The Effects And Mechanisms Of Peptidyl Arginine Deiminase 4 On Lipopolysaccharide Induced Acute Lung Injury

BackgroundAcute lung injury(ALI)/acute respiratory distress syndrome(ARDS)is a common critical and severe disease in clinic,especially in ICU.Its common causes include trauma,sepsis,pneumonia,shock,and multiple factors lead to the destruction of the "blood-air barrier" of lung tissue,which is clinically manifested as intractable hypoxemia and acute non-cardiogenic pulmonary edema.The common pathological basis of ALI/ARDS is the pulmonary inflammation mediated by various inflammatory cells and the increased pulmonary microvascular permeability caused by the uncontrolled inflammatory response,which leads to pulmonary interstitial and alveolar edema.which are the common pathophysiological basis of ALI/ARDS.Despite the widespread use of advanced treatments,such as mechanical ventilation,the prognosis is poor,and mortality rates as high as 40%or more.Therefore,further studies on the pathophysiology of ALI/ARDS will provide new therapeutic targets and treatments.Peptidyl arginine deiminases 4(PAD4)is one of the key enzymes involved in the post-translational modification of proteins through citrulination.PAD4 has become a potential target for the treatment of autoimmune diseases,tumors and other diseases.Previous studies have shown that inhibition of PAD4 expression can improve organ dysfunction,and thus improve the survival rate of mice with hemorrhagic shock and sepsis.Inhibition of PAD4 reduced the increase of endothelial cell permeability induced by intraperitoneal injection of lipopolysaccharide(LPS).Studies have shown that PAD4 participates in the formation of neutrophil extracellular traps(NETs),thereby regulating the pathophysiological processes involved in the development of ALI/ARDS.Although PAD4 has been shown to be involved in the development of a variety of diseases,the role and mechanism of PAD4 in the direct induction of acute lung injury by LPS have not been fully elucidated.Clarifying its mechanism of action is beneficial to the treatment of acute lung injury.ObjectiveIn this study,the expression of PAD4 in LPS-induced acute lung injury were firstly observed.Secondly,the protective effect of PAD4 inhibitor TDFA(Thr-Asp-F-amidine)on LPS-induced acute lung injury was clarified.Finally,the mechanism of inhibiting PAD4 in alleviating LPS-induced acute lung injury was further explored.MethodsPart ?:To observe the expression of PAD4 in lung tissue after acute lung injury induced by LPSIn this experiment,24 adult(6-8 weeks)SPF C57BL/6 mice with a body weight of about 20-25 g were randomly divided into Control group,Sham group,LPS 6 h group and LPS 24 h group.Each group of 6 animals were treated in different experiments.Control group:Mice didn't receive any treatment;Sham group:Mice were intratracheal injected with 50?l normal saline;LPS 6 h group:Acute lung injury model was established by intratracheal administration of 50 ?l LPS(1 ?g/g),detection were performed 6 h after modeling;LPS 24 h group:Acute lung injury model was established by intratracheal administration of 50?l LPS(1 ?g/g),detection were performed 24 h after modeling.Sham group were detected 6 h after intratracheal injection,Control group was detected at the same time.qRT-PCR and Western blot were used to detect and compare the mRNA and protein levels of PAD4 in lung tissues of four groups of mice.Immunohistochemical staining was used to detect and compare the changes of immunofluorescence intensity of PAD4 in lung tissues of four groups of mice.Part ?:Explore the protective effect of PAD4 inhibitor TDFA on LPS induced acute lung injuryIn vivo experiment:54 adult(6-8 weeks)SPF C57BL/6 mice weighing about 20-25g were selected and randomly divided into Sham group,LPS group and PAD4 inhibitor TDFA group,with 18 mice in each group,for experimental treatment respectively.Sham group:mice were intraperitoneally injected with normal saline 100 ?l 1h before modeling,and mice were intratracheal injected with normal saline 50 ?l.LPS group:100 ?l normal saline was injected intraperitoneally 1 h before modeling,and then mice were intratracheal injected with 50 ?l LPS(1 ?g/g).TDFA preconditioning group:100 ?l TDFA(20 mg/kg)was given intraperitoneally 1 h before the establishment of the model,and then 50 ?l LPS(1 ?g/g)was given intratracheal.The following indexes were detected and compared 6 h after trachea injection:the expression level of PAD4 protein and Cit-H3(Citrullined histone 3)in the lung tissue of the three groups;Lung tissue injury and pathological score of lung tissue injury;Wet-dry ratio of lung tissue;The number of Ly6G+neutrophils in lung tissue,the total number of activated cells and the number of neutrophils in alveolar lavage fluid;The levels of oxidative stress indexes in lung tissues included superoxide dismutase(SOD),malondialdehyde(MDA)and glutathione(GSH).The mRNA level and protein expression level of junction proteins in lung tissues:including Zonula occludens 1(ZO-1),claudin-4 and Occludin.In addition,30 SPF C57BL/6 mice were randomly divided into Sham group,LPS group and TDFA treatment group according to the experimental design.Each group of 10 mice were treated with the following experiments.Sham group:mice were intraperitoneally injected with 100 ?l normal saline,1 h later,intratracheal injected with 50?l normal saline.LPS group:mice were intraperitoneally injected with 100?l normal saline,and 1 h later,mice were intratracheal injected with 50 ?l LPS(40 ?g/g).TDFA treatment group:mice were intraperitoneally injected with 100?l TDFA(20 mg/kg),and 1 h later,mice were intratracheal injected with 50 ?l LPS(40 ?g/g).The survival rate of each group was recorded every 12 h within 72 h after lethal dose of LPS.In vitro experiment:MLE-12 cells were divided into Sham group,LPS group and TDFA-treated group.According to the above groups,MLE-12 cells were prestimulated with the same amount of normal saline or 100 nM TDFA.After 1 h,MLE-12 cells were stimulated with normal saline or LPS(100 ng/ml)according to the group.After 3 hours of LPS stimulation,the following indexes of MLE-12 cells in the three groups were detected and compared:the levels of MDA,GSH and SOD;The mRNA level and protein expression level of junction proteins ZO-1,Claudin-4 and Occludin;The transepielectrical resistance(TEER)values of MLE-12 cells were measured and compared at different time points(0 h,1 h,3 h).Part ? To explore the mechanism of LPS induced acute lung injury after TDFA was administratedIn vivo experiment:27 SPF C57BL/6 mice were randomly divided into Sham group,LPS group and PAD4 inhibitor TDFA group.Each group had nine animals.The treatment of the three groups of mice was the same as that of the mice in vivo in Part ?.The levels of pro-inflammatory cytokines tumor necrosis factor ?(TNF-?),interleukin 6(IL-6)and interleukin 1?(IL-1?)in alveolar lavage fluid of mice in the three groups were detected and compared 6 h after tracheal injection.The expression levels of P65 in the nucleus(n-p65),total P65(P65)and phosphorylated P65(p-p65)of lung tissues of the three groups were detected and compared 6 h after tracheal injection.In vitro experiment:MLE-12 cells were divided into Sham group,LPS group and TDFA-treated group.According to the above groups,MLE-12 cells were prestimulated with the same amount of normal saline or 100 nM TDFA.After 1 h,MLE-12 cells were stimulated with normal saline or LPS(100 ng/ml)according to the group.Samples of each group were collected 3 h after LPS stimulation for subsequent detection.The mRNA levels of pro-inflammatory cytokines TNF-?,IL-6 and IL-1? in MLE-12 cells of three groups were detected and compared by qRT-PCR.The expression level of Cit-H3 in MLE-12 cells of the three groups was detected and compared by Western blot.The expression levels of n-p65,P65 and p-p65 of MLE-12 cells in the three groups were detected and compared.In addition,the level of nuclear translocation of P65 was determined by confocal laser microscopy.ResultsPart ?:To observe the expression of PAD4 in lung tissue after acute lung injury induced by LPSThe mRNA expression and protein level of PAD4 in lung tissue of Sham group were not significantly different from that of Control group.The mRNA and protein levels of PAD4 in lung tissues of mice in LPS 6 h and LPS 24 h groups were significantly increased compared with those in Sham group.Immunohistochemical staining and average optical density analysis showed that compared with the Control group,the expression level and average optical density of PAD4 in the lung tissue of the Sham group were not significantly increased.Compared with Sham group,the expression level and average optical density of PAD4 in lung tissue of mice in LPS 6 h and LPS 24 h groups were significantly increased.There was no significant difference in the expression level and average optical density of PAD4 in lung tissue of mice between the LPS 6 h group and the LPS 24 h group.Part ?:Explore the protective effect of PAD4 inhibitor TDFA on LPS induced acute lung injuryIn vivo experiments showed that compared with the LPS group,TDFA significantly inhibited the expression of PAD4 protein and the level of citruline of histone H3;TDFA group significantly improved the severity of lung pathological injury and pathological score of lung tissue injury in LPS-induced acute lung injury mice;TDFA group significantly reduced the wet/dry ratio of lung tissue and reduced the degree of lung tissue edema;The number of Ly6G+neutrophils in lung tissue and the number of total activated cells and neutrophils in alveolar lavage fluid were significantly decreased in TDFA group;TDFA group significantly improved the levels of MDA,GSH and SOD in lung tissue.TDFA group significantly reversed the decrease of mRNA and protein expressions of ZO-1,Claudin-4 and Occludin in lung tissue.TDFA group significantly improved the survival rate of mice with lung injury induced by lethal dose of LPS.In vitro experiments showed that compared with LPS group,TDFA group significantly improved the levels of MDA,GSH and SOD in MLE-12 cells.TDFA group significantly improved the mRNA and protein expressions of intracellular junction proteins ZO-1,Claudin-4 and Occludin.TDFA group significantly reversed the decreasing trend of TEER value in MLE-12 cells.Part ?:To explore the mechanism of LPS induced acute lung injury after TDFA was administratedIn vivo,compared with LPS group,TDFA group significantly reduced the contents of pro-inflammatory cytokines TNF-?,IL-6 and IL-1? in alveolar lavage fluid;TDFA group significantly inhibited the high expression level of n-p65 of lung tissue;TDFA significantly inhibited the high expression of p-p65 induced by LPS;There was no significant difference in the expression level of P65 in the lung tissues of the three groups.In vitro,TDFA significantly inhibited the mRNA levels of pro-inflammatory cytokines TNF-?,IL-6 and IL-1? of MLE 12 compared with LPS group;TDFA group significantly inhibited the level of histone H3 citrullinate in MLE-12 cells;In addition,TDFA significantly inhibited the high expression of n-p65 of MLE-12;In vitro stimulation with LPS significantly up-regulated the expression of p-p65,while pretreatment in vitro did not significantly affect the expression of p-p65 in the TDFA group;There was no significant difference in the expression level of P65 in MLE-12 cells between the three groups.In addition,confocal laser microscopy confirmed that P65 was distributed in the cytoplasm of the Sham group.In the LPS group,P65 was mainly distributed in the nucleus.In the TDFA group,P65 was partially distributed in the nucleus and partially distributed in the cytoplasm.ConclusionIn vivo experiments showed that mRNA and protein levels of PAD4 were significantly increased in lung tissues of acute lung injury model animals induced by LPS.In vivo experiments,TDFA pretreatment was used:1.To improve the severity of pathological injury and edema of lung tissue in mice;2.Reduces neutrophils in mouse lung tissue and inflammatory cell infiltration of alveolar lavage fluid;3.Improve the survival rate of mice with lung injury induced by lethal dose of LPS.In vivo and in vitro experiments,TDFA was used to pretreat:1.Reduction of Cit-H3 protein levels in mouse lung tissues and MLE-12 cells;2.Improve the levels of MDA,GSH and SOD in lung tissue and MLE-12 cells;3.Reverse the decrease of mRNA and protein expressions of the junction proteins ZO-1,Claud in-4 and Occludin in lung tissue and MLE-12 cells;4.Reduce the contents of pro-inflammatory cytokines TNF-?,IL-6 and IL-1? in mice alveolar lavage fluid and the mRNA levels of pro-inflammatory cytokines in MLE-12 cells;5.Inhibit the high expression level of n-p65 in mouse lung tissue and MLE-12 cells;6.In vivo TDFA inhibited the LPS-induced high expression of p-p65 in mouse lung tissue,and in vitro TDFA did not significantly affect the LPS-induced high expression of p-p65 in MLE-12 cells.In vitro experiment,TDFA was used to pretreat:1.LPS-induced down-regulation of TEER in MLE-12 cells was reversed;2.Confocal laser microscopy confirmed that TDFA significantly inhibited the nuclear translocation level of P65 in MLE-12 cells.Research suggested that administration of TDFA can regulate the nuclear translocation of P65 by affecting the Citrulline level of histone H3 and thus affect the release of inflammatory cytokines in alveolar epithelial cells,and improve the prognosis of LPS-induced ALI after endotracheal injection.The Citrullination of NF-?B-p65 by PAD4 may be the potential cause of LPS-induced lung injury.This study suggests that TDFA is an effective method for the treatment of ALI,and inhibition of PAD4 may become an important target for the treatment of LPS-induced ALI.