Ginsenoside Rg1 protects against hydrogen peroxide induced damage to bone marrow-derived endothelial progenitor cells

doi:10.3969/j.issn.2095-4344.0646

Abstract

Abstract:

BACKGROUND: Studies have confirmed that ginsenoside Rg1 has protective effects on vascular endothelial cells, but its underlying mechanism is still unclear.
OBJECTIVE: To investigate the mechanism by which ginsenoside Rg1 protects against hydrogen peroxide induced injury of rat bone marrow-derived endothelial progenitor cells.
METHODS: Endothelial progenitor cells derived from bone marrow of Sprague-Dawley rats were isolated and cultured by direct adherent method. The endothelial progenitor cells in logarithmic growth period were divided into three groups: control group, no intervention but conventional culture for 24 hours; cell injury group, cells were cultured in EMG-2 medium containing 100 μmol/L hydrogen peroxide for 24 hours; combined intervention group, cells were pretreated with ginsenoside Rg1 (64 μmol/L) for 2 hours, then cultured in EMG-2 medium containing 100 μmol/L hydrogen peroxide for 22 hours. Cell proliferation, apoptosis and migration were detected using cell counting kit-8, TUNEL, and cell scratch test, respectively. The expression of Akt, p-Akt, Bax, and Bcl-2 proteins was measured using western blot assay. The levels of intracellular superoxide dismutase, malondialdehyde and nitric oxide were determined using ELISA method.
RESULTS AND CONCLUSION: (1) Compared with the cell injury group, the cell survival rate and cell migration ability were significantly increased, while the number of apoptotic cells reduced significantly in the combined intervention group (all P < 0.05). (2) Compared with the cell injury group, the levels of intracellular superoxide dismutase and nitric oxide were significantly increased, while the level of malondialdehyde was significantly reduced in the combined intervention group (all P < 0.05). (3) No significant difference in the expression of Akt protein was detected among the three groups, but the expression of p-Akt protein was significantly higher in the combined intervention group than the cell injury group (P < 0.05). Moreover, significantly increased Bcl-2 expression and decreased Bax expression were observed in the combined intervention group as compared with the cell injury group. In conclusion, ginsenoside Rg1 can antagonize hydrogen peroxide-induced damage to rat bone marrow-derived endothelial progenitor cells, increase cell viability, inhibit cell apoptosis, and reduce oxidative stress injury via the Erk signaling pathway.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Panax, Saponins, Endothelial Cells, Hydrogen Peroxide, Tissue Engineering

CLC Number:

R394.2

Jiang Wen-jie, Liang Xue-mei. Ginsenoside Rg1 protects against hydrogen peroxide induced damage to bone marrow-derived endothelial progenitor cells[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(33): 5338-5343.

Figures/Tables 1

2.1 骨髓来源内皮祖细胞的培养及鉴定结果原代培养48 h后，镜下可见少量细胞贴壁，以小的圆形细胞为主。随着培养时间延长，细胞体积逐渐增大，呈梭形。培养至第六七天时，形成细胞集落，可见部分细胞由梭形变为菱形(图1A)；荧光显微镜下双染色显示，DiI-Ac-LDL染色后细胞呈红色(图1B)，FITC-UEA-1染色后细胞呈绿色(图1C)，双染细胞叠加呈黄色(图1D)；流式细胞仪检测结果显示，细胞高表达CD133、CD34及KDR，阳性表达率分别为(78.6±9.9)%，(58.7±10.5)%，(88.6±6.8)%，以上结果提示实验获得的为早期内皮祖细胞。 2.2 各组细胞增殖能力经100 μmol/L过氧化氢干预后，细胞损伤组的细胞存活率较空白对照组明显降低，差异有显著性意义(P < 0.05)；经过人参皂苷Rg1预干预后，人参皂苷Rg1组细胞存活率明显高于细胞损伤组，差异有显著性意义(P < 0.05)，见图2。 2.3 各组细胞凋亡数量见图3。Tunnel检测显示，经 100 μmol/L过氧化氢干预后，细胞损伤组的细胞凋亡数量较空白对照组明显增多，差异有显著性意义(P < 0.05)；经过人参皂苷Rg1预干预后，人参皂苷Rg1组细胞凋亡数量明显少于细胞损伤组，差异有显著性意义(P < 0.05)；空白对照组、细胞损伤组、人参皂苷Rg1组细胞凋亡率分别为(2.33±0.36)%，(18.46±4.55)%，(10.29±3.97)%，组间两两比较差异均有显著性意义(P < 0.05)。 2.4 各组细胞超氧化物歧化酶、丙二醛、一氧化氮水平见表1。与空白对照组相比较，细胞损伤组超氧化物歧化酶、一氧化氮水平明显降低，丙二醛浓度显著升高，差异均有显著性意义(P < 0.05)；与细胞损伤组相比较，人参皂苷Rg1组超氧化物歧化酶、一氧化氮水平明显升高，丙二醛水平显著降低，差异均有显著性意义(P < 0.05)。 2.5 各组细胞迁移能力划痕距离越大说明细胞迁移能力越差。空白对照组、细胞损伤组、人参皂苷Rg1组24 h的划痕距离分别为(0.32±0.01) mm，(0.63±0.02) mm，(0.49±0.02) mm；与空白对照组比较，细胞损伤组内皮祖细胞迁移能力明显下降，差异有显著性意义(P < 0.05)；与细胞损伤组比较，人参皂苷Rg1组细胞迁移能力有明显提高，差异有显著性意义(P < 0.05)。 2.6 各组细胞相关蛋白表达 Western Blot检测结果显示，3组间Akt蛋白表达无差异，p-Akt蛋白表达有差异，细胞损伤组p-Akt蛋白表达明显低于空白对照组，人参皂苷Rg1组p-Akt蛋白表达明显高于细胞损伤组，差异有显著性意义(P < 0.05)，见图4。各组细胞凋亡相关蛋白表达，见图5。结果显示，细胞损伤组Bcl-2蛋白表达低于空白对照组(P < 0.05)，Bax蛋白表达高于空白对照组(P < 0.05)；人参皂苷Rg1组Bcl-2蛋白表达高于细胞损伤组(P < 0.05)，Bax蛋白表达低于细胞损伤组(P < 0.05)。"

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