摘要
目的观察BAPTA-AM抗D-氨基半乳糖(D-GalN)诱导MDCK细胞损伤作用,探讨其作用机制。方法采用MTT法,Annexin V-EGFP/PI与Hoechst33342/PI荧光染色,罗丹明123(Rhodamine 123)荧光染色,Caspase-8、Caspase-9活性测定及钙离子载体A23187诱导细胞内高钙等方法,分别测定D-GalN攻击下MDCK细胞活性,细胞凋亡状况,线粒体膜电位(△Ψm)、Caspase-8、Caspase-9活性和细胞内游离钙浓度([Ca2+]i)变化等。结果 BAPTA-AM能明显抑制D-GalN所致的细胞活力下降、减少细胞凋亡、维持△Ψm,抑制Caspase-8、Caspase-9的激活,减轻A23187所致细胞损伤,降低[Ca2+]i。结论 BAPTA-AM通过减轻钙超载,保护线粒体、抑制外源及内源性凋亡通路的激活,发挥抗肾细胞凋亡作用。
Aim To investigate the protective effects and its mechanism of BAPTA-AM on D-GalN induced apoptosis in MDCK cells.Methods MDCK cells were pretreated with BAPTA-AM at concentrations ranged from 0.5 to 50 nmol·L-1 and followed the D-GalN（20 mmol·L-1） attack.The viabilities of MDCK cells were measured by MTT assay.Cell apoptosis was evaluated by Annexin V-EGFP/PI or Hoechst 33342/PI labeling methods.Mitochondrial membrane potential（△Ψm）,Caspase-8,-9 were also tested to explore the inhibitory mechanisms.Results D-GalN attack resulted in MDCK cell injury characterized by the decrease of the cell viability,cell death（especially apoptosis）,collapse of mitochondrial membrane potential,and the activation of Caspase-8 and Caspase-9.BAPTA-AM could significantly protect the cells against the injuries induced by D-GalN attack in a dose-dependnt manner.In addition,BAPTA-AM could markedly inhibit MDCK cell injury by intracellular calcium overload induced by A23187/CaCl2 and attenuate intracellular free Ca2＋ level（i）.Conclusion BAPTA-AM is a promising MDCK cell protector against D-GalN and A23187/CaCl2 induced injuries.Its mechanism of action is related to the decreasing i and in turn,protection of mitochondria and down-modulation of apoptotic signal pathways.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2012年第2期255-260,共6页
Chinese Pharmacological Bulletin
