Sang Hai-qiang, Gu Jun-fei, Yuan Jia-rui, Zhang Ming-hua, Jia Xiao-bin, Feng Liang
Department of Cardiology, The First Affiliated Hospital of Zhengzhou University, Henan, Zhengzhou 450052, PR China.
Key Laboratory of Delivery Systems of Chinese Meteria Medica, Jiangsu Provincial Academy of Chinese Medicine, Jiangsu, Nanjing 210028, PR China; College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, PR China.
J Ethnopharmacol. 2014 Aug 8;155(1):785-95. doi: 10.1016/j.jep.2014.06.028. Epub 2014 Jun 19.
Smilax glabra Roxb. (SGR) is a traditional Chinese herb that has been used in folk for the treatment of diabetic vascular complications. Advanced glycation end products (AGEs)-induced endothelial dysfunction has been thought to be a major cause of diabetic vascular complications. The present study was conducted to investigate the protective effect of SGR extract on AGEs-induced endothelial dysfunction and its underlying mechanisms.
Human umbilical vein endothelial cells (HUVECs) were exposed to 200 μg/ml AGEs to induce endothelial dysfunction. Acridine orange/ethidium bromide (AO/EB) fluorescence assay and Annexin-V/PI double-staining were performed to determine endothelium apoptosis. Dihydroethidium (DHE) fluorescence probe, SOD and MDA kits were used to evaluate oxidative stress. The effect of SGR extract on AGEs-induced TGF-beta1 expression was determined by immunocytochemistry and western blotting. Attenuations of SGR extract on receptor for AGEs (RAGE) expression, extracellular regulated protein kinases (ERK1/2) activation and NF-κB phosphorylation were determined by immunofluorescence assay and western blotting. The blockade assays for RAGE and ERK1/2 were carried out using a specific RAGE-antibody (RAGE-Ab) or a selective ERK1/2 inhibitor PD98059 in immunofluorescence assay.
The pretreatment of SGR extract (0.125, 0.25 and 0.5 mg crude drug/ml) significantly attenuated AGEs-induced endothelium apoptosis, and down-regulated TGF-beta1 protein expression in HUVECs. It was also well shown that SGR extract could down-regulate dose-dependently ROS over-generation, MDA content, TGF-beta1 expression, ERK1/2 and NF-κB activation whereas increase significantly SOD activity. Furthermore, the AGEs-induced ERK1/2 activation could be attenuated by the blockade of RAGE-Ab (5 μg/ml) while the NF-κB activation was ameliorated by ERK1/2 inhibitor PD98059 (10 μM).
These results indicated that SGR extract could attenuate AGEs-induced endothelial dysfunction via RAGE-ERK1/2-NF-κB pathways. Our findings suggest that SGR extract may be beneficial for attenuating endothelial dysfunction in diabetic vascular complications.
菝葜是一种传统的中草药，民间用于治疗糖尿病血管并发症。晚期糖基化终产物（AGEs）诱导的内皮功能障碍被认为是糖尿病血管并发症的主要原因。本研究旨在探讨菝葜提取物对AGEs诱导的内皮功能障碍的保护作用及其潜在机制。
将人脐静脉内皮细胞（HUVECs）暴露于200μg/ml AGEs中以诱导内皮功能障碍。采用吖啶橙/溴化乙锭（AO/EB）荧光测定法和膜联蛋白V/碘化丙啶双染色法测定内皮细胞凋亡。使用二氢乙锭（DHE）荧光探针、超氧化物歧化酶（SOD）和丙二醛（MDA）试剂盒评估氧化应激。通过免疫细胞化学和蛋白质印迹法测定菝葜提取物对AGEs诱导的转化生长因子-β1（TGF-β1）表达的影响。通过免疫荧光测定法和蛋白质印迹法测定菝葜提取物对AGEs受体（RAGE）表达、细胞外调节蛋白激酶（ERK1/2）激活和核因子-κB（NF-κB）磷酸化的抑制作用。在免疫荧光测定中，使用特异性RAGE抗体（RAGE-Ab）或选择性ERK1/2抑制剂PD98059进行RAGE和ERK1/2的阻断试验。
菝葜提取物（0.125、0.25和0.5mg生药/ml）预处理可显著减轻AGEs诱导的内皮细胞凋亡，并下调HUVECs中TGF-β1蛋白表达。还表明，菝葜提取物可剂量依赖性地下调活性氧（ROS）过度生成、MDA含量、TGF-β1表达、ERK1/2和NF-κB激活，同时显著提高SOD活性。此外，RAGE-Ab（5μg/ml）阻断可减弱AGEs诱导的ERK1/2激活，而ERK1/2抑制剂PD98059（10μM）可改善NF-κB激活。
这些结果表明，菝葜提取物可通过RAGE-ERK1/2-NF-κB途径减轻AGEs诱导的内皮功能障碍。我们的研究结果表明，菝葜提取物可能有助于减轻糖尿病血管并发症中的内皮功能障碍。
