| BackgroundDue to its high incidence and harmfulness, non-alcoholic steatohepatitis(NASH)attracts more attention and becomes one of the hottest issues in medical research. The pathogenesis of NASH is still unclear now. Insulin resistance and genetic susceptibility are closely related to the main pathogenesis. Activative and proliferative Hepatic satellite cells(HSC) are considered to be one of the core steps in development of liver fibrosis. Under the stimulus of various factors, activated HSC, transformes into muscle fibroblasts and gets the ability of proliferation and secretion of cytokines and a large amount of extracellular matrix(ECM). Be the main source of liver secretion of ECM, HSC is thought to be important target cells for the prevention and treatment of chronic liver disease.Meanwhile, apoptosis of hepatocyte also has close relationship with NASH. Apoptosis of hepatocyte in liver tissue specimens from NASH patients was significantly upregulated,and increased with the degree of hepatic fibrosis and hepatitis inflammation.NF-E2-related factor 2(Nrf2) is an important transcription factor for cell defense against oxidative stress reaction. Nrf2 plays an important role in the process of resisting exogenous or endogenous oxidative stress. Nrf2 plays a clearly protective role in NASH,but the specific mechanism and target cells are still not clear.p66Shc plays an important role in the process of cell oxidative stress and apoptosis.Studies show that p66 Shc knockout mice has enhanced antioxidant ability and extended life compared to normal group. The expression of p66 Shc in heart, liver, spleen and lung were different, and its expression is very high in animal fat tissue, probably related to the cell regulation of accumulation of lipids and formation of fat tissue. Oxidative stress is one of the most important factors leading to NASH fibrosis, and anti-oxidation treatment is helpful for inhibiting liver inflammation and fibrosis.IQGAP1, a kind of Ras GTPase activating protein, plays an important role in the formation of the cytoskeleton and adhesion. Researches show that increase the activity of IQGAP1 can increase Nrf2 related anti-oxidation abitlity and up-regulate the expression of HO-1. While reduced IQGAP1 expression can lead to a drop stability of Nrf2 and reduced expression of phase II enzyme, result in reduced anti-oxidation ability.ObjectiveThis experiment builded stably transfected rat HSC-T6 and hepatocytes(BRL-3A)with up or down Nrf2 expression by lentiviral transfection technique. Then we used glucose oxidase(GO) to buid cell oxidative stress model and observed the two stably transfected cells antioxidant ability, and further explored the expression of p66 Shc and IQGAP1 protein in order to clarify thepotential protective mechanism of the Nrf2 in NASH.Methods1. We designed and synthesized 4 kinds of Nrf2 gene sh RNA lentiviral recombinant plasmid and the Nrf2 expression lentiviral plasmid vector. Then we packaged, sequenced and determined titer the lentiviral vector, and transfected them into rat HSC-T6 and BRL-3A cells. The real time PCR(RT-PCR) and Western blot were used to confirm theexpression level of Nrf2 after transfected at 96 h. At last, stably transfected cells were got after treated with the lethal concentration of drug and expression of Nrf2 were detected.2. The stably transfected HSC-T6 and BRL-3A were divided into normal group and oxidative stress stimulation group, each group with 5 subgroups. Take HSC-T6 as an example, there are five subgroups: HSC-T6(BLANK), HSC-T6-sh RNA-NC, HSC-T6-sh RNA(down regulation of Nrf2), HSC-T6-Nrf2-NC, and HSC-T6-Nrf2(up regulation of Nrf2). The normal group trained cells with routine culture-medium, while oxidative stress stimulation group was added 100U/L GO into cells culture-medium for 2 hours in order to prepare the oxidative stress model. Then, ROS content were detected by flow cytometry.And survival rate of cells and levels of MDA, LDH, and SOD were detected by ELISA.3. Expression level of p66 Shc and IQGAP1 protein in cells form experiment 2 were detected by Western blot and their relationship with Nrf2 were also Analysised.Results1. Nrf2 sh RNA and over-expression plasmid was successfully constructed confirmed by sequencing. After transfected 96 h, RT-PCR and Western blot showed that the recombinant lentiviral vector can effectively decrease/increase the m RNA and protein levels of Nrf2 gene expression. Then transfected cells were treated with lethal concentrations drug, and stably transfected HSC-T6 and BRL-3A were gained with down/up expression of Nrf2 expression.2. In HSC-T6 cells, there were no difference among different subgroup in the normal group for all detections. But the ROS, MDA and LDH levels of oxidative stress group were significantly increased than the normal group, while the survival rate and SOD level were lower(P<0.05). In oxidative stress stimulation group: compared with parental cells and negative control, MDA, LDH, and ROS levels were decreased in group of the up-regulation of Nrf2, while the survival rate and the level of SOD increased(P<0.05),and in the group of the down-regulation of Nrf2 got the opposite result(P<0.05). The same conclusion is also obtained in BRL-3A cells.3.In HSC-T6 cells, compared with parental cells and negative control, p66 Shc levelwas decreased in group of the up-regulation of Nrf2, while the IQGAP1 increased(P<0.05), and in the group of the down-regulation of Nrf2 got the opposite result(P<0.05).After oxidative stress,the expression levels of p66 Shc in oxidative stress group were increased significantly than the normal group, while IQGAP1 was lower(P<0.05). In oxidative stress stimulation group: compared with parental cells and negative control,p66 Shc level was decreased significantly in group of the up-regulation of Nrf2, while the IQGAP1 increased significantly(P<0.05), and in the group of the down-regulation of Nrf2 got the opposite result(P<0.05). The same conclusion is also obtained in BRL-3A cells.ConclusionWe successfully constructed stably transfected BRL-3A and HSC-T6 cells with up/down regulation of Nrf2 expression. Oxidative stress model showed that Nrf2 has antioxidative ability in both two cells, probably through down regulation of p66 Shc and upregulation of IQGAP1. |
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