Objective To observe the regulation of PI3KC3/Beclin1 on silica dust-induced autophagy in NR8383 cells.
Methods NR8383 cells were randomly divided into four groups:control group, silica dust group, 3-MA group, and 3-MA intervention group. The samples were collected at 1 h, 3 h, 6 h, 12 h, and 20 h after incubation. Western blot was used to detect microtube associated protein light chain 3 (LC3), autophagy substrate p62, as well as autophagy related proteins Beclin1 and PI3KC3. Laser scanning confocal microscopy was used to observe the green fluorescence emitted by autophagy activity after varied incubation durations. Co-immunoprecipitation was used to detect the expression of Beclin1 complex.
Results The results of immunofluorescence showed that the green fluorescence spot of the silica dust group was enhanced first and then decreased, the fluorescence intensity was highest at 6 h, and the fluorescence intensity of the silica dust group was elevated at all selected time points compared with the control group and the 3-MA intervention group. In the 3-MA intervention group, the LC3 fluorescent marker green spot was first enhanced and weakened, and was weaker than the silica dust group, but stronger than the control group. After adding chloroquine diphosphate (CDP), the LC3 green fluorescence signal of each group showed an increasing trend with time. Western blot results showed that the value of LC3-Ⅱ/LC3-Ⅰ in the silica group increased first and then decreased, and was higher than the control group at each time point (P < 0.05). The value ofLC3-Ⅱ/LC3-Ⅰ was highest at 6 h, when the value of the silica dust group was 248% of the control group and 372% of the 3-MA group. The value ofLC3-Ⅱ/LC3-Ⅰ in the 3-MA intervention group increased first and then decreased, and was lower than the silica dust group at each time point, but was higher than the control group (P < 0.05). At 6 h whenLC3-Ⅱ/LC3-Ⅰ value was highest, the value of the 3-MA intervention group was 90% of the silica dust group and 223% of the control group. The trend of Beclin1 and PI3KC3 protein expressions were consistent withLC3-Ⅱ/LC3-Ⅰ, and p62 protein expression was not. Co-immunoprecipitation results showed that the combination of Beclin1 and PI3K was increased in the silica dust group, and reduced after 3-MA intervention.
Conclusion Silica-induced autophagy activity in NR8383 cells changes over time, and can be down-regulated by 3-MA, suggesting that PI3KC3/Beclin1 signaling pathway participates in the autophagy of alveolar macrophages induced by silica dust.
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