Effects Of Nrf2on Insulin Resistance And Expression Of Oxidative Stress Related Genes In The NAFLS Model

Non-alcoholic fatty liver disease (NAFLD) is one of the important causes of chronic liver damage without a history of significant alcohol consumption which histologically resembles alcohol-induced liver damage. It is described as the hepatic manifestation of the metabolic syndrome and the spectrum of this disease ranges from simple steatosis to steatohepatitis. liver fibrosis, cirrhosis and eventually hepatocellular carcinoma. In recent years, with the development of people’s living level, the incidence of NAFLD increased quickly year by year, so more and more people take the disease seriously, especially in the western societies. Now the biological mechanism of NAFLD and progression to non-alcoholic steatohepatitis is not entirely understood. Now someone had proposed that the cause of the disease is "two-hit hypothesis". The "first hit" is attributed to insulin resistance and the "second hit" entails oxidative stress. Research reports that NF-E2related factor2(Nrf2) plays an important role in the pathogenesis of NAFLD, and it is a key transcription factor that plays a central role in cellular defense against oxidative and electrophilic insults. Nrf2is sequestered in the cytoplasm under physiological conditions by Kelch-like ECH-associated protein1(Keapl). Upon oxidative stress, Nrf2translocates to the nucleus, and it regulates the transcriptional activation of a battery of cytoprotective genes. So Nrf2induces expression of antioxidant related genes, such as NQO1, HO-1, GCLC, SOD-1and so on by binding to antioxidant responsive elements.In our study we use high fat diet to establish the mouse model of NAFLD and to analyze the progress of insulin resistance and oxidative stress in the disease. We use the wild-type (WT) mice and lack Nrf2(KO) mice to feed in the same conditions to explore the expression of Nrf2and oxidative stress related genes. The present study was aimed to investigate whether insulin resistance, oxidative stress and Nrf2play a role in the NAFLD.In the study, fourty male ICR mice were randomly divided into four groups, each group contained10mice, respectively4w control group (WT-4w-NC),4w model group (WT-4w-HFD),8w control group (WT-8w-NC).8w model group (WT-8w-HFD). Fourty male Nrf2-null mice were randomly divided into four groups, each group contained10mice, respectively4w control group (KO-4w-NC),4w model group (KO-4w-HFD),8w control group (KO-8w-NC),8w model group (KO-8w-HFD). The control groups were placed on standard diet and the model groups were fed with high fat diet. We should observe the mice and weigh the body weight. Thereafter, they were placed on either the HFD diet or the control diet for4weeks and8weeks and again given free access to water, before being sacrificed by removal of the mice eyes. After death, the blood samples were collected and centrifuged (3,000×g) at4℃for15min to collect serum. The serum was used to detect the serum biochemical indexes, the level of leptin and adiponectin. The liver from each mouse was removed immediately and weighed. Part of the liver tissue was used to detect the relevant indicators of oxidative stress. Liver histological changes were observed by HE staining. Real-time fluorescent quantitative PCR was done to determine hepatic expression levels of the oxidative stress related genes.The results showed that compared with the normal group, the mice hair was greasy and the liver was claybank in the HFD groups. Compared with the control groups, the HFD groups had a significantly high level of liver weight/body weight ratio, related biochemical indexes, such as ALT, AST, ALP, TC and LDL. These observations indicate that the high fat diet can establish the mouse model of NAFLD successfully. Compared with the normal group, the HFD groups had a significantly high level of HOMA-IR, serum leptin and a significantly low level of adiponectin. Moreover, the amount of MDA was significantly increased, content of GSH was significantly decreased and activity of SOD was significantly decreased in the mice liver of the HFD groups. Compared with the HFD groups in wild-type mice, the level of HOMA-IR, serum leptin, content of MDA were significantly increased, the level of adiponectin, the activity of SOD and content of GSH were decreased significantly in liver of HFD groups of Nrf2-null mice. These results indicate that NAFLD can induce insulin resistance and oxidative stress. And impairment of Nrf2activity may represent a major risk factor for the evolution of NAFLD. Compared with the control groups in wild-type mice, the mRNA levels of the Nrf2gene was up-regulated in the liver of wild-type HFD groups. And the Nrf2-targeted cytoprotective genes such as SOD-1, HO-1, NQO1, Gsta-1. GCLC were up-regulated in the liver of wild-type HFD groups. Compared with the HFD groups in wild-type mice all the Nrf2-targeted cytoprotective genes were down-regulated in the liver of Nrf2-null HFD groups. All the results indicated that livers of Nrf2-null mice on the HFD diet suffered more oxidative stress and insulin resistance than their wild-type counterparts. Nrf2may up-regulated the expression of antioxidant genes and phase Ⅱ detoxification enzyme genes and delay the NAFLD disease process.