The Role And Mechanism Of Itaconate In Sepsis-Induced Acute Lung Injury

Background: Sepsis is a syndrome of systemic inflammatory response secondary to severe infection,with a high incidence and mortality rate,affecting approximately 19 million people worldwide each year.Sepsis can cause dysfunction of multiple organs via the circulatory system。As an important site for oxygenation and gas exchange of blood,lung is highly susceptible to infection and impact,appearing acute lung injury(ALI)and even progression to acute respiratory distress syndrome(ARDS),leading to severe respiratory failure.As the cascade from ALI to ARDS is a complex process,its pathogenesis and mechanisms are numerous and complicated.Up to now,targeted prevention and treatment of ALI remains quite limited.Current studies have shown that during the pathophysiology of sepsis-induced ALI,Gramnegative bacterial endotoxins,represented by lipopolysaccharide(LPS),activate the natural immune response through various molecular mechanisms,activating alveolar macrophages and inducing the release of inflammatory mediators and chemokines,while further recruiting and activating immune cells,mainly macrophages and neutrophils,in large numbers.Although the over-activated inflammatory response can clear infectious agents,also causing severe tissue damage.Numerous studies have shown that a large number of synthesis and release of inflammatory factors,increased production of ROS and oxidative stress are the most critical factors that exacerbate lung injury.The exploration of drugs or genetic intervention targets with anti-inflammatory and anti-oxidative stress activities is important for the clinical management of sepsis-induced ALI.Itaconate is a metabolite synthesized in the tricarboxylic acid cycle by the action of aconitate decarboxylase 1.4-Octyl Itaconate(4-OI),an exogenous itaconate derivative with cell permeability,has been found to increase glutathione(GSH)levels and reduce ROS levels,significantly improving cerebral ischaemia-reperfusion-induced neurological dysfunction.It also reduced the inflammatory response in a mouse model of urate-induced acute peritonitis by reducing the secretion of interleukin-1β(IL-1β).In addition,4-OI also alkylates multiple cysteine residues of Kelch-like ECH-associated protein 1(KEAP1),resulting in its inability to interact with nuclear factor erythroid 2-related factor 2(NFE2L2 or Nrf2)and its degradation,thereby increasing Nrf2 protein and promoting its nuclear transcription to exert an anti-oxidative stress effect.Due to excessive inflammatory activation,massive release of inflammatory factors and increased oxidative stress are the main factors exacerbating acute lung injury,and many studies have shown that itaconate and its exogenous derivatives can exert anti-inflammatory and antioxidative stress effects,thus itaconate and its derivatives have great potential to combat sepsisinduced ALI.Alveolar macrophages are one of the predominant immune cells in the lungs and act as the first line of defense against pathogenic microorganisms.When the lungs are stimulated by pathogenic microorganisms,resident alveolar macrophages are immediately activated,synthesis and release inflammatory cytokines,chemokines and virulence molecules in large numbers,and recruit large numbers of neutrophils and macrophages from the peripheral blood into the lung tissue.These recruited macrophages are activated and release pro-inflammatory mediators again during the immune defense process,further recruiting inflammatory cells and mediating tissue damage,eventually leading to a positive feedback loop between macrophage death and tissue inflammation,resulting in an excessive accumulation of inflammatory cytokines and macrophages and aggravating lung tissue damage.Numerous studies have confirmed the involvement of multiple classical cell death modalities in sepsis-induced ALI,such as pyroptosis,apoptosis,and necroptosis.Targeted modulation of alveolar macrophages death signaling by pharmacological or genetic interventions is increasingly recognized as a promising strategy for the treatment of ALI/ARDS.Ferroptosis is a novel form of iron-dependent cell death discovered in recent years.Unlike other regulated cell death ways such as apoptosis,autophagy,pyroptosis and necroptosis,ferroptosis is a form of regulated cell death driven by phospholipid peroxidation,whose most biochemical features are mainly iron accumulation and increased lipid peroxidation.When intracellular redox homeostasis is disrupted,ROS accumulate in excess and lipids in cell membranes and organelle membranes are peroxidized.Polyunsaturated fatty acids contain a large number of methylene groups,and the hydrogen atoms in them can easily react with intracellular ROS to form lipid radicals and peroxyl radicals,eventually forming lipid peroxidation.Nrf2,an important intracellular redox transcriptional regulator,has been shown to directly transcribe and regulate many ferroptosis associated genes.In view of previous studies,since 4-OI can exert anti-inflammatory and anti-oxidative stress effects and has the ability to regulate Nrf2,and since ferroptosis is a cell death closely related to oxidative stress and is also tightly regulated by Nrf2,it is worth exploring whether 4-OI can regulate ferroptosis through Nrf2 and exert a protective effect against lung injury.Part I: Protective effects of 4-OI in sepsis-induced ALIObjective: 4-OI is an exogenous derivative of the anti-inflammatory metabolite itaconate produced by macrophages,and previous studies have shown that 4-OI has anti-inflammatory and antioxidant activity.This part of the study focused on investigating the protective effect of 4-OI in sepsis-induced ALI.Methods: Male C57BL/6 mice aged 6-8 weeks were used to establish a mouse model of ALI from sepsis by intraperitoneal injection of LPS(10 mg/kg).The experimental groups were Control group,4-OI group,LPS group and 4-OI+LPS group.4-OI(25 mg/kg)was administered intraperitoneally 2 h before LPS injection and 12 h after LPS injection,all mice were anesthetized and executed uniformly.HE staining was used to detect lung tissue morphology and lung injury score,Masson staining was used to detect collagen deposition in lung tissue,and immunofluorescence staining was used to detect macrophage infiltration in lung tissue.Results: In the LPS-induced ALI mouse model,compared with the Control group,the alveolar integrity of the LPS group was destroyed,the alveolar wall was significantly thickened,the interstitial edema and collagen deposition increased,and the tissue damage was obvious.4-OI+LPS group showed significantly less lung damage compared with the LPS group.The lung wet to dry weight ratio showed that the level of pulmonary oedema was significantly reduced in the 4-OI+LPS group compared to the LPS group.The levels of inflammatory factors IL-1β,TNF-α and IL-6 were significantly lower in the 4-OI+LPS group compared to the LPS group.Immunofluorescence staining results showed that 4-OI treatment significantly reduced macrophage infiltration in lung tissue.Conclusion: The itaconate derivative 4-OI significantly reduced LPS-induced ALI,lung tissue inflammatory factor levels and macrophage infiltration.Part II: 4-OI inhibits ferroptosis in sepsis-induced ALI through regulating Nrf2Objective: Ferroptosis,a form of death caused by lipid peroxidation,has been shown to play an important role in ALI and is regulated by Nrf2,and 4-OI can inhibit Nrf2 degradation.In this part of the study,we investigated whether 4-OI could inhibit ferroptosis in ALI by regulating Nrf2.Methods: The mouse model of ALI and experimental grouping were as in Part I.Bioinformatics was used to analyze the differential expression of the genes related to ferroptosis in macrophages after treatment with itaconate(ITA).The m RNA and protein expression levels of PTGS2,GPX4,Nrf2,GCLM,HO-1 and SLC7A11 in mouse lung tissues were measured by real-time quantitative PCR and western blotting.The levels of tissue iron,MDA,GSH/GSSG ratio in the lung tissues of the mice were measured using the kit.Immunohistochemical staining was used to detect the expression of 4-HNE in the lung tissues of each group of mice,and immunofluorescence was used to detect ROS levels in the lung tissues of mice.Results: Differential gene enrichment analysis showed that several ferroptosis inhibitory genes were significantly upregulated in macrophages in the ITA+LPS group compared to the LPS group.In the LPS-induced ALI mouse model,the m RNA and protein expression levels of PTGS2 in the LPS group were significantly higher than those in the Control group,whereas the PTGS2 levels in the 4-OI+LPS group were significantly lower compared with those in the LPS group.The trend of GPX4 protein and m RNA levels in the LPS and 4-OI+LPS groups was opposite to that of PTGS2 and GPX4 significantly decreased in LPS group.In addition,the levels of iron and MDA in lung tissue were significantly higher in the LPS group compared to the Control group,while the levels of iron and MDA in the 4-OI+LPS group were significantly lower compared to the LPS group.Immunohistochemical results showed the same trend in 4-HNE levels and MDA levels.Further investigation into the mechanism of inhibition of ferroptosis by 4-OI revealed that the m RNA level of Nrf2 in lung tissue of mice in the LPS group was significantly decreased compared to the Control group,and there was no significant change in the 4-OI+LPS group compared to the LPS group,while Western blot showed that the Nrf2 protein level in lung tissue of the 4-OI+LPS group was significantly increased compared to the LPS group.In addition,the levels of GCLM,HO-1 and SLC7A11,the target genes of Nrf2,were significantly higher in the 4-OI+LPS group compared to the LPS group.The results of immunofluorescence suggested that LPS stimulation significantly increased ROS levels in mouse lung tissue,while ROS levels in the 4-OI+LPS group were significantly lower compared to the LPS group.Conclusion: 4-OI significantly increased the protein level of Nrf2 and promoted the transcriptional expression of its target genes GCLM,HO-1,GPX4 and SLC7A11,increased the synthesis of reduced GSH,reduced ROS levels and inhibited LPS-induced ferroptosis in ALI in mice.Part III.4-OI attenuates ALI by inhibiting LPS-induced ferroptosis in macrophages through Nrf2Objective: Previous results suggest that 4-OI inhibits sepsis-induced ferroptosis in ALI by increasing Nrf2.As an anti-inflammatory metabolite produced by macrophages,it remains to be investigated whether 4-OI acts directly on alveolar macrophages to exert protective effects against lung injury.In addition,whether 4-OI inhibits LPS-induced ferroptosis in THP-1 cells and mouse ALI through an Nrf2-dependent pathway still needs to be further verified at the genetic level.Methods: A model was constructed using Nrf2-KO mice,and the ALI model and experimental grouping were as in Part I.THP-1 monocyte cell line was used and induced into macrophage phenotype using 100 ng/ml PMA and divided into Control group,4-OI group,LPS group,4-OI(0.125 m M)+LPS group,4-OI(0.25 m M)+LPS group and Fer-1+LPS group.An Nrf2 knockdown THP-1 cell model was constructed using si RNA.The MDA as well as GSH and GSSG levels in each group were measured using the kit,the ROS levels in each group were measured using immunofluorescence,and the PTGS2,GPX4,Nrf2,GCLM,HO-1,GCLM and HO-1 levels in mouse lung tissues were measured using real-time quantitative PCR and Western blot.The m RNA and protein expression levels of PTGS2,GPX4,Nrf2,GCLM,HO-1 and SLC7A11 were measured by real-time quantitative PCR and western blotting.HE staining to detect lung tissue morphology and lung injury score,Masson staining to detect collagen deposition in mouse lung tissues,and immunohistochemical staining to detect 4-HNE levels in lung tissues.Results: In the LPS-induced THP-1 cell injury model,compared with the Control group,the ferroptosis-related proteins GPX4,SLC7A11,GSH and GSH/GSSG ratio were significantly lower and the MDA level was significantly higher in the LPS group.4-OI exerted the same antiferroptosis effect as the ferroptosis inhibitor Fer-1,and the ferroptosis-related proteins in the 4-OI+LPS and Fer-1+LPS groups were significantly lower.Both 4-OI and Fer-1 interventions inhibited the increase in cell death and ROS levels caused by LPS treatment.Knockdown of Nrf2 by si RNA showed that the protective effect of 4-OI was completely inhibited due to the knockdown of Nrf2.After knockdown of Nrf2,the protein and m RNA levels of Nrf2 target genes GCLM,HO-1 and GPX4 were not significantly changed in the 4-OI+LPS group compared to the LPS group.The Nrf2-KO mice were used to construct a model of sepsis-induced ALI.HE and Masson staining showed no significant improvement in lung injury in the Nrf2-KO mice in the 4-OI+LPS group compared to the LPS group,and immunohistochemistry and fluorescence also showed no significant changes in the levels of 4-HNE and ROS in lung tissue compared to the LPS group.The m RNA levels of GPX4,PTGS2,HO-1 and SLC7A11 in the lung tissues of 4-OI+LPS mice did not change significantly after Nrf2 knockdown compared to the LPS group.Conclusion: 4-OI inhibited LPS-induced ferroptosis in macrophages by promoting the transcriptional expression of Nrf2 target genes GCLM,HO-1,GPX4 and SLC7A11.The antiferroptosis effect of 4-OI was significantly suppressed in both cellular and mice models after knockdown or knockout of Nrf2,indicating that 4-OI inhibited macrophage ferroptosis in an Nrf2-dependent manner and ultimately attenuates ALI.