Guo X Y, Zhou X Q, Xie W G
Institute of Burns, Tongren Hospital of Wuhan University & Wuhan Third Hospital, Wuhan 430060, China.
Zhonghua Shao Shang Za Zhi. 2021 Sep 20;37(9):869-874. doi: 10.3760/cma.j.cn501120-20200720-00352.
To investigate the effects and related mechanism of estrogen receptor β (ERβ) agonist on the migration and oxidative stress of human umbilial vein endothelial cells (HUVECs) under high glucose condition. The experimental research method was adopted. HUVECs were routinely cultured and passaged, and then cells of the logarithmic growth phase were collected for the subsequent experiments. The cells were divided into three groups according to the random number table, including normal control group (cultured with Roswell Park Memorial Institute 1640 cell culture medium (the same cell culture medium below) containing 5.5 mmol/L D-glucose), high glucose alone group (cultured with cell culture medium containing 25.0 mmol/L D-glucose alone), and high glucose+ERβ agonist diarylpropionitrile (DPN) group (cultured with cell culture medium containing 25.0 mmol/L D-glucose and 10 μmol/L DPN). Scratch test was conducted to detect the cell migration rate in the 3 groups at 24 h post scratching. Fluorescent probe method was used to detect the reactive oxygen species (ROS, denoted by red fluorescence intensity) of cells in the 3 groups on 5 d post culture. Western blotting was used to detect the protein expression levels of vascular endothelial growth factor (VEGF) and superoxide dismutase 2 (SOD2) of cells in the 3 groups on 5 d post culture. In the above-mentioned experiments, cells were grouped and cultured correspondingly as before, the number of samples in each group was 5. Data were statistically analyzed with one-way analysis of variance and least significant difference test. At 24 h post scratching, the cell migration rate in high glucose alone group was (36±5)%, which was significantly lower than (76±4)% of normal control group and (65±5)% of high glucose+DPN group (=14.511, 9.603, <0.01), and the cell migration rate in high glucose+DPN group was significantly lower than that in normal control group (=3.943, <0.01). On 5 d post culture, the level of ROS of cells in high glucose alone group (1.81±0.12) was significantly increased compared with normal control group and high glucose+DPN group (1.00±0.14, 0.91±0.15, =9.679, 10.549, <0.01), while the level of ROS of cells in normal control group and high glucose+DPN group were close (=1.031, >0.05). On 5 d post culture, the protein expression levels of VEGF and SOD2 of cells in high glucose alone group were significantly lower than the levels of normal control group (=14.175, 13.787, <0.01) and high glucose+DPN group (=6.321, 17.750, <0.01). The protein expression level of VEGF of cells in high glucose+DPN group was significantly lower than the level of normal control group (=7.206, <0.05), while the protein expression level of SOD2 of cells in high glucose+DPN group was significantly higher than the level of normal control group (=2.890, <0.05). The activation of ERβ can improve the inhibition of HUVECs migration induced by high glucose and alleviate oxidative stress injury induced by high glucose, which may be achieved by promoting the expression of VEGF and SOD2.
探讨雌激素受体β(ERβ)激动剂对高糖条件下人脐静脉内皮细胞(HUVECs)迁移及氧化应激的影响及其相关机制。采用实验研究方法。将HUVECs常规培养及传代,然后收集对数生长期细胞用于后续实验。根据随机数字表将细胞分为三组,包括正常对照组(用含5.5 mmol/L D -葡萄糖的罗斯威尔帕克纪念研究所1640细胞培养基(以下同)培养)、单纯高糖组(仅用含25.0 mmol/L D -葡萄糖的细胞培养基培养)和高糖+ERβ激动剂二芳基丙腈(DPN)组(用含25.0 mmol/L D -葡萄糖和10 μmol/L DPN的细胞培养基培养)。划痕试验检测三组细胞划痕后24 h的迁移率。荧光探针法检测培养5 d后三组细胞的活性氧(ROS,以红色荧光强度表示)。蛋白质印迹法检测培养5 d后三组细胞血管内皮生长因子(VEGF)和超氧化物歧化酶2(SOD2)的蛋白表达水平。在上述实验中,细胞按之前方法分组并相应培养,每组样本数为5。数据采用单因素方差分析和最小显著差法检验进行统计学分析。划痕后24 h,单纯高糖组细胞迁移率为(36±5)%,显著低于正常对照组的(76±4)%和高糖+DPN组的(65±5)%(F = 14.511、9.603,P < 0.01),且高糖+DPN组细胞迁移率显著低于正常对照组(t = 3.943,P < 0.01)。培养5 d后,单纯高糖组细胞的ROS水平(1.81±0.12)与正常对照组和高糖+DPN组(1.00±0.14、0.91±0.15)相比显著升高(F = 9.679、10.549,P < 0.01),而正常对照组和高糖+DPN组细胞的ROS水平相近(t = 1.031,P > 0.05)。培养5 d后,单纯高糖组细胞VEGF和SOD2的蛋白表达水平显著低于正常对照组(F = 14.175、13.787,P < 0.01)和高糖+DPN组(F = 6.321、17.750,P < 0.01)。高糖+DPN组细胞VEGF的蛋白表达水平显著低于正常对照组(t = 7.206,P < 0.05),而高糖+DPN组细胞SOD2的蛋白表达水平显著高于正常对照组(t = 2.890,P < 0.05)。ERβ的激活可改善高糖对HUVECs迁移的抑制作用并减轻高糖诱导的氧化应激损伤,这可能是通过促进VEGF和SOD2的表达来实现的。