Protective Effect And Mechanism Of ?-lipoic Acid On Autophagic Death Induced By Sodium Arsenite In Rat Insulinoma Cells

Objective:1.To investigate the role and molecular mechanism of autophagy in the death of rat insulinoma?INS-1?cells induced by sodium arsenite?NaAsO₂?;2.To investigate the effect of?-lipoic acid??-LA?on NaAsO₂-induced INS-1 The protective effect and molecular mechanism of autophagic death in cells.Methods:1.NaAsO₂-induced autophagic death of INS-1 cells:INS-1 cells were taken as the research object,negative control?Ctrl?group and NaAsO₂ groups with different concentrations?120,60,30,15,5?mol/L?were set up,?1?The proliferation inhibition rate of INS-1 cells after 24h of NaAsO₂ action on INS-1 cells was detected by CCK8 method.According to CCK8 results,they were divided into negative control group and NaAsO₂ group?30,15,5?mol/L?.?2?after NaAsO₂ acted on INS-1cells for 24h,the ultrastructure of INS-1 cells was detected by transmission electron microscope.?3?The expression of fluorescence in INS-1 cells transfected with plasmid GFP-LC3 was observed by Laser scanning confocal microscope.?4?The negative control group,NaAsO₂ group?30,15,5?mol/L?,autophagy inhibitor chloroquine group?CQ,10?mol/L?,CQ and NaAsO₂ combine group?CQ+NaAsO₂ 30?mol/L?.After INS-1 cells were treated for 24h,the autophagy-related proteins LC3?P62?Beclin-1?mTOR?PI3K and AKT were detected by Western-Blot method.2.Experimental study on the protective effect of?-LA on the autophagic death process of INS-1 cells caused by NaAsO₂:INS-1 cells were taken as the research object,and the autophagic injury model of INS-1 cells was established by selecting 30?mol/L NaAsO₂ concentration according to the results of Experiment 1.The experiment consisted of negative control group,Model group?NaAsO₂ 30?mol/L?and?-LA?200,100,50?mol/L?pretreatment group.INS-1 cells were pretreated with?-LA at different concentrations for 3h and then NaAsO2?30?mol/L?was given.NaAsO2group was treated with corresponding concentrations and cultured for 24h.?1?The proliferation inhibition rate of INS-1 cells was detected by CCK8 colorimetry.?2?Ultrastructure of INS-1 cells was observed by transmission electron microscope.?3?The expression of fluorescence in INS-1 cells transfected with plasmid GFP-LC3was observed by Laser scanning confocal microscope.?4?The expression of autophagy related proteins LC3?P62?Beclin-1?mTOR?PI3K and AKT were detected by Western-Blot.Results:1.NaAsO₂ induced autophagic death in INS-1cells:?1?NaAsO₂ can significantly inhibit the proliferation of INS-1 cells,and the IC50 of NaAsO₂ for 24h was 23.28?mol/L.?2?The double-layer membrane autophagosomes and autophagic vacuoles were observed in INS-1 cells of NaAsO₂group by transmission electron microscopy.?3?After transfection of plasmid GFP-LC3,the aggregation of green dot-like fluorescent spots in INS-1 cells was observed under the laser confocal microscopy of NaAsO₂ group.?4?Western-Blot results showed that compare with negative control group LC3-II/LC3-I protin?0.89±0.04?,P62 protin?0.50±0.04?,Beclin-1 protin?2.00±0.09?,mTOR protin?1.48±0.04?,PI3K protin?1.41±0.02?,AKT protin?1.38±0.04?,NaAsO₂ group?30,15,5?mol/L?LC3-II/LC3-I protein expression respectively?1.36±0.02,1.12±0.02,1.09±0.04?were increased,the expression of P62 protein respectively?2.78±0.03,2.71±0.06,1.14±0.11?were increased,the expression of Beclin-1 protein respectively?1.36±0.07,1.47±0.05,1.87±0.06,?were decreased,the expression of mTOR protein respectively?0.69±0.04,0.82±0.02,0.89±0.08?were decreased,the expression of PI3K protein respectively?0.61±0.04,0.70±0.03,0.89±0.05?were decreased,the expression of AKT protein respectively?0.94±0.01,1.43±0.06,1.43±0.04?were decreased,except NaAsO₂ 5?mol/L group LC3,Beclin-1 protein and15 and 5?mol/L group AKT protein expression was not statistically significant,the other differences were statistically significant?P<0.05?.Compared with the negative control group,treated with 10?mol/L CQ group,the expression of P62 protein?0.96±0.06?was increased,the difference was statistically significant?P<0.05?,the expression of LC3II/LC3I protein?1.03±0.07?),the expression of Beclin-1 protein?2.17±0.08?,mTORprotein?1.14±0.09?,PI3Kprotein?1.39±0.06?,AKT protein?1.36±0.04?,the difference There was no statistical difference.Compared with30?mol/L NaAsO₂ group,CQ and NaAsO₂ combine group,The expression of LC3-II/LC3-I protein?1.22±0.02?was decreased,the expression of P62 protein?3.00±0.02?was increased,the expression of Beclin-1 protein?1.74±0.01?was increased,the expression of mTOR protein?1.07±0.04?was increased,the expression of PI3K protein?1.08±0.06?was increased,the expression of AKT expression?1.16±0.05?was increased,the differences were statistically significant?P<0.05?.2.Protective effect of?-LA on NaAsO₂-induced autophagic death in INS-1 cells?1?After treatment with sodium arsenite.The CCK8 results showed that the cytotoxicity induced by sodium arsenite was reversed by?-LA,increasing the viability of sodium arsenite-treated INS-1 cells.?2?The results of transmission electron microscopy showed that?-LA pretreatment attenuated the increase in autophagosome accumulation induced by sodium arsenite.?3?After transfection of plasmid GFP-LC3,the green spot-like fluorescent spot aggregation was observed in the NaAsO₂ group by laser scanning confocal microscopy.There was no obvious fluorescence spot aggregation in each of the?-LA administration groups.?4?Western-Blot results showed that compare with negative control group LC3-II/LC3-I protein?2.10±0.10?,P62 protein?0.26±0.05?,Beclin-1 protein?1.35±0.05?,mTOR protein?1.12±0.07?,PI3K protein?0.88±0.05?,AKT protein?0.97±0.05?,NaAsO₂ 30?mol/L group the expression of LC3-II/LC3-I protein?3.94±0.14?was increased,the expression of P62 protein?0.97±0.03?was increased,the expression of Beclin-1protein?1.02±0.02?was decreased,the expression of mTOR protein?0.77±0.03?was decreased,the expression of PI3K protein?0.54±0.02?was decreased,the expression of AKT protein?0.68±0.01?was decreased,the difference was statistically significant?P<0.05?;compared with model group?NaAsO₂ 30?mol/L?,?-LA 200,100,50?mol/L pretreatment group LC3-II/LC3-I protein respectively?1.77±0.12,2.65±0.08,2.63±0.15?were decreased,the expression of P62 protein respectively?0.64±0.03,0.61±0.03,0.68±0.02?were decreased,the expression of Beclin-1 protein respectively?1.25±0.04,1.26±0.04,1.08±0.02?were increased,the expression of mTOR protein respectively?1.15±0.08,1.39±0.05,0.90±0.01?were increased,the expression of PI3K protein respectively?0.89±0.07,0.79±0.05,0.59±0.03?were increased,The expression of AKT protein respectively?1.88±0.05,1.16±0.03,0.79±0.01?were increased,except that the expression of Beclin-1 protein in the 50?mol/L?-LA pretreatment group was not statistically significant,the other differences were statistically significant?P<0.05?.Conclusion:1.NaAsO₂ can induce autophagic death in INS-1 cells,and its mechanism may be related to the regulation of PI3K/AKT-mTOR signaling pathway.2.?-LA may play a protective role in NaAsO₂-induced autophagic death of INS-1cells by mediating the PI3K/AKT-mTOR signaling pathway.