Chen Ziwei, Wu Xiang
Department of Cardiology, Affiliated Hospital of Nantong University, Nantong 226001, China.
Department of Cardiology, Affiliated Hospital of Nantong University, Nantong 226001, China. Email:
Zhonghua Xin Xue Guan Bing Za Zhi. 2014 Apr;42(4):327-33.
Endothelial oxidative stress plays an important role in the pathogenesis of cardiovascular disease. Salidroside, a phenylpropanoid glycoside isolated from Rhodiola rosea L, could exert potent antioxidant properties. In this study, we investigated the protective effects, and related mechanism of salidroside against high glucose (33 mmol/L)-induced cell damage in human umbilical vein endothelial cells (HUVECs).
HUVECs were cultured in normal glucose (5.5 mmol/L), high glucose (33 mmol/L), high salidroside (10 µg/ml+33 mmol/L glucose), moderate salidroside (4 µg/ml+33 mmol/L glucose), low salidroside (1 µg/ml+33 mmol/L glucose) and very low salidroside (0.1 µg/ml+33 mmol/L glucose) for 48 h. Cell viability, the level of malondialdehyde (MDA) , reactive oxygen species (ROS) , nitric oxide (NO) , [Ca(2)+]i, calmodulin (CaM) , calmodulin-dependent kinase (CaMK) IIδ, endothelial nitric oxide synthase (eNOS) , active caspase-3 protein expression and eNOS ser 1177 phosphorylation of HUVECs post various treatments were measured. The cell viability was assessed with MTT assay, and the level of ROS, and [Ca(2)+]i was analyzed using flow cytometry. Nitric oxide and MDA was detected by Nitric Oxide Assay Kit and MDA Assay Kit. Western blot was performed to detect the protein expressions of eNOS, active caspase-3 and eNOS ser 1177 phosphorylation.
Comparing to the normal glucose group, high glucose treatment increased the cell damage, the level of NO and [Ca(2)+]i (P < 0.05) , downregulated CAMKIIδ, eNOS expression and eNOS ser 1177 phosphorylation (P < 0.05), elevated the concentration of MDA and ROS (P < 0.05) in HUVECs. Salidroside treatment significantly attenuated high glucose-induce cell damage on cultured HUVECs in a dose-dependent manner. Comparing to the high glucose group, 10 µg/ml Salidroside significantly increased cell viability (P < 0.05) , inhibited high glucose-induced release of MDA , generation of ROS, active caspase 3 protein expression (P < 0.05) , upregulated the release of nitric oxide and [Ca(2)+]i by HUVECs (P < 0.05) , enhanced CaM, CAMKIIδ, eNOS expression and eNOS ser 1177 phosphorylation in HUVECs (P < 0.05) .
These findings suggeste that salidroside could attenuate high glucose induced apoptosis in HUVEC, partly through activating the Ca(2)+/CaM/CAMKIIδ/eNOS pathway.
内皮细胞氧化应激在心血管疾病发病机制中起重要作用。红景天苷是从红景天中分离出的一种苯丙素糖苷，具有强大的抗氧化特性。在本研究中，我们探究了红景天苷对高糖（33 mmol/L）诱导的人脐静脉内皮细胞（HUVECs）损伤的保护作用及相关机制。
将HUVECs分别在正常葡萄糖（5.5 mmol/L）、高糖（33 mmol/L）、高剂量红景天苷（10 µg/ml + 33 mmol/L葡萄糖）、中剂量红景天苷（4 µg/ml + 33 mmol/L葡萄糖）、低剂量红景天苷（1 µg/ml + 33 mmol/L葡萄糖）和极低剂量红景天苷（0.1 µg/ml + 33 mmol/L葡萄糖）条件下培养48小时。检测各种处理后HUVECs的细胞活力、丙二醛（MDA）水平、活性氧（ROS）、一氧化氮（NO）、[Ca(2)+]i、钙调蛋白（CaM）、钙调蛋白依赖性激酶（CaMK）IIδ、内皮型一氧化氮合酶（eNOS）、活化的半胱天冬酶 - 3蛋白表达以及eNOS第1177位丝氨酸磷酸化水平。采用MTT法评估细胞活力，使用流式细胞术分析ROS和[Ca(2)+]i水平。通过一氧化氮检测试剂盒和MDA检测试剂盒检测一氧化氮和MDA。采用蛋白质印迹法检测eNOS、活化的半胱天冬酶 - 3和eNOS第1177位丝氨酸磷酸化的蛋白表达。
与正常葡萄糖组相比，高糖处理增加了细胞损伤、NO水平和[Ca(2)+]i（P < 0.05），下调了CAMKIIδ、eNOS表达及eNOS第1177位丝氨酸磷酸化水平（P < 0.05），升高了HUVECs中MDA和ROS浓度（P < 0.05）。红景天苷处理以剂量依赖方式显著减轻了高糖对培养的HUVECs的损伤。与高糖组相比，10 µg/ml红景天苷显著提高了细胞活力（P < 0.05），抑制了高糖诱导的MDA释放、ROS生成、活化的半胱天冬酶3蛋白表达（P < 0.05），上调了HUVECs释放的一氧化氮和[Ca(2)+]i（P < 0.05），增强了HUVECs中CaM、CAMKIIδ、eNOS表达及eNOS第1177位丝氨酸磷酸化水平（P < 0.我们的发现表明，红景天苷可以减轻高糖诱导的HUVEC凋亡，部分是通过激活Ca(2)+/CaM/CAMKIIδ/eNOS途径实现的。
这些发现表明红景天苷可部分通过激活Ca(2)+/CaM/CAMKIIδ/eNOS途径减轻高糖诱导的HUVEC凋亡。
