Salidroside Protect The Endothelial Progenitor Cells And Endothelial Cells Damaged Induced By Ionizing Radiation

Objective: To explore the protective effects of salidroside on endothelial progenitor cells(EPCs) and vascular endothelial cells damaged by ionizing radiation and its underlying mechanisms.Methods: 1. The effects of salidroside on EPCs damaged induced by ionizing radiationIn vitro isolation and culture of healthy human peripheral blood-derived EPCs, and was identified with UEA-N and Di LDL double staining. The experiments were divided into normal control group, ionizi ng radiation group, salidroside group(salidroside + ionizing radiation) and LY294002 group(salidroside + ionizing radiation + LY294002). Ionizing radiation group, salidroside group and LY294002 group were treated with 4.0Gy 60 Co gamma ray radiation. Using CCK8 method, Tranwell method, Annexin FITC-V/PI double staining to observe the effect of salidroside on the proliferation, adhesion, migration and the percentage of apoptosis of EPCs after ionizing radiation. In addition, we also performed western blotting to analyze the possible molecular mechanism of salidroside. 2. The effects of salidroside on vascular endothelial cells damaged induced by ionizing radiationHuman umbilical vein endothelial cells(HUVECs) were cultured in vitro and treated with 10.0Gy 60 Co gamma ray radiation after pretreatment with salidroside(100umol/L) for 18 h. we performed CCK8 method, Transwell method, cell-cell adhesion and flow cytometry to detect the effect of salidroside on the proliferation, migration, the adhesion between HUVECs and inflammatory cell, apoptosis, reactive oxygen species(ROS) formation and mitochondrial membrane potential, and analyzed the effect of phosphoinositide 3-kinase/protein kinase-B(PI3K/Akt) with western blotting.Results:1. The protective effects of salidroside on EPCs under ionizing radiation.After exposed 4.0Gy 60 Co gamma radiation dose, the cell proliferation ability of the ionizing radiation group was decreased at 24 h, and were significantly decreased at 48 h and 72 h compared with the control group. Pretreatment with salidroside(80umol/L) for 1h before exposed ionizing radiation can improve the proliferation of EPCs. However LY294002(20umol/L) can inhibit the improve effect of salidroside on radiated EPCs. Transwell results showed that the migration rate of the ionizing radiation group was decreased by 69% than the control group, the salidroside group was 1.8 times than the ionizing radiation, while the LY29400 group was significantly decreased compared with the salidroside group. The number of adherent cell in the ionizing radiation group was 30% in the control group, the salidroside group was 2.1 times that of the ionizing radiation group, and the number of adherent cells was less in the LY294002 group than the salidroside group. The percentage of apoptosis by flow cytometry of the control group, ionizing radiation group, salidroside group and LY294002 group were(2.73±0.78)%、(80.20±2.40)%、(24.03±1.43)%�'�(58.67±5.28)% respectively. These results indicated that ionizing radiation could decrease the proliferation, migration and adhesion ability of EPCs and increase the apoptosis of cells. However, salidroside can significantly decrease the EPCs damage induced by ionizing radiation, leaded a protective effect. Western blotting results showed that ionizing radiation can inhibit the phosphorylation of Akt(p-Akt), implying inhibit the activation of PI3 K / Akt signal pathway. Pretreatment with salidroside can activate the PI3 K / Akt signaling pa thway, increase the p-Akt expression; LY294002 as a specific inhibitor of PI3 K / Akt signal pathway, can inhibit the activation of salidroside on PI3 K / Akt signaling pathway, reduce p-Akt expression. Above results indicated that the protective effects of salidroside on the radiated EPCs were related to activate the PI3K/Akt signaling pathway.2. The protective effects of salidroside on HUVECs under ionizing radiation.HUVECs were C ultured in vitro and pretreatment with salidroside for 18 h,following exposed 10.0Gy 60 Co gamma radiation dose and continued culture for 30 h.Ionizing radiation were significantly decreased cell activity, migration and were appeared apoptosis morphological changes of the nuclear condensation, nuclear fragmentation and increased the percentage of apoptosis of HUVECs. Pretreatment with salidroside was significantly increased cell activity, migration and decreased apoptosis. However, LY294002 can block the improvement effects of salidroside on radiation injury of HUVECs. Ionizing radiation were also increase the adhesion of U937 cells and HUVECs, the levels of intracellular reactive oxygen species(ROS) and the decrease of mitochondrial membrane potential, salidroside ca n significantly reduce the adhesion between HUVECs and inflammatory cells, decrease reactive oxygen species(ROS) formation and recovery the decrease of mitochondrial membrane potential, as to improve the survival. Western blotting results showed that ionizing radiation can inhibit the phosphorylation of Akt(p-Akt), implying inhibit the activation of PI3 K / Akt signal pathway. Pretreatment with salidroside can activate the PI3 K / Akt signaling pathway, increase the p-Akt expression. However, LY294002 can reduce p-Akt expression to inhibit the activation of PI3 K / Akt signal pathway of salidroside. These results indicated that the protective effect of salidroside on HUVECs under ionizing radiation were related to the inhibition of inflammation, the decrease of reactive oxygen species formation, the recovery of mitochondrial function and the activation of PI3K/Akt pathway. Conclusions:1 Ionizing radiation can induce apoptosis of endothelial progenitor cells and vascular endothelial cells.2 The protective effects of salidroside on the radiated EPCs were related to activate the PI3K/Akt signaling pathway.3 The protective effects of salidroside on HUVECs under ionizing radiation were related to the inhibition of inflammation response, the decrease of reactive oxygen species formation, the recovery of mitochondrial function and the activation of PI3K/Akt pathway.4 Salidroside can as a drug to prevent or treat radiation related diseases.