Nrf2 Regulates Hepatocytes Ferroptosis In Sepsis Through IL-6/HAMP-Hepicidin Pathway

Sepsis is a life-threatening organ dysfunction caused by the host’s dysregulated response to infection.In severe cases,it can develop into septic shock or even multiple organ failure.The rich blood supply and important functions such as metabolism,detoxification,and immunity of liver makes it one of the target organs that are easily damaged by inflammatory factors in sepsis.Dysfunction after liver damage will affect the prognosis of patients with sepsis.Inflammatory cells such as macrophages are over-activated during sepsis,causing the release of inflammatory factors,such as interleukin-6(Interleukin-6,IL-6),interleukin-1β(Interleukin-1β,IL-1β)and tumor necrosis factor-α(Tumor necrosis factor-α,TNF-α),into the blood in large quantities.Recent studies have demonstrated that,in addition to apoptosis and pyroptosis,there is also ferroptosis in septic liver injury.Ferroptosis is a type of cell death caused by iron-dependent lipid peroxidation and the accumulation of reactive oxygen species.Previous studies have shown that ferroptosis is involved in the process of sepsis liver injury.In addition,patients with sepsis have abnormal iron metabolism.Hepcidin is increased and serum iron is decreased.Hepcidin is mainly synthesized and secreted by hepatocytes.Hepcidin can internalize and degrade the iron export protein 1(Ferroportinl,FPN1)of hepatocyte membrane,so that iron cannot be transported out of hepatocytes,resulting in iron overload in hepatocytes.In inflammatory diseases such as sepsis,IL-6 could increase the transcription of hepcidin-encoding gene(HAMP).It is not yet reported that,during the development of liver injury in sepsis,whether the iron overload and liver ferroptosis are affected by IL-6-induced HAMP transcription and hepcidin secretion with subsequent FPN1 protein degradation.Nuclear factor erythroid-2 related factor 2(Nrf2)is a transcription factor that acts on the antioxidant response elements(AREs)of target genes and has antioxidant effects.At the same time,it can act on the promoter regions of inflammatory factors such as IL-6 and IL-1β inhibit their transcription,and exert an anti-inflammatory effect.This study aims to explore whether the up-regulation of Nrf2 during sepsis could increase the protein content of FPN1 by inhibiting the IL-6/HAMP-hepcidin pathway,thereby promoting iron transport in hepatocytes and reducing iron overload and ferroptosis.Objective:To investigate the effect of Nrf2 on hepatic ferroptosis in sepsis and its possible molecular mechanism.Methods:C57/BL6 mice were used to establish an animal model of sepsis liver injury by cecal ligation and puncture(CLP).Lipopolysaccharide(LPS)stimulated RAW264.7 cells were used to mimic the activation state of macrophages in sepsis.RAW264.7 cell supernatant was applied to treat primary mouse hepatocytes for mimicing of in vivo situation of sepsis-activated macrophage secretion of inflammatory factors and the subsequent effects on hepatocytes.Animals were randomly divided into four groups,sham operation group(Sham),CLP group,CLP+SFN(Sulforaphane,a Nrf2 agonist)group,CLP+DFO(Deferoxamine,an iron chelator)group.IL-6 mRNA,HAMP mRNA,FPN1 protein,non-heme iron,serum IL-6,hepcidin,and ferroptosis-related indicators,including PTGS2 mRNA,NADPH,MDA were detected by using western blotting,real-time fluorescent quantitative PCR,enzyme-linked immunosorbent assay,hematoxylin-eosin staining and other methods.Liver function indicators ALT and AST and the morphological changes of liver tissue were also observed.After the macrophages were stimulated with LPS and with or without Nrf2 agonist SFN,the supernatant of the macrophage culture medium was collected to stimulate primary hepatocytes.By using cellular immunofluorescence,real-time fluorescent quantitative PCR,western blotting,live cell staining and other related methods,the translocation of Nrf2,the transcription and expression of IL-6 in macrophages,the transcription and expression of HAMP and hepcidin,as well as the expression of FPN1 in primary hepatocytes were detected.The levels of ferroptosis indicators,such as reactive oxygen species,PTGS2 mRNA and ferrous iron ions were measured as well.Results:1.Sepsis causes liver injury,iron overload and ferroptosisCompared with Sham group,the arrangement of the hepatic cord structure in the liver tissue of mice in CLP group was disordered,the hepatic sinusoids were narrowed,and the inflammatory cells were infiltrated.Serum levels of ALT and AST of CLP mice were significantly increased.These results suggest that CLP leads to morphological and functional impairment of liver tissue.Compared with Sham group,the content of non-heme iron in the liver tissue of CLP mice was significantly increased,and the levels of NADPH,MDA and PTGS2 mRNA were also significantly increased.Compared with CLP group,the liver tissue non-heme iron、serum ALT and AST content in CLP+DFO group was significantly decreased,and the levels of NADPH,MDA and PTGS2 mRNA were significantly increased.The electron microscope results of liver tissue showed that,compared with Sham group,the mitochondria of hepatocytes in CLP group shrank,and the mitochondrial cristae decreased or disappeared,while CLP+DFO group had no significant change.These results suggest that sepsis causes iron overload and ferroptosis in mouse liver tissue.2.IL-6/HAMP/FPN1 pathway is involved in hepatic iron overload in sepsisAt the animal level,compared with Sham group,the mRNA contents of IL-6 and HAMP in the liver tissue of mice in CLP group were significantly increased.Serum IL-6 and hepcidin contents in mice of CLP group were significantly increased,while the content of FPN1 protein in the liver tissue was significantly reduced.At the cellular level,the results showed that in macrophage RAW264.7,the expression of IL-6 mRNA and protein were significantly increased after LPS stimulation.Compared with primary hepatocytes in control group or macrophage control culture supernatant-treated group(Control conditioned medium,Ctrl-CM group),the levels of HAMP mRNA and hepcidin content in primary hepatocytes were significantly increased,FPN1 protein level was decreased and intracellular Fe2+level were elevated in LPS-stimulated macrophage culture supernatant-treated group(LPS conditioned medium,,LPS-CM).Compared with the LPS-CM group,intracellular Fe2+ leve of lprimary hepatocytes were decreased in co-treated with LPS-CM and deferoxamine group(LPS-CM+DFO group).These results suggest that the iron overload may be related to the activation of IL-6/HAMP-hepcidin pathway in macrophages/hepatocytes and the subsequent decrease of FPN1 content in hepatocytes,leading to the impairment of iron transport in hepatocytes.3.Nrf2 regulates IL-6/HAMP/FPN1 pathway to ameliorate iron overload and ferroptosis in hepatocytesAt the cellular level,compared with control,Nrf2 protein content in the cytoplasm and nucleus of RAW264.7 cells treated with LPS was significantly reduced.Compared with those in LPS-treated group,the levels of IL-6 mRNA and protein expression in RAW264.7 cells treated with LPS+SFN were significantly decreased.Compared with those in LPS-CM-treated group,the HAMP mRNA and hepcidin contents in LPS+SFN-CM-treated primary hepatocytes were significantly decreased,while FPN1 protein level in the hepatocytes was increased,and the levels of ferroptosis indicators,such as reactive oxygen species and ferrous iron ions were significantly reduced in primary hepatocytes treated with culture supernatant from LPS+SFN-stimulated macrophage(LPS+SFN conditioned medium,LPS+SFN-CM).At the animal level,compared with those in CLP group,the expression of Nrf2 protein was increased,and the IL-6 and HAMP mRNA levels were significantly decreased in the liver tissue of mice in CLP+SFN group(administered with SFN after CLP).The levels of IL-6 and hepcidin in the serum were significantly decreased,while FPN1 protein level in liver tissue was increased.The content of non-heme iron was significantly decreased,the levels of NADPH,MDA and PTGS2 mRNA were also significantly decreased in CLP+SFN group.The normal mitochondrial morphology and structure in hepatocytes were preserved.These data suggest that the up-regulation of Nrf2 could improve liver ferroptosis in septic mice,which may be related to the inhibition of IL-6/HAMP-hepcidin pathway in macrophages/hepatocytes and the subsequent increase of FPN1 content in hepatocytes and the final mitigation of iron overload.Conclusion1.Sepsis induced inflammatory cell infiltration and dysfunction,and iron overload and ferroptosis in the liver;2.The IL-6/HAMP-hepcidin/FPN1 pathway is involved in liver iron overload in sepsis;3.Sepsis reduced Nrf2 expression and activity in macrophages,activation of Nrf2 can inhibit the IL-6/HAMP-hepcidin pathway,maintain the level of FPN1,and improve liver iron overload and ferroptosis.