Inorganic arsenic (As) exposure is known to induce pulmonary fibrosis, yet the underlying mechanisms remain incompletely understood. This study investigated the crosstalk between sequestosome 1 (SQSTM1/p62) and nuclear factor erythroid 2-related factor 2 (Nrf2) in regulating cellular senescence and epithelial-mesenchymal transition (EMT) in As-exposed alveolar type II (AT2) cells. RNA sequencing analysis initially suggested that As exposure promotes EMT and senescence in MLE-12 cells. Subsequent experiments demonstrated that As exposure progressively decreased the epithelial marker E-cadherin while increasing the mesenchymal marker N-cadherin in MLE-12 cells, a mouse AT2 cell line, and murine lung tissues. In addition, elevated expression of transforming growth factor (TGF)-β and α-smooth muscle actin (SMA) was observed. Meanwhile, As exposure increased the number of SA-β-galactosidase (SA-β-gal)-positive MLE-12 cells and enhanced the transcriptional activity of senescence-associated secretory phenotype (SASP)-related genes, including Cxcl1, Cxcl11, Cxcl16, Ccl20, Mcp-1, Tgf-β, and Gm-csf. Moreover, the expression of senescence-associated markers p16 and p21 was upregulated in both As-exposed MLE-12 cells and murine lungs. Notably, Nrf2 knockdown exacerbated cellular senescence, as evidenced by increased SA-β-gal-positive cells, enhanced SASP gene expression, and elevated p16 and p21 levels, along with augmented EMT marker expression. To further verify the protective role of Nrf2, As-exposed MLE-12 cells were treated with the Nrf2 activator dimethyl fumarate (DMF). DMF activates Nrf2 by modifying Keap1, thereby disrupting Keap1-mediated Nrf2 degradation and promoting Nrf2 stabilization and nuclear translocation. DMF markedly reversed As-induced senescence, as shown by decreased SASP-related gene, p16, and p21 expression, while attenuating EMT, evidenced by restored E-cadherin and reduced N-cadherin and α-SMA. Moreover, pharmacological disruption of the p62-Keap1 interaction using K67 restored Keap1-mediated suppression of Nrf2 and further aggravated senescence and EMT phenotypes. Collectively, these findings demonstrate that As exposure induces cellular senescence and EMT in MLE-12 cells and murine lungs. The p62-Keap1-Nrf2 positive feedback loop functions as an essential cytoprotective axis that attenuates As-evoked senescence and EMT in AT2 cells, underscoring its potential as a therapeutic target for arsenic-associated pulmonary fibrosis.
