| Background:High Altitude Pulmonary Edema?HAPE?is an acute severe acute mountain sickness which occurs in the special environment of high altitude.HAPE has the characteristics of acute onset,rapid progression and high incidence.If processing is not seasonable,it threatens the life of people who rapidly ascend to high altitude.It is different in the incidence of HAPE between the research reports of China and the foreign,because there are many factors can cause HAPE,such as altitude,ethnic,initial entry into the plateau or return to the plateau,and the way to enter the plateau,occupational categories and labor intensity,seasonal and climate,individual and family susceptibility,upper respiratory tract infections and so on.At present,the pathogenesis of HAPE is still not fully understood.Some researchers believe that elevated pulmonary hypertension and hemodynamic changes are the main mechanisms of HAPE,and some researchers believe that the pathogenesis of HAPE is related to fluid retention and fluid transfer disorders in the body.However,more and more evidences have confirmed that inflammatory reactions play an important role in the occurrence of HAPE,such as:1)Upper respiratory tract infection is an important cause of HAPE;2)Anti-inflammatory treatment with dexamethasone has obvious effect on HAPE;3)The levels of inflammatory factor expression in blood samples of HAPE patients is significantly increased and the blood routine indicates white blood cell count increase significantly;4)Autopsy results of HAPE patients found that there are many inflammatory exudative components,cellulose deposition and transparent membrane formation in the alveolar cavity;5)Alveolar lavage fluid of HAPE patients is a high-protein inflammatory infiltration Effluent,which includes a large amount of protein,inflammatory cells and red blood cells,immunoglobulins and complement components.In recent years,high-throughput sequencing technology has provided a comprehensive study of the complex molecular mechanisms of individual diseases through comprehensive analysis of genomics,transcriptomics and proteomics data,and has been used in diseases such as tumors and diabetes.The pathogenesis of HAPE is complex,involving gene transcription,protein translation processes and their interaction with hypoxic environments,and its molecular basis is complex.The study of the pathogenesis of HAPE from a systematic perspective can better find the proteins that play an important role in its occurrence and the key signaling pathways involved in it,providing a new perspective for the in-depth study of the pathogenesis of HAPE.In the first part,the clinical data and blood inflammatory factor test results of healthy control group?Control group?and HAPE group were compared.The blood samples of HAPE patients were detected by transcriptomics and proteomics techniques,and the differential genes and proteins in the occurrence of HAPE were screened.And use the Gene Oncology?GO?and Kyoto Encyclopedia of Genes and Genomes?KEGG?database to analysis the important biological processes and key signaling pathways which involved in differential genes and proteins of HAPE.In the second part,based on the combined analysis of transcriptomics and proteomics,the inflammatory response mediated by Toll-like receptor 4?TLR4?signaling pathway may play an important role in the development of HAPE.Using acute hypoxia combined with low-dose lipopolysaccharide?LPS?could replicates a stable rat HAPE animal model and an inflammatory response-enhanced alveolar macrophage model.Gene chip technique were used to detect the differentially expressed genes of bronchoalveolar lavage fluid?BALF?cells in rat HAPE model.Functional annotation show these differential genes mainly involved in inflammatory responses and immune responses regulate biological processes and so on,and the TLR4 signaling pathway is one of the key signaling pathways.The inflammatory response mediated by the TLR4signaling pathway was then verified in an alveolar macrophage model.In the third part,it has been reported that the protein level of hypoxia inducible factor-1??HIF-1??in peripheral blood associate with the susceptibility of HAPE.We found that there was a significant positive correlation between LPS and TLR4 of two groups in the plains,but they have no correlation in high altitude.Our results suggest that there may be other factors in the development of HAPE that affect the activation of the TLR4 signaling pathway.The differentially expressed gene TLR4?up-regulated?in the HAPE group was significantly negatively correlated with HIF-1??down-regulated?,suggesting that there may be some association between HIF-1?and TLR4 signaling pathways in HAPE.Therefore,we used HIF-1?inhibitors,agonists,siRNA interference sequences and overexpression plasmids to regulate the expression of HIF-1?in alveolar macrophages to study the inflammation responses induced by acute hypoxia combined with low-dose LPS.We enhanced the expression of HIF-1?in rats by pretreatment and tested the effect of pre-increased HIF-1?expression in vivo on lung injury in rat HAPE model.Through the above research,it reveals the key mechanism in the occurrence of HAPE,and provides effective targets and new ideas for the prevention and treatment of HAPE in the future.Methods:1.Collection the clinical examination data and blood sample,detection the omics data of people in Control group and HAPE group?1?The study subjects in Control group are healthy male adult,and HAPE group are HAPE patients who were diagnosed according to hospital diagnostic criteria and cli nical examination results.?2?Collecting the hospital case data,clinical examinations and blood samples of the study subjects?3?Perform transcriptomics and iTRAQ proteomics testing and bioinformatics analysis?4?Detection the various inflammatory factors,endotoxin?LPS?and LPS binding protein?LBP?in serum?5?Verification the important differential genes screened by transcriptomics using RT-PCR2.The experimental conditions,detection indexes and intervention treatment of rat HAPE animal model?1?The study subjects were 8-week-old male Sprague-Dawley?SD?rats weighing 200±20 g?2?Rats were randomly divided into 4 groups:?I?normoxia control group?CTL group?,?II?LPS stimulation group?LPS group?,?III?exposed to acute hypobaric hypoxia group?HPO group?,?IV?Acute hypobaric hypoxia combined with LPS treatment group?COMB group??3?The samples of rats in the HPO group and the COMB group were taken at 5000 m altitude?4?Measure the average pulmonary artery pressure,collection the abdominal aortic blood,measure the oxygen saturation of abdominal aortic blood,collect the rat BALF,pathological sectioning of lung tissue,detect the lung wet-to-dry weight ratio,and detect the levels of IL-1?,IL-6 and TNF-?in BALF3.Experimental conditions,detection indexes and intervention treatment of the alveolar macrophage model?1?The study object is rat alveolar macrophage cell line?NR8383,Chinese Academy of Sciences Cell Bank,Shanghai,China??2?NR8383 cells were divided into 4 groups:?I?CTL group,normal control;?II?LPS group,treated with 10 ng/ml LPS medium under normoxia for 6 hours,?III?HPO group,the cells were placed in an incubator containing 5%O2 for 6 hours,?IV?COMB group,treated with 10 ng/ml LPS medium in an incubator containing 5%O2 for 6 hours?3?Detection the expression changes of the TLR4 promoter which be transfected into cells by luciferase reporter gene assay?4?Using TLR4 inhibitor?TAK242?to act on NR8383,then to detect the expression of TLR4 in cells and the expression of IL-1?,IL-6 and TNF-?in cells and culture medium4.The condition and test index of the regulation of intracellular HIF-1?and preconditioning of rats with HIF-1?agonist?1?NR8383 cells were treated with HIF-1?inhibitor?PX478?,HIF-1?agonist?DMOG?,HIF-1?siRNA interference sequence and HIF-1?overexpression plasmid to regulate the levels of HIF-1?protein expression in cells.The experiments for each treatment condition were divided into two large groups:the blank control group and the intervention HIF-1?group,and each large group contained 4 groups?consistent with the previous grouping??2?Detect the expression changes of TLR4 promoter,TLR4,IL-1?,IL-6 and TNF-?after regulation the protein expression level of HIF-1??3?Pretreatment with HIF-1?agonist?DMOG?in order to enhance the protein expression level of HIF-1?in rats under normoxic conditions,and then to replicat the rat HAPE model.The mean pulmonary artery pressure was measured,abdominal aortic blood was collected,blood oxygen saturation of abdominal aorta blood was measured,rat alveolar lavage fluid?BALF?was collected,pathological sections of lung tissue,lung wet-to-dry weight ratio,and the IL-1?,IL-6 and TNF-?expression changes in plasma were measured.Results:1.The inflammatory response mediated by TLR4 signaling pathway is involved in the occurrence of HAPE?1?The inflammatory response in HAPE patients was significantly enhanced compared with the control group.HAPE patients have obvious cough,cough,difficulty breathing,coughing white or pink foam,and auscultation with wet rales in the lungs,accompanied by elevated body temperature and increased respiratory rate.The chest tissue X-ray light suggests a structural change of somewhat flaky or cloud-like infiltrated in the lung tissue.Blood routine examination of HAPE patients found that the number of white blood cells increased significantly,and the expression levels of various inflammatory factors,such as IL-1?,TNF-?,IL-6,IL-8 was also significantly increased in serum.?2?The differential genes and proteins in HAPE patients are mainly involved in biological processes such as inflammatory response and immune response regulation.A total of 3352 differentially expressed genes were screened by transcriptomics,including 2112 up-regulated genes and 1240 down-regulated genes.These differentially expressed genes are mainly involved in immune response-related biological processes;Inflammatory-related biological processes;molecular regulation related to bacterial infection;autophagy;oxidative phosphorylation-related biological processes;energy metabolism related biological processes.Many of these differentially changed biological functions are the downstream functions of the inflammatory response and immune response activation,which are the defensive response occur when the body against external stimuli such as bacteria,LPS and antigenic substances.There were 58 upregulated proteins and 37 downregulated proteins in the serum of the HAPE group.These differentially expressed proteins are mainly involved in immune system processes,defense responses to bacteria,cell activation,stress response,neutrophil activation,interleukin-8 production,leukocyte degranulation,cytokine production and other biological functions.In addition,the C-reactive protein?CRP?and?1-acid glycoprotein??1AG?were significantly elevated in the serum of HAPE patients suggesting that acute inflammatory reactions are involved in HAPE.?3?The inflammatory response mediated by TLR4 signaling pathway may play an important role in the development of HAPEThe differentially expressed genes in HAPE patients are mainly involved in inflammatory signaling pathways involved in antigen recognition,such as Toll-like receptor signaling pathway,NF-?B signaling pathway,NOD-like receptor signaling pathway;or inflammatory-related signaling pathways,such as TNF Signaling pathways,antigen processing and presentation;and other signaling pathways involved in immune response activation,such as T cell receptor signaling pathways,complement and coagulation cascades,cytokine-cytokine receptor interactions,and cell adhesion molecules?CAMs?;organism resist Gram-negative bacteria,such as Mycobacterium tuberculosis,Salmonella,Escherichia coli,Legionella signal-regulating pathways;organism against Gram-positive bacteria,such as Staphylococcus aureus.Many of the differentially changed signaling pathways are the downstream signaling pathways after inflammatory response activation,or the defense response pathways when organism resist foreign bodies,such as bacteria.According to the degree of change,enrichment factor and the involved number of differential genes,we found that Toll-like receptor signaling pathway may play an important role in the occurrence of HAPE.The differentially expressed proteins in HAPE patients are mainly involved in Toll-like receptor signaling pathways,systemic lupus erythematosus,IL-17 signaling pathway,vasopressin-regulated water reabsorption,asthma,glycosylphosphatidylinositol?GPI?-anchor Biosynthetic,acute myeloid leukemia,viral carcinogenesis,adrenergic signaling in cardiomyocytes,programmed necrosis,NOD-like receptor signaling pathways,etc.In addition,the LPS content,lipopolysaccharide binding protein?LBP?,Pentraxin-related protein PTX3 and calcium-binding protein S100A8?S100A8?were significantly increased in the serum of patients with HAPE;The TLR4 signaling pathway genes,such as CD14,TLR4,MD2,MYD88 and IRAK4 screened by transcriptomics were significantly increased,suggesting that TLR4 signaling pathway may be an important signaling pathway in HAPE.2.Hypoxia exacerbates the inflammatory response mediated by TLR4 signaling pathway that may plays a key link in the occurrence of HAPE?1?Acute hypoxia combined with low dose LPS can replicate a stable rat HAPE modelCompared with the CTL group,the LPS group and the HPO group,H.E staining showed that the number of inflammatory cells in the lung tissue of the COMB group increased more significantly,the alveolar septum filling increased significantly and the alveolar structure was severely damaged,and the lung tissue was wet and dry.The weight ratio was also increased more significantly,and the protein concentration of the alveolar lavage fluid was also significantly increased.In addition,RT-PCR and gene chip detection the inflammation-related genes in the COMB group,such as LBP,CD14,CCL3,NF-kBia,TNF-?and IL-1?were significantly higher than the CTL group,the LPS group and the HPO group.The levels of BALF and plasma inflammatory cytokines in COMB group were also significantly higher than that in CTL group,the LPS group and the HPO group.There were 1595 up-regulated genes,including 1489 in the COMB group,293 in the LPS group,and 221 in the HPO group;and 1213 down-regulated genes,of which the COMB group contained 1171 and the LPS group contained 158,suggesting that the COMB group expressed more up-regulated and down-regulated differential genes.In summary,the COMB group showed significant pulmonary edema that acute hypoxia combined with low-dose LPS can induce rat HAPE model.?2?Differentially expressed genes in the rat HAPE model are mainly involved in inflammatory and immune response-related functions,and the TLR4 signaling pathway is the key signaling pathway.GO functional enrichment analysis found that the differentially expressed genes of BALF cells in rat HAPE model mainly involved immune responses,inflammatory reactions,cell adhesion,LPS responses and other biological functions related to inflammation and immune response,such as response to IL-1,positive regulation of IL-8,production of IL-6,positive regulation of NF-?B signaling,neutrophil chemotaxis,production of cytokines and chemokines,cell adhesion,and so on.The KEGG pathway enrichment analysis found that the differentially expressed genes of BALF cells in rat HAPE model mainly involved the signaling pathways involved in Toll-like receptor signaling pathway,cytokine-cytokine receptor interaction,antigen processing and presentation signaling pathway.Moreover,gene expression changes associated with LPS reactions are evident,and these differentially expressed genes are involved in signaling pathways including Toll-like receptor signaling pathways,cytokine-cytokine receptor interactions,cell adhesion molecules,complement and coagulation cascades.The TLR4 signaling pathway and its downstream inflammatory response may play an important role in hypoxia-enhanced LPS-mediated inflammatory responses.?3?Hypoxia exacerbates LPS-induced inflammatory response via the TLR4signaling pathway in alveolar macrophageCompared with the CTL group,LPS group,and HPO group,the mRNA and protein levels of inflammatory factors such as TNF-?,IL-1?,and IL-6 in the NR8383 cells and the culture medium in the COMB group were significantly increased.In addition,the mRNA and protein levels of TLR4 gene in COMB group were significantly increased,and the activity of TLR4 promoter in COMB group was also significantly enhanced,suggesting that acute hypoxia combined with low-dose LPS can significantly increase the expression of TLR4 gene and its downstream inflammatory factors in alveolar macrophages.The TLR4inhibitor?TAK-242?was used to inhibit the expressin of TLR4 in NR8383 that TLR4mRNA and protein expression were significantly inhibited in LPS group and COMB group,and TAK-242 reduced the levels of inflammatory factors with a dose-dependent manner in LPS group and COMB group.Taken together,the TLR4 signaling pathway may be an important hub for acute hypoxia exacerbates low-dose LPS-induced inflammatory responses in alveolar lung macrophages.3.HIF-1?protects against HAPE by inhibiting the inflammatory response mediated by TLR4 signaling pathway?1?The interaction between HIF-1?and TLR4 may be involved in the occurrence of HAPECorrelation analysis showed that the differential expression gene TLR4 in the Control group was significantly positively correlated with the LPS content in the serum,but there was no significant correlation between LPS and TLR4 in the HAPE group,suggesting that there may be other factors that may affects the activation of TLR4 signaling pathway in the occurrence of HAPE.In addition,the correlation analysis indedicate that there hava a significant negative correlation between the up-regulated TLR4 and the down-regulated HIF-1?,suggesting that There may be some links between them.?2?HIF-1?regulates the inflammatory response of alveolar macrophages by regulating the expression level of TLR4First,using PX478 and siRNA interference sequences to reduce the level of HIF-1?in NR8383 can enhance TLR4 promoter activity;conversely,using DMOG and HIF-1?overexpression plasmid to increase the level of HIF-1?in NR8383 can inhibit TLR4promoter activity.Second,using PX478 and siRNA interference sequences to reduce the level of HIF-1?in NR8383 can enhance the transcription and translation levels of TLR4,and the TNF-?expression of cells is increased;conversely,using DMOG and HIF-1?overexpression plasmid to increase the level of HIF-1?in NR8383 can inhibit the transcription and translation levels of TLR4,and the TNF-?expression of cells is attenuated.Taken together,HIF-1?regulates the inflammatory response of alveolar macrophages by modulating the expression level of TLR4.?3?Pre-increased the levels of HIF-1?in rats can alleviate the damage in rat lung tissue caused by inflammatory response which mediated by acute hypoxia combined with low-dose LPSCompared with the untreated COMB group,the H.E staining results showed that the number of inflammatory cells and membrane congestion level in the alveolar space of DMOG pretreated COMB group were significantly decreased,the wet-to-dry weight ratio of lung tissue was significantly reduced,and the arterial oxygen saturation was significantly increased.In addition,the levels of inflammatory factors,such as TNF-?,IL-1?and IL-6 in the plasma of the DMOG pretreatment COMB group were significantly reduced.In conclusion,pre-increased the levels of HIF-1?in rats can alleviate the damage in rat lung tissue caused by inflammatory response which mediated by acute hypoxia combined with low-dose LPS.Conclusion:?1?The inflammatory response in HAPE patients was significantly enhanced compared with the control group.?2?The differential genes and proteins in HAPE patients are mainly involved in biological processes such as inflammatory response and immune response regulation.?3?The inflammatory response mediated by TLR4 signaling pathway may play an important role in the development of HAPE?4?Acute hypoxia combined with low-dose LPS can replicate a stable rat HAPE model.The differentially expressed genes in rat HAPE model are mainly involved in inflammatory and immune-related functions,and TLR4 signaling pathway is a key signaling pathway.?5?Hypoxia exacerbates LPS-induced inflammatory response via the TLR4 signaling pathway in alveolar macrophage?6?HIF-1?regulates the inflammatory response of alveolar macrophages by regulating the expression level of TLR4.Pre-increased the levels of HIF-1?in rats can alleviate the damage in rat lung tissue caused by inflammatory response which mediated by acute hypoxia combined with low-dose LPS. |
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