HMGB1-Mediated Cell Death-A Crucial Element in Post-Hepatectomy Liver Failure

Abstract

Liver resection (LR) is the primary treatment for hepatic tumors, yet posthepatectomy liver failure (PHLF) remains a significant concern. While the precise etiology of PHLF remains elusive, dysregulated inflammatory processes are pivotal. Therefore, we explored the theragnostic potential of extracellular high-mobility-group-box protein 1 (HMGB1), a key damage-associated molecular pattern (DAMP) released by hepatocytes, in liver recovery post LR in patients and animal models. Plasma from 96 LR patients and liver tissues from a subset of 24 LR patients were analyzed for HMGB1 levels, and associations with PHLF and liver injury markers were assessed. In a murine LR model, the HMGB1 inhibitor glycyrrhizin, was administered to assess its impact on liver regeneration. Furthermore, plasma levels of keratin-18 (K18) and cleaved cytokeratin-18 (ccK18) were quantified to assess suitability as predictive biomarkers for PHLF. Patients experiencing PHLF exhibited elevated levels of intrahepatic and circulating HMGB1, correlating with markers of liver injury. In a murine LR model, inhibition of HMGB1 improved liver function, reduced steatosis, enhanced regeneration and decreased hepatic cell death. Elevated levels of hepatic cell death markers K18 and ccK18 were detected in patients with PHLF and correlations with levels of circulating HMGB1 was observed. Our study underscores the therapeutic and predictive potential of HMGB1 in PHLF mitigation. Elevated HMGB1, K18, and ccK18 levels correlate with patient outcomes, highlighting their predictive significance. Targeting HMGB1 enhances liver regeneration in murine LR models, emphasizing its role in potential intervention and prediction strategies for liver surgery.

Keywords: HMGB1; caspase-cleaved keratin-18; partial hepatectomy; post hepatectomy liver failure.

Figure 1

High mobility group box protein-1 (HMGB1) levels correlate with post-hepatectomy liver failure (PHLF) and liver injury markers ALT (alanine-aminotransferase) and AST (aspartate-aminotransferase). (A) Immunofluorescence staining and quantification of HMGB1 in liver biopsies before and after resection, with or without PHLF. (Two-way ANOVA: * p < 0.05, ** p < 0.01). Regeneration-induced effects depicted as difference after-before resection (∆ postOP-preOP) (unpaired t-test: * p < 0.05). (B) Plasma HMGB1 levels before and after partial hepatectomy from patients with and without PHLF (Two-way ANOVA: ** p < 0.01). (C) ROC (receiver operation curve) analysis comparing HMGB1 predictive potential with PHLF. (D) Correlation of plasma HMGB1 with AST and ALT levels on POD1.

Figure 1

High mobility group box protein-1 (HMGB1) levels correlate with post-hepatectomy liver failure (PHLF) and liver injury markers ALT (alanine-aminotransferase) and AST (aspartate-aminotransferase). (A) Immunofluorescence staining and quantification of HMGB1 in liver biopsies before and after resection, with or without PHLF. (Two-way ANOVA: * p < 0.05, ** p < 0.01). Regeneration-induced effects depicted as difference after-before resection (∆ postOP-preOP) (unpaired t-test: * p < 0.05). (B) Plasma HMGB1 levels before and after partial hepatectomy from patients with and without PHLF (Two-way ANOVA: ** p < 0.01). (C) ROC (receiver operation curve) analysis comparing HMGB1 predictive potential with PHLF. (D) Correlation of plasma HMGB1 with AST and ALT levels on POD1.

Figure 2

Inhibiting high mobility group box protein-1 (HMGB1) with glycyrrhizin enhances liver regeneration. (A) Alanine-aminotransferase (ALT) and aspartate-aminotransferase (AST) levels in 15-week-old mice on a fast food diet, treated with glycyrrhizin or DMSO control, 24 h post partial hepatectomy (PHx) (unpaired t-test: * p < 0.05, ** p < 0.01). (B,C) Oil Red O Staining and Hematoxylin & eosin (H&E), 48 h post PHx (Two-way ANOVA: ** p < 0.01). (D) Gene expression of KI67, PCNA, and Cyclin D1 before and 48 h post PHx (Two-way ANOVA: ** p < 0.01, *** p < 0.001, **** p < 0.0001). (E,F) Immunofluorescence quantifications of KI67 and cleaved caspase 3 (cCasp3) and p21 pre- and post-PHx (Two-way ANOVA: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Figure 2

Inhibiting high mobility group box protein-1 (HMGB1) with glycyrrhizin enhances liver regeneration. (A) Alanine-aminotransferase (ALT) and aspartate-aminotransferase (AST) levels in 15-week-old mice on a fast food diet, treated with glycyrrhizin or DMSO control, 24 h post partial hepatectomy (PHx) (unpaired t-test: * p < 0.05, ** p < 0.01). (B,C) Oil Red O Staining and Hematoxylin & eosin (H&E), 48 h post PHx (Two-way ANOVA: ** p < 0.01). (D) Gene expression of KI67, PCNA, and Cyclin D1 before and 48 h post PHx (Two-way ANOVA: ** p < 0.01, *** p < 0.001, **** p < 0.0001). (E,F) Immunofluorescence quantifications of KI67 and cleaved caspase 3 (cCasp3) and p21 pre- and post-PHx (Two-way ANOVA: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Figure 3

High mobility group box protein-1 (HMGB1) levels are associated with cell death marker keratin-18 (K18) and cleaved caspase cytokeratin-18 (ccK18). (A) Plasma Keratin-18 (K18) and cleaved caspase cytokeratin-18 (ccK18) levels pre- and post- partial hepatectomy (PHx) in patients with and without post-hepatectomy liver failure (PHLF) (Two-way ANOVA: * p < 0.05, ** p < 0.01). (B) Receiver operating characteristic (ROC) curve comparing HMGB1, K18 and ccK18 postoperative day 1 (POD1) with PHLF. (C) Correlation of plasma HMGB1 with K18 and ccK18 on POD1. ROC curves of HMGB1 were compared to ROC curves of PDR and R15 in a subset of 64 patients (D) and in a subset of 39 patients ROC curves of HMGB1 and APRI+ALBI were compared (E).

Figure 3

High mobility group box protein-1 (HMGB1) levels are associated with cell death marker keratin-18 (K18) and cleaved caspase cytokeratin-18 (ccK18). (A) Plasma Keratin-18 (K18) and cleaved caspase cytokeratin-18 (ccK18) levels pre- and post- partial hepatectomy (PHx) in patients with and without post-hepatectomy liver failure (PHLF) (Two-way ANOVA: * p < 0.05, ** p < 0.01). (B) Receiver operating characteristic (ROC) curve comparing HMGB1, K18 and ccK18 postoperative day 1 (POD1) with PHLF. (C) Correlation of plasma HMGB1 with K18 and ccK18 on POD1. ROC curves of HMGB1 were compared to ROC curves of PDR and R15 in a subset of 64 patients (D) and in a subset of 39 patients ROC curves of HMGB1 and APRI+ALBI were compared (E).