Fig 2: ApoJ overexpression exacerbates liver fibrosis in TAA-induced mice. (A) Schematic illustration of the experimental protocol in which mice were treated with TAA for 6 weeks following intravenous injection with AAV-ApoJ or AAV-GFP as a control (upper panel). Serum levels of ALT and AST as indicators of liver injury (lower panel). (B) Representative liver histology using H&E, Masson’s trichrome, and Sirius Red staining to assess the extent of fibrosis (left panel). Scale bar = 100 μm. Quantification of the positive staining areas for Masson’s trichrome and Sirius Red is presented (right panel). (C) Immunostaining for α-SMA and F4/80 in the liver tissue of mice treated with AAV-ApoJ or AAV-GFP (left panel). Scale bar = 100 μm. Quantification of positive staining areas for α-SMA and F4/80 is displayed (right panel). (D) Real-time qPCR analysis of mRNA levels for fibrotic genes in liver tissue. (E) Western blot analysis showing the protein expression levels in the livers of mice treated with AAV-ApoJ or AAV-GFP. n = 6 per group. (F) Quantification of protein expression levels for α-SMA, TGF-β1, pSTAT3/STAT3 in Figure 7E. Data are presented as mean ± SEM. Statistical significance is denoted as follows: ∗P < .05 and ∗∗P < .01.

Fig 3: ApoJ loss in hepatocytes inhibition HSCs activation in vitro. (A) Schematic depiction of the experimental protocol. PMHs were harvested from Flox and ApoJ HKO mice exposed to CCl4 for 6 weeks, followed by the preparation of CM. Subsequently, the prepared CM samples were utilized to treat mHSCs. (B) Determination of TGF-β1 and ApoJ levels within the CM via ELISA assay (n = 6). (C) qPCR analysis of the mRNA levels of HSCs activation markers in mHSCs treated with indicated CM. (D) Immunofluorescent staining of α-SMA. scale bar: 50 μm. (E) Growth curve in mHSCs cultured in the hepatocyte medium with and without the addition of CM. (F) Western blot analysis of α-SMA, TGFβR1, and pSmad3/Smad3 expression in mHSCs treated with the indicated CM. (G) Quantification of protein expression levels for α-SMA, TGFβR1, and pSmad3/Smad3 in Figure 8F. (H) qPCR analysis of the mRNA levels of HSCs activation markers in mHSCs treated with recombinant ApoJ (rmApoJ). (I) Western blot analysis of α-SMA in mHSCs treated with the rmApoJ (left panel). The intensities of α-SMA were quantified (right panel). Data are presented as mean ± SEM. Statistical significance is denoted as follows: ∗P < .05; ∗∗P < .01; and ∗∗∗P < .001.

Fig 4: Regulation of ApoJ expression in liver cirrhosis and fibrosis models. (A) Serum ApoJ levels across different stages of liver fibrosis in patients and the Pearson correlation coefficient between serum ApoJ levels and the severity of liver fibrosis. (B) The ROC curve assessing the diagnostic performance of serum ApoJ for liver fibrosis. (C) ApoJ expression in liver tissues from rat treated with CCL4 for 3 and 9 weeks. Data were obtained from the GEO database, accession number GSE73499. (D) tSNE plot illustrating the clustering of liver cells from the patient with cirrhosis (upper panel). scRNA-seq analysis of ApoJ expression in various cell types from cirrhosis patients (lower panel). Data were retrieved from the GEO database, accession number GSE136103. (E) UMAP plot depicting the clustering of liver cells from mice treated with CCL4 (upper panel). The boxplot illustrates the expression levels of the ApoJ gene in the indicated cell clusters (lower panel). Data are from GSE216836. (F) Co-localization of Alb (Hepatocyte marker), Desmin (HSC marker), and ApoJ in the liver sections of liver fibrosis patient was assessed by immunofluorescent confocal microscopy (left panel). Quantification of positive staining areas for Sirius Red, and α-SMA in liver sections (right panel). Scale bar, 100 μm (n = 10). (G) Photomicrographs of liver sections from human in the indicated groups, stained with H&E, Sirius Red for collagen, α-SMA for activated stellate cells, CD68 for macrophages, and ApoJ (left panel). Quantification of positive staining areas for CD68, and ApoJ in liver sections (right panel). Scale bar, 100 μm (n = 10). Data are presented as mean ± SEM. Statistical significance is indicated as follows: ∗P < .05; ∗∗P < .01; and ∗∗∗P < .001.

Fig 5: Protective effect of ApoJ deficiency on chronic CCL4-induced liver fibrosis. (A) Construction strategy of hepatocyte-specific ApoJ knockout mouse using Cre-LoxP system. (B) PCR validation of the genotyping for ApoJ-flox and Alb-cre mice. The PCR results confirm the presence of the targeted alleles in the mouse models. (C) qPCR analysis of ApoJ expression in various organs in WT and ApoJ HKO mice. (D) Schematic representation of the experimental protocol for WT and ApoJ HKO mice treated with CCl4 for 6 weeks (1 mL/kg, i.p. injection twice a week). (E) ALT and AST levels in serum of WT and ApoJ HKO mice treated with CCl4 for 6 weeks. (F) Assessment of liver fibrosis of WT and ApoJ HKO mice treated with CCl4 for 6 weeks by H&E, Masson’s trichrome, and Sirius Red staining (left panel). Quantification of the fibrotic area in Masson’s trichrome staining and Sirius Red staining is shown (right panel). Scale bar = 100 μm. (G) Immunostaining for α-SMA and F4/80 in liver tissue of WT and ApoJ HKO mice (left panel). Quantification of the α-SMA-positive area and F4/80-positive area is shown (right panel). Scale bar = 100 μm. (H) Real-time qPCR analysis of mRNA levels for fibrotic genes in liver tissue of WT and ApoJ HKO mice. (I) Western blot analysis of protein levels of ApoJ, α-SMA, TGF-β1, pSTAT3, and STAT3 in liver tissue of WT and ApoJ HKO mice. Actin is shown as a loading control. (J) Quantification of protein intensity for the ratio of pSTAT3 to total STAT3, α-SMA, and TGF-β1 in the samples corresponding to Figure 3I. (K) The levels of MDA, reduced GSH, and SOD in liver of WT and ApoJ HKO mice. Data are presented as mean ± SEM. ∗Indicates a significant difference with P < .05.