Expression and significance of Nrf2/ARE pathway ralated factors in the HepG2 cell model of steatosis / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To explore a new method of establishing HepG2 cell model of steatosis and observe the expression and significance of nuclear factor erythroid-2p45-related factor 2(Nrf2)/antioxidative response element (ARE) pathway related factors in HepG2 cells of steatosis.</p><p><b>METHODS</b>HepG2 cells were induced with DMEM containing 25% fetal bovine serum, 0.1% MCT/LCT Fat Emulsion and 0.1 mmol/L free fatty acid (FFA) at different stages and the control group cells were cultured with normal DMEM medium. After the cell models were successfully established, lipid droplets in cytoplasm were observed with Oil Red 0 staining, and the triglyceride (TG) accumulation in HepG2 cells were tested by biochemical assay. Intracellular reactive oxygen species (ROS) concentration were detected by flow cytometry. Nitric oxide (NO), superoxide dismutase(SOD), malonyldialdehyde(MDA) and glutathione peroxidase(GSH-Px) were tested by biological reagent kit, while the protein expression of nuclear factor erythroid-2p45-related factor 2(Nrf2), heme oxygenase-1 (HO-1) and</p><p><b>NAD(P)H</b>quinone oxidoreductase-1(NQO1) were analyzed by Western blot.</p><p><b>RESULTS</b>Compared with that in the control group, red cytoplasmic lipid droplets were visible in model group; TG,ROS, NO, MDA concentration (P < 0.05, P < 0.01) and the protein expression of Nrf2, HO-1 and NQO1 (P < 0.05, P < 0.01)were significantly higher in model group, while SOD, GSH-Px concentration reduced significantly (P < 0.01).</p><p><b>CONCLUSION</b>The in vitro cell model of steatosis and oxidative stress was successfully established. The activation of Nrf2/ARE pathway related factors maybe relevant to the overreaction of oxidative stress in HepG2 cells of steatosis.</p>

The pathway of PI3k/Akt-aPKCiota/zeta-Nrf2 regulating the expression of gamma-glutamylcysteine synthetase in the bronchial epithelial cells of rats / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To observe the effect of the signal pathway of phosphoinositol-3-kinase (PI3K)/Akt-antypical protein kinase C(iotazeta) (aPKC(iotazeta))-Nuclear factor-E2 related factor (Nrf2) on gamma-glutamylcysteine synthetase (gamma-GCS) of the bronchial epithelial cells of rats after exposure to cigarette smoke extracts (CSE).</p><p><b>METHODS</b>gamma-GCS, Nrf2, p-Akt and p-aPKC(iotazeta) proteins were semi-quantified by Western blot. gamma-GCS protein expression was assessed by immunocytochemistry. gamma-GCS mRNA expression was detected by reverse transcription-polymerase chain reaction (RT-PCR). Nrf2 protein was observed by immunofluorescence. The rate of the cells expressed p-Akt were analyzed by flow cytometry. GSH content and gamma-GCS activity were measured.</p><p><b>RESULTS</b>GSH content, Nrf2 protein of nucleus, p-aPKC(iotazeta) protein, p-Akt protein and positive cells, gamma-GCS protein and mRNA and activity were significantly increased after exposure to CSE for 3 hours. aPK(iotazeta) inhibitor RO813220 significantly reduced the expression of p-aPKC(iotazeta) protein, gamma-GCS protein and mRNA and activity, but enhanced Nrf2 protein of cytoplasm expression, had no effect on p-Akt. p-Akt inhibitor LY294002 and RO813220 + LY294002 decreased p-aPKC(iotazeta) protein, p-Akt protein and positive cells, gamma-GCS protein and mRNA and activity expression, increased Nrf2 protein of cytoplasm expression. The correlation analysises demonstrated that there were a positive correlation between Nrf2 and gamma-GCS, p-Akt, p-aPKC(iotazeta), between p-Akt and Nrf2, p-aPKC(iotazeta), gamma-GCS, between p-aPKC(iotazeta) and Nrf2, p-Akt, gamma-GCS.</p><p><b>CONCLUSION</b>CSE might upregulate gamma-GCS expression through PI3K/Akt-aPKC(iotazeta)-Nrf2 signaling pathway in the bronchial epithelial cells of rats.</p>

Regulation glutathione synthesizing and regenerating enzymes by xenobiotics in the brain of mice with targeted deletion of the NRF2 transcription factor gene gwqrzo, M.Y

Basal expression of certain antioxidant enzymes was shown to be lower in the livers of Nrf2[-/-] mice [Hhyes et al., 2000]. In order to investigate the role of Nrf2 in the regulation of GSH synthesizing and regenerating enzymes in the brain, cytbsols from the brains of Nrf2[-/-] mice treated with the antioxidant agents ethoxyquin, oltipraz, kahweol palmitate or indole-3-carbinol were analysed for glutatltione content, expression and activity of certain antioxidant enzymes. The analysis enzyme activities indicated reduction of 6 phosphogluconate dehydrogenase and Glutathione reductase activities by almost 14 and 20% in Nrf2 null compared with the wild type control respectively Treatment with chemopreventive agents increased the levels of these enzymes in Nrf2 [-/-] mice brain. But only 6-phosphogluconate dehydrogenase activity was increase in both Nif2[+/+] and Nrf2[-/-] mice brain. However, deletion of Nrf2 did not affect the protein levels of glutmyl cysteine ligase [GCL] and glutathione synthetase [GS] enzymes. Despite lack of apparent effect of the deficiency of Nr12 on the levels of proteins of GCL and GS, upon treatment with chemical additives, there was an increase in the level of GSH in both Nrf2[+/+] and Nrf2[-/-] mice brain upon treatment with chemical additives

Comparative study of PM2.5 - and PM10 - induced oxidative stress in rat lung epithelial cells

Accurate estimation of the exposure-response relationship between ambient urban particulate matters (PM) and public health is important for regulatory perspective of ambient urban particulate matters (PM). Ambient PM contains various transition metals and organic compounds. PM10 (aerodynamic diameter less than 10 microgram) is known to induce diverse diseases such as chronic cough, bronchitis, chest illness, etc. However, recent evaluation of PM2.5 (aerodynamic diameter less than 2.5 microgram) against health outcomes has suggested that the fine particles may be more closely associated with adverse respiratory health effects than particles of larger size. This study was performed to evaluate PM2.5-induced oxidative stress in rat lung epithelial cell in order to provide basic data for the risk assessment of PM2.5. PM2.5 showed higher cytotoxicity than PM10. Also, PM 2.5 induced more malondialdehyde (MDA) formation than PM10. In Hoechst 33258 dye staining and DNA fragmentation assay, apopotic changes were clearly detected in PM2.5 treated cells in compared to PM10. Expression of catalase mRNA was increased by PM2.5 rather than PM10. PM2.5 induced higher Mth1 mRNA than PM10. In pBR322 DNA treated with PM2.5, production of single strand breakage of DNA was higher than that of PM10. In Western blot analysis, PM2.5 induced more Nrf-2 protein, associated with diverse transcriptional and anti-oxidative stress enzymes, compared to PM10. Our data suggest that PM2.5 rather than PM10 may be responsible for PM-induced toxicity. Additional efforts are needed to establish the environmental standard of PM2.5.