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

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.