红景天苷通过激活钙离子／钙调蛋白／钙调蛋白激酶δ／内皮型一氧化氮合酶通路抑制高糖对人脐静脉内皮细胞的损伤

Salidroside attenuates high glucose.induced apoptosis in human umbilical vein endothelial cells via activating the Ca2＋/CaM/CAMKⅡδ/eNOS pathway

目的研究红景天苷对高糖诱导的内皮细胞损伤的保护作用及其机制。方法将人脐静脉内皮细胞（HUVEC）分为6组，分别为正常血糖组（5．5mmol／L葡萄糖进行干预）、高糖组（33mmol／L葡萄糖进行干预）、红景天苷10μg/m1组（10μg／ml红景天苷＋33mmol／L葡萄糖进行干预）、红景天苷4μg／ml组（4μg／ml红景天苷＋33mmol／L葡萄糖进行干预）、红景天苷1μg／ml组（1μg/ml红景天苷＋33mmol／L葡萄糖进行干预）、红景天苷0．1μg/ml组（红景天苷0．1μg／ml＋33mmol／L葡萄糖进行干预）。各组HUVEC培养48h后，MTY法检测其细胞存活率，分别使用丙二醛（MDA）检测试剂盒及一氧化氮（NO）检测试剂盒检测细胞内MDA、NO浓度，Westernblot法检测活性半胱氨酸天冬氨酸蛋白酶－3（caspase．3）、钙调蛋白（CaM）、钙调蛋白激酶δ（CAMKⅡδ）、总内皮型一氧化氮合酶（eNOS）的蛋白表达水平，以及检测eNOSserll77位点的磷酸化水平，并使用流式细胞仪检测细胞内ca2＋浓度和活性氧（ROS）的水平。结果细胞培养后48h，高糖组HUVEC存活率显著低于正常血糖组（P〈0．05），细胞NO含量少于正常血糖组（P〈0．05），MDA、ROS及活性caspase．3的表达水平均显著高于正常血糖组（P均〈0．05），细胞内ca2＋浓度显著低于正常血糖组（P〈0．05），CaM、CAMKⅡδ、总eNOS的表达水平均显著低于正常血糖组（P均〈0．05），eNOSserll77位点的磷酸化水平亦显著低于正常血糖组（P〈0．05）。红景天苷10μg/ml组细胞存活率显著高于高糖组（P〈0．05），细胞内NO含量显著高于高糖组（P〈0．05），细胞内MDA、ROS及活性caspase一3的表达水平均显著低于高糖组（P均〈0．05），细胞内Ca2＋浓度显著高于高糖组（P〈0．05），CaM、CAMKⅡ8和总eNOS的表达水平均显著高于高糖组（P均〈0．05），eNOSserll77位点的磷酸化水平亦显著高于高糖组（P〈0．05）。结论红景天苷对高糖诱导的内皮细胞损伤具有保护作用，其机制可能与Ca2＋／CaM／CAMKⅡδ／eNOS信号通路有关。

Objective 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 retool/L）-induced cell damage in human umbilical vein endothelial cells （HUVECs）. Methods 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） , [ Ca2＋ ]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 withMTF assay, and the level of ROS, and [ Ca2 ＋}]I 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 pbosphorylation. Results Comparing to the normal glucose group, high glucose treatment increased the cell damage, the level of NO and [ Ca2＋ ] i （P 〈 0. 05 ）, downregulated CAMK Ⅱδ, eNOS expression and eNOS set 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 Ixg/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 [ Ca2 ＋ ] i by HUVECs （ P 〈 0. 05 ）, enhanced CaM, CAMKⅡδ, eNOS expression and eNOS ser 1177 phosphorylation in HUVECs （P 〈 0. 05）. Conclusions These findings suggeste that salidroside could attenuate high glucose induced apoptosis in HUVEC, partly through activating the Ca2 ＋/CaM/CAMK Ⅱδ/eNOS pathway.