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To detect the effects of silica on the cell viability of RAW264.7 cells, the cells were incubated with silica (200 μg/mL) for 0, 6, 12, 24, and 48 h. After incubation, cell viability was evaluated using an MTT assay. The absorbance values derived from control or silica-exposed cells were compared. As shown in Table 1, silica decreased cell viability.
Time/OD Value (h) Control 200 μg/mL Silica Group 0 0.265 ± 0.041 0.265 ± 0.041 6 0.467 ± 0.050 0.275 ± 0.013* 12 0.594 ± 0.027 0.343 ± 0.032* 24 0.676 ± 0.016 0.431 ± 0.033* 48 0.865 ± 0.015 0.582 ± 0.029* Note.*: P < 0.05, compared with control. Table 1. Effect of Silica (200 μg/mL) on Cell Viability of RAW264.7 Cells
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To investigate the effect of silica on the apoptosis of RAW264.7 cells, we detected apoptotic cells by fluorescent microscopy after DAPI staining. Silica induced prominent nuclear changes in treated cells (Figure 1). The nuclei were round, intact, and uniformly stained in control cells. However, at 24 and 48 h, silica exposure induced nuclear condensation, resulting in smaller nuclei that displayed membrane blebbing and fragmentation as the cells died. Nuclear changes were not significant at 6 and 12 h (data not shown). Next, to quantify silica-induced apoptosis, we performed flow cytometry. As shown in Figure 2, apoptotic cells were increased time-dependently by silica exposure. Furthermore, the level of cleaved caspase-3, which induces apoptosis, was detected in silica-treated cells. The expression of caspase-3 increased at 24 h and peaked at 48 h after silica exposure (Figure 3).
Figure 1. Silica treatment induced nuclear changes in RAW264.7 cells. Cells were treated with silica (200 μg/mL) for 0 h (A), 24 h (B), and 48 h (C) (magnification × 400). Morphological changes of cells nuclei were observed by DAPI staining under a fluorescence microscope.
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We performed transmission electron microscopy to observe the endoplasmic reticulum of silica-induced RAW264.7 cells. ER expansion was observed at 6 h after silica treatment and the extent of ER expansion increased gradually in a time-dependent manner in silica-treated cells (Figure 4).
Figure 4. Silica treatment induced endoplasmic reticulum expansion of RAW264.7 cells. Cells were treated with silica (200 μg/mL) for 0 h (A), 6 h (B), 12 h (C), 24 h (D), and 48 h (E). Morphological changes of the endoplasmic reticulum were observed by transmission electron microscopy (magnification × 10, 000). White arrows show expanded endoplasmic reticulum.
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To determine the effect of silica on ERS, we examined the expression of ER stress markers GRP78/BiP and CHOP. As shown inFigure 5, silica caused a notable increase in the expression of Bip mRNA and protein. Maximum induction was observed at 6 h. Additionally, silica induced CHOP expression, which was increased in a time-dependent manner (Figure 5).
Figure 5. Silica treatment induced endoplasmic reticulum stress of RAW264.7 cells. Cells were treated with silica (200 μg/mL) for 0, 6, 12, 24, and 48 h. The expression of BiP and CHOP mRNA was determined by real-time PCR (A, C); *: P < 0.05, compared with 0 h group, #: P < 0.05, compared with 24 h group. The expression of BiP and CHOP proteins was determined by Western blotting (B, D).
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To identify the role of ERS in silica-induced apoptosis in RAW264.7 cells, we selected 4-PBA as an ERS inhibitor. First, we detected the effect of 4-PBA on endoplasmic reticulum expansion. The results showed that 4-PBA inhibited silica-induced endoplasmic reticulum expansion (Figure 6).
Figure 6. 4-PBA inhibited silica-induced endoplasmic reticulum expansion of RAW264.7 cells. Cells were pre-incubated with 20 μmol/L 4-PBA for 1 h and then stimulated with silica (200 μg/mL) for 48 h. Morphological changes of the endoplasmic reticulum were observed by transmission electron microscopy (magnification × 10, 000). White arrows show expanded endoplasmic reticulum. (A) without silica and 4-PBA group, (B) silica group, (C) silica and 4-PBA group, (D) 4-PBA group.
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Next, we examined the effect of 4-PBA on the expression of ER stress markers BiP and CHOP in silica-stimulated cells. The results showed that 4-PBA (1, 10, 20 μmol/L) inhibited silica-induced mRNA and protein expression of Bip (Figure 7). In addition, pretreatment with 4-PBA (10, 20 μmol/L) decreased the mRNA and protein expression of CHOP (Figure 7).
Figure 7. 4-PBA inhibited silica-induced ERS of RAW264.7 cells. Cells were pre-incubated with 1, 10, and 20 μmol/L 4-PBA for 1 h and then stimulated with silica (200 μg/mL) for the indicated times. Expression of Bip and CHOP mRNA was determined by real-time PCR (A, C); Expression of Bip and CHOP proteins was determined by Western blotting (B, D). *: P < 0.05, compared with silica-stimulated group.
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Next, we investigated the effect of 4-PBA on apoptosis induction by silica. DAPI staining showed that pretreatment with 4-PBA attenuated nuclear condensation in silica-stimulated cells (Figure 8). In addition, flow cytometry analysis demonstrated that 4-PBA prevented silica-induced apoptosis of RAW264.7 cells (Figure 9). The expression of cleaved caspase-3 was also decreased by 4-PBA in silica-treated cells (Figure 10).
Figure 8. 4-PBA attenuated nuclear condensation in silica-stimulated RAW264.7 cells. Cells were pre-incubated with or without 10 μmol/L 4-PBA for 1 h and then stimulated with or without silica (200 μg/mL) for 48 h. Morphological changes of cell nuclei were observed by DAPI staining under a fluorescence microscope (magnification × 400). (A) without silica and 4-PBA group, (B) silica group, (C) silica and 4-PBA group, (D) 4-PBA group.
Figure 9. 4-PBA inhibited silica-induced apoptosis of RAW264.7 cells. Cells were pre-incubated with or without 10 μmol/L 4-PBA for 1 h and then stimulated with or without silica (200 μg/mL) for 48 h. Apoptotic cells were observed by flow cytometry. (A) without silica and 4-PBA group, (B) silica group, (C) silica and 4-PBA group, (D) 4-PBA group, (E) all histograms show the apoptotic cell populations (%), *: P < 0.05, compared with silica-stimulated group.