Thrombospondin‐1 Inhibition Reduces Nrf2 Activity and Increases Liver Injury during Acetaminophen‐Induced Hepatotoxicity

Abstract

Introduction Acetaminophen (N-Acetyl-p-Aminophenol or APAP)-induced hepatotoxicity is the most common cause of acute liver failure in the United States and Western Europe. Toxicity from APAP results from the accumulation of the toxic metabolite NAPQI, a consequence of hepatic glutathione (GSH) depletion. Therefore, pathways regulating GSH and antioxidant pathways can play a pivotal role in APAP-induced hepatotoxicity. Thrombospondin-1 (TSP1) is a homotrimeric protein that can modulate antioxidant signaling pathways and other signaling pathways involved in cell death and inflammation. Therefore, the hypothesis of this study was that TSP1 was upregulated in the liver during APAP-induced hepatotoxicity and inhibition of TSP1 can contribute to liver injury by increasing oxidative damage and hepatic cell death. Methods C57Bl/6 mice or TSP1 null mice (TSP1-/-) were administered 300 mg/kg or 600 mg/kg of APAP and were euthanized either 2-hours or 6-hours after injection. Liver injury was assessed by measuring serum ALT and AST levels as well as liver H&E and TUNEL staining. TSP1, CYP2E1, Nrf2, and SOD1 expression were assessed by real-time PCR, immunoblotting, ELISA or immunohistochemistry analyses. Liver GSH levels were measured by colorimetric assay. The GSH synthesis enzymes GCLC, GCLM and GSR were assessed by RTPCR. All data were analyzed using Graphpad Prism software with differences between two groups compared using the Student's t-test and differences between three or more groups were determined by ANOVA. Results C57Bl/6 mice administered 300 mg/kg or 600 mg/kg had a significant increase of TSP1 mRNA and protein in their livers 6-hours after injection with this effect being dose-dependent. TSP1-/- mice had a similar degree of hepatic necrosis at 2-hours and 6-hours following APAP injection compared to C57Bl/6 mice. However, TSP1-/- mice had a significant increase of serum ALT and AST levels as well as increased TUNEL staining 6-hours following APAP administration. Nrf2 and SOD1 protein expression were significantly decreased in TSP1-/-mice but not C57Bl/6 mice 6-hours after APAP injection. This was associated with decreased GSH, GCLC, GCLM and GSR in APAP-treated TSP1-/- mice compared to C57Bl/6 mice administered APAP. Conclusions Together, these data demonstrate that elimination of TSP1 protein in APAP-treated mice reduced hepatic Nrf2 expression and GSH activity leading to worse liver injury. Treatment strategies aimed at inducing Nrf2 activity or increasing TSP1 expression may be beneficial to improve outcomes during APAP-induced liver injury.