Effects Of Molybdenum And Cadmium Co-exposure On Apoptosis Of Primary Duck Renal Tubular Epithelial Cells

In order to investigate the effect of molybdenum（Mo）and cadmium（Cd）combined exposure to duck renal tubular epithelial cells on the apoptosis,10-15 day-old ducklings were selected to established the in vitro culture model of duck renal tubular epithelial cells by mesh filtration and enzymatic digestion.The experiment was divided into seven groups:control group（serum-free medium culture）,LCd group（2.5μmol/L Cd in serum-free medium）,HCd group（5μmol/L Cd in serum-free medium）,LMo group（480μmol/L Mo in serum-free medium）,HMo group（960μmol/L Mo in serum-free medium）,LMoCd group（2.5μmol/L Cd+480μmol/L Mo in serum-free medium）,HMoCd group（5μmol/L Cd+960μmol/L Mo added to serum-free medium）,all of which exposed for 12h.The activity of ATPase(Ca²⁺-ATPase,Na⁺/K⁺-ATPase)in duck renal tubular epithelial cells were detected.Apoptosis rate,intracellular calcium ion level,cellular reactive oxygen species level,intracellular pH,and cellular mitochondria membrane potential were determined by flow cytometry.At the same time,the mRNA expression levels of related apoptosis genes Bcl-2,Bak-1 and Caspase-3 were determined;in addition,apoptosis was observed by acridine orange（AO）/ethidium bromide（EB）staining.The results are as follows:1.The activity of ATPase(Ca²⁺-ATPase,Na⁺/K⁺-ATPase)in treated groups was significantly（P<0.01）lower than that in the control group.The activity of ATPase in combined treated groups was significantly（P<0.05 or P<0.01）lower than that in alone treated groups.2.The apoptotic rate,intracellular calcium ion level and cellular ROS level in treated groups were significantly higher（P<0.01）than those in control group,and the apoptosis rate,intracellular calcium level and cellular ROS leve in combined treated groups were significantly（P<0.05 or P<0.01）higher than those in alone treated groups.The intracellular pH and cell mitochondrial membrane potential in treated groups were significantly（P<0.01）lower than those in control group.The intracellular pH and mitochondrial membrane potential in combined treated groups were significantly（P<0.05or P<0.01）lower than those in alone treated groups.3.Compared with the control group,the expression of Bcl-2 mRNA in treated groups was significantly decreased（P<0.01）,and the expression of Bak-1 and Caspase-3 mRNA in treated groups were significantly increased（P<0.01）.The expression of Bcl-2 mRNA in combined treated groups was significantly lower than that in alone treated groups,and the mRNA expression of Bak-1 and Caspase-3 in combined treated groups were significantly higher（P<0.01）than those in alone treated groups.4.Acridine orange（AO）/ethidium bromide（EB）staining showed that apoptotic cells were present in treated groups,and the number of apoptosis in the combined treated groups were higher than those in alone treated groups.In conclusion,Mo and Cd exposure increased levels of reactive oxygen species and calcium ions in duck renal tubular epithelial cells,decreased ATPase activity,cell pH and mitochondrial membrane potential,down-regulated Bcl-2 expression,and up-regulated Bak-1 and Caspase-3 expression.Mo and Cd induced apoptosis of duck renal tubular epithelial cells through oxidative stress-mediated apoptosis pathway.Mo and Cd had dose-effect relationship on duck renal tubular epithelial cells,and it showed a synergistic effect between them.