The Jieduan-Niwan Formula Reduces Inflammatory Responses in Acute-on-Chronic Liver Failure Rats by Inhibiting HMGB1-Induced Hepatocyte Pyroptosis

Background: Acute-on-chronic liver failure (ACLF) is a global intractable disease. HMGB1-induced hepatocyte Pyroptosis expanding inflammatory responses contributes to the pathogenesis of ACLF. The JDNW formula (JDNWF) has a significant clinical effect on ACLF, but its hepatoprotective mechanisms remain elusive.

Purpose: To explore the potential molecular mechanisms of the JDNWF in ACLF by HMGB1-induced hepatocyte Pyroptosis.

Methods: Rats were divided into normal, ACLF, Caspase-1 inhibitor, HMGB1 inhibitor, JDNW, JDNW+Caspase-1 inhibitor and JDNW+HMGB1 inhibitor groups. The ACLF rat model was established by 40% carbon tetrachloride-induced liver fibrosis, followed by intraperitoneal injection of D-galactosamine and lipopolysaccharide. The liver function, coagulation function, liver pathological damage and ultrastructural changes of hepatocytes were evaluated. Triple-immunostaining of active Caspase-1, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and albumin were performed to evaluate the percentage of pyroptotic hepatocytes. Western blot, immunofluorescence, enzyme-linked immunosorbent assay (ELISA) and quantitative Real-Time PCR (RT-qPCR) were used to analyze the expressions of key genes and proteins in HMGB1-induced Pyroptosis pathways and the level of inflammatory factors.

Results: The JDNWF improved liver function, coagulation function and liver pathological damage, reduced the percentage of pyroptotic hepatocytes and inflammatory responses, and down-regulated the expressions of key genes and proteins in the HMGB1-induced Pyroptosis pathways in ACLF rats. The effect of the JDNWF was better than those of HMGB1 inhibitor (glycyrrhizin) and Caspase-1 inhibitor (VX-765). Compared with glycyrrhizin or VX-765, there were no significant differences in the above indicators after the JDNWF in combination with glycyrrhizin or VX-765. These results indicated that the JDNWF inhibited hepatocyte Pyroptosis and liver inflammation in ACLF rats through the HMGB1-induced Pyroptosis pathways.

Conclusion: The JDNWF protects the livers of ACLF rats by inhibiting HMGB1-induced hepatocyte Pyroptosis reducing inflammatory responses, suggesting that HMGB1-induced hepatocyte Pyroptosis may be a potential therapeutic target of ACLF.