Study On The Role Of MAPK4 In Lipopolysaccharide-induced Murine Acute Lung Injury

This study consists of two parts as described below:Objective:Part 1 To detect the expression of MAPK4 in murine acute lung injury?ALI?;to observe the effect of MAPK4 deficiency in murine ALI and explore the possible mechanism.Part 2 To observe the effect of MAPK4 interference on the pathology of murine ALI.Methods:Part 11.WT mice?C57BL/6?was challenged with intraperitoneal injection of 10mg/kg LPS to induce murine ALI model;Real-time PCR was used to detect the mRNA level of MAPK4;Western blot?WB?was utilized to detect the protein level of MAPK4;immunofluorescence?IF?was performed to detect the expression and localization of MAPK4.2.Murine ALI model was established in WT or MAPK4^-/-mice by intraperitoneal injection of LPS;the survival ratio was recorded;the weight index,lung weight index and lung wet-dry ratio were detected after 24h of LPS treatment;BCA assay was used to analyze the total protein concentration of bronchoalveolar lavage fluid ?BALF?;HE staining was used to measure the pathologic injury of lung tissues;Real-time PCR and ELISA were utilized to analyze the expression levels of inflammatory cytokines,such as IL-1?and TNF-?,in lung tissues;FCM was performed to detect the compositions and cell counts of immune cells,such as Gr-1⁺neutrophils,F4/80⁺macrophages and NK1.1⁺cells,in BALF;Western blot and IHC were utilized to detect the expression levels of related signaling pathways,such as p-AKT,p-ERK1/2 and p-JNK,in lung tissues.3.Genomic DNA was extracted from lung tissues of control and ALI mice.The Sequenom MassARRAY was used for quantitative analysis of CpG methylation level of MAPK4 promoter.4.Versions of MAPK4 promoter vectors were constructed by 3'deletion assay.Then,these vectors were transiently transfected into RAW264.7 macrophages and the relative luciferase activity was measured to analyze the core sequence of MAPK4 promoter;Bioinformatics was utilized to predict the presumed critical transcriptional factors of core sequence in MAPK4 promoter;Real-time PCR and Western blot were used to detect the expression levels of candidate transcriptional factors;EMSA was performed to verify the bindings of transcriptional factors and MAPK4 promoter.Part 21.Murine ALI model was established by intraperitoneal injection of LPS in WT mice,then ALI mice was treated with MAPK4-shRNA to silence the expression of MAPK4 by intratracheal instillation;Real-time PCR and Western blot were used to measure the expression level of MAPK4;HE staining was used to detect the pathologic injury of lung tissues.2.Real-time PCR was utilized to analyze the expression levels of inflammatory cytokines,such as IL-1?and TNF-?,in lung tissues after MAPK4-shRNA treatment;Western blot was used to detect the expression levels of related signaling pathways,such as p-AKT,p-ERK1/2 and p-JNK,in lung tissues;lastly,control and MAPK4-shRNA-treated mice were treated with large dose of LPS to observe the survival time.Results:Part 11.Real-time PCR data showed that the expression of MAPK4 increased obviously in the lung tissues of ALI mice and reached the peak at 24 hours post LPS challenge ?P<0.05?;Western blot data showed that the protein level of MAPK4 significantly increased in ALI mice?P<0.05?;IF data showed that the expression level of MAPK4 protein was also elevated in the lung tissues of ALI mice,and macrophages could express MAPK4.2.After LPS treatment,MAPK4^-/-mice had a prolonged survival time compared with LPS-treated WT mice?P<0.05?;The body weight index and lung weight index of LPS-treated MAPK4^-/-mice did not change significantly?P>0.05?;Importantly,the lung edema of MAPK4^-/-mice reduced obviously?P<0.05?;HE staining data also showed that the infiltration of inflammatory cells and alveolar interstitium thickening in lungs also reduced significantly in LPS-treated MAPK4^-/-mice;Real-time PCR and ELISA data showed that the expression levels of pro-inflammatory cytokines,including IL-1?,IL-6 and TNF-?,markedly decreased in LPS-treated MAPK4^-/-mice?P<0.05?,while the expression levels of anti-inflammatory cytokines,including IL-4,IL-10 and TGF-?,increased significantly?P<0.05?;FCM data showed that the total cell counts of BALF in LPS-treated MAPK4^-/-mice obviously decreased?P<0.05?;The proportions and cell counts of Gr-1⁺neutrophils,??T⁺cells and CD11c⁺DCs obviously decreased in LPS-treated MAPK4^-/-mice?P<0.05?;Although the proportions of F4/80⁺ macrophages and NK1.1⁺cells did not change significantly?P>0.05?,the cell counts of these cells obviously reduced in BALF from LPS-treated MAPK4^-/- mice?P<0.05?;The proportion of MHC II⁺on macrophages significantly increased in LPS-treated MAPK4^-/-mice?P<0.05?;The proportion and cell count of CD4⁺T cells significantly decreased in LPS-treated MAPK4^-/-mice?P<0.05?,even though the proportion of CD8⁺T cells increased?P<0.05?,the cell count of CD8⁺T cells did not change significantly?P>0.05?;The proportions and cell counts of CD62L⁺and CD69⁺on CD4⁺T cells obviously decreased in LPS-treated MAPK4^-/-mice?P<0.05?,conversely,the proportion of CD62L⁺on CD8⁺T cells significantly increased?P>0.05?;Western blot and IHC data showed that the expression levels of related signaling pathways,including p-AKT,p-JNK,p-p38 MAPK and p-MK5,significantly decreased in lung tissues from MAPK4^-/- mice?P<0.05?.3.Bioinformatics data showed that there was a CpG island in MAPK4 promoter?+72⁺382 relative to the TSS?;Sequenom MassARRAY assay data showed that the CpG methylation level of MAPK4 promoter did not change significantly between control and ALI mice?P>0.05?.3'deletion assay data showed that the luciferase activity increased significantly after 2.2kb truncated into 1.5kb in MAPK4 promoter?P<0.05?,indicating that this region might be the core sequence of MAPK4 promoter;Transcription factor binding sites?TFBS?prediction databases?TRANSFAC and JASPAP?analysis showed that there were 5 candidate transcription factors,including Sp1,Egr-1,PU.1,NR3C1 and NFKB1;Real-time PCR data showed that,compared with those in control mice,the mRNA levels of NFKB1,NR3C1 and Egr-1 were obviously lower in ALI mice,while the mRNA levels of Sp1 and PU.1 significantly increased?P<0.05?,indicating that NFKB1 and NR3C1 might be critical transcriptional factors in regulating MAPK4 expression in ALI;Western blot data also showed that the protein levels of NFKB1 and NR3C1 decreased obviously in lung tissues of ALI mice?P<0.05?;EMSA data further showed that NFKB1 and NR3C1 could directly bind to the core sequence of MAPK4 promoter.Part 21.Real-time PCR and Western blot data showed that,compared with that in control group,the expression of MAPK4 markedly decreased in MAPK4-shRNA treatment group?P<0.05?;The lung edema and inflammatory injury of lung tissues significantly reduced in MAPK4-shRNA treatment group?P<0.05?2.Real-time PCR data showed that the levels of pro-inflammatory cytokines,including IL-1?and TNF-?,significantly decreased?P<0.05?,while the level of anti-inflammatory cytokine IL-10 obviously increased in MAPK4-shRNA treatment group compared with those in control group?P<0.05?;Western blot data further showed that the expression levels of p-AKT,p-JNK and p-p38 MAPK significantly decreased in MAPK4-shRNA treatment mice;Of note,MAPK4-shRNA treatment could significantly prolong the survival time of ALI mice?P<0.05?.Conclusion:1.MAPK4 is highly expressed in the lung of ALI mice.MAPK4 deficiency can significantly reduce the pathology of ALI,accompanied with the altered transduction of related signaling pathways,such as AKT and JNK pathway.2.The up-regulation of MAPK4 in ALI maybe not related with DNA methylation in its promoter region,which is mainly negatively regulated by transcriptional factor NFKB1 and NR3C1.3.The interference of MAPK4 could significantly reduce the pathology of ALI mice and prolong the survival time of ALI mice.