Abstract
BACKGROUND: Acetaminophen, or N-acetyl-para-aminophenol (APAP), causes severe liver damage and acute liver failure when overdosed. Oligodeoxynucleotides containing CpG motifs (CpG ODN) can regulate the function of macrophages, which play an important role in drug-induced liver injury. It is unclear whether CpG ODN-treated macrophages play an immune regulation role in APAP-induced liver injury. In the present study, we aim to explore the role of CpG ODN-activated macrophages in APAP-induced liver injury and the underlying mechanism in protecting against the cytotoxicity of APAP. METHODS: In vivo, C57BL/6 mice were treated with APAP (300 mg/Kg) or/and CpG ODN (ODN 1826, 1.65 mg/Kg) by intraperitoneal injection, then survival rate, histopathological evaluation, and inflammatory factors were observed to ascertain the protective effect of CpG ODN. Then, CpG ODN-treated macrophages were reinfused into the animal model to determine the effector cells. In vitro, RNA sequencing and untargeted metabolomics detection were performed to illustrate the underlying mechanism. Last, Acod1 siRNA interference was used to clarify the role of IRG1 in resistance to APAP cytotoxicity by ROS and apoptosis indicator detections. RESULTS: We found that CpG ODN showed a protective effect against APAP cytotoxicity by stimulating macrophages rather than hepatic parenchymal cells. In particular, reinfusion of CpG ODN-treated macrophages to mice can alleviate APAP-induced liver injury. Transcriptome and metabolome analysis revealed that the expression of aconitate decarboxylase 1 (Acod1; also known as immune responsive gene 1, IRG1) and the metabolite itaconate generated by IRG1 catalysis increased after CpG ODN stimulation. In addition, we found that the mechanism of this protective effect is ascribed to the increased expression of Acod1 and the antioxidative function of itaconate by the activation of the TLR9/NF-κB signaling pathway. CONCLUSION: CpG ODN alleviated liver injury induced by APAP through the activation of the TLR9/NF-κB signaling pathway in macrophages, upregulating the expression of IRG1 protein, promoting the production of endogenous metabolite itaconate, and inhibiting macrophage apoptosis which was regulated by upregulating the expression of Nrf2 to inhibit ROS production. This study sheds new light on CpG ODN as a therapeutic strategy in resistance to APAP-induced liver injury.