白藜芦醇通过激活PI3K/AKT通路抑制内皮细胞凋亡的研究

目的观察白藜芦醇对脂多糖作用下人脐静脉血管内皮细胞凋亡的影响,并探讨其作用机制。方法体外培养人脐静脉血管内皮细胞,实验分为5组:空白组给予单纯培养基培养;模型组培养基中加入1000 ng/mL脂多糖孵育24 h;5μmol/L白藜芦醇组和10μmol/L白藜芦醇组培养基中分别先加入5μmol/L、10μmol/L白藜芦醇孵育12 h,再加入1000 ng/mL脂多糖孵育24 h;PI3K抑制剂组培养基中先加入10μmol/L白藜芦醇和10μmol/L PI3K抑制剂LY294002共处理12 h,再加入1000 ng/mL脂多糖孵育24 h。使用CCK-8试剂盒检测各组细胞的增殖能力;流式细胞术检测各组细胞凋亡率;DHE荧光探针检测各组细胞中活性氧(ROS)含量;ELISA检测各组细胞培养液中一氧化氮(NO)、肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)水平;Western blot法检测各组AKT的磷酸化情况。结果与空白组相比,模型组细胞增殖能力明显降低,细胞凋亡率和细胞中ROS含量及TNF-α、IL-6水平均明显升高,NO水平及P-AKT/AKT表达水平明显降低,差异均有统计学意义(P均<0.05)。与模型组和PI3K抑制剂组相比,10μmol/L白藜芦醇组细胞增殖能力明显升高,细胞凋亡率和细胞中ROS含量及TNF-α、IL-6水平明显下降,NO水平及P-AKT/AKT表达水平明显升高,差异均有统计学意义(P均<0.05)。结论白藜芦醇能够提升脂多糖作用下人脐静脉血管内皮细胞的增殖能力,抑制细胞凋亡,减轻细胞的氧化应激和炎性反应,恢复细胞的分泌功能,可能与其激活PI3K/AKT通路而发挥保护作用有关。

Study on the inhibition of resveratrol apoptosis of endothelial cell through activating PI3K/AKT pathway

Objective It is to observe the effect of resveratrol on apoptosis of human umbilical vein endothelial cells under the action of lipopolysaccharide and to explore its mechanism.Methods Human umbilical vein vascular endothelial cells were cultured in vitro.The experiment was divided into 5 groups:the blank group was cultured with simple medium;the model group was added with 1000 ng/mL lipopolysaccharide and incubated for 24 h;5μmol/L resveratrol group and 10μmol/L resveratrol group were respectively added with 5μmol/L resveratrol and 10μmol/L resveratrol and incubated for 12 h,then they were added with 1000 ng/mL lipopolysaccharide and incubated for 24 h;the PI3K inhibitor group was treated with 10μmol/L resveratrol and 10μmol/L PI3K inhibitor LY294002 for a total of 12 hours,and then was added with 1000 ng/mL lipopolysaccharide and incubated for 24 hours.CCK-8 kit was used to detect the cell proliferation ability of each group;flow cytometry was used to detect the apoptosis rate of each group;DHE fluorescent probe was used to detect the content of reactive oxygen species(ROS)in each group;ELISA was used to detect the levels of nitric oxide(NO),tumor necrosis factor-α(TNF-α),and interleukin-6(IL-6)in cell culture fluid of each group;Western blot was used to detect the phosphorylation of AKT in each group.Results Compared with the blank group,the cell proliferation ability of the model group was significantly reduced,the apoptotic rate and the ROS content and the levels of TNF-αand IL-6 in the cells were significantly increased,and the levels of NO and P-AKT/AKT expression were significantly increased,the differences were statistically significant(all P<0.05).Compared with the model group and the PI3K inhibitor group,the cell proliferation ability of the 10μmol/L resveratrol group was significantly increased,the apoptosis rate and the ROS content and the levels of TNF-αand IL-6 in the cells were significantly decreased,the expression of P-AKT/AKT was significantly increased,and the differences were statistically significant(all P<0.05).Conclusion Resveratrol can enhance the proliferation of human umbilical vein vascular endothelial cells under the action of lipopolysaccharide,inhibit apoptosis,reduce oxidative stress and inflammatory response of cells,restore cell secretion function,and the mechanism may be related with its protective role by activating PI3K/AKT pathways.