Study Of Arsenic Induced Autophagosomes Accumulation Regulatory Mechanism In MLTC-1 Cells

[Objective] Arsenic is a common environmental contaminant,arsenic pollution exists in many countries.The effect of arsenic on the male reproductive system has become into a consensus.In order to the reproductive toxicity mechanism of arsenic,in this study,we use mouse Leydig cells line?MLTC-1?as the research object to establish a model of arseniasis in vitro.We detected the effect of arsenic on autophagy related genes and proteins in MLTC-1,and further to detect the regulatory factors regulation of autophagosomes formation on arsenic exposure.We hope we can provide a theoretical basis for further reveal the reproductive toxicity mechanism of arsenic through autophagy,and provide ideas for possible mitigation and treatment of arsenic toxicity.[Method] The mouse Leydig cells line?MLTC-1?was preserved by our lab.Cells were under recovered,cultured and passaged,and chose the cells in good condition then treated on different concentrations of As₂O₃.First of all,cell proliferation rate was detected by MTT to determine the test concentrations of As₂O₃,the test concentrations were 0,3,6,9 ?M.Arsenic treated 24 h later,autophagosomes were detected by Lyso-Tracker Red and MDC;and extracted total RNA and total protein,q PCR and Western Blot to detected the autophagy related genes and proteins Beclin1,LC3,Atg7 and p62 expression level.Subsequently,m TOR and Vps34 was inhibited by rapamycin and 3-MA respectively,and Beclin1 was silence by si RNA,after inhibited by these three autophagy regulators followed 9?M arsenic treated,then detected the autophagy related proteins LC3,Atg7 and p62 expression level.[Results] 1.Lyso-Tracker Red and MDC staining shows,after different concentrations?0,3,6,9 ?M?of As₂O₃ treated 24 h,the higher concentration of As₂O₃ the stronger of red and green fluorescence intensity.This result showed that with increase of As₂O₃ concentration treated,autophagosomes accumulation.2.The autophagy related genes mRNA expression levels were detected by q PCR: compared with the control group,LC3 mRNA expression level was increased significantly in 9 ?M As₂O₃ treated group?P < 0.001?,Beclin1 and Atg7 mRNA expression level was increased significantly in 9 ?M As₂O₃ treated group?P < 0.05?,p62 mRNA expression level was increased significantly in 6?M As₂O₃ treated group?P < 0.05?.3.Western Blot was used to detected the autophagy related proteins expression level: compared with the control group,the ratio of LC3-II/LC3-I was increased significantly in 9 ?M As₂O₃ treated 24h?P < 0.05?,and Beclin1,Atg7 and p62 expression levels were significant increased in 9 ?M As₂O₃ treated 24h?P < 0.05,P < 0.01,P < 0.01?.4.Before As₂O₃ treated cells,20nM rapamycin?m TOR inhibitor?was treated for 1h,as results displayed,m TOR inhibition was up-regulated significantly Atg7 expression level?P < 0.05?,and down-regulated significantly p62 expression level?P < 0.001?;m TOR inhibitor pre-treated then As₂O₃ treated was unchanged the ratio of LC3-II/LC3-I and the expression level of p62.5.RNA interference was used to silence Beclin1.Beclin1 expression level was decreased when si RNA transfection group?P < 0.05?,and ratio of LC3-II/LC3-I was also down-regulated?P < 0.05?,and also p62 expression level was up-regulated?P < 0.05?;after Beclin1 was silence,Beclin1 and p62 expression levels and radio of LC3-II/LC3-I were not significant change when followed arsenic treatment.6.Vps34 mRNA expression level was significant up-regulated in 9 ?M As₂O₃ treated group?P < 0.05?,immunofluorescence results showed that Vps34 protein expression was up-regulated under arsenic exposure.3-MA,the inhibitor of Vps34,was significant decreased the ratio of LC3-II/LC3-I?P < 0.05?and significant increased p62 expression level?P < 0.001?;Vps34 inhibition?5m M 3-MA pre-treated for 1h?then 9 ?M As₂O₃ treated for 24 h,the ratio of LC3-II/LC3-I was increased significantly compared with Vps34 inhibition group?P < 0.05?,but lower than 9 ?M As₂O₃ treated group?P < 0.05?,for the p62 expression level was not change compared with Vps34 inhibition group but higher than 9 ?M As₂O₃ treated group?P < 0.05?.7.The m TOR pathway and the Beclin1-Vps34 complex autophagy regulator genes mRNA expression levels were detected by q PCR.m TOR and Atg13 mRNA expression level were not change in the control and arsenic treatment group,ULK1 mRNA expression level was significant increased in 9 ?M As₂O₃ treated group?P < 0.05?;arsenic exposure was no effect on Vps15 mRNA expression level,Atg14 and UVRAG mRNA expression was increased significantly in 9 ?M As₂O₃ treated group?P < 0.05,P < 0.01?,Rubicon mRNA expression level was no significant changed compare with control group.[Conclusion] In this study,arsenic exposure can up-regulated autophagy related genes and proteins,resulting the increase of autophagosomes in MLTC-1 cells.The m TOR pathway and the Beclin1-Vps34 complex play a regulatory role.Arsenic induced accumulation of autophagosomes,on the one hand,it's due to arsenic promote autophagosomes formation,on the other hand,is block autophagosomes degradation.It's perhaps an embodiment of arsenic reproductive toxicity.