Fig 2: Analysis of GSTM3 levels in the serum and liver tissues of patients with fibrosis.(A) H&E and Masson’s trichrome staining and representative immunohistochemical staining of GSTM3 in liver sections of patients with fibrosis (n = 44) at different stages of fibrosis (S0: n = 9; S1–2: n = 14; S3–4: n = 21). (B) ELISA analysis of serum GSTM3 levels in HC (n = 99) and patients with fibrosis (n = 180) at different stages of fibrosis (S1–2: n = 104; S3–4: n = 76). (C–F) Stratified analyses of serum GSTM3 levels in patients with liver fibrosis at different stages. (G) ROC curve analysis of serum GSTM3 for identifying significant liver fibrosis (S ≥ 2). (H) Correlation analysis of serum GSTM3 levels and the fibrosis index, FIB-4. (I) ELISA analysis of serum GSTM3 levels in HC (n = 99), patients with liver fibrosis (n = 40), and patients with liver cirrhosis (n = 56). (J, K) ROC curve analyses of serum GSTM3 levels in patients with chronic liver disease at different disease stages. Comparisons between two groups were performed using the Mann–Whitney U test, and comparisons among multiple groups were performed using the Kruskal–Wallis H test. Correlation analysis was conducted using Spearman’s rank correlation. Data are presented as mean ± SEM (ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001). P-values were obtained using an unpaired t-test. GSTM3, glutathione S-transferase Mu 3; H&E, hematoxylin and eosin; ELISA, enzyme-linked immunosorbent assay; HC, healthy controls; ROC, receiver operating characteristic; FIB-4, Fibrosis-4; SEM, standard error of the mean; ns, not significant.

Fig 3: Effects of GSTM3 knockdown on HSC activation and ROS levels in LX-2 cells.(A–C) LX-2 cells were transfected with si-GSTM3 for 24 h (n = 3). (A) RT-qPCR analysis of Gstm3 mRNA expression levels. (B) Western blot analysis of GSTM3 protein levels. (C) Western blot analysis of TIMP1, COL1A1, and fibronectin levels. (D–H) LX-2 cells were transfected with si-GSTM3 for 12 h and then treated with or without TGF-β for 24 h (n = 3). (D) RT-qPCR analysis of Timp1, Col1a1, and α-Sma mRNA expression levels. (E) Western blot analysis of TIMP1, elastin, COL1A1, and fibronectin levels. (F, G) Immunofluorescence staining for α-SMA and COL1A1. (H) ROS levels in LX-2 cells. Data are presented as mean ± SEM (*P < 0.05; **P < 0.01; ***P < 0.001). P-values were obtained using an unpaired t-test. GSTM3, glutathione S-transferase Mu 3; HSC, hepatic stellate cell; ROS, reactive oxygen species; RT-qPCR, reverse transcription quantitative polymerase chain reaction; TIMP1, tissue inhibitor of metalloproteinases 1; COL1A1, collagen type I alpha 1 chain; TGF-β, transforming growth factor-β; α-SMA, α-smooth muscle actin; SEM, standard error of the mean; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Fig 4: Effects of GSTM3 overexpression on HSC activation and ROS levels in LX-2 cells.LX-2 cells were transfected with the pcDNA3.1-GSTM3 plasmid for 24 h (n = 3). (A) Western blot analysis of GSTM3 levels. (B) Western blot analysis of TIMP1, COL1A1, and α-SMA levels. (C) RT-qPCR analysis of Timp1 mRNA expression levels. (D, E) Immunofluorescence staining for α-SMA and COL1A1. (F) ROS levels in LX-2 cells. Data are presented as mean ± SEM (*P < 0.05; **P < 0.01; ***P < 0.001). P-values were obtained using an unpaired t-test. GSTM3, glutathione S-transferase Mu 3; HSC, hepatic stellate cell; ROS, reactive oxygen species; TIMP1, tissue inhibitor of metalloproteinases 1; COL1A1, collagen type I alpha 1 chain; α-SMA, α-smooth muscle actin; RT-qPCR, reverse transcription quantitative polymerase chain reaction; SEM, standard error of the mean; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Fig 5: Effects of GSTM3 knockdown on HSC activation via the PPARγ signaling pathway.LX-2 cells were transfected with si-GSTM3 for 12 h and then treated with or without TGF-β for 24 h (n = 3). (A, B) Heatmap and volcano plots of DEGs in LX-2 cells. (C) KEGG enrichment analysis of DEGs and a list of significantly enriched pathways based on P-values. (D) GO functional annotation analysis of DEGs. (E) Western blot analysis of PPARγ, FABP4, and CD36 levels. “+” and “−” indicate the presence and absence of si-GSTM3 transfection, respectively. Data are presented as mean ± SEM (*P < 0.05; **P < 0.01; ***P < 0.001). P-values were obtained using an unpaired t-test. GSTM3, glutathione S-transferase Mu 3; HSC, hepatic stellate cell; PPARγ, peroxisome proliferator-activated receptor γ; TGF-β, transforming growth factor-β; DEGs, differentially expressed genes; KEGG, Kyoto Encyclopedia of Genes and Genomes; GO, Gene Ontology; FABP4, fatty acid-binding protein 4; CD36, cluster of differentiation 36; SEM, standard error of the mean; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.