Ferroportin1 Deficiency In Hepatocytes Aggravate Drug-induced Liver Injury Through Ferroptosis

Background and objectionThe liver is the largest detoxification and metabolism organ in human body,and it is also the main target organ of drug injury.At present,drug-induced liver injury is difficult in clinical treatment,which has the characteristics of high incidence and large recurrence,and can be developed into cirrhosis or liver failure.Acute liver injury is the initial link and common pathway in liver diseases,early detection and intervention is an important means to treat drug-induced liver injury.Iron plays a very important role in mammalian cells.As a necessary metal ion for in vivo oxidoreductase reaction,iron is involved in almost all the redox reactions in the body.Ferroportin（FPN1）is the only membrane transporter known to transfer intracellular iron out of the cell,playing an important role in maintaining iron balance between cells and systems.Meanwhile,in addition to the iron transport function,FPN1 has been confirmed to be associated with tumor and fibrosis.Ferroptosis is a recently-recognized form of regulated cell death caused by an iron-dependent accumulation of lipid reactive oxygen species.It is obviously different from other forms of regulatory cell death,such as apoptosis,necrosis and autophagy.The essence of ferroptosis is the metabolic disorder of intracellular lipid oxide,and then abnormal metabolism under the catalysis of iron ions,produces a large amount of lipids,disrupts the intracellular redox balance,attacks biological macromolecules,and triggers cell death.FPN1 is highly expressed in iron-transport-related cells,such as hepatocytes and duodenal epithelial cells,which are closely linked to irontransport,but the association with ferroptosis remains unknow.In drug-induced liver injury,the level of FPN1expression and severity of liver injury and its mechanism are also unclear.In this study,a transgenic mouse model of FPN1-/-hepatocyte specific knockout was constructed at the animal level,and an L02 cell model of FPN1 knockdown was constructed at the cell level.Combined with the drug-induced liver injury model,the role of FPN1 in drug-induced liver injury was studied,providing a theoretical basis for the rational utilization of FPN1in clinic.This study aimed to construct a liver injury model in vivo and vitro through transgenic mice and FPN1 knockdown cell lines,and analyze the correlation between FPN1 and drug-induced liver injury.Combined with ferroptosis inducers and inhibitors,the association between FPN1 and ferroptosis was investigated at animal and cellular levels.Based on the animal model of drug-induced liver injury,the role of ferroptosis in drug-induced liver injury was explored,providing theoretical basis for FPN1 as a marker to prevent drug-induced liver injury in clinic,and providing guidance for rational clinical use.MethodsThis study is divided into three parts:Part 1:1)Indicated knockdown L02 cells were treated with Sorafenib（2-12μM）,Cisplatin（1-14μM）,Epirubicin（3-18μM）,Cyclophosphamide（30-180μM）,APAP（30-150μM）,Paclitaxel（4.375-70n M）for 24 hours and cell viabilities were assayed.2)Liver HE staining and ALT,AST,SOD content in serum in Drug-induced liver injury mouse modle.3)Liver sections were incubated with antibodies against capase3,c-capase3,capase7,c-capase7,capase9,c-capase9,cytochrome c,RIPK1,RIPK3,MLKL,LC3,P62 in WT and FPN1^-/-mouse.Part 21)Indicated knockdown L02 cells were treated with RSL3（6μM）,Sorafenib（5μM）for 24 hours and cell viabilities in the presence or absence of 20μM ferrostatin-1 and 200μM DFO were assayed,Cells were subsequently fifixed and stained with crystal violet.2)L02-SCR and L02-ShRNA cells were treated with RSL3 or Sorafenib for 24 hours,then measured MDA,GSH content and examined using transmission electron microscopy.Mitochondrial length（along the long axis）was measured and summarized.3)Liver HE staining and ALT,AST,SOD content in serum in ferroptosis mouse modle.4)WT and FPN1^-/-primary hepatocytes were treated with Sorafenib or DFO for 24hours,then measured MDA,GSH,NADPH,mRNA content of PTGS2 and examined using transmission electron microscopy.Mitochondrial length（along the long axis）was measured and summarized.Part 31)WT and FPN1-/-primary hepatocytes were treated with CCL4 or APAP for 48hours,then measured MDA,GSH,NADPH,mRNA content of PTGS2.ResultsPart 1 Decreased FPN1 in hepatocytes aggravate drug-induced liver injuryThe viability results of L02 cells using multiple clinical drugs showed that knockdown of FPN1 by lentivirus（L02-ShRNA）enhance cell death（p<0.05）,suggesting that FPN1 may be associated with drug-induced liver injury.Subsequently,in order to confirm the idea,FPN1 knockout mice were used in this study,the models of drug-induced liver injury were constructed in transgenic mice by CCL4 and APAP.Hepatic HE staining showed that the hepatic pathological injury of FPN1^-/-mice were more serious than that of WT group,ALT and AST level were increased and SOD content was decreased in serium（P<0.05）.This results verified that the decreased expression of FPN1 in hepatocytes aggravate drug-induced liver injury.Meanwhile,immunohistochemical results showed that there was no obvious phenomenon of apoptosis,necrosis and autophagy in the liver.Collectively,these findings suggest that decreased FPN1 in hepatocytes aggravate drug-induced liver injury.Part 2 Decreased FPN1 induced ferroptosis in hepatocytesCombining the association of FPN1 with iron transport,we hypothesized that FPN1aggravate drug-induced liver injury through ferroptosis.To investigate the interaction of FPN1 and ferroptosis,growth inhibition and crystal violet staining were used,The results showed that compared to L02-SCR,the ferroptosis inducer RSL3 and Sorafenib could aggravate the death of L02-ShRNA while being reversed to some extent by ferroptosis inhibitors,indicating a negative correlation between FPN1 and ferroptosis at the cell level（P<0.05）.Furthermore,the transmission electron microscopy showed the mitochondria of L02-ShRNA decreased significantly.Based on previous reports,malondialdehyde（MDA）content can be used as a measure of lipid peroxidation.Compared with L02-SCR,L02-ShRNA had increased levels of lipid peroxidation and and reduced GSH content,which indicated FPN1 was associated with ferroptosis.Next,we investigated whether FPN1 knockout induces ferroptosis in vivo by measuring ferroptosis in transgenic mice.To establish a mouse model of ferroptosis,WT and FPN1-/-mice were fed Sorafenib or DFO,Similarly,knockout of FPN1 promoted sorafenib-induced growth inhibition with increased ferroptotic events including GSH depletion,Mitochondrial length reduction,and an increase of lipid MDA.While ferroptosis inhibitors（DFO）significantly reversed growth inhibition in the absence of FPN1.Serum ALT levels in FPN1^-/-mice were higher than in wild-type mice and DFO-treated mice had significantly lower ALT levels compared to untreated FPN1^-/-mice.Taken together,these findings indicate that FPN1 downregulation induces ferroptosis.Part 3 Ferroptosis induction in mouse models of drug-induced liver injuryDrug-induced liver injury（DILI）is the common condition with drug-associated tissue damage in clinic.To investigate whether ferroptosis is associated with DILI,we measured ferroptosis in two classic mouse models of DILI（CCL4 and APAP）.Both CCL4and APAP mice had significantly higher hepatic MDA levels,as well as lower hepatic GSH and NADPH content,compared to both wild-type and FPN1^-/-mice with PBS.On the other hand,significant difference was found between wild-type and FPN1^-/-mice injected with CCL4 and APAP.Taken together,these findings indicate that DILI induces ferroptosis in mice.Conclusion:1.Decreased FPN1 in hepatocytes aggravate drug-induced liver injury.2.Decreased FPN1 induced ferroptosis in hepatocytes.3.Ferroptosis induction in drug-induced liver injury.