[Mechanism of MDA5 in macrophages in SiO(2)-induced epithelial-mesenchymal transition]

Objective: To investigate the regulatory role and molecular mechanisms of macrophage-derived melanoma differentiation-associated protein 5 (MDA5) in SiO(2)-induced epithelial-mesenchymal transition (EMT) in lung epithelial cells. Methods: In March 2024, 16 male C57BL/6 mice were selected to establish a silicosis mouse model by intratracheal instillation of 50 μl of silica dust suspension (50 mg/ml). The control group and silica dust group each consisted of 8 mice. After successful establishment of the silicosis mouse model, lung tissues from 2 randomly selected mice in each group were analyzed by single-cell transcriptome sequencing, while lung tissues from the remaining 6 mice were used for subsequent experiments. Single-cell RNA sequencing (scRNA-seq) of mouse lung tissue was performed to characterize MDA5 expression profiles and pathway enrichment in macrophage subpopulations. THP-1 cells were treated with silica dust suspension at concentrations of 0, 50, 100, 150, 200, and 250 μg/ml, followed by detection of MDA5 expression via Western blot and quantitative reverse transcription polymerase chain reaction (qRT-PCR). THP-1 cells were first induced to differentiate into adherent macrophages using 1 μmol/L phorbol 12-myristate 13-acetate (PMA). Following differentiation, the cells were divided into four groups based on treatment conditions, each with three replicates, and all treatments lasted for 24 hours. The groups were as follows: control group, treated only with RPMI-1640 medium, without silica suspension or siRNA transfection; silica group, treated with 250 μg/ml silica suspension, without siRNA transfection; silica+siIFIH1 control group, first transfected with 20 nmol/L negative control siRNA (non-targeting sequence for IFIH1) using a cell transfection kit, and after 48 hours, co-treated with 250 μg/ml silica suspension; silica+siIFIH1 group, first transfected with 20 nmol/L IFIH1-specific siRNA (siIFIH1 #3, with optimal knockdown efficiency) using the transfection kit, and after 48 hours, co-treated with 250 μg/ml silica suspension. After the above THP-1 cell treatments were completed, the original culture medium was discarded, and the cells were washed with PBS. Then, serum-free RPMI-1640 medium was added to continue culturing the THP-1 cells for another 24 hours. Finally, the serum-free RPMI-1640 culture supernatants from the four THP-1 cell groups were collected and used to treat A549 cells for 24 hours. MDA5 knockdown in THP-1 cells was achieved using siRNA to evaluate changes in the expression of inflammatory factors including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), C-C motif chemokine ligand 2 (CCL2), and C-C motif chemokine ligand 3 (CCL3). A549 cells were treated with conditioned medium from THP-1 cells to examine the effect of MDA5 knockdown on the expression of EMT markers [E-cadherin, Vimentin, etc.] in A549 cells. For statistical analysis, comparisons between two groups were conducted using two-tailed unpaired t-tests, while comparisons among multiple groups were performed using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. Results: Lung tissue scRNA-seq revealed significant upregulation of MDA5 expression (P<0.05) and enhanced pathway enrichment (P<0.05) in macrophages from silica-exposed mice. In THP-1 cells, both protein and transcriptional levels of MDA5 demonstrated dose-dependent elevation (P<0.05) following SiO(2) exposure. Knocking down MDA5 substantially attenuated the expression of pro-inflammatory mediators (P<0.05) in THP-1 cells. Conditioned media derived from THP-1 cells treated with SiO(2) promoted EMT in A549 cells while knocking down MDA5 in THP-1 cells inhibited the above effects. Conclusion: SiO(2) exposure upregulates MDA5 expression in macrophages, triggering a pro-inflammatory cascade that induces EMT in lung epithelial cells, thereby driving the progression of silica-induced pulmonary fibrosis.