1.The pathway of PI3k/Akt-aPKCiota/zeta-Nrf2 regulating the expression of gamma-glutamylcysteine synthetase in the bronchial epithelial cells of rats.
Chinese Journal of Applied Physiology 2011;27(1):115-119
OBJECTIVETo 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).
METHODSgamma-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.
RESULTSGSH 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.
CONCLUSIONCSE might upregulate gamma-GCS expression through PI3K/Akt-aPKC(iotazeta)-Nrf2 signaling pathway in the bronchial epithelial cells of rats.
Animals ; Bronchi ; cytology ; enzymology ; Environmental Exposure ; adverse effects ; Epithelial Cells ; enzymology ; GA-Binding Protein Transcription Factor ; metabolism ; Glutamate-Cysteine Ligase ; genetics ; metabolism ; Isoenzymes ; metabolism ; Male ; Oncogene Protein v-akt ; metabolism ; Phosphatidylinositol 3-Kinases ; metabolism ; Protein Kinase C ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Tobacco Smoke Pollution ; adverse effects
2.Expression and significance of Nrf2/ARE pathway ralated factors in the HepG2 cell model of steatosis.
Yu-pei ZHANG ; Yi-lin KONG ; Qin-he YANG ; Ling JIN ; Yin-ji LIANG ; Yi-fang HE ; Yuan-jun DENG ; Yuan-yuan LI ; Guan-long WANG ; Shao-bing CHENG
Chinese Journal of Applied Physiology 2016;32(1):13-17
OBJECTIVETo 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.
METHODSHepG2 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
NAD(P)Hquinone oxidoreductase-1(NQO1) were analyzed by Western blot.
RESULTSCompared 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).
CONCLUSIONThe 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.
Antioxidant Response Elements ; Culture Media ; Fatty Acids, Nonesterified ; Fatty Liver ; metabolism ; GA-Binding Protein Transcription Factor ; Glutathione Peroxidase ; metabolism ; Heme Oxygenase-1 ; metabolism ; Hep G2 Cells ; Humans ; Malondialdehyde ; metabolism ; NAD(P)H Dehydrogenase (Quinone) ; metabolism ; NF-E2-Related Factor 2 ; metabolism ; Nitric Oxide ; metabolism ; Oxidative Stress ; Reactive Oxygen Species ; metabolism ; Superoxide Dismutase ; metabolism ; Triglycerides ; metabolism
3.Comparative study of PM2.5 - and PM10 - induced oxidative stress in rat lung epithelial cells.
Jin Hyuk CHOI ; Jun Sung KIM ; Young Chul KIM ; Yoon Shin KIM ; Nam Hyun CHUNG ; Myung Haing CHO
Journal of Veterinary Science 2004;5(1):11-18
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.
Air Pollutants/chemistry/*toxicity
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Animals
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Apoptosis/physiology
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Benzimidazoles/metabolism
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Blotting, Western
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Cell Line
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Cell Survival/physiology
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DNA Fragmentation/physiology
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DNA Repair Enzymes/genetics/metabolism
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DNA-Binding Proteins/metabolism
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Epithelial Cells/drug effects/enzymology/pathology
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Formazans/metabolism
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GA-Binding Protein Transcription Factor
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Lipid Peroxides/metabolism
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Lung Diseases/*chemically induced/enzymology/pathology
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Oxidative Stress/*physiology
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RNA, Messenger/chemistry/genetics
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Rats
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Reverse Transcriptase Polymerase Chain Reaction
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Tetrazolium Salts/metabolism
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Transcription Factors/metabolism