Mid-zone hepatocytes exhibit the most pronounced proliferative decline in early acute liver injury.

Wildtype C57BL/6J mice were intraperitoneally injected with 300 mg/kg APAP for 0, 3, 6, 12 and 24 h. (A) Immunohistochemical staining of Ki67 to detect proliferating hepatocytes in liver sections at 0, 3, 6, 12 and 24 h post-APAP. Representative images of pericentral (PC), middle (Mid) and periportal (PP) are shown. Red arrows indicate Ki67-positive hepatocytes. (B) Quantitative analysis of Ki67+ hepatocytes across liver zones (PC, Mid, PP) at 0, 3, 6, 12, and 24 h post-APAP. (C) Quantitative analysis of Ki67+ hepatocytes across 0, 3, 6, 12, and 24 h post-APAP in each liver zone (PC, Mid, PP). (D) Changes of Ki67-positive cells across liver zones (PC, Mid, PP) at 0, 3, 6, 12 and 24 h post-APAP. Data are presented as log2-value change (number of Ki67-positive cells at x h post-APAP – the number of Ki67-positive cells at 0 h post-APAP). (a) denotes significance between PC and Mid regions, (b) denotes significance between PC and PP regions, and (c) denotes significance between Mid and PP regions. Data points represent mean hepatocyte counts from six high-power fields per zonal layer (n=3 mice/group) in B and C. Data are represented as means ± SD; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.

Mid-zone hepatocytes show distinct gene expression profiles with reduced proliferative activity during early acute injury.

(A) Heatmap showing the gene expression of the top ten differentially expressed genes in hepatocyte zones (zones PP, Mid, and PC) at 0, 3, 6, 24 h post-APAP. The major functions of hepatocytes in each zone are labeled on the left side for 0, 6, and 24 h post-APAP, respectively. (B) Volcano plot illustrating the DEGs in Mid-zone at 6 h compared to 0 h post-APAP. Gray dots denote genes that are not statistically significant. Red and blue dots represent genes that are upregulated and downregulated, respectively, in the sample tissue, at least a 0.5-fold difference from the matched control, with a false discovery rate (FDR) threshold of 0.05. (C) Scatter plot shows the dynamic changes of Sqstm1 mean expression level in hepatocyte zones (zones PP, Mid, and PC) at 0 and 6 h post-APAP. (D) Immunohistochemical staining of p62 (protein product of Sqstm1) to detect the dynamic changes of p62 across hepatocyte zones at 0 and 6 h post-APAP. Red arrows indicate p62-positive hepatocytes. (E) Percentage of Ki67-positive spots analyzed at 0, 3, 6 and 24 h post-APAP. (F) Average module score for S-phase (up) and G2/M-phase (down) genes across hepatocyte zones (zones PP, Mid, and PC) at 0, 3, 6, 24 h post-APAP. (G) Expression levels of the S-phase gene Nasp (up) and the G2/M phase gene Cks1b (down) in each hepatocyte zone (zones PP, Mid, and PC) at 0, 3, 6, 24 h post-APAP.

Zonal metabolism of APAP leads to stress response in Mid-zone hepatocytes.

(A) Schematic figure illustrating the metabolic process of liver injury caused by APAP. (B) Dot plot displays the scaled gene expression levels of the Cyp family in hepatocyte zones (zones PP, Mid, and PC) at 0, 3, 6, 24 h post-APAP. (C) Immunohistochemical staining of Cyp1a2 to detect its dynamic changes in hepatocyte zones (from CV to PV) at 0, 3, 6, 24 h post-APAP. Injured area is outline by black dashed lines. (D) Quantification of zonal distribution of Cyp1a2-positive cells in liver sections at 0, 3, 6, and 24 h post-APAP, respectively. (E) Schematic figure illustrating expression changes of Cyp1a2 before and after APAP. (F) Immunofluorescence staining of Chop protein (green) to observe the dynamic changes in its expression across hepatocyte zones (from CV to PV) at 6 h post-APAP with doses of 100, 300, or 500 mg/kg. Cyp1a2 protein (red) staining highlights the area around the CV. Cell nuclei are stained with DAPI (blue). (G) Quantification of zonal distribution of Chop-positive cells in liver sections from mice treated with varying doses of APAP– 100, 300, or 500 mg/kg. (H) Schematic figure illustrating zonal metabolism of APAP results in the gene expression shift in Mid hepatocytes following necrosis. Data are represented as means ± SD; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.

The Atf4-Ddit3 axis emerges as pivotal in Mid-zone during early acute injury.

(A) Heatmap displays the area under the curve (AUC) scores of transcription factor (TF) motifs, estimated per dot by Single-Cell Regulatory Network Inference and Clustering (SCENIC), highlighting gene regulatory networks and differentially activated TF motifs in hepatocyte zones (PP, Mid, PC) at 6 h post-APAP. Columns represent TF motifs, rows represent dots, and color intensity indicates AUC scores. (B) Heatmap shows the activity of transcription factors (TFs) across different zonal regions at 6 h post-APAP. TF activity was quantified using AUCell scoring. The color key represents regulon activity scores. (C) Violin plots shows transcriptional activity of Atf4 and Ddit3 in each zonation at 6 h post-APAP. (D) GO pathway analysis reveals the top 15 enriched pathways for genes co-regulated by Atf4 and Ddit3 in mid hepatocyte zones at 6 h post-APAP. (E) Heatmap showing the target genes expression of Atf4 (left) and Ddit3 (right) in hepatocyte zones (zones PP, Mid, and PC) at 6h post-APAP. (F) Immunohistochemistry staining of p-eIF2A in liver sections from mice at 0 h and 3 h post-APAP. p-eIF2A-positive hepatocytes are indicated by red arrows. The number of peIF2A-positive cells per field of view (FOV) is quantified. n=3 mice/group. (G) Heatmap displays stress response and ER stress-related genes from differentially expressed genes (DEGs) identified in Mid hepatocyte zones at 0 and 6 h post-APAP. Data are represented as means ± SD; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.

The Atf4-Ddit3 axis protects hepatocytes from liver injury.

(A) Immunohistochemical staining of Atf4 in liver sections at 0 and 6 h post-APAP. Red arrows indicate Atf4-positive hepatocytes. Zonal distribution of Atf4-positive cells in liver sections at 6 h post-APAP is quantified. The statistic is the percentage of Atf4-positive hepatocytes in each layer over the total number of Atf4-positive hepatocytes. n=3 mice. (B) Schematic figure illustrating the overexpression of Atf4 via AAV in hepatocytes of wildtype C57BL/6J mice. Overexpression of EGFP is used as a control. (C) H&E staining showing liver morphology from mice overexpressing AAV-TBG-EGFP or AAV-TBG-Atf4 at 6 h post-APAP. The injured area is outlined by black dashed lines. The percentage of injured area is quantified. n=3 mice/group. (D) Serum levels of ALT is measured in mice overexpressing AAV-TBG-EGFP or AAV-TBGAtf4 at 6, 24, 48, and 72 h post-APAP. n=6 mice/group. (E) Immunohistochemistry staining of Atf4 in liver sections from mice overexpressing AAVTBG-EGFP or AAV-TBG-Atf4 at 12 h post-APAP. Atf4-positive hepatocytes are indicated by red arrows. The number of Atf4-positive cells per field of view (FOV) is quantified. n=9 mice/group. (F) Immunofluorescence staining of Ki67 (red) was performed to assess proliferating hepatocytes in mice overexpressing AAV-TBG-EGFP or AAV-TBG-Atf4 at 72 h post-APAP. Cell nuclei were stained with DAPI (blue). The number of Ki67-positive cells per FOV is quantified. n=6 mice/group. (G) Immunohistochemistry staining of p-eIF2A in liver sections from mice overexpressing AAVTBG-EGFP or AAV-TBG-Atf4 at 12 h post-APAP. p-eIF2A-positive hepatocytes are indicated by red arrows. The number of Atf4-positive cells per FOV is quantified. n=6 mice/group. Data are represented as means ± SD; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.

The Atf4-Ddit3 axis protects the liver by pausing proliferation via Btg2.

(A) Heatmap of Ddit3-binding signals in chromatin domains. (B) Overlap of Ddit3-binding genes by Cut&Run with differentially expressed genes (DEGs) in mid zone was shown. These genes were referred as Ddit3-binding DEGs. (C) Integrative Genomics Viewer (IGV) plot shows the Cut&Run peaks of Ddit3 and input on Btg2-site, Ifrd1-site, Fos-site and Egr1-site. (D) Immunohistochemical staining of Btg2 in liver sections at 0 and 6 h post-APAP. Red arrows indicate Btg2-positive hepatocytes. (E) Schematic figure illustrating overexpression of Ddit3 via AAV in hepatocytes of wildtype C57BL/6J mice. Overexpression of EGFP is used as a control. (F) Immunohistochemical staining of Btg2 in liver sections from mice overexpressing either AAV-TBG-EGFP or AAV-TBG-Ddit3 at 12 h post-APAP. Btg2-positive hepatocytes are indicated by red arrows. Btg2-positive hepatocytes in liver sections is quantified. n=4 mice/group. (G) Schematic figure illustrating overexpression of Btg2 via AAV in hepatocytes of wildtype C57BL/6J mice. Overexpression of EGFP is used as a control. (H) H&E staining shows morphology of livers from AAV-TBG-EGFP/Btg2 overexpressing mice at 6 h post-APAP. Injured area is outlined by black dashed lines. The percentage of injury area is quantified. n=3 mice/group. (I) Serum levels of ALT is measured at 6h post-APAP in AAV-TBG-EGFP/Btg2 overexpressing mice. n=3 mice/group. (J) Immunohistochemical staining of Ki67 in liver sections from mice overexpressing either AAV-TBG-EGFP or AAV-TBG-Btg2 at 72 h post-APAP. Ki67-positive hepatocytes are indicated by red arrows. Ki67-positive cells in liver sections is quantified. n=4 mice/group. (K) Immunohistochemical staining of Ki67 in liver sections from mice after knocking down Btg2 via overexpressing either IP479-EFS-sgEGFP or IP479-EFS-sgBtg2 at 72 h post-APAP. Ki67-positive hepatocytes are indicated by red arrows. Ki67-positive cells in liver sections is quantified. n=4 mice/group. Data are represented as means ± SD; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.

The Atf4-Ddit3 axis mediates integrated stress protection in mid-zone hepatocytes at the expense of proliferative capacity during early AILI.

Schematic model depicting the adaptive response of mid-zone hepatocytes to acute liver injury, wherein activation of the Atf4-Ddit3 pathway upregulates Btg2 to induce transient cell cycle arrest, balancing survival and regeneration.