The Inhibitory Effects And Mechanism Of (+)-clausenamide On Ferroptosis Against APAP-induced Liver Injury

Research on drug-induced liver injury has dramatically increased over the past decade,and it has grown up to be a hot topic for clinicians,academics,pharmaceutical companies and regulators.APAP is an antipyretic analgesics commonly used in clinical,which caused acute liver damage in many countries by its hepatotoxicity.Studies have shown that traditional forms of death,such as apoptosis and necrosis,are involved in APAP-induced hepatocyte death,but these types of death are still controversial and imperfection in their elucidation of the pathological processes and mechanisms.Therefore,elucidating the APAP-induced liver cell death type and its mechanism of death for the development of liver protection drugs is particularly crucial.Overdosing of APAP leads to a significant increase in lipid peroxidation in the liver,which is accompanied by a large accumulation of iron in the cells.These characteristics are highly compatible with the newly discovered cell death method—Ferroptosis.Therefore,we hypothesize that ferroptosis participates in the process of APAP-induced hepatocyte death.The anticipating results of this study will be significance in the pathogenesis of drug-induced liver injury and the research and development of hepatoprotective drugs.Objective:(1)To investigate whether ferroptosis is involved in APAP-induced hepatocyte death and to explore new mechanisms of APAP liver injury which providing new strategies for the clinical treatment of drug-induced liver injury;(2)APAP-induced hepatic injury model of ferroptosis was established to screen and evaluate small molecule compounds that inhibit the protective effect of ferroptosis on hepatic injury;(3)To study the mechanism of(+)-clausenamide in inhibiting the damage of ferroptosis,which can provide pharmacological and molecular biological basis for the study of hepatic protection of other ferroptosis inhibitors.Methods: At the animal level,C57BL/6 mice were used to establish the APAP liver injury model.The pharmacodynamic evaluation of the hepatoprotective effect of(+)-clausenamide was investigated from the aspects of liver function and lipid peroxidation,premeliminary investigate whether APAP induces the novel cell death of ferroptosis and the role of(+)-clausenamide.Based on this study,we used ferroptosis inducer erastin as a positive control drug to compare with APAP-induced injury.The group was set to normal group,APAP model group,erastin model group,(+)-clausenamide + APAP group,Fer-1 + APAP group,(+)-clausenamide + erastin group,Fer-1 + erastin group.(+)-Clausenamide and Fer-1(inhibitor of ferroptosis)were given oral and intraperitoneal injection,respectively.The protective effects of(+)-clausenamide on APAP-induced liver injury were verified in terms of liver function parameters,biochemical parameters and molecular biology(western blotting,q-PCR).MDA kit,4-HNE immunohistochemistry and liquid chromatography-mass spectrometry were used to detect the effect of(+)-clausenamide on lipid peroxidation-related parameters in APAP model,the expression of Ptgs2 m RNA,SLC7A11 and GPX4 protein was detected by q-PCR and western blotting to determine whether(+)-clausenamide protects APAP-induced liver injury by inhibiting the gene and protein expression of ferroptosis-related signaling pathways.At the cellular level,APAP and erastin models were screened using thiazolyl blue(MTT)and lactate dehydrogenase(LDH)release methods,and appropriate cells were selected to study the protective effect of(+)-clausenamide;the hepatoprotective effect of(+)-clausenamide on inhibiting ferroptosis was evaluated and analyzed by high performance liquid-electrochemical detection(HPLC-ECD)and western blotting.Based on this,flow cytometry and confocal imaging were used to analyze the accumulation of lipid peroxidation in the APAP and erastin models.The MDA kit was used to detect the lipid peroxidation product in the cell and the lipid peroxidation level of the ferroptosis hepatocyte model was comprehensively analyzed.Results: The animal level experimental results showed that the serum ALT and AST levels were significantly increased in APAP-induced mice,and the liver tissue was significantly pathologically injured,while the high and low doses of(+)-clausenamide(50 mg/kg and 100 mg/kg)exerted significant protective effects.At the same time,we found that APAP can significantly increase the expression of Ptgs2 m RNA in vivo model of ferroptosis,and reduce the expression of GPX4 protein,while the administration of(+)-clausenamide can be improved.The above experimental results demonstrate that(+)-clausenamide can protect APAP-induced liver injury and suggest that APAP injury may be related to cell death of ferroptosis.In contrast to the erastin-induced positive ferroptosis model,we found that(+)-clausenamide(50 mg/kg)and Fer-1(2.5 ?mol/kg)both significantly reduced APAP-induced ALT and AST levels,improved liver pathological features,reduced the level of lipid peroxidation product MDA,and increased glutathione(GSH)content.The levels of gene and protein expression of ferroptosis-related signaling pathways in(+)-clausenamide treatment hepatic tissue were significantly up-regulated and the expression of 4-HNE was decreased to exert hepatoprotective effect.The above experimental results showed that(+)-clausenamide could inhibit ferroptosis to protect APAP hepatotoxicity.Hepa RG cells were selected in cell experiments,APAP-induced hepatocyte injury model was established and a series of(+)-clausenamide concentrations were screened,and the morphology of the cells was photographed.On the APAP model,5 ?M(+)-clausenamide had a significant protective effect(p < 0.05),and 20 ?M of(+)-clausenamide had a significant protective effect on the erastin model(p < 0.05);in lipid peroxidation assays,we stained drug-treated cells with liperfluo dye and assayed by flow cytometry and found that the lipid peroxidation levels of cells in the 10 m M APAP and 40 ?M erastin model groups were significantly increased,while(+)-clausenamide can reduces the level of lipid peroxidation;then,confocal experiments with C11-BODIPY dye further demonstrated that(+)-clausenamide can reduce lipid peroxidation.At the same time,(+)-clausenamide can significantly regulate ferroptosis-related pathway proteins.In the upstream(erastin)and downstream(RSL3)inducers of ferroptosis,it was found that(+)-clausenamide protects erastin-induced injury and has no protective effect on RSL3-induced death,indicating that the target of(+)-clausenamide is upstream of ferroptosis.However,the target screening of(+)-clausenamide showed that it may act on glutathione-S-transferase(GST),so the GST enzyme activity was examined,the test result showed that(+)-clausenamide had a significant increase in the decrease of APAP and erastin-induced GST activity.Conclusion:(1)APAP induced hepatic dysfunction,hepatic pathological damage,hepatocyte deaths significantly increased,lipid peroxidation products MDA and 4-HNE increased,GSH content decreased,GPX4 protein expression and activity decreased,and increased Ptgs2 m RNA expression(a marker for assessment of ferroptosis in vivo),these data demonstrate that ferroptosis is involved in APAP-induced hepatocyte death;(2)(+)-clausenamide improved APAP and erastin-induced ferroptosis injury,greatly reduced the number of hepatocyte deaths,eliminated the accumulation of lipid peroxidation products,increased GSH content,increased GPX4 protein level and GPX4 enzyme activity,and decreased Ptgs2 m RNA expression,these indicate that(+)-clausenamide inhibits ferroptosis to protect APAP-induced liver injury,and the mechanism of this inhibition may be related to increased GST enzyme activity.