中国药理学与毒理学杂志2024，Vol.38Issue(8)：561-574,14.DOI:10.3867/j.issn.1000-3002.2024.08.001
环磷酰胺对人神经母细胞瘤SH-SY5Y细胞的损伤效应及机制
Damage effect and mechanisms of cyclophosphamide to human neuroblastoma SH-SY5Y cells
摘要
Abstract
OBJECTIVE To investigate the damage effect and mechanisms of cyclophosphamide(CTX)and its active metabolite derivative 4-hydroperoxycyclophosphamide(4-HC)to human neuroblas-toma SH-SY5Y cells.METHODS SH-SY5Y cells were treated with CTX[0(cell control),0.01,0.1,1,5,10,20,40 and 80 mmol·L-1]and 4-HC[0(cell control),0.01,0.1,1,5,10,20,40 and 80 μmol·L-1]for 48 h.Cell confluence and morphology were observed by the IncuCyte ZOOM system.Cell viability was assessed by CCK-8 assay.Lactate dehydrogenase(LDH)release was measured by LDH assay kit.SH-SY5Y cells were treated with CTX(0,1,5,10 and 20 mmol·L-1)and 4-HC(0,1,5,10 and 20 μmol·L-1)for 48 h before cell proliferation was analyzed by 5-ethynyl-2′-deoxyuridine(EdU)staining assay.Immunofluorescence was employed to assess the levels of the DNA double-strand break marker γ-H2AX and to evaluate changes in mitochondrial membrane potential.SH-SY5Y cells were treated with CTX(0,1,5 and 10 mmol·L-1)and 4-HC(0,1,5 and 10 μmol·L-1)for 48 h,and the alterations in glycolysis and oxidative phosphorylation levels were analyzed using the Seahorse XFe96 Analyzer.RESULTS Compared with the cell control group,cell confluence and cell viability were significantly reduced in the CTX and 4-HC groups(P<0.01),and the half-maximal inhibitory concentrations(IC50)for CTX and 4-HC were 4.44 mmol·L-1 and 4.78 μmol·L-1,respectively.The release rate of LDH was signif-icantly increased while the percentage of EdU+cells was significantly reduced in the CTX and 4-HC groups(P<0.01).The percentage of γ-H2AX+cells was significantly increased and mitochondrial membrane potential significantly decreased in the CTX and 4-HC group(P<0.05).Treatment with CTX and 4-HC resulted in reduced levels of maximum glycolytic capacity,glycolytic reserve,maximal respi-ration,and ATP production(P<0.05).CONCLUSION CTX and 4-HC exert significant cytotoxic effects on SH-SY5Y cells by disrupting cell membrane structure,impeding cell proliferation,and reducing cell viability.The mechanisms underlying these effects may involve intracellular DNA damage,disturbance of energy metabolism and mitochondrial dysfunction.关键词
环磷酰胺/4-过氧化氢环磷酰胺/神经毒性/能量代谢Key words
cyclophosphamide/4-hydroperoxycyclophosphamide/neurotoxicity/energy metabolism分类
医药卫生引用本文复制引用
李佳佳,王娇,肖文一,韦冬晖,张永祥,蒋宁,周文霞..环磷酰胺对人神经母细胞瘤SH-SY5Y细胞的损伤效应及机制[J].中国药理学与毒理学杂志,2024,38(8):561-574,14.基金项目
National Key Research and Develop-ment Program of China(2022YFC3500304) 国家重点研发计划(2022YFC3500304) （2022YFC3500304）
