Deficiencies in vitamins or other factors(B6,B12,folic acid, betaine)and genetic disorders for the metabolism of the non-protein amino acid-homocysteine(Hcy)lead to hyperhomocysteinemia(HHcy).HHcy is an integral compo...Deficiencies in vitamins or other factors(B6,B12,folic acid, betaine)and genetic disorders for the metabolism of the non-protein amino acid-homocysteine(Hcy)lead to hyperhomocysteinemia(HHcy).HHcy is an integral component of several disorders including cardiovascular disease,neurodegeneration,diabetes and alcoholic liver disease.HHcy unleashes mediators of inflammation such as NFκB,IL-1β,IL-6,and IL-8,increases production of intracellular superoxide anion causing oxidative stress and reducing intracellular level of nitric oxide(NO),and induces endoplasrnic reticulum(ER)stress which can explain many processes of Hcy-promoted cell injury such as apoptosis, fat accumulation,and inflammation.Animal models have played an important role in determining the biological effects of HHcy.ER stress may also be involved in other liver diseases such as α_1-antitrypsin(α_1-AT)deficiency and hepatitis C and/or B virus infection.Future research should evaluate the possible potentiative effects of alcohol and hepatic virus infection on ER stress-induced liver injury,study potentially beneficial effects of lowering Hcy and preventing ER stress in alcoholic humans,and examine polymorphisrn of Hcy metabolizing enzymes as potential risk-factors for the development of HHcy and liver disease.展开更多
OBJECTIVE:To investigate effects of the extracts from soothing-liver and invigorating-spleen formulas on the injury due to oxidative stress,mediated by the Nuclear factor-like 2(Nrf2)-Antioxidant response element(ARE)...OBJECTIVE:To investigate effects of the extracts from soothing-liver and invigorating-spleen formulas on the injury due to oxidative stress,mediated by the Nuclear factor-like 2(Nrf2)-Antioxidant response element(ARE) pathway,in the hepatocytes of rats with non-alcoholic fatty liver disease(NAFLD) induced by high-fat diet.METHODS:Soothing-liver and invigorating-spleen formula mixtures were prepared for five groups:normal,model,soothing-liver formula group(SLG),invigorating-spleen formula group(ISG),integrated formula group(IG).The rat model of NAFLD was induced by feeding rats a high-fat diet(HFD).After 16 weeks,the hepatic tissue was examined following Haematoxylin-Eosin(H&E) staining and with Transmission electron microscopy(TEM).Levels of hepatic lipids,serum lipids and hepatic functions were measured using a biochemical analyser.Hepatocytes were isolated from the livers of rats and were identified by cellar immunohistochemistry,cellular immunofluorescence and flow cytometry.The expression levels of Nrf2,Kelch-like epichlorohydrin-associated protein 1(Keap-1),haeme oxygenase-1(HO-1) and NAD(P)H:quinone oxidoreductase 1(NQO1) m RNAs were assessed by real-time fluorescence quantitative PCR.Nrf2,Keap-1,HO-1 and NQO1 proteins were measured by Western blotting.Finally,the levels of oxidative stress factors Superoxide Dismutase(SOD),malonaldehyde(MDA) and Glutathione peroxidase(GSH-Px) in hepatocytes were measured by WST-1,TBA and colorimetry.RESULTS:The H & E and TEM results showed that the NAFLD model rats successfully reproduced typical pathogenetic and histopathological features.The liver function and levels of hepatic lipids and serum lipids from the model rats were dramatically increased.Compared with the model group,the levels of hepatic lipids,serum lipids and hepatic function in the treatment groups were ameliorated to different degrees.The yields of purified hepatocytes in each rat were 4-5 × 108.The viability of the isolated hepatocytes was higher than 95%,with a purity over 93.2%.Cellular immunohistochemistry analysis showed that the hepatocytes were brown,while in the cellular immunofluorescence analysis,the hepatocytes showed green fluorescence.The expression levels of Nrf2,Keap-1,HO-1 and NQO1 m RNA and protein in the hepatocytes were significantly higher in the model group than in the normal group(P < 0.05,P < 0.01).Compared with the model group,the expression of Nrf2,Keap-1,HO-1 and NQO-1 m RNAs and proteins in all treatment groups increased,especially in the IG(P < 0.01).CONCLUSION:The extracts from soothing-liver and invigorating-spleen formulas may protect the liver against the injury induced by oxidative stress in hepatocytes by influencing the Nrf2-ARE pathway,which may be the mechanism having the potential for prevention and treatment of NAFLD.展开更多
基金Supported by the U.S.National Institute of Alcohol Abuse and Alcoholism,R01 AA014428 and by the Robert E.and May R.Wright Foundation,No.263
文摘Deficiencies in vitamins or other factors(B6,B12,folic acid, betaine)and genetic disorders for the metabolism of the non-protein amino acid-homocysteine(Hcy)lead to hyperhomocysteinemia(HHcy).HHcy is an integral component of several disorders including cardiovascular disease,neurodegeneration,diabetes and alcoholic liver disease.HHcy unleashes mediators of inflammation such as NFκB,IL-1β,IL-6,and IL-8,increases production of intracellular superoxide anion causing oxidative stress and reducing intracellular level of nitric oxide(NO),and induces endoplasrnic reticulum(ER)stress which can explain many processes of Hcy-promoted cell injury such as apoptosis, fat accumulation,and inflammation.Animal models have played an important role in determining the biological effects of HHcy.ER stress may also be involved in other liver diseases such as α_1-antitrypsin(α_1-AT)deficiency and hepatitis C and/or B virus infection.Future research should evaluate the possible potentiative effects of alcohol and hepatic virus infection on ER stress-induced liver injury,study potentially beneficial effects of lowering Hcy and preventing ER stress in alcoholic humans,and examine polymorphisrn of Hcy metabolizing enzymes as potential risk-factors for the development of HHcy and liver disease.
基金a grant from National Natural Science Foundation of China:Mechanism of Nrf2/ARE Pathway Mediated by p38 MAPK regulating Oxidative Stress-Related Genes in NAFLD Rats and Effect of Soothing Liver and Invigorating Spleen Recipes(No.81273617)
文摘OBJECTIVE:To investigate effects of the extracts from soothing-liver and invigorating-spleen formulas on the injury due to oxidative stress,mediated by the Nuclear factor-like 2(Nrf2)-Antioxidant response element(ARE) pathway,in the hepatocytes of rats with non-alcoholic fatty liver disease(NAFLD) induced by high-fat diet.METHODS:Soothing-liver and invigorating-spleen formula mixtures were prepared for five groups:normal,model,soothing-liver formula group(SLG),invigorating-spleen formula group(ISG),integrated formula group(IG).The rat model of NAFLD was induced by feeding rats a high-fat diet(HFD).After 16 weeks,the hepatic tissue was examined following Haematoxylin-Eosin(H&E) staining and with Transmission electron microscopy(TEM).Levels of hepatic lipids,serum lipids and hepatic functions were measured using a biochemical analyser.Hepatocytes were isolated from the livers of rats and were identified by cellar immunohistochemistry,cellular immunofluorescence and flow cytometry.The expression levels of Nrf2,Kelch-like epichlorohydrin-associated protein 1(Keap-1),haeme oxygenase-1(HO-1) and NAD(P)H:quinone oxidoreductase 1(NQO1) m RNAs were assessed by real-time fluorescence quantitative PCR.Nrf2,Keap-1,HO-1 and NQO1 proteins were measured by Western blotting.Finally,the levels of oxidative stress factors Superoxide Dismutase(SOD),malonaldehyde(MDA) and Glutathione peroxidase(GSH-Px) in hepatocytes were measured by WST-1,TBA and colorimetry.RESULTS:The H & E and TEM results showed that the NAFLD model rats successfully reproduced typical pathogenetic and histopathological features.The liver function and levels of hepatic lipids and serum lipids from the model rats were dramatically increased.Compared with the model group,the levels of hepatic lipids,serum lipids and hepatic function in the treatment groups were ameliorated to different degrees.The yields of purified hepatocytes in each rat were 4-5 × 108.The viability of the isolated hepatocytes was higher than 95%,with a purity over 93.2%.Cellular immunohistochemistry analysis showed that the hepatocytes were brown,while in the cellular immunofluorescence analysis,the hepatocytes showed green fluorescence.The expression levels of Nrf2,Keap-1,HO-1 and NQO1 m RNA and protein in the hepatocytes were significantly higher in the model group than in the normal group(P < 0.05,P < 0.01).Compared with the model group,the expression of Nrf2,Keap-1,HO-1 and NQO-1 m RNAs and proteins in all treatment groups increased,especially in the IG(P < 0.01).CONCLUSION:The extracts from soothing-liver and invigorating-spleen formulas may protect the liver against the injury induced by oxidative stress in hepatocytes by influencing the Nrf2-ARE pathway,which may be the mechanism having the potential for prevention and treatment of NAFLD.