雌激素受体β激动剂对高糖作用下人脐静脉内皮细胞迁移和氧化应激的影响及其机制

郭晓雨; 周雪情; 谢卫国

doi:10.3760/cma.j.cn501120-20200720-00352

雌激素受体β激动剂对高糖作用下人脐静脉内皮细胞迁移和氧化应激的影响及其机制

doi: 10.3760/cma.j.cn501120-20200720-00352

武汉大学同仁医院暨武汉市第三医院烧伤研究所　　430060

基金项目:

国家自然科学基金面上项目 81772097

详细信息

通讯作者:
谢卫国，Email：wgxie@hotmail.com

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出版历程
- 收稿日期: 2020-07-20

Effects and mechanism of estrogen receptor β agonist on the migration and oxidative stress of human umbilical vein endothelial cell under high glucose condition

Institute of Burns, Tongren Hospital of Wuhan University＆Wuhan Third Hospital, Wuhan 430060, China Corresponding author: Xie Weiguo, Email: wgxie@hotmail.com

Funds:

General Program of National Natural Science Foundation of China 81772097

摘要

摘要: 目的探讨雌激素受体β（ERβ）激动剂对高糖作用下人脐静脉内皮细胞（HUVEC）迁移和氧化应激的影响及相关机制。方法采用实验研究方法。将HUVEC常规培养传代，取对数生长期细胞进行后续实验。将细胞按随机数字表法分为3组，正常对照组采用含5.5 mmol/L D-葡萄糖的RPMI 1640细胞培养基（下同）培养，单纯高糖组采用仅含25.0 mmol/L D-葡萄糖的细胞培养基培养，高糖+二芳基丙腈（DPN）组采用含25.0 mmol/L D-葡萄糖+10 μmol/L DPN的细胞培养基培养。采用划痕试验检测划痕后24 h 3组细胞迁移率，荧光探针法检测培养5 d 3组细胞内活性氧水平（以红色荧光强度表示），蛋白质印迹法检测培养5 d 3组细胞内血管内皮生长因子（VEGF）及超氧化物歧化酶2（SOD2）的蛋白表达。以上每个检测均取细胞同前行相同分组及相应培养，每个指标检测每组细胞样本数均为5。对数据行单因素方差分析、LSD-t检验。结果划痕后24 h，单纯高糖组细胞迁移率［（36±5）%］明显低于正常对照组和高糖+DPN组［（76±4）%、（65±5）%，t=14.511、9.603，P<0.01］，高糖+DPN组细胞迁移率明显低于正常对照组（t=3.943，P<0.01）。培养5 d，单纯高糖组细胞内活性氧水平（1.81±0.12）明显高于正常对照组、高糖+DPN组（1.00±0.14、0.91±0.15，t=9.679、10.549，P<0.01），高糖+DPN组细胞内活性氧水平与正常对照组相近（t=1.031，P>0.05）。培养5 d，单纯高糖组细胞内VEGF和SOD2蛋白表达均明显低于正常对照组（t=14.175、13.787，P<0.01）和高糖+DPN组（t=6.321、17.750，P<0.01）；高糖+DPN组VEGF蛋白表达明显低于正常对照组（t=7.206，P<0.01），SOD2蛋白表达明显高于正常对照组（t=2.890，P<0.05）。结论激活ERβ可通过促进VEGF和SOD2的表达，明显改善高糖对HUVEC迁移的抑制，缓解高糖诱导的氧化应激损伤。
- 雌激素受体β /
- 内皮细胞 /
- 细胞迁移分析 /
- 氧化性应激 /
- 高糖
Abstract: Objective 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. Methods 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 t test. Results 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 (t=14.511, 9.603, P<0.01), and the cell migration rate in high glucose+DPN group was significantly lower than that in normal control group (t=3.943, P<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, t=9.679, 10.549, P<0.01), while the level of ROS of cells in normal control group and high glucose+DPN group were close (t=1.031, P>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 (t=14.175, 13.787, P<0.01) and high glucose+DPN group (t=6.321, 17.750, P<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 (t=7.206, P<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 (t=2.890, P<0.05). Conclusions 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.
- Estrogen receptor beta /
- Endothelial cells /
- Cell migration assays /
- Oxidative stress /
- High glucose
所有作者均声明不存在利益冲突

烧伤治疗仍面临高代谢、肌肉消耗、胰岛素抵抗的严峻挑战。然而，在缺乏具体指南的情况下，目前仍不清楚该如何调控这些代谢靶点。比利时Antwerp大学医学与健康科学学院MOVANT研究组Ulrike Van Daele教授团队近期在《Burns & Trauma》发文《Status of adult inpatient burn rehabilitation in Europe: are we neglecting metabolic outcomes?》，此文旨在展示欧洲烧伤住院患者的康复现状。经由欧洲烧伤协会向欧洲各地烧伤中心发放电子调查问卷，问卷包含针对治疗师、医师、营养师的一般和专业问题，涉及运动处方、代谢管理以及治疗的优先次序、动机和烧伤引起的代谢序贯事件的知识。通过计算优势比来分析优先治疗的反应和烧伤代谢序贯事件知识之间的相关性。最终，8个欧洲国家91个烧伤中心中18个有响应（响应率19.8%），包括59名有（12.3±9）年烧伤专业经验的临床医师。只有42%的治疗师向插管患者提供了抗阻运动，有38%提供了有氧运动，在能下床活动的患者中分别为87%和65%，而在能离开病房的患者中则分别为97%和83%。通过间接测热法、肌肉消耗和胰岛素抵抗评估静息能量消耗的受访者分别只有40.7%、15.3%和7.4%，在使用频率和方法上差异明显。在高代谢（59.3%）、肌肉消耗（70.4%）或胰岛素抵抗（44.4%）的病例中，并非所有临床医师都改变了治疗方案，提示管理策略存在较大差异。在治疗目标重要性评分、动机和烧伤代谢序贯事件知识方面存在明显的跨学科差异。代谢序贯事件的预防是最不重要的治疗目标，而功能状态的恢复则是最重要的。对烧伤代谢序贯事件的了解与将代谢序贯事件作为重要治疗目标的优先性分级有关（优势比=4.63，95%置信区间=1.50~14.25，P<0.01）。此项调查揭示了，在欧洲烧伤住院患者康复治疗中存在不一致性，尤其是对代谢结果的潜在忽视。研究结果为针对住院烧伤患者制订康复实践指南提供了必要的基础。

杨敏，编译自《Burns & Trauma》，2021，9:tkaa039；韩春茂，审校

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1 划痕试验观察3组人脐静脉内皮细胞划痕后各时间点迁移情况光学显微镜×40。1A、1B、1C.分别为正常对照组、单纯高糖组、高糖+二芳基丙腈（DPN）组划痕后即刻；1D、1E、1F.分别为正常对照组、单纯高糖组、高糖+DPN组划痕后24 h，图1D、1F剩余划痕面积较小，图1E剩余划痕面积较大

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2 3组人脐静脉内皮细胞培养5 d活性氧水平（红色荧光）观察 DCFH-DA×400。2A.正常对照组活性氧水平较低；2B.单纯高糖组活性氧水平高；2C.高糖+二芳基丙腈组活性氧水平较低，且与图2A相近

注：2'，7'-二氯二氢荧光素二乙酸酯（DCFH-DA）荧光探针阳性为红色荧光

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3 蛋白质印迹法检测3组人脐静脉内皮细胞培养5 d VEGF、SOD2的蛋白表达。3A.条带图；3B.条图（x¯±s，样本数为5）

注：VEGF为血管内皮生长因子、SOD2为超氧化物歧化酶2、DPN为二芳基丙腈；条带图上方与条图横坐标1、2、3分别为正常对照组、单纯高糖组、高糖+DPN组；3组间VEGF、SOD2总体比较，F=100.381、148.302，P<0.001；与正常对照组比较，^aP<0.01，^cP<0.05；与单纯高糖组比较，^bP<0.01

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