Effect Of PI3K/mTOR On Expression Of Autophagy In NR8383 Cells Exposed To Silica Dust

Objectives In order to investigate the effect of the PI3 K / Akt / m TOR signaling pathway in rat NR8383 cells esposed to silica dust and regulating effect of antioxidant NAC whether through the pathway to influence the occurrence and development of silicosis, we use PI3 K inhibitor LY294002, m TOR inhibitor rapamycin(RAPA) and NAC, and then observe the changes of autophagy activity of the cells.Methods ELISA was used to access the amounts of TNF-α、TGF-βⅠin supernatants of cultured cells that exposed to dust 3h、6h、12h、20h,24 h, and then determined the optimal time of cells exposed to dust. At the determined optimal time we used LY294002、RAPA and NAC, ELISA method was used to detect production of TNF-α、TGF-βⅠin cell culture supernatants, and the analysis of ANOVA or Kruskal-Wallis H method. The levels of Akt and p-m TOR protein were determined by Western blot; Immunofluorescence assay and Western Blotting were used to observe and determine the occurrence of autophagy, and the analysis use ANOVA or Kruskal-Wallis H method.Results 1 NR8383 cells exposed to dust 3h, 6h, 12 h, 20 h, 24 h, and ELISA method to detect the expression of TNF-α、TGF-βⅠ.The expression of TGF-βⅠ in 20 h was higher than 6h, 12h(P<0.05), 24 h and 20 h expression was not significant. The expression of TNF-α in the supernatant of the cells was higher than that in 12 h, 6h(P<0.05), 20 h was higher than that of 3H, 6h, 12h(P<0.05), 24 h and 20 h there was no difference(P>0.05). 2 Effect of different intervention on the expression of TNF-α and TGF-βⅠ in supernatant of NR8383 cells. The expression levels of TNF-α、TGF-βⅠ in LY294002 silica dust group were higher than those in the silica dust group, the difference was statistically significant(P<0.05); The secretion level of TNF-α in RAPA+silica group was lower than that of silica dust group(P<0.05) while the secretion level of TGF-βⅠ was increased in the RAPA+silica group(P<0.05). The content of TNF-α in NR8383 cell supernatant of NAC+silica group was lower than that of silica group, the difference was statistically significant(P<0.05). 3 Laser confocal microscopy observed NR8383 cells autophagy activity. Compared with the control group, the LC3 protein of NR8383 cells in the silica group was enhanced; Compared with the silica group, the fluorescence signal of LY294002+silica group was obviously weak; Compared with the silica group, fluorescence signal was weaker in RAPA+silica group. There was no significant change in the fluorescence signal of NAC+silica group compared with the silica dust group. Groups add lysosomal inhibitor CDP LC3 level higher than without CDP. 4 Western blotting detected expression of Akt and p-m TOR protein. Akt protein expression of LY294002+silica group weaker than that the control group and silica group. P-m TOR in expression of Rapa+silica group weaker than that in the control group and silica group. Pm TOR expression in NAC+silica group had no significant change compared with control group and silica group. 5 Western blotting detected autophagy activity in NR8383 cells. Compared with the control group, the expression of LC3 protein in silica group was increased, and the difference was statistically significant(P<0.05); The expression of LC3 protein in LY294002+silica group was lower than that in the silica group, and the difference was statistically significant(P<0.05); Compared with the silica group, the expression of LC3 in RAPA+silica group was decrease, but the difference was not statistically significant(P>0.05); Compared with the silica group, the difference of LC3expression in RAPA +silica group was not statistically significant(P>0.05).Conclusions 1 Autophagy increased in rat NR8383 cells induced by Si O2 dust. 2 PI3K/Akt signaling pathway is involved in the silica dust mediated alveolar macrophage autophagy activities. 3 There was no significant association between the m TOR signaling pathway and the autophagy activities in the NR8383 cells exposed to silica dust for 20 h. 4 Antioxidant NAC through m TOR signaling pathway effects autophagy of cells exposed to silica dust is weak and it has a certain inhibition effect on the inflammatory response mediated by dust alveolar macrophages.