Molecular Mechanism Research Of Ethanol Extract Of Herpetospermum Pedunculosum Seeds Alleviating Acetaminophen-induced Liver Injury

H.pedunculosum seeds are the dry mature seeds of the annual climbing herb Herpetospermum pedunculosum(Ser.)C.B.Clarke of the Cucurbitaceae family,mainly distributed in southwestern China,India and Nepal,in hillside shrubs at an altitude of 2300-3500 meters.Known for its liver-protecting effect,it exists in 60% of Tibetan medicine prescriptions for liver diseases,and is a commonly used drug in Tibetan medicine for liver and gallbladder diseases.Acetaminophen(N-acetyl-p-aminophenol,APAP),also known as paracetamol,is a drug widely used to treat pain and fever.Overdose can lead to severe liver damage,even acute liver failure.NAC(N-Acetyl-Cysteine)is currently the only approved antidote for APAP overdose,but due to the inherent limitations of NAC treatment,new drugs must be developed for the treatment of APAP-induced liver injury.Traditional records and modern pharmacological studies suggest that H.pedunculosum seeds have the potential to alleviate APAP-induced liver injury.Therefore,this work focuses on investigating the pharmacological efficacy and molecular mechanism of ethanol extract of the seeds of H.pedunculosum(HPEE),which is rich in lignans,on the protection against APAP-induced liver injury.Objective of the study: To assess the protective effect and mechanism of HPEE on APAPinduced liver injury in vitro and in vivo.Materials and methods: We established BRL-3A cell and mouse models of APAP-induced liver injury,and obtained HPEE by extracting the seeds of H.pedunculosum with absolute ethanol.Cell viability assay,biochemical index detection,reactive oxygen species(ROS)detection,immunofluorescence assay,RT-qPCR,histological observation,Western Blot,immunohistochemical analysis and molecular docking were used to study HPEE alleviates APAP-induced liver injury efficacy and mechanism of action.Results: HPEE attenuated APAP-induced liver injury both in vitro and in vivo.In vitro,HPEE significantly increased cell viability,reduced the activity of alanine aminotransferase(ALT),aspartate aminotransferase(AST)in cell culture supernatant and the content of malondialdehyde(MDA)in cells,increased glutathione(GSH)levels,and reduced APAPinduced ROS accumulation.HPEE also inhibited APAP-induced apoptosis by downregulating Bax and Cleaved-Caspase 3.Preliminary mechanistic exploration suggests that HPEE activates Nrf2 to regulate the transcriptional expression of downstream target genes,including HO-1 and NQO1,thereby inhibiting oxidative stress and apoptosis caused by APAP overdose.In vivo,pretreatment of mice with HPEE significantly inhibited elevated serum ALT and AST activity,improved hepatocyte necrosis,reduced apoptosis,upregulated the protein expression of Nrf2,HO-1 and NQO1 in liver tissues,and resisted oxidative damage.Molecular docking showed that the compounds in HPEE include dehydrodiconiferylalcohol(1),herpetolide A(2),threoBuddlenol E(3),picrasmalignan A(4),herpetrione(5)can interact with kelch-like ECHassociated protein 1(Keap1),suggesting that the components interfere with the interaction of Nrf2 and Keap1 to activate Nrf2 for efficacy.Conclusion: HPEE can alleviate liver injury caused by excessive APAP by activating Nrf2 signaling pathway to inhibit oxidative stress and apoptosis.