Analysis Of LncRNA Expression Profiling In Lung Tissue In A Mouse Model Of Septic Lung Injury And Functional Study Of GM33647 In Alveolar Macrophages

Objective sepsis is a kind of immune response disorder caused by infection,which leads to life-threatening multiple organ dysfunction,which is characterized by sustained excessive inflammation and immune suppression.The symptoms in the early stage of sepsis are not typical and the timely diagnosis and treatment is difficult,which leads to adverse prognosis and high mortality.Though the progress of modern medical treatment and the development of new drugs have improved the symptoms and prognosis of sepsis,sepsis is still one of the main causes of death in intensive care units.Acute lung injury(ALI)is the most common complication of sepsis,which is characterized by decreased compliance,hypoxemia and pulmonary edema,which leads to poor prognosis of sepsis.The biomarkers of septic acute lung injury are helpful for diagnose and treatment in septic acute lung injury in time and improving the prognosis of sepsis patients.Exploring detailed mechanism would provide theoretical basis and potential therapeutic targets for the treatment of septic acute lung injury.Therefore,the establishment of septic acute lung injury mouse model could provide material basis for the study.Methods c57b1/6 male mice is the research object.40 mice aged 6-8 weeks were randomly divided into sham operation group and septic model group,among which 10 mice were used in each group for survival analysis,and 10 mice in each group were used for the collection of samples.In this study,mouse model was established by cecal ligation and puncture(CLP).The operation in sham operation group was consistent with sepsis model group except for no ligation and puncture.The survival rate curve was drawn by continuous observation for 7 days.The mice were sacrificed at 24 hours after operation:(1)the expression levels of IL-6,IL-10,AST,ALT,BUN and Cre in serum and IL-6,IL-10 and IL-1β in bronchoalveolar lavage fluid of each group were detected by ELISA.A bicinchoninic acid(BCA)protein assay kit was used to evaluate the protein concentration of the BALF supernatants.The number of cells in bronchoalveolar lavage fluid was measured by cell count.(3)The lung,liver and kidney tissues of mice were collected and histology was analyzed by hematoxylin and eosin staining(H&E).Results The survival rate of mice in sepsis model group decreased dramatically between 12h and 48h,approximately 80%.The survival rate is 60%at 24h.The survival rate of sham operation group is 100%.The expression levels of IL-6 and IL-10 in the serum of sepsis model group were significantly elevated compared to sham operation group.The expression levels of IL-6,IL-1β and IL-10 and the protein concentration and cell number in bronchoalveolar lavage fluid were significantly increased in septic mouse model.The expression level of AST,ALT,BUN and Cr in the serum was also significantly increased in sepsis mouse model.The histology analyses showed substantial inflammatory cell infiltration into the alveolar space,thickening of the peribranchial wall and a congested vasculature in lung tissues and edema and disorder of cells,necrosis and infiltration of inflammatory cells in liver tissues and the brush edge of renal tubules disappeared,renal tubular hemorrhage and renal tubular cell necrosis accompanied by inflammatory cells Infiltration in kidney tissues were observed in sepsis mouse model.Conclusion The mortality rate of sepsis model reached 80%in 12-48 hours and the 24-hour survival rate was about 60%.The expression of inflammatory factors in serum was significantly increased.The histology of liver and kidney tissues showed acute injury characteristics.The damage related proteins in serum were significantly increased.The abnormal histomorphology,infiltrated inflammatory cells and necrosis were observed,suggesting the existence of multiple organ dysfunction.Besides,lung morphology was changed with inflammatory cells infiltration in sepsis model group,which indicated that the model of septic acute lung injury was successfully established.Objective The detailed mechanism of septic acute lung injury is still unclear.Recently,researchers have proved that long non coding RNA(lncRNA)played an important role in the development of sepsis.Some of the lncRNAs have been confirmed as regulatory molecules in sepsis related liver injury,kidney injury and lung injury.In view of the abundance of lncRNA in human tissues and blood,lncRNAs are a suitable molecule as biomarkers.Some studies have shown that lncRNAs are involved in every biological process.However,the expression profile of lncRNAs in lung tissue has not been explored in septic acute lung injury.Comprehensive analysis of lncRNA profiles would provide a theoretical basis for the mechanism in septic acute lung injury.Besides,exploring the regulatory mechanism of lncRNAs would provide a potential therapeutic target for the treatment in septic acute lung injury.Therefore,this study intends to explore the lncRNA expression profile by microarray sequencing,and predict the biological function and potential mechanism of lncRNA through bioinformatic analysis.Methods LncRNA and mRNA expression profiles in lung tissues from the septic mouse model were detected via an Agilent microarray.Heat maps and Volcano plots were used to assess the differentially expressed mRNAs and lncRNAs in mice.Enrichment analysis of Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis were implemented for predicting the potential mechanism of differentially expressed mRNAs and lncRNAs.Co-expression networks of lncRNA and mRNA were established to explore potential hub-gene.The GO and KEGG pathways of the hub-gene were analyzed to provide the basis for further verification.QPCR was applied to verify the reliability of the microarray data.Results A total of 353 differentially expressed lncRNAs(233 upregulated and 120 downregulated)and 3116 differentially expressed mRNAs(1378 upregulated and 1738 downregulated)were detected.Go terms enrichments was mainly in inflammatory response and cytokine activation;KEGG pathways analysis was enriched in Staphylococcus infection and cytokine-cytokine receptor interaction;Go analysis of differentially expressed of lncRNA was enriched in metal ion binding,transcriptional regulation and transcription process;KEGG pathways analysis was enriched in Notch signaling pathway,cell adhesion molecules and mTOR signaling pathway.Co-expression networks showed that lncRNA GM33647 may be the hub-gene;(4)Go analysis of GM33647 was enriched in LPS related reactions,neutrophil chemotaxis,inflammatory cell migration and inflammation;KEGG pathways analysis was enriched in TNF signaling pathway,PI3K-Akt signaling pathway and NF-kappa B signaling pathway.QPCR showed that the results of differentially expressed lncRNAs in lung tissues of septic ALI mouse model were consistent with microarray data.Conclusion This study explored the expression profiles of lncRNA in sepsis induced lung injury.In the lung tissues of sepsis mouse model,qPCR results showed that the microarray results were reliable.GO and KEGG pathways analysis suggested that lncRNAs may be involved in the inflammatory pathway in the process.The co-expression network implied lncRNA GM33647 as a hub-gene and the target for further investigation.Objective studies have shown that in the process of sepsis,macrophages secrete inflammatory factors,causing cell damage.The apoptosis and necrosis of macrophages will have adverse effects on the prognosis of sepsis.Therefore,studies focus on the related functions of macrophages in sepsis.In the process of septic ALI,alveolar macrophages have a significant impact on alveolar cells,lung tissue fibroblasts and so on.A large number of inflammatory factors secreted by alveolar macrophages continue to damage lung tissue.Therefore,it is very important to study the mechanism of alveolar macrophages in septic ALI.We have selected lncRNA GM33647 as the research object.In order to further exploration we chose mouse alveolar macrophage line as a subject and determine the functions and potential mechanism of lncRNA GM33647 in septic ALI.Method LPS was used to stimulate mouse alveolar macrophage line(MH-S)to establish a septic cell model.CCK8 and MTT were used to detect cell proliferation.QPCR was used to detect the expression level of IL-6 and cell morphological changes were observed by microscope.LPS and Nigericin sodium salt were used for NLRP3 inflammasome activated cell model.QPCR was used to verify the expression levels of IL-1β,IL-18 and caspase-1.CCK8 was used to detect cell proliferation.The expression level of lncRNA GM33647 was verified in cell model via qPCR.Then GM33647 was knocked down by small RNA interference and the optimal concentration of siRNA transfection and liposome was explored by cell fluorescence.The efficiency of siRNA on GM33647 was verified by qPCR.Thus,the optimal concentration of siRNA and liposome was selected.The expression levels of IL-6,IL-10 and TNF-α were detected via qPCR.The mitochondrial membrane potential was analyzed by JC-1 flow cytometry and the number of mitochondria was determined by fluorescence confocal microscopy.In NLRP3 inflammasome activated cell model,the expression levels of IL-1β,IL-18 and caspase-1 were verified by qPCR.Results In MH-s with LPS treatment,IL-6 expression level was elevated significantly.In MH-s with LPS/Nigericin treatment to activate the NLRP3 inflammasome,the results showed that the expression levels of IL-1β,IL-18 and caspase-1 were significantly up-regulated.In the sepsis cell model,the expression level of lncRNA GM33647 was significantly decreased,which was consistent with the sequencing results.However,in the NLRP3 inflammasome activated cell model,the expression level of lncRNA GM33647 was significantly increased.The concentration of siRNA at 50 nM and liposome at 3 μL/well(6-well plate)were taken as optimal transfection condition with transfection efficiency above 90%.QPCR verified that the knockdown level was more than 70%,which could be used in this experiment.Knockdown GM33647 in sepsis cell model increased the expression levels of IL-6,IL-10 and TNF-α significantly.JC-1 flow cytometry showed that the mitochondrial membrane potential was significantly decreased and the mitochondrial area was significantly decreased when transfected with siRNA of GM33647.In NLRP3 inflammasome activated cell model,the expression levels of IL-1β,IL-18 and caspase-1 were significantly increased when transfected with siRNA of GM33647.Conclusion Knockdown of lncRNA GM33647 would up-regulate the expression of inflammatory factors induced by LPS,affect the function of mitochondria and reduce the production of mitochondria.The expression of IL-1β,IL-18 and caspase-1were increased by silencing GM33647.The decrease of GM33647 in sepsis may aggravate septic acute lung injury and affect the prognosis by regulating the above process.