To investigate the mechanism of tetramethylpyrazine (TMP) in alleviating CCl₄-induced liver fibrosis in mice by regulating ferroptosis through the SLC7A11/GPX4 signaling axis.

Thirty male C57BL/6 mice were randomly divided into control group, CCl₄ model group, and low-, medium-, high-dose TMP groups (50, 100, and 150 mg/kg). Except for the control group, mice were intraperitoneally injected with a mixture of CCl₄ and olive oil (1 : 10, 5 mL/kg) twice weekly for 8 weeks. From week 5, mice in the TMP groups were administered TMP by gavage once daily for 4 weeks. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured to evaluate liver function. Histopathological changes were assessed by hematoxylin-eosin (HE), Masson's trichrome, and Sirius red staining. The expression of α-SMA, COL1A1, Fibronectin, SLC7A11, and GPX4 was detected by immunohistochemistry and Western blot, and their mRNA levels were determined by qPCR. Fe²⁺, malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH) levels were measured using commercial assay kits. One-way analysis of variance (ANOVA) was used for comparison among multiple groups, and Tukey's post-hoc test was used for pairwise comparison between groups. If the data did not conform to normal distribution or homogeneity of variance, Kruskal-Wallis H test was adopted, and Dunn's test was used for pairwise comparison between groups.

Compared with the model group, the levels of ALT [(251.70 ± 10.80) vs (450.00 ± 14.49) U/L] and AST [(236.70 ± 10.80) vs (427.50 ± 12.14) U/L], the degree of collagen fiber deposition in liver tissue [Masson: (1.10 ± 0.07)% vs (1.72 ± 0.17) %, Sirius Red: (0.17 ± 0.02) % vs (0.45 ± 0.04) %], the protein levels of α-SMA (1.17 ± 0.27 vs 8.08 ± 0.48), COL1A1 (4.30 ± 0.73 vs 18.78 ± 0.93) and Fibronectin (1.60 ± 0.32 vs 7.75 ± 0.60), the contents of Fe²⁺ [(2.60 ± 0.14) vs (3.63 ± 0.19) μmol/g protein] and MDA [(0.72 ± 0.03) vs (1.12 ± 0.07) nmol/mg protein] were decreased in the high-dose TMP group, while the protein levels of SLC7A11 (0.98 ± 0.05 vs 0.29 ± 0.03) and GPX4 (0.73 ± 0.08 vs 0.14 ± 0.03), the activity of SOD [(1 413.00 ± 47.19) vs (1 147.00 ± 42.74) U/mg protein] and the content of GSH [(1.60 ± 0.07) vs (1.15 ± 0.07) μmol/g protein], the mRNA levels of SLC7A11 (0.80 ± 0.07 vs 0.10 ± 0.03) and Gpx4 (0.75 ± 0.07 vs 0.37 ± 0.05) were increased, all these differences were statistically significant (all P < 0.05).

TMP may alleviate CCl₄-induced liver fibrosis in mice by regulating the SLC7A11/GPX4 signaling axis to inhibit ferroptosis, which has potential anti-fibrotic application value.