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Metformin alleviates high glucose-mediated oxidative stress in rat glomerular mesangial cells by modulation of p38 mitogen-activated protein kinase expression in vitro.

Molecular medicine reports (2015-03-12)
Xin-Ming Yao, Shan-Dong Ye, Chun-Chun Xiao, Jun-Fei Gu, Di Yang, Shan Wang
ABSTRACT

The aim of the current study was to investigate the effects and mechanism of metformin in oxidative stress and p38 mitogen-activated protein kinase (p38MAPK) expression in rat glomerular mesangial cells (MCs) cultured in a high glucose medium. Rat glomerular MCs (HBZY-1) were cultured in complete medium and divided into the following five groups: Normal control (NC), high glucose (HG), metformin-treated, SB203580-treated (SB) and N-acetylcysteine-treated (NAC). The production of intracellular reactive oxygen species (ROS) in rat glomerular MCs was measured using flow cytometry. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in the supernatant was detected using colorimetric analysis and an ELISA, respectively. p22phox mRNA levels in rat glomerular MCs were determined using reverse transcription-quantitative polymerase chain reaction. The levels of p22phox protein and phosphorylated p38 mitogen-activated protein kinase (p-p38MAPK) protein in rat glomerular MCs were determined by western blot analysis. Compared with the NC group, the activity of SOD in the supernatant was significantly reduced, whereas the levels of MDA in the supernatant, intracellular p22phox mRNA and protein, p-p38MAPK protein in addition to ROS production in rat glomerular MCs were significantly increased in the HG group (P<0.05). When metformin was added to the high glucose medium, the activity of SOD in supernatant fluid was increased significantly, whereas a significant reduction (P<0.05) was observed in the levels of MDA in the supernatant, intracellular p22phox mRNA and protein, p-p38MAPK protein in addition to ROS production in rat glomerular MCs. These results were similar to those obtained when SB203580 or N-acetylcysteine was added to the high glucose medium (P<0.05). In conclusion, metformin was suggested to alleviate high glucose-induced oxidative stress and p-p38MAPK protein expression in rat glomerular MCs, which may contribute to its reno‑protective abilities in diabetes.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
N-Acetyl-L-cysteine, Sigma Grade, ≥99% (TLC), powder
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Thiazolyl Blue Tetrazolium Bromide, powder, BioReagent, suitable for cell culture, suitable for insect cell culture, ≥97.5% (HPLC)
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N-Acetyl-L-cysteine, suitable for cell culture, BioReagent
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Thiazolyl Blue Tetrazolium Bromide, 98%
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Phenylmethanesulfonyl fluoride, ≥98.5% (GC)
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4,4′-Diaminodiphenylmethane, ≥97.0% (GC)
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Phenylmethanesulfonyl fluoride, ≥99.0% (T)
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N-Acetyl-L-cysteine, BioXtra, ≥99% (TLC)
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Amyloid Protein Non-Aβ Component, ≥80% (HPLC)