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 ( Δψm ), 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 Ca level ([ Ca2+ ]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 [ Ca2+ ]i and in turn, protection of mitochondria and down-modulation of ap-optotic signal pathways.%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.
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