hepatocyte cell line L-02 was used. Cells were seeded in multi-well plates. Then the cells are divided into 6 groups that would finally become the normal group(Normal),the model group(Model),3 EPx groups(LEPx?MEPx?HEPx)and positive control group(Vit E). Cells are cultured in the base culture medium for 24 h.To produce the Normal group, cells were cultured in the RPMI-1640 culture medium for4 h.To produce the Model group, cells were cultured in the base culture medium supplemented with 15 mmol/L CCl4(final concentration) for 4 h.To produce the EPx groups, cells were cultured in the base culture medium supplemented with 15 mmol/L CCl4 and 10-8 g/m L?10-7 g/m L?10-6g/m L EPx(final concentration) for 4 h.To produce the Vit E group, cells were cultured in the base culture medium supplemented with 15mmol/L CCl4 and 50?mol/L Vit E(final concentration) for 4 h.Then cell viability was assessed by the CCK8 assay. The activities of ALT?AST?SOD?GSH-Px?GSH and MDA in cell culture supernatant or cell were determined using commercial kits.Gene expression of SOD and GPx were analysis by RT-PCR.Protein expression of CYP2E1 was analysis by western-bolting.3.In vivo study,male ICR mouse was used. The animals were randomly divided into six groups with each consisting of 12 mice. The 6 groups are the normal group(Normal),the model group(Model),3 CEP groups(LCEP?MCEP?HCEP) and positive control group(Silymarine). Mice in the 3 CEP groups were treated with CEP at a dose of 150, 300 and 450 mg/kg body weight for three consecutive weeks(once per day),and mice in Silymarine group were treated with silymarine at a dose of 100mg/kg,while mice in normal group and model group were treated with physiological saline.The mice were injected with CCl4(10 ml/kg i.p. of 0.1% CCl4 solution in olive oil) one hour after the last administration, except for the normal group, which was given only olive oil injection. The animals were sacrificed 16 h after the CCl4 and olive oil treatment, respectively. The activities of ALT?AST?SOD?GSH-Px?GST?GSH and MDA in serum or liver were determined using commercial kits.Gene expression of SOD and GPx were analysis by RT-PCR.Protein expression of CYP2E1 was analysis by western-bolting. An extra sample of liver were stained with hematoxylin-eosin(HE) and examined under light microscope for general histopathology examination.Result:1. The extraction, separation and purification of EP CEP is a palegreen powder solid,while EPx is a white powder solid. With phenol-sulfuric acid method, the polysaccharide content of CEP is 53.93%, while EPx is97.49%. With gelatin-barium sulfate turbidimetry, the sulfate radical content of CEP is30.44%, while EPx is 22.49%.2. Protective effect of EPx against CCl4 induced hepatotoxicity in vitro Compared with the Normal,the Model had a lower cell ciability(P<0.01).And the activities of ALT?AST in the culture supernatant were higher(P<0.01).The activities of GPx?SOD in cell were lower,the level of GSH was lower(P<0.01) and the level of MDA was higher(P<0.01).The expression of GPx?Cn/Zn SOD gene and CYP2E1 protein were higher(P<0.01).Compared with the Model, the cell ciability of LEPx?MEPx?HEPx were higher(P<0.05),and the effect was found to be dose-dependent.The activities of ALT in the culture supernatant was lower(P<0.01).The activities of GPx of 3 EPx groups were significantly higher than that of Vit E, and the effect was found to be dose-dependent. The activities of GPx of MEPx?HEPx were higher(P<0.01) than Model except LEPx. The activities of SOD of LEPx?MEPx?HEPx were higher than Model(P<0.01),and MEPx was the most highest.Vit E also showed higher in the activities of SOD(P<0.01),but lower than EPx.The level of GSH of LEPx?MEPx?HEPx were higher than Model(P<0.01),and closed to the level of Normal(P>0.05).And the level of MDA of LEPx?MEPx?HEPx were lower than Model(P<0.01), and the effect was found to be dose-dependent.The expression of CYP2E1 protein of MEPx?HEPx and Vit E were lower than Model(P<0.01),and closed to the level of Normal(P>0.05).The expression of the gene of SOD of MEPx was lower than Model(P<0.05).And the expression of the gene of GPx of LEPx was lower than Model(P<0.05). The expression of the gene of SOD and GPx of Vit E was significantly lower than Model(P<0.01).3. Protective effect of CEP against CCl4 induced hepatotoxicity in vivo Compared with Normal,the liver index of Model was heavier(P<0.01).And the activities of ALT?AST in serum were significantly higher(P<0.01).The activities of GPx?GST?SOD in liver were significantly lower,the level of GSH was lower(P<0.01)and the level of MDA was higher(P<0.01).The expression of GPx?Cn/Zn SOD gene and CYP2E1 protein were higher(P<0.01). And histopathological examination of livers challenged with CCl4 showed vacuole formation and inflammatory infiltration.The hepatocytes were markedly edematous and the cytoplasm of hepatocytes was loose.Compared with the Model, the liver index of LCEP?MCEP?HCEP were lighter but had no ststistically significant(P>0.05).The activities of ALT and AST in serum were significantly lower(P<0.01).The activities of GPx of 3 CEP groups and Silymarin were significantly higher than that of Model(P<0.01), and MCEP had the highest activities of GPx. The activities of GST of MCEP?HCEP were higher(P<0.01).The activities of SOD of LCEP?MCEP?HCEP were higher than Model(P<0.01),and MCEP was the highest.Silymarin also showed higher in the activities of GPx?GST and SOD(P<0.01),but lower than EPx.The level of GSH of LCEP?MCEP?HCEP were higher than Model(P<0.01),and closed to the level of Normal(P>0.05).And the level of MDA of LCEP?MCEP?HCEP were lower than Model(P<0.01), and closed to the level of Normal. The expression of CYP2E1 protein of MCEP?HCEP and Silymarin were lower than Model(P<0.01).The expression of the gene of SOD and GPx of LCEP?MCEP?HCEP were lower than Model(P<0.05). The expression of the gene of SOD and GPx of Silymarin was significantly lower than Model(P<0.05).The liver sections of mice pretreated with the CEP before CCl4 challenge showed that the areas of normal liver architecture and patches of inflammatory infiltration and necrotic hepatocytes. Livers of mice pretreated with the dose of 300 mg/kg showed well-preserved architecture.Conclusion:1. The CEP was extracted by cellulose enzymatic.Then purify and separate the CEP to get EPx. The polysaccharide content of CEP is 53.93%, while EPx is 97.49%. The sulfate radical content of CEP is 30.44%, while EPx is 22.49%.2. The EPx was effective in preventing CCl4-induced damage to hepatocyte in vitro.And the effects of EPx was better than Vit E. The hepatoprotective effects of EPx may be due to inhibited lipid peroxidation, effective recovery of the antioxidative defense system and inhibition the expression of CYP2E1 protein.3. The CEP was effective in preventing CCl4-induced damage to hepatocyte in vivo. The hepatoprotective effects of CEP may be due to inhibited lipid peroxidation, effective recovery of the antioxidative defense system and inhibition of the expression of CYP2E1 protein to protect liver from radical.4. The hepatoprotective effects of EP may be due to inhibition of the expression of CYP2E1 protein, effective recovery of the antioxidative defense system and radical scavenging. |
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