| Background:Sepsis is a systemic inflammatory response and progressive organ injury syndrome caused by severe infection with a high morbidity and mortality,the pathophysiology of which mainly includes inflammation,immune dysfunction and coagulopathy.Although studies have confirmed that the immunosuppression in the advanced disease stage is the leading death cause of patients with sepsis,septic shock caused by"cytokine storm"and cardiac dysfunction is also an important death cause of young patients with toxic shock syndrome or Neisseria meningitidis infection in the early stage of sepsis.As an critical pump organ of the human body,the heart plays a vital role in the occurrence and development of sepsis.In recent years,with the rapid development of Doppler tissue imaging,radionuclide myocardial perfusion imaging and hemodynamic monitoring technology,the definition of sepsis-induced cardiomyopathy（SIC）has gradually become clear.SIC is defined as a global（systolic and diastolic）but reversible cardiac function caused by myocardial depressants released from pathogen and host during septic shock.The pathogenesis of SIC mainly involves preload deficiency during distributed shock,cardiopulmonary interaction during mechanical ventilation,and right ventricular-pulmonary artery coupling during vascular compression.A retrospective cohort study reported that SIC occurred in 13.8%of patients with sepsis,and SIC can be regarded as a prognostic indicator in patients with sepsis.The mortality of patients with sepsis can be as high as 70-90%when SIC occurs,however,the mortality is only 20%when there is no SIC.Therefore,it is of great significance to further explore and clarify the mechanism underlying SIC and find molecular targeted therapeutic drugs for the clinical treatment of SIC.Ferroptosis is a new type of cell death pattern discovered in recent years.Unlike other regulated cell death（RCD）like apoptosis,necroptosis,autophagy,and pyroptosis,ferroptosis is an iron-and reactive oxygen species（ROS）-dependent oxidizing RCD,which is characterized by the reduction or vanishing of mitochondria crista,presence of smaller mitochondria with condensed mitochondrial membrane densities,as well as outer mitochondrial membrane rupture.Ferroptosis can be induced by specific chemical compounds such as Erastin,Ras-selective lethal small molecule 3（RSL3）,butionine sulfoximine,sulfasalazine,sorafenib,as well as artesunate.Mechanistically,the activation of mitochondrial voltage-dependent anion channels,mitogen-activated protein kinases（MAPK）and endoplasmic reticulum stress,and the inhibition cystine/glutamate antiporter can induce ferroptosis,the process of which is characterized by the accumulation of lipid peroxidation products and lethal reactive oxygen species（ROS）triggered by iron metabolism.Recent studies have proposed that ferroptosis can be regulated by nuclear receptor coactivator4（NCOA4）-mediated autophagy,which was also called ferritinophagy.NCOA4 is in essence a selective autophagy cargo protein,which could release ferrous iron and disrupt intracellular iron homeostasis by indirectly degrading ferritin via autophagy,and finally induce ferroptosis.The roles of ferroptosis are being uncovered recently.In doxorubicin-induced cardiotoxicity,Nrf2-mediated heme degradation can lead to systemic non-heme iron accumulation in mice.A large amount of free iron enters the mitochondria of cardiomyocytes and induces an increase in lipid peroxidation of mitochondrial membranes,eventually leading to ferroptosis of cardiomyocytes.In the mice model of myocardial ischemia/reperfusion（I/R）injury,ferroptosis inhibitor（Fer-1）or iron chelator（dxrazoxane）can significantly reduce myocardial infarct size and cardiac injury.Studies have proved that in sepsis induced cardiac injury,oxidative stress in cardiomyocytes is activated and a large amount of ROS is generated,but the role of ferroptosis and its potential mechanisms have not been reported.The definition of ferroptosis is still controversial for the reason that the mechanism of ferroptosis is very complicated.The signaling pathways and mechanisms are different under different types of tissues or cells within different stimulation conditions.This study intends to establish a mice model of sepsis-induced cardiac injury and a cell model of H9c2 cardiomyocyte injury induced by lipopolysaccharide（LPS）,and to observe the effects of ferroptosis inhibitors and agonists on cardiac function and cardiac injury in mice.The mechanism of ferroptosis during sepsis-induced cardiac injury was further explored in order to provide new targets and therapy idea for the prevention and treatment of SIC in the future.Methods:Part one:In vivo experiments:In this study,male C57BL mice（8-23 weeks old）weighing23-25 g were used.LPS（10 mg/kg）was injected intraperitoneally to establish a mice model of sepsis-induced cardiac injury.The experimental groups were:Saline group（Control）,LPS group,LPS+Fer-1 group,and LPS+dexrazoxane group（LPS+DXZ）.Fer-1 was injected at a dose of 1 mg/kg/d and DXZ was injected at a dose of 20mg/kg/d for 2 consecutive days before LPS injection.After LPS injection for 12 hours,mice heart and peripheral blood were collected.The m RNA and protein expression levels of PTGS2 in the cardiac tissue in mice were detected using real-time quantitative PCR and western blotting.The protein expression of 4-HNE in the cardiac tissue in mice was detected using immunohistochemical staining.The expression of MDA in the serum and cardiac tissue were detected using commercial kits.Tissue and serum non-heme iron levels were measured using the chromogen method.Iron content cardiac tissue was detected using Prussian blue staining.Mitochondrial morphology and size of cardiomyocytes by transmission electron microscopy.The ROS level in the cardiac tissue was detected by ROS fluorescent probe.Cardiac function was assessed via echocardiography.Lactate dehydrogenase（LDH）kit and aspartate aminotransferase（AST）ELISA kits were used to detect the serum LDH and AST contents of mice.Creatine kinase isoenzymes（CK-MB）level in serum was detected using automatic biochemical analyzer.Cardiac tissue morphology and inflammatory cell infiltration conditions were assessed by H&E staining.The protein expression levels of CD45 and CD68 in cardiac tissue were detected using immunohistochemical staining.The m RNA expression levels of inflammatory factors including TNF-α,IL-1β,HMGB1,and MCP-1 in cardiac tissue was detected using real-time quantitative PCR.The percent survival was calculated within 7 days after LPS injection.In vitro experiments:The cell line used in this study was rat H9c2 cardiomyocyte.H9c2 cardiomyocytes were randomly divided into 5 groups:Control group（PBS）,LPS group,LPS+Erastin group,LPS+Sorafenib group and LPS+Fer-1 group.The dose of LPS,erastin,sorafenib and Fer-1 was 10μg/m L,5μM,and 1μM and 1μM.Cell or medium samples were collected 6 hours after stimulation for subsequent detection.The m RNA and protein expression levels of PTGS2 in cardiomyocytes were detected using real-time quantitative PCR and western blotting.Cell morphology of cardiomyocytes were observed using phase contrast microscope.Cell viability was detected using CCK8 assay kit.The malondialdehyde（MDA）and reduced nicotinamide adenine dinucleotide phosphate（NADPH）activities of cardiomyocytes were detected using commercial oxidative stress assay kits.LDH content in cell culture medium was detected using LDH cytotoxicity detecting kit.Flow cytometry and BODIPY 581/591 fluorescent probe staining were used to detect the levels of ROS and lipid peroxidation of cardiomyocytes,respectively.JC-1staining was used to evaluate the mitochondrial membrane potential of cardiomyocytes.Part two:In vitro experiments:The cell line used in this study was rat H9c2 cardiomyocyte.To begin with,the protein expression levels of Ferritin and NCOA4 were detected using western blot after LPS（10μg/m L）stimulation for 0,3,6,9,and 12 hours.The intracellular iron content of H9c2 was assessed using colorimetric method after LPS stimulation for 3,6,9 and 12 hours.H9c2 cardiomyocytes were then randomly divided into 4 groups:Control si RNA group,LPS+Control si RNA group,NCOA4si RNA group,and LPS+NCOA4 si RNA.H9c2 cardiomyocytes were transfected with NCOA4 double-stranded small-molecule RNA to knock down the NCOA4 gene in vitro.Control si RNA group was transfected with negative control si RNA as a blank control.After 36 hours of NCOA4 transfection,LPS stimulation was performed.Cell or medium samples were collected after 6 hours for subsequently detection.PGSK fluorescent probe was used to label the Fe2+of cardiomyocytes.Cell morphology of cardiomyocytes were observed using phase contrast microscope.Cell viability was detected using CCK8 assay kit.LDH content in cell culture medium was detected using LDH cytotoxicity detecting kit.BODIPY 581/591 fluorescent probe staining was used to detect the levels of lipid peroxidation.Western blot was used to detect the protein expression levels of Ferritin and NCOA4 in 4 groups.The binding of Ferritin to NCOA4 was assessed using co-immunoprecipitation after LPS stimulation for 0and 6 hours.In addition,the autophagy inhibitor 3-Methyladenine（3-MA）（5μM）and Atg5si RNA were used to inhibit cell autophagy.To further exclude intracellular ferritin degradation from the proteasome degradation pathway,MG132（20μM）was used to inhibit intracellular proteasomes.Cell viability,LDH content in cell culture medium,cell morphology and levels of lipid peroxidation were detected.Part three:In vitro experiments:The cell line used in this study was rat H9c2 cardiomyocyte.H9c2 cardiomyocytes were randomly divided into 4 groups:Control si RNA group,LPS+Control si RNA group,SFXN1 si RNA group,and LPS+SFXN1si RNA.H9c2cardiomyocytes were transfected with SFXN1 double-stranded small-molecule RNA to knock down the SFXN1 gene in vitro.Control si RNA group was transfected with negative control si RNA as a blank control.After 24 hours of SFXN1 transfection,LPS stimulation was performed.Cell or medium samples were collected after 6 hours for subsequently detection.Real-time quantitative PCR,western blot and immunohistochemical staining were used to detect the m RNA and protein expression levels of SFXN1 in cardiomyocytes.Iron chelator deferoxamine was used to eliminate the intracellular Fe2+.Cell viability,LDH content in cell culture medium,cell morphology and levels of lipid peroxidation were detected.Additionally,Mito-Ferro Green fluorescent probe and Mito-SOX probe were used to label mitochondrial iron as well as mitochondrial ROS.In vivo experiments:In this study,male C57BL mice（8-23 weeks old）weighing23-25 g were used.LPS（10 mg/kg）was injected intraperitoneally to establish a mice model of sepsis-induced cardiac injury.The experimental groups were:Saline group（Control）,LPS group and LPS+Deferiprone group（LPS+DEF）.DEF was dissolved in drinking water（1 mg/ml）for 14 consecutive days before LPS injection.After LPS injection for 12 hours,mice heart and peripheral blood were collected.Immunohistochemical staining was used to detect the protein expression of Ferritin,NCOA4,SFXN1 and PTGS2 in cardiac tissue of control group and LPS group.Cardiac function,mitochondrial morphology of cardiomyocytes,the protein expression of 4-HNE,the m RNA expression levels of inflammatory factors including TNF-αand IL-1β,serum LDH content and ROS level in the cardiac tissue were detected of LPS group and LPS+DEF group.Finally,the percent survival was also calculated within 7 days after LPS injection.Results:Part one:1.In the mice model of sepsis-induced cardiac injury,the m RNA and protein expression levels of PTGS2 were significantly higher after LPS injection for 12 hours than those in control group,however,the m RNA and protein expression levels of PTGS2 in LPS+Fer-1 group and LPS+DXZ group were significantly lower than those of LPS group.Compared with the mice in LPS group,the levels of 4-HNE,serum MDA,and cardiac MDA in LPS+Fer-1 group and LPS+DXZ group significantly decreased.Compared with the mice in control group,LPS injection can significantly increase the content of non-heme iron in cardiac tissue and serum.The content of non-heme iron in cardiac tissue in LPS+DXZ group was significantly lower than that in LPS group,while the content of non-heme iron in serum was significantly higher than that in LPS group.Prussian blue staining showed that iron density of cardiac tissue in LPS group was significantly increased,however,iron density of cardiac tissue in LPS+Fer-1 group and LPS+DXZ group was significantly lower than that in LPS group.Transmission electron microscopy results showed that the mitochondrial Flameng score of cardiomyocytes in LPS+Fer-1 group and LPS+DXZ group was significantly lower than that in LPS group.Additionally,the ROS fluorescence density of cardiomyocytes in LPS+Fer-1 group and LPS+DXZ group was significantly lower than that in LPS group.2.After LPS stimulation for 6 hours,H9c2 cardiomyocyte viability in LPS group was significantly reduced,and LDH content in the medium was significantly increased.H9c2 cardiomyocyte activity in LPS+Erastin group and LPS+Sorafenib group was significantly reduced compared to LPS group,and LDH content in the medium was significantly increased.However,cell viability in LPS+Fer-1 group was significantly increased,and LDH content in the medium was significantly reduced compared with LPS group.The m RNA and protein levels of PTGS2 in LPS+Erastin group and LPS+Sorafenib group were significantly higher than those in LPS group,and the expression of PTGS2 in LPS+Fer-1 group was significantly lower than that in LPS group.Flow cytometry and BODIPY 581/591 probe staining results showed that the levels of ROS and lipid peroxidation in LPS+Erastin group and LPS+Sorafenib group were significantly higher than that in LPS group,while oxidative stress in LPS+Fer-1 group was significantly inhibited compared with LPS group.The PGSK fluorescence intensity in LPS+Erastin group and LPS+Sorafenib group was significantly lower than that in LPS group,while the fluorescence intensity was significantly increased in LPS+Fer-1 group.The mitochondrial damage score in LPS+Erastin group and LPS+Sorafenib group was significantly higher than that in LPS group,while the mitochondrial damage score in LPS+Fer-1 group was significantly reduced compared with LPS group.3.Compared with the mice in control group,the 7-day survival rate in LPS group was significantly reduced,while the 7-day survival rates in the LPS+Fer-1 group and LPS+DXZ group were significantly higher than that in LPS group.The levels of cardiac injury biomarkers including LDH,CK-MB as well as AST in LPS+Fer-1group and LPS+DXZ group were significantly lower than those in LPS group.Cardiac function of the mice in LPS+Fer-1 group and LPS+DXZ group was significantly improved compared with the mice in LPS group,which was mainly evidenced by a marked increase in left ventricular ejection fraction and fraction shorting.Compared with LPS group,the infiltration level of inflammatory cells and the m RNA levels of inflammatory factors in cardiac tissue were significantly reduced in LPS+Fer-1 group and LPS+DXZ group.Part two:1.After LPS stimulation for 0,3,6,9,and 12 hours,the protein level of Ferritin in H9c2 cardiomyocytes decreased in a time-dependent manner,while NCOA4expression level increased at first and then decreased,reaching a peak after stimulation for 9 hours.After LPS stimulation for 3,6,9 and 12 hours,the iron content in H9c2 cardiomyocytes increased in a time-dependent manner.After LPS stimulation for 6 hours,the number of intracellular Fe2+,LDH content in the culture medium as well as lipid peroxidation level significantly increased while cell viability significantly decreased.However,NCOA4 knockdown can significantly reduce the number of intracellular labile iron pools,LDH content in the culture medium as well as lipid peroxidation level.Western blot showed that NCOA4 knockdown can significantly inhibit the degradation of intracellular Ferritin.Co-immunoprecipitation results showed that there was only a small amount of binding between intracellular NCOA4 and Ferritin when without LPS stimulation while the interaction between NCOA4 and Ferritin significantly enhanced after LPS stimulation for 6 hours.2.After LPS stimulation for 6 hours,3-MA or Atg5 knockdown can significantly inhibit LPS-induced decrease of Ferritin protein expression,enhance cell activity,reduce LDH content in the medium,and inhibit lipid peroxidation.After LPS stimulation for 6 hours,the inhibition of intracellular proteasome by MG132 did not block the protein degradation of intracellular Ferritin,and at the same time,it had no significant effects on cardiomyocytes damage and lipid peroxidation.Part three:1.After LPS stimulation for 6 hours,the m RNA and protein levels of SFXN1significantly increased,however the NCOA4 knockdown had no significant effect on the alteration of SFXN1 expression induced by LPS.Iron chelator deferoxamine significantly inhibited the increased expression of SFXN1 induced by LPS.After LPS stimulation for 6 hours,SFXN1 knockdown can significantly improve cell activity,alleviate cell damage,and inhibit lipid peroxidation.Meanwhile,NCOA4 knockdown can significantly inhibit LPS-induced mitochondrial iron overload and mitochondrial ROS production.2.Compared with control group,the protein expression level of Ferritin in cardiac tissue in LPS group was significantly reduced,while the levels of NCOA4,SFXN1and PTSG2 were significantly increased.3.Compared with LPS group,the cardiac function and mitochondrial damage of the mice in LPS+DEF group was significantly improved,the levels of oxidative stress and inflammatory factor were significantly reduced,meantime the 7-day survival rate was also significantly improved.Conclusion:（1）After LPS stimulation,ferroptosis in cardiac tissue and H9c2 cardiomyocytes is activated,the inhibition of which can significantly improve cardiac function and survival rate.（2）NCOA4-mediated ferritinophagy may be one of the causes of ferroptosis induced by LPS in cardiomyocytes.（3）Ferritinophagy-induced ferroptosis in cardiomyocytes is related to the activation of mitochondrial membrane anchoring protein SFXN1. |
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