冬凌草甲素对脐静脉内皮细胞损伤的保护作用

目的 研究冬凌草甲素（oridonin）对高糖高脂诱导的人脐静脉内皮细胞（HUVECs）损伤的保护作用机制.方法 体外培养HUVECs,噻唑蓝法（MTT）确定oridonin作用的最佳浓度;将细胞分为空白对照组、高糖高脂＋oridonin预处理组（以下简称为oridonin预处理组,oridonin预处理细胞24 h）、高糖高脂组（高糖高脂的浓度为33.3 mmol/L葡萄糖＋500 μmol/L棕榈酸,处理48 h）,利用流式细胞仪检测细胞凋亡率,观察各组细胞凋亡情况;酶联免疫吸附法（ELISA）检测上清液中肿瘤坏死因子仅（TNF-α）、白介素6（IL-6）的浓度,以观察细胞的炎症情况;实时定量聚合酶链反应（RT-PCR）分析HUVECs内TAp63、沉默信息调节因子1（Sirt1）mRNA的表达水平;Western blotting分析TAp63、Sirt1及凋亡相关基因磷酸化细胞外调节蛋白激酶（p-ERK）、B淋巴细胞瘤2（Bcl-2）的蛋白表达水平.两组间比较采用￡检验,多组间采用单因素方差分析.结果 Oridonin作用于HUVECs细胞24 h的最佳浓度为5 μmol/L.高糖高脂组HUVECs细胞内TAp63（mRNA及蛋白）、Sirt1 （mRNA及蛋白）、Bcl-2蛋白水平明显低于空白对照组（t=-22.50、-6.02、-16.22、-6.72、-7.71,均P＜0.05）,HUVECs凋亡率、细胞内p-ERK蛋白表达及上清中炎症因子TNF-α、IL-6浓度明显高于空白对照组（t=14.34、12.59、17.37、17.90,均P＜0.05）.与高糖高脂组相比,oridonin预处理组HUVECs内TAp63 mRNA和蛋白、Sirt1mRNA和蛋白、Bcl-2蛋白表达明显升高（t=5.85、5.06、4.55、3.04、5.81,均P＜0.05）,HUVECs凋亡率、细胞内p-ERK蛋白表达及上清中炎症因子TNF-α及IL-6的浓度明显下降（t=-7.11、-7.81、-10.51、-10.36,均P＜0.05）.结论 冬凌草甲素能够抑制高糖高脂诱导的HUVECs的凋亡,可能与其抑制高糖高脂诱导的炎症反应和（或）增加TAp63、Sirt1、Bcl-2的表达,且降低p-ERK的表达有关.

Protective effect of oridonin against the injuries in endothelial cells

Objective To investigate the protective role of oridonin in the vascular endothelial cells dysfunction induced by high glucose and lipid.Methods The optimum concentration of oridonin on human umbilical vein endothelial cells（HUVECs）were determined by MTT assay.The model of HUVECs was established and divided into 3 groups：control group,oridonin group（oridonin treated for 24 h,with high glucose and high palmitic）,high glucose and palmitic acid group 33.3 mmol/L glucose.HUVECs were pretreated with oridonin under the condition of 33.3 mmol/L glucose and 500 μmol/L palmitic acid,while other groups received 33.3 mmol/L glucose and 500 μmol/L palmitic acid and dimethyl sulfoxide（DMSO）.After 48 h treatment,the concentration of tumor necrosis factor-α （TNF-α）and interleukin-6 （IL-6） in the supernatant was detected by the enzyme-linked immunosorbent assay （ELISA） to observethe inflammation state while flow cytometry was used to detect the cell apoptosis of each group,the mRNA expression of TAp63 and Silent information regulator 1 （Sirt1） in HUVECs was detected by the real time polymerase chain reaction（real time PCR）.And the protein expression of TAp63,Sirt1,phosphorylated extracellular regulated protein kinases （p-ERK） and B-cell lymphoma 2（Bcl-2） were determined by Western-blotting.Result The optimum concentration of oridonin treating on HUVECs was 5 μmol/L when effect for 24 h.Compared with the control group,the apoptosis rate,the protein expression of phosphated ERK,and the concentration of inflammation factors（TNF-α and IL-6） in the supernatant were significantly higher （t=14.34,12.59,17.37,17.90,P＜0.05 all above） while the expression of TAp63 mRNA and protein,Sirt1 mRNA and protein,Bcl-2 protein was significantly lower in the HUVECs in the high glucose and palmitic acid group （t=-22.50,-6.02,-16.22,-6.72,-7.71,all P＜0.05）.But in the oridonin group which were treated with 33.3 mmol/L glucose and 500 μmol/L palmitic acid,the pretreatment with oridonin sig