title: Over-production of nitric oxide by oxidative stress-induced activation of the TGF-β1/PI3K/Akt pathway in mesangial cells cultured in high glucose.
abstract: OBJECTIVE
  To investigate whether NO over-production in rat mesangial cells cultured in high glucose (HG) is related to activation of the TGF-β1/PI3K/Akt pathway.
  
  
  METHODS
  Rat mesangial cells line (HBZY-1) was exposed to HG (24.44 mmol/L) or H2O2 (10 μmol/L) for 16 h. NO release was quantified using the Griess assay. The TGF-β1 level was measured using ELISA. The protein expression of p-Akt, t-Akt, Bim, and iNOS was examined by Western blotting. The mRNA levels of TGF-β1 and Bim were measured using RT-PCR. The cell proliferation rate was estimated using a BrdU incorporation assay.
  
  
  RESULTS
  Treatment of the cells with HG, H2O2, or TGF-β1 (5 ng/mL) significantly increased the NO level that was substantially inhibited by co-treatment with the NADPH oxidase inhibitor diphenylene iodonium (DPI), TGF-β1 inhibitor SB431542, or PI3K inhibitor LY294002. Both HG and H2O2 significantly increased the protein and mRNA levels of TGF-β1 in the cells, and HG-induced increases of TGF-β1 protein and mRNA were blocked by co-treatment with DPI. Furthermore, the treatment with HG or H2O2 significantly increased the expression of phosphorylated Akt and iNOS and cell proliferation rate, which was blocked by co-treatment with DPI, SB431542, or LY294002. Moreover, the treatment with HG or H2O2 significantly inhibited Bim protein and mRNA expression, which was reversed by co-treatment with DPI, SB431542, or LY294002.
  
  
  CONCLUSIONS
  The results demonstrate that high glucose causes oxidative stress and NO over-production in rat mesangial cells in vitro via decreasing Bim and increasing iNOS, which are at least partially mediated by the TGF-β1/PI3K/Akt pathway.
authors.affiliation: Key Laboratory of New Drugs and Clinical Application, Xuzhou Medical College, Xuzhou 221004, China.
authors.forename: Yun-peng
authors.initials: YP
authors.lastname: Zhai
authors.forename: Qian
authors.initials: Q
authors.lastname: Lu
authors.forename: Yao-wu
authors.initials: YW
authors.lastname: Liu
authors.forename: Qian
authors.initials: Q
authors.lastname: Cheng
authors.forename: Ya-qin
authors.initials: YQ
authors.lastname: Wei
authors.forename: Fan
authors.initials: F
authors.lastname: Zhang
authors.forename: Cheng-lin
authors.initials: CL
authors.lastname: Li
authors.forename: Xiao-xing
authors.initials: XX
authors.lastname: Yin
mesh_terms: D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D000072224:Bcl-2-Like Protein 11; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D005947:Glucose / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D050527:Mesangial Cells / Q000187:drug effects / Q000378:metabolism*; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D009569:Nitric Oxide / Q000235:genetics / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects / Q000235:genetics*; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D053773:Transforming Growth Factor beta1 / Q000235:genetics / Q000378:metabolism*
chemical_list: D051017:Apoptosis Regulatory Proteins; D000072224:Bcl-2-Like Protein 11; C000606778:Bcl2l11 protein, rat; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D053773:Transforming Growth Factor beta1; D009569:Nitric Oxide; D006861:Hydrogen Peroxide; D019255:NADPH Oxidases; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D005947:Glucose