关键词: 当归红芪超滤物, 血管内皮细胞, 细胞凋亡, X射线, 辐射, 放射治疗

Abstract:

Objective： To investigate the protective effect of ultra-filtration extract from Angelica Sinensis Radix and Hedysari Radix （UFE-AH） on the injury of human umbilical vein endothelial cells （HUVECs） induced by X-ray，and to clarify its possible mechanism. Methods The HUVECs were cultured in vitro and irradiated with different doses （0，2，4，6，8 and 10 Gy） of X-ray； the optimal radiation dose （6 Gy） was selected. The HUVECs were intervened with different concentrations （0，50，100，200，400，600，800 and 1 000 μg·L^－1） of UFE-AH， and the optimal proliferation promoting concentrations （100，200 and 400 μg·L^－1） were selected.The experiment was divided into blank group， model group （6 Gy X-ray）， low dose of UFE-AH group （6 Gy X-ray + 100 μg·L^－1 UFE-AH）， medium dose of UFE-AH group （6 Gy X-ray+200 μg·L^－1 UFE-AH） and high dose of UFE-AH group （6 Gy X-ray + 400 μg·L^－1 UFE-AH）.The survival rates of cells in various groups were measured by CCK-8 assay， the apoptotic rates of cells in various groups were measured by flow cytometry，the ultrastructures were observed by transmission electron microscope， and the expression levels of phosphatidylinositol 3-kinase （PI3K）， protein kinase B （Akt）， phosphorylated Akt（p-Akt），vascular endothelial growth factor （VEGF），B-cell lymphoma-2（Bcl-2），Bcl-2 associated X protein （Bax） and cysteinyl aspartate specific proteinase-3（caspase-3） proteins in the cells in various groups were measured by Western blotting method. Results The CCK-8 assay results showed that compared with 0 Gy X-ray，the inhibitory rates of HUVECs irradiated by X-ray were increased with the increase of radiation dose when the indiation dose was greater than 2 Gy at 48 and 72 h after radiation （P<0.05 or P<0.01）， and the increasing of inhibition rates at 6 Gy X-ray and above were more significant（P<0.01）. Compared with 0 μg·L^－1 UFE-AH， the survival rates of cells after treated with 100，200，400 and 600 μg·L^－1UFE-AH were increased（P<0.05 or P<0.01），especially treated with 100，200 and 400 μg·L^－1 UFE-AH for 24，48 and 72 h （P<0.01）. Compared with blank group，the survival rate of the cells in model group was decreased（P<0.01）；compared with model group， the cell survival rates in medium and high doses of UFE-AH groups were significantly increased（P<0.01）. Compared with blank group，the apoptotic rate of the cells in model group was significantly increased（P<0.01）；compared with model group， the apoptotic rates of the cells in different doses of UFE-AH groups were significantly decreased（P<0.01）.The transmission electron microscope results showed that the cell membrane in blank group was intact and the cytoplasm was more uniform， the mitochondria were oval； no obvious swelling was observed， and a small amount of lysosomes were observed in the cells；compared with blank group， the cells in model group had severe swelling， multiple damage of the cell membrane， loose cytoplasm and many vacuolar areas， mildly irregular nuclei， the significantly reduced mitochondria， mild or moderate swelling， shallower and uneven matrix， local breakage and shortening of a small part of the cristae of mitochondria， and a large amount of autolysosomes （ASS） in the cells； compared with model group， the cell swelling in low， medium， and high doses of UFE-AH groups was alleviated，intracellular organelle vacuoles were gradually reduced， mitochondrial swelling was alleviated， the mitochondrial number was increased， and a certain amount of ASS were observed， but they still did not return to normal. Compared with blank group，the expression levels of PI3K， p-Akt， Bcl-2 and VEGF proteins in the cells in model group were significantly decreased（P<0.01）， and the expression levels of Bax and caspase-3 proteins and the Bax/Bcl-2 ratio were significantly increased（P<0.01）； compared with model group， the expression levels of PI3K， p-Akt， Bcl-2 and VEGF proteins in the cells in medium and high doses of UFE-AH groups were significantly increased（P<0.01），and the expression levels of Bax and caspase-3 proteins and the Bax/Bcl-2 ratio were significantly decreased（P<0.01）. Conclusion UFE-AH at a certain dose has a protective effect on the X-ray-induced injury of HUVECs， and its mechanism may be related to the effect of UFE-AH on the PI3K， Akt， p-Akt， VEGF， Bcl-2， Bax and caspase-3 protein expressions in the HUVECs.

Key words: Ultra-filtration extract from Angelica Sinensis Radix and Hedysari Radix, Vascular endothelial cells, Apoptosis, X-ray, Radiation, Radiation therapy