Effects Of Inhibiting Autophagy On Apoptosis Of Duck Renal Tubular Epithelial Cell Induced By Cadmium

In order to investigate the effect of Cd exposure on the autophagy and the relationship between autophagy inhibition and cadmium-induced apoptosis of duck renal tubular epithelial cells.10-15-day-old ducklings were selected to establish an in vitro culture model of duck renal tubular epithelial cells by tissue digestion.Cadmium sulfate（3Cd SO₄·8H₂O）was used as Cd source.The experiment was divided into six groups.They are control group（Control group）,1.25μmol/L Cd group（2%serum medium plus 1.25μmol/L Cd）,2.5μmol/L Cd group（2%serum medium plus 2.5μmol/L Cd）,5μmol/L Cd group（2%serum medium plus 5μmol/L Cd）,3-MA group（2%serum medium plus 2.5μmol/L 3-MA）,Cd+3-MA group（2%serum medium supplemented with 5μmol/L Cd+2.5μmol/L 3-MA）,After 12 hours of exposure,detected the Cd contents inside and outside the cell,acidic autophagy vesicle,autophagosome and LC3 accumulation point were observed,the expression level of autophagy-related genes（LC3A,LC3B,Beclin-1,m TOR,p62,Atg5,Dynein）and apoptosis-related genes（Caspase-3,Cyt C,Bak-1,Bax,Bcl-2,Bcl-2/Bax）,autophagy-related protein（LC3II/LC3I,Beclin-1,P62）and apoptosis-related protein（Caspase-3,cleaved Caspase-3）were measured,cell morphology was observed.Apoptosis and mitochondrial membrane potential（MMP）were measured.The results are as follows:1.Compared to the control group,the concentration of Cd ions in the cells and the cell supernatant increased significantly in a concentration-dependent manner in the Cd treatment group of different concentrations（P<0.001）.2.Monodansylcadaverine（MDC）staining showed that acidic autophagy vesicles increased with the Cd concentration in the treatment group,and flow cytometry was consistent with staining observation;electron microscopic observation showed that autophagosome and autophagy lysosomes increased with the Cd concentration in the treatment group;LC3 immunofluorescence staining showed that LC3 immunofluorescence spots increased with the Cd concentration in the treatment group;the level of autophagy in5μmol/L Cd group was more significant than that in the other two groups;3.Compared to the control group,the m RNA expression levels of LC3A,LC3B,ATG5and Beclin-1 were significantly increased in each exposed group（P<0.05 or P<0.01）,the m RNA expression levels of m TOR,p62 and Dynein were significantly decreased（P<0.05or P<0.01）;the protein expression levels of Beclin-1 and LC3II/LC3I in each Cd-exposed group significantly increased（P<0.01）;the protein expression level of p62was significantly decrease（P<0.01）;the expression of genes and proteins in 5μmol/L Cd group was more significant than that in the other two groups;4.Compared to 5μmol/L Cd group,the protein expression levels of Beclin-1 and LC3II/LC3I were significantly reduced（P<0.05）;the protein expression level of p62 was extremely significantly increased（P<0.001）in the Cd+3-MA group;5.Compared to 5μmol/L Cd group,the cell injury was more serious,the cell apoptosis rate was significantly increased（P<0.05）;mitochondrial membrane potential was significantly reduced（P<0.01）in the Cd+3-MA group;6.Compared to 5μmol/L Cd group,the m RNA expression levels of Caspase-3,Cyt C,Bak-1 and Bax were significantly or extremely significantly increased（P<0.05 or P<0.001）;The m RNA expression levels of Bcl-2 and Bcl-2/Bax are extremely significantly decreased（P<0.001）in the Cd+3-MA group;7.Compared to the 5μmol/L Cd group,the protein expression of Caspase-3 and cleaved Caspase-3 increased（P<0.05）in the Cd+3-MA group;Conclusion:Cd could induce autophagy in duck renal tubular epithelial cells,inhibiting autophagy aggravates the apoptosis by Cd through mitochondrial-mediated pathways.