Function Analysis Of Nuclear Factor Of The Activated T Cell 5 In Smoke Inhalation Acute Lung Injury

According to statistics,About 5% ~ 10% of burn patients complicated with inhalation lung injury,the mortality rate of severe inhalation injury as high as 80%,One of the important causes of death in patients with early burns,Although the study of inhalation injury has been the focus of research both at domestic and abroad,but it is a key factor is always failed to get the breakthrough.Resulting in high mortality,Therefore,it is necessary to study the exact pathogenesis of lung injury and effective intervention measures.More and more studies have proved that: Nuclear factor kappa B(NF-?B)plays an important role in the pathogenesis of acute lung injury.Blocking the activation of NF-?B signaling pathway can theoretically inhibit the cascade amplification effect of inflammatory response.Thus,Inhibitors of the NF-?B signaling pathway have not been successful.microRNAs(miRNAs)technology provides the possibility of solving this problem.MiRNAs are regulators of small interfering RNAs.The miRNA can specifically bind to 3'-UTRs of target cellular mRNA in turn triggering mRNA degradation or inhibition of translation.miRNAs Play an important role in cell development and tumorigenesis.However,the role of miRNAs in smoke inhalation acute lung injury is rarely reported.Nuclear factor of the activated T cell 5(NFAT5)is a human gene that encodes a transcription factor.Due to participate in the regulation of osmotic pressure by everyone to understand.In addition,NFAT5 can induce proinflammatory expression,and promote the development of inflammatory diseases.Does NFAT5 also be associated with the pathogenesis of smoke inhalation acute lung injury? The correlation studies have not been reported at home and abroad.Its related research has not been reported.In this study,the pathogenesis of smoke inhalation acute lung injury was studied at the gene and molecular level by using the model of acute lung injury in rats with smoke inhalation.Explore:(1)Does NFAT5 participate in the pathogenesis of smoke inhalation lung injury?(2)The role of NFAT5 in the pathogenesis of smoke inhalation acute lung injury(3)Screening and validation of NFR5 regulatory factor(miR-155).We first established animal models of smoke inhalation acute lung injury and found differentially expressed miRNAs and genes(miR-155 and NFAT5)by miRNA chip detection and expression profiling.To study the function of NFAT5 in smoke inhalation acute lung injury by both in vivo and in vitro experiments.The specific research methods are as follows:1.Smoke inhalation acute lung injury Rat Animal Model Establishment: The model of acute lung injury was established by using the self-made smoke inhalation injury instrument.The lung tissue HE staining,lung wet-dry ratio and COX2 Western blot were used to validate the model.2.Screening and Correlation Analysis of miR-155,NFAT5: Compare the differential expression of microRNAs in the normal and injury groups and analyze the differences of related proteins and signal pathways by microRNA microarray and microarray(MiRNA4.0 chip data and RTA chip data),The miRNAs and the corresponding target genes with differentially expressed genes were screened out;3.Study on the relationship between miR-155 and NFAT5 targeting at cell level:The target gene was expressed by bioinformatics method.The target gene and luciferase reporter plasmid were constructed by genetic engineering.The miRNA and the target gene mRNA 3'-UTR were identified by double luciferase assay.4.The study of miR-155 function:The rats were divided into A: normal control group: smoke inhalation injury group + placebo(saline),B: smoke inhalation injury group + miRNA-155 control,C: smoke inhalation injury group + miRNA-155 mimics,D: smoke inhalation injury group + miRNA-155 inhibitors,24 hours after transduction treatment,by pathologic HE staining,enzyme-linked immunosorbent assay(Tumor Necrosis Factor-alpha(TNF-?)? monocyte chemotactic peptide-1(MCP-1)?(Interleukin-IL)-1??IL-8)),Real time PCR(TNF-?,IL-1?)?Western blot of COX2,and so on.The results showed that RT-PCR To investigate whether miR-155 can reduce lung injury and reduce the expression of inflammatory molecules in lung tissue.5.NFAT5 in the study of smoke inhalation of lung injury: The expression of target gene and the expression of target gene were detected by Western blot and RT-PCR in the smokers group and the normal control group.And the expression of target gene and related signal pathway protein was detected by Western blot in Step 3.Simultaneously,we selected gene knockout and normal mice.Pathological examination and Western blot were used to detect the effect of target genes in smoke inhalation lung injury.Results:1.Successful Establishment of Rat Smoke Inhalation Acute Lung Injury Animal Model:The Histopathology results showed that the inflammatory cell infiltration,hyperemia and alveolar changes were observed in the smoke-injured group.The wet-to-dry ratio and the gray value of COX2 showed that the smoke-induced injury group was larger than the normal group,the difference was statistically significant(with P values below 0.01);2.MiR-155,NFAT5 is involved in the smoke inhalation of acute lung injury,and miR-155 has a targeted regulatory trend for NFAT5:The Affymetrix miRNA 4.0 Array were screened for 47 differentially expressed miRNAs(with P values below 0.05).The up-regulated and down-regulated miRNAs were 24 and 23,respectively.More than 2-fold differentially expressed miRNAs included miR-449a-5R,miR-205,miR-155-5p and down-regulated miR-92b-5p,miR-667-5p,miR-708-5p.(with P values below 0.05).The up-regulated expression of miR-155 was up-regulated by 3.45-fold,Affymetrix Rat transcriptome Array1.0 screened a total of 30 kinds of gene expression differences were statistically significant(with P values below 0.05),there are upregulated AKR1B10?CXCL13?VSIG4 and downregulated PLP2?SCD1?NFAT5?ATP6VB2 etc.and MiRNA4.0 chip data and RTA chip dat common analysis showed that miR-155 had a tendency to control the expression of NFAT5.3.miR-155 possesses a target effect on 3'UTR of NFAT5:TargetScan?Miranda?PicTar shared the results that NFAT5 has the complementary binding sites with 3'UTR of miR-155.And luciferase reporter vectorwas constructed.therefore the result of sequencing and double digesting of recombined plasmid were completely correct.Dual-luciferase reporter assay showed that miR-155 possesses a target effect on 3'UTR of NFAT5.4.Mi R-155 attenuated lung injury and systemic inflammatory response in smoke inhalation acute lung injury:Histopathological observation showed less inflammatory cell infiltrations in,hyperemia,swelling in group of the smoke inhalation injury+miRNA-155 mimics than in other groups,the smoke inhalation injury+miRNA-155 mimics was significantly.protein levels of COX2,Tumor Necrosis Factor-alpha(TNF-?),monocyte chemotactic peptide-1(MCP-1),(Interleukin-IL)-1?,IL-8(with P values below 0.05)bying ELISA,Western blot,RT-PCR.and Show that using the miR-155 can effectivery alleviate the lung inflammation in mouse with acute lung injury induced by smoke inhalation.5.NFAT5 Increases Smoke Inhalation Acute Lung Injury:The expression of NAFT5 was detected by Western blot and RT-PCR in the smokers group and the normal control group.The results showed that the results of the two tests were higher than those of the normal control group,the difference was statistically significant(with P values below 0.01).At the same time,The group of the smoke inhalation injury+miRNA-155 mimics western blot analysis showed the relative expression value of NFAT5,P-P65,P65,COX2 were significantly lower then other groups(with P values below 0.05).Histopathological observation showed less inflammatory cell infiltrations in,hyperemia,swelling in NFAT5-knockdown mice than normal C57 mice.The group of the NFAT5-knockdown mice western blot analysis showed the relative expression value of NFAT5,P-P65,COX2 were significantly lower then other groups(with P values below 0.05).Conclusions:1.NFAT5 play an important role in smoke inhalation acute lung injury by mediating activation of NF-?B pathway.2.inhibition of NFAT5 can attenuate smoke inhalation acute lung injury,miR-155 is the negative regulation of NFAT5,which can attenuate acute lung injury.