Cai Xiaxia, Bao Lei, Ren Jinwei, Li Yong, Zhang Zhaofeng
Department of Nutrition and Food Hygiene, School of Public Health, Peking University, Beijing, P. R. China.
Department of Clinical Nutrition, Peking University International Hospital, Beijing, P. R. China.
Food Funct. 2016 Feb;7(2):805-15. doi: 10.1039/c5fo01062d.
Grape seed procyanidin B2 (GSPB2) was reported to have protective effects on diabetic nephropathy (DN) as a strong antioxidant. Our previous studies demonstrated that GSPB2 was effective in ameliorating podocyte injury in rats with DN. However, little is known about the benefits of GSPB2 in protecting against podocyte apoptosis and its molecular mechanisms in vitro. In the present study, we investigated whether GSPB2 could protect podocytes from high glucose-induced apoptosis and explored the possible mechanism. Cell viability and apoptosis were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry, respectively. The intracellular reactive oxygen species (ROS) level was measured using a dichlorofluorescein diacetate (DCFH-DA) fluorescent probe. Real-time reverse transcription-PCR was used to determine the gene expression of nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (TFAM), and quantitative real-time PCR was used to detect mitochondrial DNA (mtDNA) copy number. Western blots were carried out for the related protein expression in podocytes. Our results showed that GSPB2 significantly inhibited high glucose-induced podocyte apoptosis and increased the expression of nephrin and podocalyxin. GSPB2 treatment also suppressed intracellular ROS production and oxidative stress. The mRNA expressions of NRF-1, TFAM and mtDNA copy number were markedly increased, and mitochondrial swelling was effectively reduced in podocytes cultured under high glucose after GSPB2 treatment. The AMPK-SIRT1-PGC-1α axis was also activated by GSPB2 intervention. In conclusion, GSPB2 protected podocytes from high glucose-induced mitochondrial dysfunction and apoptosis via the AMPK-SIRT1-PGC-1α axis in vitro, suggesting a potential role of GSPB2 in the treatment of DN.
据报道，葡萄籽原花青素B2（GSPB2）作为一种强抗氧化剂，对糖尿病肾病（DN）具有保护作用。我们之前的研究表明，GSPB2对改善DN大鼠的足细胞损伤有效。然而，关于GSPB2在体外保护足细胞免受凋亡及其分子机制的益处知之甚少。在本研究中，我们研究了GSPB2是否能保护足细胞免受高糖诱导的凋亡，并探讨了可能的机制。分别通过3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）和流式细胞术检测细胞活力和凋亡。使用二氯荧光素二乙酸酯（DCFH-DA）荧光探针测量细胞内活性氧（ROS）水平。采用实时逆转录PCR测定核呼吸因子1（NRF-1）和线粒体转录因子A（TFAM）的基因表达，采用定量实时PCR检测线粒体DNA（mtDNA）拷贝数。对足细胞中的相关蛋白表达进行蛋白质印迹分析。我们的结果表明，GSPB2显著抑制高糖诱导的足细胞凋亡，并增加nephrin和podocalyxin的表达。GSPB2处理还抑制细胞内ROS产生和氧化应激。GSPB2处理后，在高糖培养的足细胞中，NRF-1、TFAM的mRNA表达和mtDNA拷贝数显著增加，线粒体肿胀有效减轻。GSPB2干预还激活了AMPK-SIRT1-PGC-1α轴。总之，GSPB2在体外通过AMPK-SIRT1-PGC-1α轴保护足细胞免受高糖诱导的线粒体功能障碍和凋亡，提示GSPB2在DN治疗中具有潜在作用。
