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
Yun-peng ZHAI#, Qian LU#, Yao-wu LIU, Qian CHENG, Ya-qin WEI, Fan ZHANG, Cheng-lin LI, Xiao-xing YIN*
Key Laboratory of New Drugs and Clinical Application, Xuzhou Medical College, Xuzhou 221004, China
Aim: 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.
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/PI3K/Akt pathway.
Keywords: mesangial cells; high glucose; oxidative stress; NO; iNOS; Bim; TGF-β1; PI3K/Akt pathway
We thank Miss Hao GUO for help with the manuscript. The work was supported by the National Natural Science Foundation of China (No 81173104), the Jiangsu University Natural Science Foundation of China (No 11KJD310004), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), China.
#These authors contributed equally to this work.
* To whom correspondence should be addressed.
E-mail yinxx@xzmc.edu.cn
Received 2012-07-26 Accepted 2012-12-20
Keywords:
Key Laboratory of New Drugs and Clinical Application, Xuzhou Medical College, Xuzhou 221004, China
Aim: 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.
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/PI3K/Akt pathway.
Keywords: mesangial cells; high glucose; oxidative stress; NO; iNOS; Bim; TGF-β1; PI3K/Akt pathway
We thank Miss Hao GUO for help with the manuscript. The work was supported by the National Natural Science Foundation of China (No 81173104), the Jiangsu University Natural Science Foundation of China (No 11KJD310004), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), China.
#These authors contributed equally to this work.
* To whom correspondence should be addressed.
E-mail yinxx@xzmc.edu.cn
Received 2012-07-26 Accepted 2012-12-20