RESUMO
Background Lipid metabolism imbalance is tightly linked to the development and progression of multiple diseases. The phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway is important for the regulation of lipid metabolism. However, whether silicosis is associated with lipid metabolic abnormalities has yet to be explored. Objective To observe the changes of lipid deposition, cholesterol, and phosphorylated proteins of PI3K/AKT/mTOR pathway in silicon dioxide (SiO2)-induced MLE-12 cells and to explore potential mechanism of lipid composition regulated though the pathway. Methods (1) MLE-12 cells were stimulated with 50 mg·L−1 SiO2 suspension, and divided into fourgroups: a control group and three SiO2 groups (12, 24, and 48 h of stimulation). (2) Cellproliferation was detected to determine an optimal dose of LY294002, an inhibitor of PI3K protein. LY294002 at 5 μmol·L−1 was used for further study, in which MLE-12 cells cultured for 48 h were divided into four groups: a control group; a 50 mg·L−1 SiO2 suspension stimulation group; a 50 mg·L−1 SiO2 suspension and 5 μmol·L−1 LY294002 treatment group; a 5 μmol·L−1 LY294002 treatment group. Total cholesterol (TC), free cholesterol (FC), cholesterol ester (CE; total cholesterol minus free cholesterol), and triglycerides (TG) were measured with enzyme assay kits. Lipid deposition was observed using Oil Red O staining. The expressions of p-PI3K, p-AKT, and p-mTOR proteins were detected by Western blotting. Results (1) The contents of TC, FC, and CE in the 50 mg·L−1 SiO2-induced MLE-12 cells were increased compared to those of the control group in a time-dependent manner by trend analysis, and the increment at 24 and 48 h were significant. By 48 h, the contents of cholesterol indicators were all elevated: TC from (2.242±0.181) mg·g−1 to (5.148±0.544) mg·g−1, FC from (1.923±0.158) mg·g−1 to (4.168±0.433) mg·g−1, and CE from (0.318±0.067) mg·g−1 to (0.978±0.134) mg·g−1, compared with the control group (P<0.01). The changes of TG were not significant (P>0.05). The SiO2 suspension induced orange-red particle deposition in the MLE-12 cells, especially at 48 h (P<0.01). The protein expression levels of p-PI3K, p-AKT, and p-mTOR in SiO2-stimulated MLE-12 cells were higher than those of the control groups with the prolongation of stimulation time, which peaked at 48 h (P<0.01). (2) The contents of TC, FC, and CE in MLE-12 cells of the SiO2 + LY294002 group were decreased, comparing to those of the SiO2 stimulation only group (P<0.01), companied with less orange-red lipid deposition, and suppressed protein expression levels of p-PI3K, p-AKT, and p-mTOR (P<0.01). Conclusion SiO2 could induce increases of cholesterol and lipid deposition through activation of PI3K/AKT/mTOR signaling pathway in MLE-12 cells.
RESUMO
microRNA (miR)-142-3p is implicated in malignancy and has been identified as a biomarker for aggressive and recurrent lung adenocarcinomas. This study aimed to evaluate the inhibitory effect of miR-142-3p on apoptosis and inflammation induced by bleomycin in MLE-12 cells. MLE-12 cells were first transfected either with miR-142-3p mimic or miR-142-3p inhibitor and then the cells were exposed to 50 μg/mL of bleomycin. Thereafter, cell viability, apoptosis and the expression of pro-inflammatory cytokines were assessed using CCK-8, flow cytometry, RT-PCR and western blot analyses. Cox-2, PI3K, AKT and mTOR expressions were detected by western blotting after bleomycin was administered together with NS-398 (an inhibitor of Cox-2). As a result, cell viability was significantly decreased, as well as apoptosis and the expression of IL-1 and TNF-α were remarkably increased after 50 and 100 μg/mL of bleomycin administration. miR-142-3p overexpression alleviated bleomycin-induced apoptosis and overproduction of these two pro-inflammatory cytokines, while miR-142-3p suppression exhibited completely opposite results. Up-regulation of Cox-2 and inactivation of PI3K/AKT/mTOR were found in bleomycin-pretreated cells, while these abnormal regulations were partially abolished by miR-142-3p overexpression and NS-398. In conclusion, this study demonstrated that miR-142-3p overexpression protected bleomycin-induced injury in lung epithelial MLE-12 cells, possibly via regulating Cox-2 expression and PI3K/AKT/mTOR signaling pathway. These findings provide evidence that miR-142-3p may be a therapeutic strategy for idiopathic pulmonary fibrosis (IPF) treatment.