| BackgroundNon-alcoholic fatty liver disease (NAFLD) is a kind of clinicopathological syndrome which has the same histopathological change with alcoholic hepatitis, but no medical history of excessive drink. The spectrum of NAFLD ranges from non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis(NASH) and cirrhosis. The histopathological character of NASH is hepatocyte injury and steatosis, hepatic inflammation and hepatic fibrosis. NASH is the critical turning point in the process from steatosis to hepatic fibrosis and even cirrhosis. In recent years, following the lifestyle and diet changes, the morbidity of NASH has increased significantly in China. However, pathogenesis of NASH have not been clearly clarified. The"two hit"hypothesis suggests that the oxidative insults could be the key factor in the occurrence and development of NASH.It is considered that the activation and proliferation of hepatic stellate cell (HSC) is the center link in the progress of hepatic fibrosis. The major function of HSC is stockpiling fat and vitamin A in physiological status. In the pathologic condition, HSC is activated and transformed to myofibroblast which has the character of proliferation, secreting cell factor, synthetizing extracellular matrix(ECM). Activated HSC is the primary source of ECM, therefore it is identified as the target cell in research of chronic liver disease.NF-E2-related factor 2 (Nrf2) is an important transcription factor in the process of anti-oxidative stress response. Nrf2 can be combined with antioxidant-response element (ARE), which can up-regulate the expression of numerous antioxidant enzymes and phase-Ⅱdetoxifying enzymes, and enhance oxidation resistance. Recent studies suggest that Nrf2 is central regulator of cellular antioxidation. Nrf2 can maintain the oxidation/antioxidation balance, reduce the sensitivity of the cell to death signals, and it plays an important role against exogenous or endogenous oxidatie stress. Nrf2 has an significant part in regulating oxidative stress, but most studies focus on the nervous and respiratory systems. The effect and mechanism of Nrf2 in HSC have seldom been reported. This experiment used rats HSC-T6 as research object, and the cell model of oxidative stress was established. The aim was to observe the expression of Nrf2 in HSC and the influence of oxidatie stress on its nuclear translocation. Furthermore, it was to research the protective effect of Nrf2 on HSC against oxidative insults by up-regulating the level of Nrf2 nuclear translocation.ObjectiveThis experiment prepared oxidative stress model of HSC with glucose oxidase(GO), and aimed to investigate the influence of oxidation on nuclear translocation of Nrf2 in HSC. Furthermore, we presume that if curcumin should promote Nrf2 nuclear translocation and up-regulate the expression of antioxidant enzymes and phase-Ⅱdetoxifying enzymes, which probably could protect the liver against injury. It will provide a new experimental and theoretical basis for the prevention and treatment of NASH.Methods1.HSC-T6s were divided into negative control group and oxidative group. HSC-T6s in oxidative group were treated with 100 mU/ml GO for 2 h, and HSC-T6s in negative control group were incubated with nothing. Then the expressions of total Nrf2 and nuclear Nrf2 were evaluated with Western blot. HSC-T6s were analyzed by immunocytochemistry to observe the nuclear translocation of Nrf2 protein. The reactive oxygen species(ROS)level was analyzed on a flow cytometer by using Dihydroethidium (DHE). The levels of malondialdehyde (MDA) and glutathione(GSH) were measured by spectrophotometry.2.HSC-T6s were separated into three groups. HSC-T6s in the negative control group were incubated with DMEM. HSC-T6s in the oxidative group were incubated with 100 mU/ml GO for 2 h before each experimental manipulation. HSC-T6s in the curcumin intervention group were incubated with 30 uM curcumin for 3 h and then incubated with 100 mU/ml GO for 2 h before each experimental manipulation. The expression of Nrf2 in HSC-T6 was observed using the methods of immunocytochemistry and Western blot analysis. The activation of HSC was observed by indirect immunofluorescence. The extent of ROS was determined by flow cytometry. The levels of MDA and GSH were determined by spectrophotometry using commercially available reagent kits. The levels of extracellular matrix secretion(ECM) were assessed by radioimmunoassay.Results1. Oxidative stress model of HSC-T6 was established with GO successfully. The levels of ROS and MDA in oxidative group were higher significantly than negative control group(P<0.01).2. The expression of total Nrf2 in negative control group and oxidative group had no obvious change which detected by Wstern blot, and the expression of nuclear Nrf2 in oxidative group was higher than negative control group. Immunocytochemistry assay showed that the Nrf2 protein of negative control group was expressed only in cytoplasm, but the Nrf2 protein of oxidative group was expressed both in cytoplasm and in nucelus. Compared with control group the levels of GSH in oxidative group showed manifest increase(P<0.01).3. Western blot assays demonstrated that the expression of total Nrf2 in every group had no significant change, while the expression of cytoplasmic Nrf2 decreased slightly in the oxidative group, and the level of cytoplasmic Nrf2 in the curcumin intervention group significantly decreased compared to the negative control group. Immunohistochemistry experiments also showed that in the curcumin intervention group, the amount of positive brown staining was abundant in the nucleus, as compared to the oxidative group, in which only a little positive brown staining was observed, and the negative control group, in which there was no positive brown staining in the nucleus.4. The expression of desmin in each group was positive, and there was no obvious difference among groups. There was no obvious expression ofα-SMA in the negative control group, whereas there was abundant expression in the oxidative group and a small quantity of expression in the curcumin intervention group.5. The fluorescence intensity of DHE in the oxidative group was strongest, the fluorescence intensity of DHE in the negative control group was weak, and the curcumin intervention group displayed intermediate fluorescence intensity(P<0.01). The level of MDA in the oxidative group was significantly enhanced compared with the negative control group(P<0.01), while the MDA level was attenuated but remained higher than the negative control group after curcumin pretreatment(P<0.01). Compared to the negative control group, the level of GSH increased slightly in the oxidative group(P<0.01), and the GSH level in the curcumin intervention group was significantly elevated(P<0.01). 6. The levels of PCⅢ, CⅣ, LN and HA in the supernatant of HSC-T6 culture medium were enhanced significantly after oxidative stres(sP<0.01), and the ECM levels in the curcumin intervention group were lower than oxidative group but higher than negative control group(P<0.01).ConclusionOxidative stress model of HSC-T6s with GO was successfully established. The study shows that oxidative stress can activate the nuclear translocation of Nrf2 to develop its biological effects in HSCs. Meanwhile, curcumin upregulates Nrf2 nuclear translocation, which could decrease the intracellar ROS level, alleviate the oxidative insults, inhibit HSC activation and reduce the secretion of extracellular matrix. The findings point out that the translocation of Nrf2 could be a target for the prevention and treatment of NASH. |
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