Fig. 4

Panx1-targeting nanobodies interact with the NLRP3 inflammatory cascade in acetaminophen-overdosed mice. a Overview of the NLRP3 signaling cascade in acetaminophen (APAP)-induced hepatotoxicity. Overdosing mice with APAP triggers oxidative metabolization catalyzed by cytochrome (CYP) P450 enzymes in hepatocytes. Associated-NAPQI formation causes necrosis, which triggers a Panx1-mediated inflammatory response in the liver. The opening of Panx1 channels leads to extracellular ATP release underlying NLRP3-mediated maturation of IL-1β. b Adult mice were overdosed with APAP (300 mg/kg) or kept untreated (UTC). After 2 h, some mice were additionally administered either nanobody (Nb1, Nb3, Nb9 or non-targeting Nb (10 mg/kg)) or N-acetylcysteine (NAC) (200 mg/kg). Sampling was performed 24 h after APAP overdosing. Hepatic protein levels of CYP2E1, Panx1, NLRP3, pro-caspase-1 and IL-1β were assessed by immunoblot analysis. Protein levels were normalised against the total protein content and expressed as relative alteration compared to APAP mice (n = 4 (UTC and APAP) or n = 12 (Nb1, Nb3, Nb9, non-targeting Nb and NAC) animals per group). All data was analysed by unpaired t-tests with Welch’s correction or parametric 1-way analysis of variance followed by post hoc tests with Bonferroni’s correction. Data were expressed as means ± S.D