Effect Of Curcumine On Intracellar Reactive Oxygen Species And Insulin Resistance In Cultured Human Hepatocytes Lo2

BackgroundNon-alcoholic fatty liver disease (NAFLD) is a kind of clinicopathological syndrome which is caused by certain reasons except alcohol or other definite factors and is characterized by diffused macrovascular hepatocyte steatosis. NAFLD is a spectrum of chronic liver diseases that can lead to cirrhosis and even hepatocellular carcinoma. Nonalcoholic steatohepatitis (NASH), is the critical turning point at which steatosis progresses to advanced stages such as hepatic fibrosis, cirrhosis and even hepatocellular carcinoma. So NASH is being paid more attention on. In recent years ,as China's lifestyle and diet changes, the incidence of NASH has increased significantly. However, pathogenic mechanisms of NASH have not been clearly defined . The"two hit"hypothesis suggests that the imbalance of oxidants and antioxidants leads to oxidative stress, resulting in lipid peroxidation that is closely related to the occurrence and development of NASH. Nowadays, although the clinical research and foundational experiments on the therapy for NASH have got more attention of the experts in our country and abroad, none has clearly been proved effective. So the research on pathogenesis and approaches to the treatment of NASH are still in need.It is reported that oxidative stress, lipid peroxidation, insulin resistance and genetic are concerned with NASH.Nowadays the research on the pathogenesis of NASH are more focused on oxidative stress, lipid peroxidation, insulin resistance.Experiments confirmed that oxidative stess can cause liver insulin resistance.The IGF-1R/IR-PI3K-Akt signaling pathway is abnormal.NF-E2-related factor 2 (Nrf2) is an important transcription factor in the process of anti-oxidative stress response. The transcription factor Nrf2 is regulated negatively by its inhibitor Keap1. This protein is located in the cytoplasm and binds Nrf2 preventing its migration to the nucleus. Nrf2- Keap1 plays an important role in fighting against exogenous and endogenous oxidative stress. It is also the hub of cellular response to oxidative stress regulator. Nrf2 regulation through interaction with ARE encodes antioxidant proteins and phaseⅡenzymes. Study found that Nrf2-ARE-Keap1 has a wide range of cellular protection in anti-cancer,neuroprotective,anti-inflammatory.Curcumin is a kind of natural antioxidant,which is belonged to polyphenol compound and is innoxious to human beings. Curcumin can inhibit many diseases resulted from peroxidation.So the people pay more attention to the antioxidant activity of curcumin .Its extensive biological activity includes anti-inflammation, anti-mutagenesis, anti-oxidation, reducing cholesterol, anti-tumor and anti-archerosclerosis etc.In this subject we use glucose oxidase to establish oxidative stress liver cell model. Then we observe the translocation of Nrf2 in LO2 using the methods of indirect immunofluorescence and western blot.We observe the extent of oxidative stress by flow cytometry .We observe the levels of MDA,GSH,AST,ALT by spectrophotometric method and kinetic rate method using commercially available reagent kits. We observe the level of IRS1 and JNK which reflect the insulin resistence in LO2 by western blot.The LO2 is treated with different concentrations of curcumin to observe the dose-effect relationship.ObjectiveInvestigate the effect of curcumine on intracellar ROS and insulin resistance(IR) induced by glucose oxidase(GO) in cultured human hepatocyte LO2 to provide a new theoretical basis of the pathogenesis of NASH.Methods1,Hepatocytes LO2 were treated with 25U/L,50 U/L,75 U/L,100U/L and 125 U/L glucose oxidase for 2 h . The levels of MDA,GSH and LDH were measured by spectrophotometric method. The levels of AST and ALT were measured by kinetic rate method using commercially available reagent kits.2,Hepatocyte LO2 grown on cover slips were treated with 15 and 30μmol/L curcumine,or vehicle(RPMI1640) as control. After 6 or 12 h of treatment, the level of nuclear translocation was analyzed by immunocytochemistry and western blot. Hepatocyte LO2 were divided into control group,model group curcumine-treated group and inhibitor group. Hepatocytes in control group were only incubated with RPMI 1640, hepatocytes in model group were treated with 100 U/L glucose oxidase(GO) for 2 h, hepatocytes in curcumine-treated group were treated with 30μmol/L curcumine for 12 h and subsequently treated with100 U/L GO for 2 h. hepatocytes in inhibitor group were treated with 0.1μmol/L Wortmannin for 1 h. Then the hepatocytes were treated as the curcumine-treated group.3,Then the Nrf2 nuclear translocation in hepatocytes was observed by immunocytochemistry and western blot. The levels of MDA and GSH were measured by spectrophotometric method. The levels of AST and ALT were measured by kinetic rate method using commercially available reagent kits. The ROS level was analyzed on a flow cytometer by using Dihydroethidium (DHE). The content of glucose remained in culture supernatant was measured by the method of glucose oxidizes peroxides (GOD-POD). The level of JNK/P-JNK,IRS1/P-IRS1 was measured by western blot.Results1,We establish oxidative stress model of hepatocytes LO2 with glucose oxidase successfully.2,Curcumine resulted in the enhanced nuclear translocation. The effect of the treatment with 30μmol/L curcumine was better than that in the treatment with 15μmol/L curcumine. While at the same concentration,the effect of 12 h treatment was better than that of 6 h treatment.3,Compared with control group, the levels of MDA,AST,ALT and the level of intracellar ROS increased significantly in model group(P<0.01). However, the levels in curcumine group decreased significantly in comparison with those in model group(P<0.01). Compared with control group, the level of GSH decreased significantly in model group(P<0.01). However, the levels of GSH in curcumine group increased significantly in comparison with those in model group(P<0.01). Curcumine resulted in the enhanced nuclear translocation.. The content of glucose remained in culture solution increased significantly in model group ( P<0.01 ) in comparison with control group. Compared with model group, the content of glucose remained in culture supernatant decreased significantly (P<0.01)in curcumine group.4,The curcumin can reduce insulin resistance in human hepatocytes LO2. The content of glucose remained in culture solution increased significantly in model group(P<0.01)in comparison with control group. Compared with model group, the content of glucose remained in culture supernatant decreased significantly (P<0.01)in curcumine group.ConclusionWe establish oxidative stress model of hepatocytes LO2 with glucose oxidase successfully and explore the antioxidant effect of curcumine. Curcumine can reduce the intracellar ROS level and alleviate the oxidative stress-mediated IR in human hepatocytes through enhanced Nrf2 nuclear translocation. Through the experiment we confirmed the damage of hepatocytes LO2 resulted from oxidative stress and insulin resistance and observe the effect of curcumin on oxidative stress and insulin resistance.The effect is related with the translocation of Nrf2 from cytoplasm to nucleus.