Abstract
Drug-induced liver injury is the major cause of acute liver failure. However, the underlying mechanisms seem to be multifaceted and remain poorly understood, resulting in few effective therapies. Here, we report a novel mechanism that contributes to acetaminophen-induced hepatotoxicity through the induction of ferroptosis, a distinctive form of programmed cell death. We subsequently identified therapies protective against acetaminophen-induced liver damage and found that (+)-clausenamide ((+)-CLA), an active alkaloid isolated from the leaves of Clausena lansium (Lour.) Skeels, inhibited acetaminophen-induced hepatocyte ferroptosis both in vivo and in vitro. Consistently, (+)-CLA significantly alleviated acetaminophen-induced or erastin-induced hepatic pathological damages, hepatic dysfunctions and excessive production of lipid peroxidation both in cultured hepatic cell lines and mouse liver. Furthermore, treatment with (+)-CLA reduced the mRNA level of prostaglandin endoperoxide synthase 2 while it increased the protein level of glutathione peroxidase 4 in hepatocytes and mouse liver, confirming that the inhibition of ferroptosis contributes to the protective effect of (+)-CLA on drug-induced liver damage. We further revealed that (+)-CLA specifically reacted with the Cys-151 residue of Keap1, which blocked Nrf2 ubiquitylation and resulted in an increased Nrf2 stability, thereby leading to the activation of the Keap1–Nrf2 pathway to prevent drug-induced hepatocyte ferroptosis. Our studies illustrate the innovative mechanisms of acetaminophen-induced liver damage and present a novel intervention strategy to treat drug overdose by using (+)-CLA.
Highlights
Materials and methodsChemicals and reagents Erastin (S7242), fer-1 (S7243), necrostatin-1(nec-1, S8037), and ZVAD-fmk (S7023) were purchased from Selleck Ltd. (Shanghai, China)
Drug-induced liver injury (DILI) is a major cause of acute liver and kidney failures
(see figure on previous page) Fig. 1 (+)-CLA protects against APAP- and erastin-induced liver lipid peroxidation in vivo. a The chemical structure of (+)-CLA. b Schematic diagram of the experimental procedures. c Histopathological changes were examined by Hematoxylin and eosin (H&E) staining and observed with microscopy
Summary
Chemicals and reagents Erastin (S7242), fer-1 (S7243), necrostatin-1(nec-1, S8037), and ZVAD-fmk (S7023) were purchased from Selleck Ltd. (Shanghai, China). Slices were washed with phosphate buffer saline (PBS, three times, 10 min) and incubated with biotinylated goat antirabbit secondary antibody for 1 h at room temperature. Liperfluo staining and flow cytometry analysis Cells were plated in 100 mm dishes and treated with (+)-CLA for 24 h. The cell lysates were diluted four-fold with the buffer including 1% Triton X-100, 150 mM NaCl, and 10 mM Tris-HCl. Protein A beads and anti-Keap[1] antibodies were added for incubation at 4 °C overnight. Immunoprecipitated proteins were subjected to western blot analysis with Nrf[2] and Keap[1] antibodies. Western blot analysis was used to analyze 20 μL cell lysates to evaluate the expression of Nrf[2]. Cells were washed and stained with PI followed Annexin V-FITC at room temperature for 15 min. Multiple groups were compared by t-tests, one-way ANOVA analysis or two-way ANOVA and a probability (P) value < 0.05 was considered statistically significant
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
