ML385 Attenuates Malignant Progression of Silica-induced Lung Adenocarcinoma Cells through the ROS/NRF2-autophagy Axis Pathway

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Abstract
Silica exposure is associated with an increased risk of lung adenocarcinoma, but its molecular mechanism remains unclear. This study aims to establish a repeatable experimental protocol to explore how the nuclear factor erythroid 2-related factor 2 (NRF2) inhibitor ML385 inhibits silica-induced malignant progression of lung adenocarcinoma by regulating the ROS/NRF2-autophagy axis pathway. Firstly, a silicosis model was established by intranasal perfusion of silica suspension in C57BL/6 mice. The degree of pulmonary fibrosis was evaluated by Masson staining, the infiltration of immune cells was analyzed by immunofluorescence, and the expressions of NRF2, cyclin-dependent kinase 1 (CDK1), and voltage-dependent anion channel 1 (VDAC1) in lung tissue were detected by immunohistochemistry. Subsequently, in in vitro experiments, the RAW264.7 macrophage cell line was treated with silica. Autophagic flux and oxidative stress levels were evaluated by LC3-lysosome co-localization and ROS probes, and intervention was carried out with the NRF2 inhibitor ML385. Finally, the effects of ML38 on migration, invasion, and apoptosis of lung adenocarcinoma cells were analyzed by scratch assay, cells passing through pores of a specific size assay, and flow cytometry. The results showed that silica could induce pulmonary fibrosis, immune cell infiltration, and upregulation of NRF2, CDK1, and VDAC1 in mice (p < 0.001), and inhibit the autophagic flux of macrophages and reduce ROS levels. ML385 can reverse these effects (p < 0.05). This protocol provides a complete experimental process from in vivo model construction to in vitro mechanism research, offering an operational technical path for studying the molecular mechanism of silica-related lung adenocarcinoma and therapeutic strategies targeting NRF2.
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