Metallothionein 1 Inhibits Ferroptosis And Sensitizes Astaxanthin To Alleviate LPS-Induced Acute Lung Injury Xue Dong

BackgroundAcute lung injury(ALI)and acute respiratory distress syndrome(ARDS)are common clinical critical diseases,and sepsis is an important cause of the development of ARDS.Although advances have been achieved in lung-protection ventilation,fluidconservative management,and hormonal therapy,accompanied by profound activation of systemic inflammatory response syndrome(SIRS)and development of multiple-organ dysfunction(MODS),the mortality is still high at 40%.Therefore,it is of great significant societal and scientific implications to better understand how to prevent and treat ARDS.The pathogenesis of ALI/ARDS has not been fully elucidated,and oxidative stress and inflammatory response play an important role in its pathogenesis.A lot of reactive oxygen species(ROS)can directly damage lung tissue,causing oxidative damage to lung parenchymal cells and pulmonary interstitial edema,but the exact mechanism is not yet clear.In recent years,there is increasing evidence that ferroptosis plays an important role in sepsis-induced multi-organ dysfunction and inhibition of ferroptosis significantly reduces ALI.Metallothionein(MT)is a metal-binding protein with low molecular mass and high cysteine content which is commonly expressed in almost all organs.MT-1 serves as a metal transporter and antioxidant protein that plays a role in metal detoxification in the body,maintaining physiological elemental homeostasis and preventing organ damage caused by metal overload.Lipopolysaccharide(LPS)has an acute induction effect on MT-1 expression in a range of tissues,including the liver,heart,kidney and brain.Currently,the role of MT-1 in LPS-induced ALI is remain unclear.Astaxanthin(AST)is a natural antioxidant with multiple biological functions that cross bilayer membranes to prevent oxidative stress and lipid peroxidation by scavenging ROS.A recent study confirmed a novel connection between AST and ferroptosis.It has been found that AST activates the Nrf2/HO-1 pathway to enhance autophagy and inhibit ferroptosis to alleviate acetaminophen-induced liver injury.However,the role and mechanism of AST in LPS-induced ferroptosis in ALI have not been elucidated.MT-1shows some similarities with AST in antioxidant stress and inhibition of inflammatory response.This study hypothesized that MT-1 could cooperate with astaxanthin to inhibit ferroptosis and enhance the protective effect on ALI.It provides options for discovering new targets and developing effective therapeutic drugs for ALI.MethodsPartⅠ: Screening and validation of hub genes for ferroptosis in ALI based on bioinformatics1.Bioinformatics analysis of LPS-induced differential expression genes(DEGs)in the lung tissue of ALI mice using the GEO database and analysis of the biological processes involved by enrichment.2.Cytoscape(v3.7.2)software was used to construct PPI networks,mi RNA-m RNAlnc RNA interaction ce RNA networks and transcription factor regulatory networks.3.R’s p RRophetic package was used to analyze the association between ALI models and different small molecule inhibitors.4.A single-sample gene-set enrichment analysis algorithm was used to quantify the relative levels of immune cell infiltration in the ALI model mice.5.RT-q PCR methods to verify the expression of hub genes in lung tissue.PartⅡ: The protective effect and molecular mechanism of MT-1 in ALI by regulating ferroptosis1.LPS-induced ALI mouse model was constructedC57BL/6 mice were selected to construct LPS induced ALI model.16 mice were randomly divided into five groups(5mice/group)as follows: the control group;LPS(5mg/kg,dissolved in saline);LPS+Erastin(10mg/kg,i.p.);LPS+Fer-1(10mg/kg,i.p.).After 24 h,the mice were sacrificed and eye blood and lung tissues were collected.The lung tissue was assessed for pathological damage by HE and Masson staining,and the W/D ratio and pathological damage score were calculated.2.LPS-induced MLE-12 cell model was constructedMLE-12 cells were randomly divided into 4 groups: the control group;LPS(20mg/L);LPS+Erastin(5μM)and LPS+Fer-1(2μM).After 24 h,cell viability was measured by CCK8,MDA and GSH kits.The expression of GPX4 protein in MLE-12 cells was detected by Western blot and the ultrastructure of intracellular mitochondria was observed by transmission electron microscopy.3.Differential expression of MT-1 in vivo and in vitroMT-1 protein expression was detected by Western blot in vivo and in vitro,respectively,and confocal microscopy was applied to observe the differences in MT-1expression in MLE-12 cells.4.MT-1 expression after si RNA transfection of MLE-12 cellsThe si RNA control(si Cont)and si Mt-1 knockdown(si Mt-1)were transfected into MLE-12 cells by liposomes,respectively,and the RNA or protein was extracted after 72 h of continued incubation.Experimental groups: si Cont group;si Cont+LPS group;si Mt-1group and si Mt-1+LPS group.MT-1 m RNA and protein expression were detected by RTq PCR and Western blot.5.Effect of MT-1 knockdown on ferroptosis in LPS-induced MLE-12 cellsThe MLE-12 cells were transfected with si Cont and si Mt-1 via liposomes and then stimulated with LPS(20mg/L)in the treated group and an equal amount of PBS in the non-treated group after 72 h of incubation.Experimental groups: si Cont group;si Cont+LPS group;si Mt-1 group and si Mt-1+LPS group.CCK8 was used to detect cell viability,MDA,GSH and ROS kits to detect oxidative stress,and western blot to detect the expression of GPX4 and SLC7A11 proteins in MLE-12 cells.Part Ⅲ: MT-1 sensitizes AST to protect against LPS-induced ALI1.LPS-induced ALI mouse model was constructed and administration of AST interventionC57BL/6 mice were selected to construct an LPS-induced ALI model.25 mice were randomly divided into five groups(5mice/group)as follows: the control group;LPS(5mg/kg,dissolved in saline);LPS+Fer-1(10mg/kg,i.p.);LPS+AST-L(10mg/kg,i.g.);LPS+AST-H(60mg/kg,i.g.).After 24 h,mice were executed and ocular blood and lung tissue were collected.The lung tissue was assessed for pathological damage by HE and Masson staining,and the W/D ratio and pathological damage score were calculated.2.LPS-induced MLE-12 model was constructed and administration of AST interventionThe murine lung epithelial cell line MLE-12 cells were randomly divided into 6groups: the control group;AST(10μM);LPS(20mg/L);LPS+Fer-1(2μM);LPS+AST-L(1μM);LPS+AST-H(10μM).After 24 h,the cell viability was measured by CCK8,the oxidative stress level was measured by MDA and GSH kits,and the expression of GPX4 protein in MLE-12 cells was measured by western blot and the ultrastructure of intracellular mitochondria was observed by transmission electron microscopy.3.Treatment with AST after MT-1 knockdownThe MLE-12 cells were transfected by liposome with si Cont and si Mt-1,respectively,and the treated group was stimulated with LPS(20mg/L)or AST(10μM)after 72 h of incubation,while the non-treated group was stimulated with an equal amount of PBS.Experimental groups: si Cont+LPS group;si Mt-1+LPS group;si Cont+LPS+ AST(10μM)group;si Mt-1+LPS+ AST(10μM)group.CCK8 was applied to detect cell viability,MDA and GSH to detect oxidative stress levels,and western blot to detect changes in GPX4 and SLC7A11 proteins in MLE-12 cells.ResultsPartⅠ: Screening and validation of hub genes for ferroptosis in ALI based on bioinformatics1.A total of 14 DEGs commonly associated with ferroptosis and inflammation were screened based on three datasets,GSE2411,GSE18341 Juvenile,and GSE18341 adult.RT-q PCR was applied to verify that Gpx2,Txnrd1,Gclc,Hif1α,Atf3,MT-1,IL-6,Tnfaip3 and Zfp36 were hub genes in ALI and ferroptosis.2.The results of enrichment analysis suggested that DEGs were closely associated with biological processes such as cytokine-mediated signaling pathways and leukocyte migration.The results of KEGG analysis showed that these DEGs were involved in biologically relevant signaling pathways such as TNF and IL-17.3.The transcription factors with high expression correlation were screened by transcription factor regulatory networks,including Junb,Hif1 a,Bcl3,Maff,Batf,Elf5,Myc,and Atf3.4.Based on the 138 different chemotherapeutic and small molecule drugs,significant differences in IC50 values were found between the model of ALI in mice and the normal model in mice for 15 small molecule drugs,especially for ABT-263,AKT.inhibitor.Ⅷ and AMG-706 drugs.Part Ⅱ : The protective effect and molecular mechanism of MT-1 in ALI by regulating ferroptosis1.LPS-induced ferroptosis in the lungs of ALI mice,and inhibition of ferroptosis using Fer-1 could attenuate lung injury and inflammatory response.2.LPS and Erastin stimulation induced upregulation of MT-1 expression in lung tissues and MLE-12 cells,and downregulation of MT-1 expression after Fer-1 treatment.3.MT-1 knockdown significantly increased LPS-induced oxidative stress and inflammatory response levels in MLE-12 cells.4.MT-1 knockdown downregulated the expression of ferroptosis-related proteins GPX4 and SLC7A11.Part Ⅲ: MT-1 sensitizes AST to protect against LPS-induced ALI1.AST significantly attenuated the inflammatory response and pathological damage in the lung tissue of ALI mice.2.AST significantly reduced the level of oxidative stress in lung tissues and MLE-12 cells of ALI mice.3.AST significantly inhibited ferroptosis in ALI mice and MLE-12 cells.4.MT-1-sensitized AST inhibited ferroptosis via GPX4 pathway.Conclusions1.We identified 14 potential ferroptosis-related hub genes(FRHGs)and validated the expression levels of 13 FRHGs by RT-q PCR,9 of which(GPX2,Txnrd1,Gclc,Hif1α,ATF3,Mt-1,IL-6,Tnfaip3,Zfp36)might play a vital role in ALI.The discovery of these genes will contribute to the understanding of ALI pathogenesis and provide biomarkers and potential therapeutic targets for the diagnosis and treatment of ALI.2.MT-1 attenuates LPS-induced ALI by inhibiting ferroptosis via activation of the SLC7A11-GSH-GPX4 signal axis,providing a novel molecular mechanism for the treatment of the disease.3.AST attenuates LPS-induced ALI by inhibiting ferroptosis via various pathways,such as reducing iron content,inhibiting lipid peroxidation,increasing antioxidant enzyme activity and regulating ferroptosis-related protein expression,and MT-1 and AST co-protect lung epithelial cells from lipid peroxidation and ferroptosis.