| BackgroundNonalcoholic fatty liver disease (NAFLD) has become a more common liverdisease. It is a clinical and pathological syndrome featured mainly as fataccumulation and fatty degeneration of liver cells without an excessive alcoholconsumption history. NAFLD is a spectrum of liver disease ranging fromhepatosteatosis, to non-alcoholic steatohepatitis (NASH), through to fibrosis andcirrhosis. NASH is a key stage in NAFLD pathogenesis, and it is related to thefurther development from steatosis to cirrhosis and even liver cancer. NASH ischaracterized mainly as fat desposion, liver cell damage, inflammation andfibrosis. In recent years, the incidence of NASH has a substantial increase withthe improvement in people’s living standards and significant changing in people’sdiet and lifestyle. At the same time, it has an upward trend globally and hasreceived more and more attention. But the pathogenesis of NASH is not fullyeluciated. The two-hit hypothesis is a commonly accepted theory which believes that the lipid peroxidation, oxidative stress and insulin resistance(IR) play animportant role in the development of NASH.The most common risk factors associated with NASH include oxidativestress, IR, lipid oxidation, heredity and so on. It is confirmed that IR often existsin the patients with NASH, and IR is the triggering factor rather than the result.Under the condition of IR, a large amount of free fatty acids(FFA) is producedand β-oxidation of fatty acid becomes weak in liver which decreases the ability ofthe synthesis of very low density lipoprotein, and results in a large number oflipid deposition in the liver cells. In these cells, the intracellular ATP reservereduces and oxidative stress enhances. All above pathogenic factors participate inthe formation of liver damage collaboratively. It is confirmed that oxidative stressis an important pathogenic factor of IR by activating a variety of redox sensitivekinase such as JNK can phosphorylate the insulin receptor substrate1(IRS-1) atSer and thereby inhibitits phosphorylationat Tyr by the insulin receptor.The transcription factor NF-E2-related factor2(Nrf2) is an important factorfor protecing cells against oxidative damage. Nrf2can combines withanti-oxidant response element (ARE) when it is stimulated by external oxidativestress, and induces the expressions of phase II antioxidant enzymes. The hemeoxygenase-1(HO-1) is a very important one and has been proved to improve IR.It can improve IR by the anti-oxidation and anti-inflammatory effects of itsproducts such as carbon monoxide (CO), free iron (Fe), biliverdin (BV) andbilirubin (BR) and so on. Previous study has confirmed that induction of HO-1expression can significantly reduce oxidative damage and improve IR in thehigh-fat diet-induced IR rat model.Our previous studies have confirmed that curcumin can increase Nrf2nuclear translocation and has protective effects on oxidative stress in hepatocytes. This study is to investigate the protective effects on the IR and NASH by theacitviaton of Nrf2/HO-1pathway induced by curcumin.ObjectiveThe high-fat-diet rat model with IR and oxidative stress model of LO2hepatocytes are used in the study. The study is to invistigate the influences ofupregulation of Nrf2/HO-1pathway by curcumin on the liver function, pathology,oxidative stress and IR and to discuss whether there will be a protection on IR inrat liver and LO2hepatocytes by the activiation of Nrf2/HO-1pathway. The aimis to supply the theoretic and experimental basis for NASH prevention andimprovement of IR through using Nrf2/HO-1pathway as a target.Methods1. Building IR rat model with high-fat diet. The rats were randomly dividedinto control group, model group and curcumin-treated group. The control groupwas given a normal diet, while the model group and curcumin-treated group weregiven high fat diet for6weeks. Then the rats in control and model groups weregiven the same while the curcumin-treated group was given high fat andintragastric gavage of curcumin200mg/kg a day for4weeks. At the end of the10th week, expressions of Nrf2, HO-1and insulin signaling pathway weredetected by Western blot, the liver function was assed with biochemical analyzerand the levels of oxidative stress was assed by the spectrophotometric method.2. The oxidative stress model of human LO2hepatocytes was prepared withglucose oxidase (GO). LO2hepatocytes were divided into control group, modelgroup, curcumin-treated group and inhibitor group. LO2hepatocytes in controlgroup were normally cultured. LO2hepatocytes in model group were treated with100U/L GO for2hours. LO2hepatocytes in curcumin-treated group weretreated as the model groups after cells were treated with30μm curcumin for12 hours. And LO2hepatocytes in the inhibitor group were treated the same as thecurcumin-treated group in the presence of0.1μM wortmannin (PI3K inhibtor) for1hour. Expressions of Nrf2, HO-1and insulin signaling pathway were detectedby Western blot. The level of cellular oxidative stress was tested by thespectrophotometric method. The levels of AST and ALT in the cell culture fluidswere checked by the rate method. And the glucose level in the culture mediumwas also determined with oxidase-peroxidase method.Results1. The IR rat model was established successfully by high-fat diet induction.The glucose infusion rate (GIR) in the model group was significantly lower thanthat the control group(P＜0.01).2. The rat liver MDA level in the model group was significantly higher thanthat in the control group (P <0.01), while the level in the curcumin-treated groupwas significantly lower than that in the model group (P <0.01). The GSH level ofrat liver in the model group was significantly lower than that in the control group(P <0.01), while the level in curcumin-treated group was significantly higher thanthat in the model group (P <0.01). The serum ALT and AST levels in the modelgroup were significantly higher than those in the control group (P <0.01) and thecurcumin-treated group were significantly lower than those in the model group (P<0.01).3. The nuclear Nrf2and HO-1expressions of rat livers in the control groupwere low, and the expressions in the model group was higher than the controlgroup, and that in the curcumin-treated group increased significantly than in boththe control group and the model group. To the expression of phosphorylated JNKlevel in rat liver, the model group was higher than in the control group, and thecurcumin-treated group was lower than in the model group. There was no significant difference in the expression of JNK level among the four groups. Tothe expression of phosphorylated IRS-1in rat liver, the model group was lowerthan the control group and the curcumin-treated group was higher than the modelgroup. There was no significant difference in IRS-1expression level among thefour groups.4. To the LO2hepatocytes liver function indicators of human liver cells andoxidative stress parameters ALT, AST and MDA, the model group was higherthan the control group(P <0.01), the curcumin-treated group was lower than themodel group(P <0.01), the inhibitor group was higher than the curcumin-treatedgroup (P <0.01). To the GSH level, the model group was lower than the controlgroup(P <0.01), the curcumin-treated group was higher than the model group(P<0.01), the inhibitor group was lower than the curcumin-treated group(P <0.01).To the glucose content in the cell culture medium, the model group was higherthan the control group (P <0.01), the curcumin-treated group was lower than themodel group(P <0.01), the inhibitor group was higher than the curcumin-treatedgroup (P <0.01).5. The expression of Nrf2nuclear translocation and HO-1of LO2hepatocytes in model group was a little higher than in the control group, thecurcumin-treated group increased significantly compared with the model groupand the inhibitor group was lower than in the curcumin-treated group. To theexpression of phosphorylated JNK level in cells, the model group was higher thanin the control group, the curcumin-treated group was lower than the model groupand the figure of the inhibitor group was between the model group and thecurcumin-treated group. To the expression of JNK level in liver cells, there wasno significant difference among the groups. To the expression of phosphorylatedIRS-1in liver cells, the model group was lower than the control group, the curcumin-treated group was higher than the model group, and the expressiondegree of inhibitor group was between the model group and the curcumin-treatedgroup. There was also no significant change in the IRS-1expression amongdifferent groups.ConclusionBy observing the effects of Nrf2inducer curcumin on the IR model ofhigh-fat diet rats, it was discovered that the expression of liver HO-1can beincreased by inducing Nrf2nuclear translocation and liver damage, oxidativestress and IR caused by the high-fat diet can be improved accordingly. Byinducing or blocking nuclear translocation of Nrf2to the oxidative stress modelof LO2hepatocytes, the study discovered that after blocking Nrf2nucleartranslocation, curcumin-induced improvements in liver function and IR arereversed partially. It also confirmed that IR caused by oxidative stress can bereduced by inducing Nrf2/HO-1pathway and its effect may be improve insulinsignaling pathway by reducing the phosphorylated JNK level and increasingphosphorylated IRS-1one. This study provides a new theoretical base forintervening NASH using Nrf2/HO-1as a target. |
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