Over-production of nitric oxide by oxidative stress- induced activation of the TGF-β1/PI3K/Akt pathway in mesangial cells cultured in high glucose

Aim： To investigate whether NO over-production in rat mesangial cells cultured in high glucose （HG） is related to activation of the TG F-β1/PI3 K/Akt pathway. Methods： Rat mesangial cells line （HBZY-1） was exposed to HG （24.44 mmol/L） or H2O2 （10 pmol/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-131 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 H202 significantly inhibited Bim protein and mRNA expression, which was reversed by co-treatment with DPI, SB431542, or LY294002. Conclusion： 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/PβK/Akt pathway.