雌激素处理的hBMSC对高糖诱导血管内皮细胞损伤的保护作用*

doi:10.12047/j.cjap.6244.2022.054

中国应用生理学杂志 ›› 2022, Vol. 38 ›› Issue (4): 289-294.doi: 10.12047/j.cjap.6244.2022.054

• 研究论文 • 下一篇

雌激素处理的hBMSC对高糖诱导血管内皮细胞损伤的保护作用^*

张晓东¹, 孙石磊¹, 王冠^1,2, 李霄凌¹, 姚立杰¹, 徐浩¹, 张鹏³, 沈雷^4△

1.黑龙江省齐齐哈尔医学院解剖学教研室, 齐齐哈尔 161006;
2.北京市首都医科大学组织胚胎学系, 北京 100069;
3.上海复旦大学基础医学院人体解剖与组织胚胎学系, 上海 200433;
4.黑龙江省齐齐哈尔医学院基础医学院, 齐齐哈尔 161006

收稿日期:2021-12-23 修回日期:2022-07-19 出版日期:2022-07-28 发布日期:2022-11-23
通讯作者: ^△Tel: 0452-2663809; E-mail: shenlei815@qmu.edu.cn
基金资助:
^*黑龙江省教育厅科学技术研究项目(2017-KYYWF-0701); 齐齐哈尔市科技计划联合引导项目(LHYD-202017); 黑龙江省自然科学基金项目(LH2021H121); 齐齐哈尔市科技创新引导项目(LHYD-2021002)

Protective effects of estrogen modified hBMSC on HG-induced injury of vascular endothelial cells

ZHANG Xiao-dong¹, SUN Shi-lei¹, WANG Guan^1,2, LI Xiao-ling¹, YAO Li-jie¹, XU Hao¹, ZHANG Peng³, SHEN Lei^4△

1. Department of Anatomy, Qiqihar Medical College, Qiqihar 161006;
2. Department of Histology and Embryology, Beijing Capital Medical University, Beijing 100069;
3. Department of Anatomy and Histo-embryology, School of Basic Medicine, Fudan University, Shanghai 200433;
4. School of Basic Medicine, Qiqihar Medical College, Qiqihar 161006, China

Received:2021-12-23 Revised:2022-07-19 Online:2022-07-28 Published:2022-11-23

摘要/Abstract

摘要： 目的: 探讨雌激素处理人骨髓间充质干细胞(hBMSC)对高糖诱导的人脐静脉血管内皮细胞(HUVEC)损伤的保护作用及机制。方法: 采用30 mmol/L葡萄糖刺激hBMSC细胞建立高糖模型并分组:以无刺激者为高糖对照组(HG组)、以20 μmol/L雌激素处理者为高糖雌激素组(HG+E2组)、以5 μmo/L蛋白激酶B(PKB/Akt)抑制剂Triciribine预处理45 min后,再以20 μmol/L雌激素处理者为高糖Akt抑制剂组(HG+E2+Triciribine组)和正常条件培养的hBMSC为正常对照组(NG组)。分别于处理12 h后,采用CCK8法检测各组hBMSC的细胞活力,硝酸还原酶法和ELISA法检测各组培养基上清中NO、VEGF和IL-8的含量(n=6),48 h后采用Western blot检测内皮型一氧化氮合酶(eNOS)和磷酸化eNOS(p-eNOS)蛋白表达水平(n=3)。此外,提取各组hBMSC的培养基上清作为条件培养基(CM)培养人脐静脉血管内皮细胞(HUVEC)并分组为:HG-CM组(HG组条件培养基处理)、HG+E2-CM组(HG+E2组条件培养液处理)、HG+E2+Triciribine-CM组(HG+E2+Triciribine组条件培养基处理)和HG-H组(高糖对照组,30 mmol/L葡萄糖终浓度处理),分别于12 h后,采用CCK8法检测各组HUVEC的细胞活力(n=6),24 h后采用流式细胞术检测各组HUVEC细胞的凋亡率(n=3);48 h后采用划痕实验观察各组HUVEC细胞的迁移率(n=3)。结果: 与NG组相比,HG组中hBMSC细胞活力和细胞内eNOS蛋白磷酸化水平降低(P<0.05),细胞培养液上清中NO、VEGF和IL-8含量减少(P<0.05);与HG组相比,HG+E2组中hBMSC的细胞活力和细胞中eNOS蛋白磷酸化水平显著增加(P<0.05),细胞培养基上清中NO、VEGF和IL-8含量增加(P<0.05),而当hBMSC细胞中Akt蛋白活性被抑制后,HG+E2+Triciribine组中上述结果指标呈反向变化(P<0.05)。此外,与HG-CM组相比,HG+E2-CM组中HUVECs的细胞活力和迁移能力显著增加(P<0.05),细胞凋亡比例降低(P<0.05),而与HG+E2-CM组相比,HG+E2+Triciribine-CM组中HUVECs的细胞活力和迁移能力降低(P<0.05),细胞凋亡比例增加(P< 0.05)。结论: 雌激素可能通过激活hBMSC细胞Akt/eNOS信号通路,促进NO、VEGF和IL-8的分泌,进而增加HUVECs的细胞活力和迁移能力,并抑制细胞凋亡的发生,对高糖诱导的HUVECs细胞损伤发挥保护作用。

关键词: 雌激素, 人骨髓间充质干细胞, 细胞损伤, 高糖条件, 人脐静脉血管内皮细胞, 细胞培养

Abstract: Objective: To investigate the protective effects and potential mechanisms of estrogen modified human bone marrow mesenchymal stem cells (hBMSC) on high glucose (HG)-induced injury of vascular endothelial cells. Methods: hBMSCs were cultured under 30 mmol/l glucose to establish a high glucose model (HG), and then were divided into four groups as following: HG group (HG control, without any treatment), HG+E2 group (cells were treated with 20 μmol/L estrogen), HG+E2+ Triciribine group (cells were pretreated with 5 μmol/L protein kinase B (PKB/Akt) inhibitor for 45 min, and then modified by 20 μmol/L estrogen), and NG group (cells were cultured under normal conditions). After 12 h treatment, the cell viability of hBMSC was detected by CCK8 assay, and the contents of NO, VEGF and IL8 in the supernatant of cultured medium in each group were detected by nitrate reductase and ELISA assay (n=6). After 48 h, the expression levels of endothelial nitric oxide synthase (eNOS) and phosphorylated eNOS (p-eNOS) were detected by Western blot (n=3). In addition, the cell supernatant of each group was further extracted as conditioned medium to culture HUVECs, and the cells were subsequently divided into HG-CM group (HUVECs were treated with HG group’s conditioned medium), HG+E2-CM group (HUVECs were treated with HG+E2 group’s conditioned medium), HG+E2+Triciribine-CM group (HUVECs were treated with HG+E2+ Triciribine group’s conditioned medium) and HG-H group (HUVEC were cultured under HG condition, which were treated with final concentration 30 mmol/l glucose). The cell viability of HUVECs in each group was detected by CCK8 assay after 12 h cultured (n=6). After 24 h treatment, the apoptosis rate of HUVECs in each group was detected by flow cytometry (n=3). Furthermore, the migration rate of HUVECs in each group was observed by wound healing assay after 48 h cultured (n=3). Results: Compared with NG group, the cell viability and eNOS protein phosphorylation level of hBMSC in HG group and the contents of NO, VEGF and IL-8 in the supernatant of cultured medium were decreased (P<0.05). Compared with HG group, the cell viability and eNOS protein phosphorylation level in HG+E2 group and the contents of NO, VEGF and IL-8 in cultured medium supernatant were increased significantly (P<0.05), whereas pre-treatment of hBMSC cells with a Akt inhibitor Triciribine, the above indexes showed reverse changes (P<0.05). Furthermore, compared with HG-CM group, the cell viability and migration ability (P<0.05) of HUVECs in HG+E2-CM group were increased significantly (P<0.05), and the proportion of apoptosis was decreased (P<0.05). While compared with HG+E2-CM group, the cell viability and migration ability of HUVECs in HG+E2+Triciribine-CM group were decreased (P<0.05), and the proportion of apoptosis was increased (P<0.05). Conclusion: Estrogen may promote the secretion of NO, VEGF and IL-8 by activating the Akt/eNOS signaling pathway of hBMSC cells, increase the cell viability and migration ability of HUVECs and inhibit the occurrence of apoptosis, play a protective role against the injury of HUVECs induced by HG condition.

Key words: estrogen, human bone marrow mesenchymal stem cells, cell injury, high glucose condition, human umbilical vein endothelial cells, cell culture

中图分类号:

张晓东, 孙石磊, 王冠, 李霄凌, 姚立杰, 徐浩, 张鹏, 沈雷. 雌激素处理的hBMSC对高糖诱导血管内皮细胞损伤的保护作用^*[J]. 中国应用生理学杂志, 2022, 38(4): 289-294.

ZHANG Xiao-dong, SUN Shi-lei, WANG Guan, LI Xiao-ling, YAO Li-jie, XU Hao, ZHANG Peng, SHEN Lei. Protective effects of estrogen modified hBMSC on HG-induced injury of vascular endothelial cells[J]. CJAP, 2022, 38(4): 289-294.

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雌激素处理的hBMSC对高糖诱导血管内皮细胞损伤的保护作用^*

Protective effects of estrogen modified hBMSC on HG-induced injury of vascular endothelial cells

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