Experimental study on expression changes of ferroptosis biomarkers in mice with NaAsO₂-induced liver injury

Objective To investigate whether ferroptosis is involved in NaAsO₂ (sodium arsenite)-induced liver injury in mice.

Methods A total of 32 male SPF-grade Balb/c mice, aged 6-8 weeks, were randomly assigned by body weight into control, low-concentration, medium-concentration, and high-concentration groups, with eight mice in each group. The mice were exposed to 0, 10, 20, and 40 mg/L NaAsO₂, respectively, via free drinking water for 12 consecutive weeks. Pathological liver injury was assessed by hematoxylin-eosin staining. Mitochondrial ultrastructure in hepatocytes was observed using a transmission electron microscope. The level of reactive oxygen species in liver tissue was measured using an enzyme-linked immunosorbent assay. The Fe²⁺ content in liver tissue was determined using a colorimetric assay. Immunohistochemistry was used to detect the positive expression of glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11), nuclear receptor coactivator 4 (NCOA4), and alpha-smooth muscle actin (α-SMA) in liver tissues. The expression levels of GPX4, SLC7A11, NCOA4, and α-SMA in liver tissues were measured by Western blot.

Results Compared with the control and low-concentration groups, the medium-and high-concentration groups exhibited decreased food intake, water consumption, autonomous activity, and body weight. Hematoxylin-eosin staining revealed scattered punctate necrosis, patchy necrosis, and inflammatory cell infiltration in the necrotic area of the liver tissue in the high-concentration group. Transmission electron microscopy demonstrated mitochondrial morphological alterations and membrane rupture in the high-concentration group. Enzyme-linked immunosorbent assay indicated significantly elevated reactive oxygen species levels in the low-, medium-, and high-concentration groups compared with the control group (t=2.72, P=0.013; t=2.24, P=0.037; t=3.63, P=0.002). The colorimetric assay showed significantly increased Fe²⁺ levels in the medium-and high-concentration groups compared with the control group (t=2.64, P=0.016; t=3.03, P=0.007). Immunohistochemistry demonstrated significantly reduced positive expression of GPX4 and SLC7A11 (t=-4.15, P=0.003; t=-4.76, P=0.001; t=-4.76, P=0.001; t=-7.97, P < 0.001) and significantly elevated positive expression of NCOA4 and α-SMA (t=2.66, P=0.029; t=3.60, P=0.007; t=5.93, P < 0.001; t=16.86, P < 0.001) in the medium-and high-concentration groups compared with the control group. Western blot showed significantly reduced SLC7A11 expression in the low-, medium-, and high-concentration groups (t=-3.04, P=0.016; t=-3.61, P=0.007; t=-5.77, P < 0.001) and significantly elevated α-SMA expression in the medium- and high-concentration groups (t=2.82, P=0.023; t=4.50, P=0.002) compared with the control group. Although the differences in GPX4 and NCOA4 expression measured by Western blot were not significant among groups (P>0.05), GPX4 expression decreased and NCOA4 expression increased with increasing exposure concentrations.

Conclusion High-concentration NaAsO₂ exposure can cause liver injury in mice, while reducing SLC7A11 and GPX4 levels and increasing ferroptosis in liver tissue.