Yao Xin-Ming, Ye Shan-Dong, Xiao Chun-Chun, Gu Jun-Fei, Yang Di, Wang Shan
Department of Endocrinology, Anhui Provincial Hospital Affiliated to Anhui Medical University, Hefei, Anhui 230001, P.R. China.
Mol Med Rep. 2015 Jul;12(1):520-6. doi: 10.3892/mmr.2015.3446. Epub 2015 Mar 6.
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.
本研究旨在探讨二甲双胍对高糖培养基中培养的大鼠肾小球系膜细胞（MCs）氧化应激及p38丝裂原活化蛋白激酶（p38MAPK）表达的影响及其机制。将大鼠肾小球MCs（HBZY-1）培养于完全培养基中，并分为以下五组：正常对照组（NC）、高糖组（HG）、二甲双胍处理组、SB203580处理组（SB）和N-乙酰半胱氨酸处理组（NAC）。采用流式细胞术检测大鼠肾小球MCs内活性氧（ROS）的产生。分别采用比色分析法和酶联免疫吸附测定法检测上清中超氧化物歧化酶（SOD）活性和丙二醛（MDA）含量。采用逆转录-定量聚合酶链反应测定大鼠肾小球MCs中p22phox mRNA水平。通过蛋白质免疫印迹分析测定大鼠肾小球MCs中p22phox蛋白和磷酸化p38丝裂原活化蛋白激酶（p-p38MAPK）蛋白水平。与NC组相比，HG组上清中SOD活性显著降低，而大鼠肾小球MCs上清中MDA水平、细胞内p22phox mRNA和蛋白、p-p38MAPK蛋白水平以及ROS产生均显著升高（P<0.05）。当向高糖培养基中添加二甲双胍时，上清中SOD活性显著升高，而大鼠肾小球MCs上清中MDA水平、细胞内p22phox mRNA和蛋白、p-p38MAPK蛋白水平以及ROS产生均显著降低（P<0.05）。这些结果与向高糖培养基中添加SB203580或N-乙酰半胱氨酸时获得的结果相似（P<0.05）。总之，提示二甲双胍可减轻高糖诱导的大鼠肾小球MCs氧化应激和p-p38MAPK蛋白表达，这可能有助于其在糖尿病中的肾脏保护作用。
