Ezetimibe protects against alpha-amanitin-induced hepatotoxicity by targeting the NTCP receptor: Mechanistic insights from in vitro and in vivo models.

BACKGROUND

α-Amanitin, the primary lethal toxin of Amanita phalloides, induces irreversible hepatotoxicity by selectively inhibiting RNA polymerase II, leading to transcriptional arrest. Despite advancements in managing mushroom poisoning, a targeted antidote remains unavailable. The sodium taurocholate co-transporting polypeptide (NTCP), a hepatic bile acid transporter, facilitates α-amanitin entry into hepatocytes. Pharmacological blockade of NTCP represents a promising therapeutic strategy.

OBJECTIVE

To evaluate ezetimibe, an NTCP inhibitor, as a protective agent against α-amanitin-induced hepatotoxicity.

METHODS

Transcriptomic profiling of α-amanitin-exposed mouse liver tissues (NCBI accession: PRJNA809431) was conducted using DESeq2. Molecular docking simulations assessed interactions between NTCP, α-amanitin, and ezetimibe. Therapeutic efficacy was evaluated in vivo (mouse models) and in vitro (cultured hepatocytes). Key outcomes included survival rates, liver injury markers (ALT, AST), apoptosis (Bax/Bcl-2 ratio), and oxidative stress parameters.

RESULTS

NTCP expression was upregulated in α-amanitin-exposed livers. Molecular docking revealed α-amanitin binding at NTCP residue VAL-160, whereas ezetimibe interacted with LEU-14 and ASN-17. Ezetimibe (50 mg/kg) improved survival rates from 25 % to 80 % in α-amanitin-exposed mouse models (p < 0.01), reduced serum ALT (68 ± 5 U/L vs. 165 ± 12 U/L; p < 0.05) and AST (72 ± 6 U/L vs. 158 ± 10 U/L; p < 0.05), and attenuated apoptosis (60 % decrease in Bax/Bcl-2; p < 0.05). In vitro, ezetimibe restored hepatocyte viability 2.1-fold (p < 0.05) and reduced oxidative stress (40 % decrease in malondialdehyde; p < 0.05). Transcriptomic analysis linked α-amanitin toxicity to p53-mediated apoptosis.

CONCLUSION

Ezetimibe protects against α-amanitin hepatotoxicity by blocking NTCP-mediated uptake, supporting its potential clinical repurposing as a targeted antidote.