Yan Hai-dong, Li Xue-zhu, Xie Jun-mei, Li Man
Department of Nephrology, Eastern Hospital, Tongji University, Shanghai 200120, China.
Chin Med J (Engl). 2007 May 5;120(9):787-93.
Advanced glycation end products (AGEs) play a critical role in the development of diabetic nephropathy. Reactive oxygen species (ROS) may play a critical role in AGEs induced growth factor expression. In this study, the effects of AGEs on transforming growth factor beta1 (TGF-beta1), connective tissue growth factor (CTGF) and fibronectin (Fn) mRNA expression and oxidative stress in cultured NRK-49F cells were examined.
NRK-49F cells were incubated with medium containing different doses of AGEs (50, 100 or 200 microg/ml) for 24 hours, or with AGEs 100 microg/ml for different times (0, 12, 24 or 48 hours). Cells in the serum-free medium or medium containing 25 mmol/L glucose were controls. Cells were treated with 25 mmol/L glucose and 100 microg/ml AGEs for 24 hours to determine the effects between AGEs and glucose. We clarified the role of antioxidant by pretreating cells with N-acetylcysteine (10 mmol/L), ginkgo biloba extract (50 or 100 mg/L) for 24 hours and with 100 microg/ml AGEs for further 24 hours. Alamarblue dye assay was used to analyze cell growth; intracellular ROS generation was measured by flow cytometry; intracellular glutathione by fluorescence spectrophotometry; expressions of TGF-beta1, CTGF and Fn mRNA by semiquantitative RT-PCR.
AGEs significantly increased the expressions of TGF-beta1, CTGF, Fn mRNA and intracellular ROS generation, and decreased the glutathion level in NRK-49F cells in dose- and time-dependent manners. High glucose and AGEs together significantly increased the expression of TGF-beta1, CTGF and Fn mRNA, compared with AGEs and high glucose separately. Preincubation with N-acetylcysteine or ginkgo biloba extract increased GSH level, suppressed AGEs-induced oxidative stress and TGF-beta1, CTGF and Fn mRNA overexpression.
AGEs can significantly increase expression of TGF-beta1, CTGF, Fn mRNA in NRK-49F cells through enhancement of oxidative stress. The accumulation of AGEs may play a pivotal role in the pathogenesis of tubulointerstitial fibrosis in diabetic nephropathy. Suppression of AGEs induced TGF-beta1, CTGF and Fn mRNA overexpression in renal fibroblasts through inhibition of oxidative stress may be a mechanism underlying effect of ginkgo biloba extract in diabetic nephropathy. In addition, antioxidant therapy may help prevent AGEs accumulation and its induced damage.
晚期糖基化终末产物（AGEs）在糖尿病肾病的发展中起关键作用。活性氧（ROS）可能在AGEs诱导生长因子表达中起关键作用。本研究检测了AGEs对培养的NRK-49F细胞中转化生长因子β1（TGF-β1）、结缔组织生长因子（CTGF）和纤连蛋白（Fn）mRNA表达及氧化应激的影响。
将NRK-49F细胞与含不同剂量AGEs（50、100或200μg/ml）的培养基孵育24小时，或与100μg/ml AGEs孵育不同时间（0、12、24或48小时）。无血清培养基或含25mmol/L葡萄糖的培养基中的细胞作为对照。用25mmol/L葡萄糖和100μg/ml AGEs处理细胞24小时，以确定AGEs与葡萄糖之间的相互作用。通过用N-乙酰半胱氨酸（10mmol/L）、银杏叶提取物（50或100mg/L）预处理细胞24小时，再用100μg/ml AGEs处理24小时，阐明抗氧化剂的作用。采用Alamarblue染料法分析细胞生长；通过流式细胞术检测细胞内ROS生成；用荧光分光光度法检测细胞内谷胱甘肽；用半定量RT-PCR检测TGF-β1、CTGF和Fn mRNA的表达。
AGEs以剂量和时间依赖性方式显著增加NRK-49F细胞中TGF-β1、CTGF、Fn mRNA的表达及细胞内ROS生成，并降低谷胱甘肽水平。与单独的AGEs和高糖相比，高糖和AGEs共同作用显著增加TGF-β1、CTGF和Fn mRNA的表达。用N-乙酰半胱氨酸或银杏叶提取物预孵育可提高谷胱甘肽水平，抑制AGEs诱导的氧化应激及TGF-β1、CTGF和Fn mRNA的过表达。
AGEs可通过增强氧化应激显著增加NRK-49F细胞中TGF-β1、CTGF、Fn mRNA的表达。AGEs的蓄积可能在糖尿病肾病肾小管间质纤维化的发病机制中起关键作用。通过抑制氧化应激来抑制AGEs诱导的肾成纤维细胞中TGF-β1、CTGF和Fn mRNA的过表达可能是银杏叶提取物在糖尿病肾病中发挥作用的潜在机制。此外，抗氧化治疗可能有助于预防AGEs的蓄积及其诱导的损伤。
