Effects of PI3K / Akt signaling pathway on the autophagy of rat alveolar macrophages exposed to dust / 中国职业医学

ABSTRACT

OBJECTIVE: To determine the regulating role of phosphatidylinositol 3-kinase( PI3K) / protein kinase B( Akt)signaling pathway in the autophagy activity of rat NR8383 cells exposed to silicon dioxide( SiO_2). LY294002 was used to block PI3 K pathway. METHODS: i) The normal NR8383 cells were used and divided into blank group and silica exposure group( final concentrations of SiO_2 suspension were 0 and 50 mg / L respectively). They were cultured for 3,6,12,20 and24 hours. The enzyme linked immunosorbent assay( ELISA) was used to assess the amount of tumor necrosis factor-α( TNF-α) and transforming growth factor-β1( TGF-β1) in supernatants of cultured cells,and then the optimal time of cells exposed to dust was determined. ii) NR8383 cells were divided into control group( treated with a same volume of F-12 K medium without serum),silica group( treated with SiO_2 suspension,final concentration 50 mg / L) and intervention group( treated with SiO_2 suspension and PI3 K inhibitor LY294002,final concentration 50 mg / L and 20 μmol / L,respectively).Cells were harvested following incubation. ELISA was used to detect the levels of TNF-α and TGF-β1 at the time point of20 hours after incubation. To reveal the autophagy status of cells,Western blotting was used to detect Akt and microtubuleassociated proteins 1 light chain 3( LC3) protein at time point of 20 hours; laser scanning confocal microscope( LSCM)was used to observe the immunofluorescence expression of autophagy at time points of 3,6,12 and 20 hours. The cells were also treated with the lysosomal inhibitor chloroquine diphosphate( CDP) at the same time of SiO_2 treatment. RESULTS: i) The time point of 20 hours was confirmed to be the best dust exposure time for in vitro cell model of NR8383 cells.ii) The levels of TNF-α and TGF-β1 of supernatant in the silica group were higher than those of the control group( P <0. 05). The levels of TNF-α and TGF-β1 of supernatant in the intervention group were higher than those of the control group and silica group( P < 0. 05). The Akt protein expression of the intervention group was lower than those in the control group and the silica group,respectively. The LC3 Ⅱ / Ⅰ protein level of the silica group was higher than those of the control group and intervention group( P < 0. 05),but no statistical significance was found between the control group and intervention group( P > 0. 05). LSCM results indicated that autophagy expression at time points of 3 and 6 hours were stronger than those of 12 and 20 hours in control group; autophagy expression at time point of 12 hours was stronger than those of 3 and 6 hours in the silica group,while the autophagy expression at time point of 20 hours was slightly weaker than that of 12 hours,but still stronger than those of 3 and 6 hours. Compared with the same time point in control group,autophagy expression at 3 and 6 hours were weaker in the silica group,while the expressions increased obviously at time points of 12 and 20 hours. Autophagy expression at all time points decreased in the intervention group compared with silica group,especially at the time point of 20 hours. The autophagy expression in each group increased in varying degrees after added with CDP blocking. CONCLUSION: Silica dust exposure can induce autophagy in rat NR8383 cells. PI3 K inhibitor LY294002 can reduce the autophagy expression indicating that the PI3 K / Akt signaling pathway might participate in the autophagy process of silica dust inducing autophagy in alveolar macrophages.