The Protective Effects And Possible Mechanisms Of Polygonatum Sibricum Instant Powder On Alcoholic Liver Disease In Mice Based On Nrf2/ARE Pathway

Objective A mouse model of alcoholic liver disease,was established by chronic alcohol feeding and acute alcohol intragastric administration,and mice were given Polygonatum sibricum instant powder（PSIP）to explore the protective effect of PSIP on alcoholic liver disease mice and its possible mechanism by intragastric administration.Methods After one week of acclimatization,the female ICR mice were randomly divided into a control group（Ctrl）,model group（EtOH）,PSIP low-dose group（0.5 g/kg,LPSIP）,PSIP medium-dose group（1.0 g/kg,MPSIP）,PSIP high-dose group（2.0 g/kg,HPSIP）,and Polygonatum sibricum polysaccharide group（0.75 g/kg,PSP）.Mice in the intervention groups were given PSIP or PSP by gavage,while mice in the control and model groups were given the same volume of solvent by gavage for 30 days.Form days16-20,all mice were fed the alcoholic controlled liquid diet ad libitum to acclimatize them to the liquid diet.Then,all mice,except the control group,were fed with an alcoholic controlled liquid diet for ten days.At day 31,the ethanol-fed and pair-fed mice were gavaged in the early morning with a single dose of ethanol（5 g/kg）or isocaloric maltose dextrin,respectively.All the were sacrificed nine h later.Serum transaminase and other related liver function indexes were detected.The liver weight was weighed,and the ratio of liver weight to body weight was calculated.Part of liver tissue was fixed in paraformaldehyde,paraffin sections were prepared for H&E staining,and frozen sections were used for oil red O staining.The residual liver tissue was packed in-80℃.The Enzyme-labelled instrument was used to test the liver malonaldehyde（MDA）and reduced glutathione（GSH）,total antioxidant capacity（T-AOC）,catalase（CAT）,superoxide dismutase（SOD）and glutathione peroxidase（GSH-Px）levels.The mRNA levels of nuclear factor erythroid-2-related factor 2（Nrf2）and its downstream protective proteins in the liver were detected by real-time fluorescence PCR.The protein levels of Nrf2 and its downstream and phosphorylated protein levels of mitogen-Activated Protein Kinase（MAPK）in the liver were detected by Western blotting.Results Mice in the model group showed significant higher levels of serum alanine aminotransferase（ALT）,aspartate aminotransferase（AST）andγ-glutamine transferase（γ-GT）activities and total bilirubin（TBIL）and triglyceride levels when compared to the control group mice（P<0.01）.Compared with the control group,a large number of fat vacuoles and lipid accumulation were observed in pathological sections of the liver of mice in the model group.Meanwhile,triglyceride（TG）and MDA levels of the model group in the liver were significantly higher than that of the control group（P<0.01）.Besides,compared with the control group,the GSH,T-AOC,CAT,SOD and GSH-Px activity of model group in the liver had lower levels（P<0.01）.Oxidative stress generating enzymes NADPH oxidase 1（NOX1）,p67phox,gp91phox and cytochrome P4502E1（CYP2E1）mRNA levels of liver were significantly increased in model group compared to that in control mice.Compared with the model group,the levels of liver injury and oxidative stress in the PSIP intervention group and the PSP group were significantly improved,which was reflected by the decreased levels of ALT,AST andγ-GT,as well as TBIL and TG in the serum.Compared with the model group,TG and MDA levels of liver were significantly decreased in the PSIP intervention group and the PSP group（P<0.05,P<0.01）.The levels of GSH,T-AOC,CAT,SOD and GSH-Px liver were significantly increased in the PSIP intervention group and the PSP group mice compared to those in the model group（P<0.05,P<0.01）.Hepatic mRNA levels of NOX1,p67phox,gp91phox and CYP2E1 were significantly decreased in the PSIP intervention group and the PSP group mice compared to the model group mice（P<0.05,P<0.01）.Mechanism studies showed that the mRNA levels of Nrf2,heme oxygenase-1（HO-1）,NADPH:quinone oxidoreductase-1（NQO1）,glutathione S transferase p1（GSTp1）and glutathione reductase（GR）in the PSIP intervention group and PSP group mice liver were higher than those in the model group（P<0.05,P<0.01）.Compared with the model group,the protein levels of Nrf2,HO-1 and NQO1 in the PSIP intervention group and PSP group mice liver were significantly increased（P<0.05,P<0.01）,and the protein levels of Nrf2 in the nucleus were also significantly increased（P<0.01）.The hepatic phosphorylation of extracellular regulated protein kinases(Erk_1/2)in the PSIP intervention group and the PSP group mice were significantly higher than those in the model group（P<0.05,P<0.01）.Conclusions PSIP can play a protective role in mice with alcoholic liver disease by improving the antioxidant defence ability and reducing the level of oxidative stress.The Nrf2/ARE pathway as potential mechanism might be activated,which could promote the expression and nuclear transport of Nrf2,and induce the expression of downstream antioxidant proteins and other protective proteins.