Ultrafine silicon dioxide nanoparticles cause lung epithelial cells apoptosis via oxidative stress-activated PI3K/Akt-mediated mitochondria- and endoplasmic reticulum stress-dependent signaling pathways

Silicon dioxide nanoparticles (SiO2NPs) are widely applied in industry, chemical, and cosmetics. SiO2NPs is known to induce pulmonary toxicity. In this study, we investigated the molecular mechanisms of SiO2NPs on pulmonary toxicity using a lung alveolar epithelial cell (L2) model. SiO2NPs, which primary particle size was 12 nm, caused the accumulation of intracellular Si, the decrease in cell viability, and the decrease in mRNAs expression of surfactant, including surfactant protein (SP)-A, SP-B, SP-C, and SP-D. SiO2NPs induced the L2 cell apoptosis. The increases in annexin V fluorescence, caspase-3 activity, and protein expression of cleaved-poly (ADP-ribose) polymerase (PARP), cleaved-caspase-9, and cleaved-caspase-7 were observed. The SiO2NPs induced caspase-3 activity was reversed by pretreatment of caspase-3 inhibitor Z-DEVD-FMK. SiO2NPs exposure increased reactive oxygen species (ROS) production, decreased mitochondrial transmembrane potential, and decreased protein and mRNA expression of Bcl-2 in L2 cells. SiO2NPs increased protein expression of cytosolic cytochrome c and Bax, and mRNAs expression of Bid, Bak, and Bax. SiO2NPs could induce the endoplasmic reticulum (ER) stress-related signals, including the increase in CHOP, XBP-1, and phospho-eIF2α protein expressions, and the decrease in pro-caspase-12 protein expression. SiO2NPs increased phosphoinositide 3-kinase (PI3K) activity and AKT phosphorylation. Both ROS inhibitor N-acetyl-l-cysteine (NAC) and PI3K inhibitor LY294002 reversed SiO2NPs-induced signals described above. However, the LY294002 could not inhibit SiO2NPs-induced ROS generation. These findings demonstrated first time that SiO2NPs induced L2 cell apoptosis through ROS-regulated PI3K/AKT signaling and its downstream mitochondria- and ER stress-dependent signaling pathways.


Animals.
To establish the SiO2NPs exposured mouse model, four-week-old C57BL/6 male mice was obtained from BioLASCO Taiwan Co., Ltd (Taipei, Taiwan). All protocols used were approved by the Institutional Animal Care and Use Committee (IACUC), and the care and use of laboratory animals conducted in accordance with the guidelines of the Animal Research Committee of China Medical University, Taiwan. Mice was housed in a room at a constant temperature of 22 ± 2°C with a 12 h light-dark cycle. The mice were randomly assigned to pretreatment groups, weighed, and administered with the indicated drugs or vehicle.
The mice were randomly distributed into four groups: (a) vehicle control, (b) SiO2NPs 6.2 mg/kg, (c) SiO2NPs 12.5 mg/kg, (b) SiO2NPs 31 mg/kg. Mice placed under deep anesthetized by intraperitoneal injection of zoletil 50 (40 mg/kg). The SiO2NPs was diluted by PBS, and 50 µL of mixture exposed to mice via intranasal route. The mortality rate of mice was observed after treated with SiO2NPs for 1 to 8 days.
Wet -to -dry weight ratio. At day 8 of the experiment completion, all lungs were dissected free of nonpulmonary tissue and weighed and then dried to a constant weight at 60°C.
Wet-to-dry (W/D) ratios will be obtained by dividing the wet weight by the final dried weight 53 .
Lipid peroxidation assay. Lung tissues were harvested from mice under zoletil 50 anesthesia (40 mg/kg i.p.). All samples were homogenized and centrifuged at 3000 g for 10 min at 4°C. The cell lysate were homogenized and centrifuged at 1000 rpm for 20 min at 4°C. 2 Collecting the supernatant and assays were carried out immediately using the lipid peroxidation assay kit (Calbiochem). Absorbance at 586 nm for malondialdehyde(MDA) and 405 nm for MPO was measured using an ELISA microplate reader 54 .

Histological evaluation.The lung tissue was graded by Animal Disease Diagnostic
Center College of Veterinary Medicine, National Chung Hsing University, Taiwan. The alteration of edema, hemorrhage, leukocyte infiltrate and necrosis in lung tissue was observed by hematoxylin and eosin (H&E) staining. Histological changes will be scored by counting the frequency of foci per field observed at 40X, using a 0-to 4-point scale with injury in 0, 25, 50, 75 or 100% of the investigated tissue. The scoring scale is as follows: 0 (absent), 1 (mild), 2 (moderate), 3 (severe) and 4 (overwhelming). The wet -to -dry weight ratio was determined after SiO2NPs treatment at day 8. (C) The alteration of MDA levels were determined after SiO2NPs treatment at day 8. All data are presented as the means ± S.D.; n = 16 for all groups. *p < 0.05 as compared to vehicle control. treatment. C57BL/6 male mice were instilled via intranasal route with PBS, and 6.2 mg/kg, 12.5 mg/kg, 31 mg/kg of SiO2NPs. The lung tissues were collected at day 8 and the mRNA 5 expression of caspase-3 (A), caspase-7 (B), and caspase-9 (C) was determined by quantitative real-time polymerase chain reaction (qPCR) analysis. All data are presented as the means ± S.D.; n = 16 for all groups. *P < 0.05 as compared to the vehicle control group. Con: control. Figure 6. The mRNA expression of lung tissues in mice after SiO2NPs treatment. C57BL/6 male mice were instilled via intranasal route with PBS, and 6.2 mg/kg, 12.5 mg/kg, 31 mg/kg of SiO2NPs. The lung tissues were collected at day 8 and the mRNA expression of CHOP (A), XBP-1 (B), Grp78 (C), Grp94 (D), and capase-12 (E) was determined by quantitative real-time polymerase chain reaction (qPCR) analysis. All data are presented as the means ± S.D.; n = 16 for all groups. *P < 0.05 as compared to the vehicle control group.