The Role Of Autophagy In Molybdenum-induced Injury Of Duck Renal Tubular Epithelial Cells

In order to explore the effect of Mo exposure on the relationship between autophagy and cell injury 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 enzyme digestion and sieve filtration.Ammonium molybdate(（NH₄）₆Mo₇O₂₄·4H₂O)was used as Mo source.The experiment was divided into six groups.They are control group（Control group）,480μmol/L Mo group（2%serum medium plus 480μmol/L Mo）,720μmol/L Mo group（2%serum medium plus 720μmol/L Cd）,960μmol/L Mo group（2%serum medium plus 960μmol/L Mo）,3-MA group（2%serum medium plus 2.5μmol/L 3-MA）,Mo+3-MA group（2%serum medium supplemented with 720μmol/L Mo+2.5μmol/L 3-MA）,After 12 hours of exposure,the cell morphology,acidic autophagy vesicle,autophagosome and LC3 accumulation point were observed,the expression level of autophagy-related genes and proteins,cell survival rate,LDH release level,intracellular SOD,CAT and GSH-Px activity,MDA and H₂O₂content,reactive oxygen species（ROS）level,mitochondrial membrane potential（MMP）,mitochondrial mass,mitochondrial RCR and OPR were measured.The results are as follows:1.Monodansylcadaverine（MDC）staining showed that acidic autophagy vesicles increased in all molybdenum exposed groups,and flow cytometry was consistent with staining observation;electron microscopic observation showed that autophagosome and autophagy lysosomes increased in all Mo exposed groups;LC3immunofluorescence staining showed that LC3 immunofluorescence spots increased in each Mo exposed group;the level of autophagy in 720μmol/L Mo group was more significant than that in the other two groups;2.Compared to the control group,the m RNA expression levels of LC3A,LC3B,ATG5 and 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<0.05 or P<0.01）;the protein expression levels of Beclin-1 and LC3II/LC3I in each Mo-exposed group significantly increased（P<0.01）;the expression of genes and proteins in 720μmol/L Mo group was more significant than that in the other two groups;3.Compared to 720μmol/L Mo group,the protein expression levels of Beclin-1and LC3II/LC3I in Mo+3-MA group was significantly decreased（P<0.01）;4.Compared to 720μmol/L Mo group,the cell injury was more serious,the cell survival rate was significantly decreased（P<0.01）and the level of LDH in culture medium was significantly increased（P<0.01）in Mo+3-MA group;5.Compared to 720μmol/L Mo group,the level of ROS,the content of H₂O₂and MDA in the culture medium of Mo+3-MA group increased significantly（P<0.01）,the activity of SOD,CAT and GSH-Px were decreased significantly（P<0.01）;6.Compared to 720μmol/L Mo group,the mitochondrial membrane potential（MMP）,mitochondrial mass,mitochondrial RCR and OPR in the Mo+3-MA group decreased significantly（P<0.01）.Conclusion:Mo could improve the autophagy level of duck renal tubular epithelial cells,and inhibition of autophagy can aggravate mitochondrial dysfunction induced by molybdenum by regulating oxidative stress.