Water extract from Herpetospermum pedunculosum attenuates oxidative stress and ferroptosis induced by acetaminophen via regulating Nrf2 and NF-icB pathways

Ethnopharmacological relevance: The seeds of Herpetospermum pedunculosum seeds is a traditional Tibetan medi-cine possessing hepatoprotective effect, but their protective effect on APAP-induced liver injury has not yet been explored.Aim of the study: This study aimed at exploring the protective effect and mechanism of the water extract from the seeds of Herpetospermum pedunculosum (HPWE) on APAP-induced liver injury in vitro and in vivo.Materials and methods: In vitro and in vivo models of liver injury were established by APAP treatment of BRL-3A cells or mice. The effect and mechanism of action of HPWE were explored by using cell viability assay, ELISA, immunofluorescence assay, RT-qPCR, histological observation and immunohistochemistry staining, western blotting and high-content imaging system.Results: In vitro experiments showed that HPWE treatment significantly promoted the cell viability, decreased ALT/AST level, and inhibited the ROS accumulation induced by APAP. Furthermore, HPWE and Fer-1 alleviated erastin-induced cell ferroptosis, upregulated GPX4 and SLC7A11 expression, and reduced lipid peroxides pro-duction. Further study showed that APAP could also downregulate the expression of GPX4 and SLC7A11, causing cell ferroptosis, and HPWE and Fer-1 counteracted this process. Our in vivo experiments showed that pretreat-ment with HPWE in APAP-treated mice significantly alleviated the serum ALT/AST level, decreased necrotic cells and inflammatory cell infiltration, upregulated the expression of GPX4 and SLC7A11. Further, it was demon-strated that HPWE treatment downregulated Nrf2 and its downstream target genes, i.e. HO-1 and NQO1 expression at the mRNA and protein levels. HPWE treatment also inhibited the activation of NF-icB p65 and downregulated its target genes, i.e. TNF-alpha and IL-1(i, expression.Conclusion: The present study showed that HPWE could relieve oxidative stress and ferroptosis via activating Nrf2 signaling pathway and inhibiting NF-icB mediated pathway.