The Effect Of Beclin1 Complex On The Autophagy Of NR8383 Induced By The Silica

Objectives In order to investigate the effect of Beclin1/PI3KC3,Beclin1/Bcl-2 complex on autophagy of NR8383 induced by the silic,PI3KC3 inhibitor 3-methyladenine(3-MA)and Bcl-2 inhibitor ABT-737 were used to observe the changes on autophagy activity of NR8383 cells.Methods CCK-8 was used to access the concentration of 3-MA and ABT-737.Then the cells were randomly divided into six groups: control group,silica group,3-MA group,3-MA intervention group(3-MA+silica dust),ABT-737 group and ABT-737 intervention group(ABT-737+ silica dust),and the drugs were used 1 hour in advance,and then added 50μg/ml silica dust to each group,incubating the cells for 1h,3h,6h,12 h and 20 h.The cells were collected.Wright-Giemsa staining was used to observe the morphology of NR8383 cells.IF was used to observe the LC3 B green fluorescence of NR8383 cells.LC3 B,p62,Beclin1,PI3KC3 and Bcl-2 were determined by Western blot.IP was used to observe the expression of Beclin1/PI3KC3,Beclin1/Bcl-2 complex.Results 1 CCK-8 was used to access the concentration of 3-MA and ABT-737.The cell survival rate at low concentrations(0,0.5,1,2,5 m M)was not statistically significant(P>0.05),and the cell survival rate at 10 m M was significant(P<0.05).After pre-experient,the concentration of 3-MA was 5m M.After treated with different concentrations of ABT-737,the cell survival rate was not statistically significant with low concentration(0,0.5,1,2.5μM)(P<0.05).When the concentration was 5μM or 10μM,the cell viability decreased(P<0.05),and the concentration of ABT-737 was determined to be 2.5μM.2 WrightGiemsa staining: the cytoplasm and nuclei of NR8383 cells were clear in control group.The cytoplasmand nuclei in silica group were darker than control group,and the cytoplasm contained similar dust-like particles.3 Immunofluorescence observation: Silica group,3-MA intervention group and ABT-737 intervention group with the time prolonged,LC3 B green fluorescent strength first enhanced and then weakened,and at the time point the fluorescence strength of LC3 B in silica group was higher than that of 3-MA intervention group,and weaker than that of ABT-737 intervention group.After addition of CDP,the degradation of autophagosome was blocked,and the LC3 B green fluorescence strength of each group was enhanced.4 The expression of the autophagy-related proteins after 3-MA intervention: There were statistical differences between different treatment groups(P<0.05),and the expression of LC3 B and Beclin1 in the silica group was higher than that in the control group and the 3-MA intervention group.The expression of p62 was lower than the control group and 3-MA intervention group,and the expression of PI3KC3 was higher than the control group and 3-MA intervention group.There were statistical differences between different time after treatment(P<0.05),and the expression of LC3 and Beclin1 at 6h was higher than that of the other four time points,and p62 expression at 6h is lower than the other four time points.The interaction of all combinations was significant(P<0.05).The results of IP was shown that Beclin1 protein was detected in PI3KC3 precipitated protein,and the level of Beclin1 was decreased after intervented by 3-MA.5 The expression of the autophagy-related proteins after ABT-737 intervention: There were statistical differences between different treatment groups(P<0.05),and the expression of LC3 B and Beclin1 in the silica group was lower than that in the ABT-737 intervention group.The expression of p62 was higher than the ABT-737 intervention group.The expression of Bcl-2 in silicon group was higher than that of ABT-737 intervention group.There were statistical differences between different time after treatment(P<0.05),and the expression of LC3 and Beclin1 at 6h was higher than that of the other four time points,and p62 expression at 6h is lower than the other four time points.The interaction of all combinations was significant(P<0.05).The results of IP was shown that Bcl-2 protein was detected in Beclin1 precipitated protein,and the level of Bcl-2 was decreased after intervented by ABT-737.Conclusions 1 autophagy induced by by silica dust increased in NR8383 cells.2 PI3KC3 inhibitor 3-MA inhibits the autophagy activity of NR8383 cells by decreasing the PI3KC3 activity and inhibiting the Beclin1/PI3KC3 complex,suggesting that the Beclin1/PI3KC3 complex may play a positive role in the autophagy of the NR8383 cells.3 Bcl-2 inhibitor ABT-737 dissociates Beclin1-Bcl-2 complex by competitive binding of Bcl-2,reducing the affinity of Bcl-2 to Beclin1,activating Beclin1,and enhances the autophagic activity of NR8383 cells,suggesting that the Beclin1/Bcl-2 complex may play a negative role in the autophagy of NR8383 cells.