Effect Of As₂O₃ On Autophagy And Apoptosis Of AML-12 Cells

[Background] Arsenic（As）is a naturally existing metalloid and a toxic element,and it has two different forms,organic and inorganic.Arsenic trioxide（As₂O₃）is one of inorganic arsenic with high toxicity.It has been reported that As₂O₃ mainly cause methylate in the liver after entering the body,and also induces liver cell apoptosis.Both apoptosis and autophagy are forms of programmed cell death with complex molecular regulations.In this experiment,we chose normal mouse hepatocytes（AML-12）,and established the As₂O₃ poisoning model.Expressions of autophagy and apoptosis-related genes and proteins were studied when inhibiting or inducing AML-12 cell autophagy.[Methods] AML-12 cells were incubated with different concentrations of As₂O₃ for different time,and the cell viability was evaluated by CCK-8 method.The expressions of p EGFP-C3-LC3 B and 7-AAD in AML-12 cells treated with As₂O₃ for 24 h was detected by fluorescence microscopy.The expressions of autophagy and apoptosis related genes and proteins were detected by q RT-PCR and Western blot.Then 8μmol/L As₂O₃ concentration was selected for further experiments.Autophagy was changed by adding autophagy inhibitor（3-MA）and autophagy inducer（Rapamycin）,and then the expression of autophagy and apoptosis were detected by plasmid transfected,7-AAD stain,q RT-PCR and Western blot.[Results]1.After treatment with 0,2,4,6,8,10,12 μmol/L As₂O₃ on AML-12 cells for 24 h or 48 h,using CCK-8 we found with the increase of As₂O₃,cell viability of AML-12 cells were significantly reduced（p<0.01,p<0.05）,and the inhibitory effect was in does and time-dependent way in a certain range.2.After treated with 0,4,6,8,10,12 μmol/L As₂O₃ on AML-12 cells for 24 h,with the increase of As₂O₃ concentration,the positive cells transfected with p EGFP-C3-LC3 B plasmid gradually increased.After 7-AAD staining,the number of nuclei labeled with red fluorescence increased with the increase of As₂O₃ concentration.3.After treatment with 0,4,6,8μmol/L As₂O₃ on AML-12 cells for 24 h,As₂O₃ can significantly increase the autophagy of AML-12 cells detect by q RT-PCR and western blot.We found that the LC3 m RNA（p < 0.05）and LC3-II/LC3-I（p < 0.05）protein level were increased.At the same time,we observed apoptosis increased,represented by increased expression of caspase3 m RNA（p < 0.01）and cleaved-caspase3（17k Da）protein（p < 0.01）.Exposure to As₂O₃ significantly altered the m RNA expression levels of both autophagy（m TOR,PI3 K and P62）and apoptosis（Bcl-2）;and the protein expression levels of autophagy（P62）and apoptosis markers（Bcl-2）.4.The AML-12 cells were treated with 0,8 μmol/L As₂O₃ for 24 h.The occurrence of autophagosomes was observed in 8 μmol/L As₂O₃-treated AML-12 cells.We found nuclear fragmentation in As₂O₃ treated cells indicating that As₂O₃ induced apoptosis in AML-12 cells.5.Autophagy was inhibited by the autophagy inhibitor 3-methyladenine（3-MA）,3-MA could significantly inhibit the autophagy and apoptosis levels induced by As₂O₃.After treatment with 3-MA,the positive cells transfected with plasmid was decreased.LC3 m RNA（p<0.01）,LC3-II/LC3-I protein expression were decreased.At the same time,7-AAD marked cells,caspase3 m RNA（p<0.01）and cleaved-caspase3（17k Da）protein（p<0.05）expression were decreased,the apoptosis level was decreased.The expression of Bcl-2 m RNA was increased and the P62 was decreased（p<0.05）.6.The autophagy level induced by As₂O₃ was enhanced by Rapamycin,and the apoptosis level was decreased at the same time.We observed that treatment with Rapamycin increased the number of autophagic positive cells compared with the As₂O₃ alone treated group,and the expression of LC3 m RNA（p<0.05）was increased,caspase3 m RNA（p<0.05）and cleaved-caspase3（17k Da）（p<0.01）protein expression were reduced.At the same time,Bcl-2 m RNA（p<0.05）was increased,but the P62 m RNA（p<0.05）expression was decreased.[Conclusion] Our study demonstrated that As₂O₃ up-regulated the levels of autophagy and apoptosis.We also found that both 3-MA and Rapamycin reduced the apoptosis induced by As₂O₃ in AML-12 cells.This ample evidence of apoptosis regulation by autophagy can be a novel therapeutic intervention against As₂O₃ induced hepatotoxicity.