| BackgroundPulmonary surfactant protein B(SP-B)is a hydrophobic protein secreted by type II alveolar epithelial cells and plays an important role in reducing alveolar surface tension and host defense.SP-B plays an important role in lung surfactant function and is associated with lung disease.SP-B deficient mice died of severe acute respiratory failure at birth.There are several polymorphisms of human SP-B gene,among which the important single nucleotide polymorphism is SNP rs1130866(SP-B 1580C/T).SP-B 1580C/T polymorphism forms two common genetic alleles,C allele has the additional N-linked glycosylation sites and T allele has not,this glycosylation site difference may affect different SP-B protein processing and functions in certain diseases or stress,this polymorphism has different ability to maintain different respiratory homeostasis and host defense functions.Studies have shown that SP-B 1580C/T gene polymorphism is associated with pulmonary disease.Adults and children who carry the C allele at locus 1580 of SP-B gene are more likely to suffer severe lung damage.Pseudomonas aeruginosa pneumonia is a common gram-negative bacterial infection in ICU,which can directly cause lung injury.In severe cases,it can cause sepsis and even life-threatening complications,resulting in a high mortality.It has little research on the susceptibility of human SP-B 1580C/T gene polymorphism to pseudomonas aeruginosa pneumonia,especially severe pneumonia.The human transgenic(hTG)mouse model is helpful to overcome the limitation of human biological research in vivo,and can study the complex physiological or pathological processes of human beings,which is an ideal tool for the study of human diseases.In this study,we used hTG SP-B 1580C/T mice to establish a pseudomonas aeruginosa pneumonia model to study the susceptibility of different alleles and explore the underlying mechanisms.Objectives1.Establish a model of pseudomonas aeruginosa pneumonia in hTG SP-B 1580C/T mice,monitor the bacterial growth and analyze the tissue damage and inflammation at the histological and molecular levels;2.Study lung SP-B changes,NF-?B/NLRP3 signal pathway and cell death in pseudomonas aeruginosa pneumonia hTG mice;3.Investigate the susceptibility of different alleles of human SP-B 1580C/T mice and explore the underlying mechanisms.Material and MethodsPart one:Establishment and observation lung injury in pseudomonas aeruginosa pneumonia hTG SP-B mice model1.Human SP-B transgenic mice(FVB/N background)and FVB/N wild type mice were selected at 8-12 weeks and weighing about 25g.DNA was extracted from the tail of all mice for PCR gene identification.According to the PCR results,the mice were divided into three groups:SP-B-T,SP-B-C and WT group.Each group was then randomly assigned to pneumonia group and control group.2.The bioluminescent pseudomonas aeruginosa Xen5 strain was cultured and the bacterial concentration was determined by measuring OD600 value and confirmed by bacteria inoculation culture on LB agar plate at 37? overnight.The bacterial solution was diluted 500 times with sterile saline.50?l of P.aeruginosa dilution solution(4×104CFU/mouse)was injected intratracheally to make pneumonia model or 50?l sterile saline for control groups.After the pneumonia model,mice conditions were regularly observed,bacterial dynamic changes were detected by in vivo imaging system and the mice mortality rate was calculated.3.After 24 hours of infection,mice were anesthetized and sacrificed.Fresh blood,bronchoalveolar alveolar lavage fluid(BALF)and lung tissue were collected.BALF was diluted and was inoculated on LB agar plate and bacterial colony count was observed.The total number of cells in BALF was counted with a hemocytometer,and the classification of macrophages and neutrophils was observed after the BALF was cytospin and stained with HEMA3.After fixation with 10%formalin for 24 hours,lung tissues were stained with HE in paraffin embedded sections,and the pathological changes were observed and lung injury score was calculated.The pathological changes of lung tissue at the cellular level were observed by transmission electron microscopy,and the differences of lamellar bodies in each group were statistically analyzed.4.Lung tissues were homogenized and centrifuged,the supernatant was collected.The total protein concentrations in lung tissue homogenate and BALF were determined by BCA method.SP-B protein levels in lung tissue homogenate and BALF were determined by western blot.The expression of SP-B in lung tissues was observed by immunofluorescence.SP-B levels in each group were compared.5.Large Aggegrates were extracted from fresh BALF after high speed secondary centrifugation and surface tension was determined by constrained drop surfactometry(CDS).The adsorbed surfactant film is compressed and expanded at a rate of 20 cycles per second at 37? and relative humidity of nearly 100%to simulate normal tidal respiration.Closed-loop Axisymmetric Drop Shape Analysis(Cl-ADSA)was used to measure the surface tension and surface area of surfactant membrane.The minimum surface tension(?min)during dynamic cycling was used as an indication of surface activity.Part two:Susceptibility and potential mechanism of human SP-B 1580C/T gene polymorphism to pneumonia model6.Immunofluorescence and western blot methods were used to observe the expression of pNF-?B in lung.Meanwhile,inflammatory cytokines TNF-?,IL-6,IL-1? levels in serum,lung tissue homogenate and BALF were determined by ELISA.7.The apoptotic cells in lung were observed by TUNEL method.The levels of apoptosis-related proteins Bcl-2 and cleaved caspase-3 in lung were determined by western blot.Differences between the groups were compared to observe apoptosis in this model.8.The expression of pMLKL in lung tissues was observed by immunofluorescence.The levels of necroptosis-related proteins RIP3,MLKL and pMLKL were determined by western blot and the differences among the groups were compared to observe cell necroptosis in this model.9.The expression of NLRP3 in lung tissues was observed by immunofluorescence.The levels of NLRP3,ASC,cleaved caspase-1 and GSDMD were determined by western blot and the differences between the groups were compared to observe cell pyroptosis in this model.10.Statistical analysis.Data were presented as mean ± SEM.All statistics were performed with SigmaStat software and GraphPad Prism was used for mapping.Continuous variables were tested for normality and equality of variances.Student's t test was used between the two groups and one-way ANOVA was used in multiple comparisons.Survival analysis was performed with Kaplan-Meier survival curves and examined statistically by the Log-rank test.When P<0.05,statistical differences were considered,and when P<0.01,statistical differences were considered to be very significant.ResultsPart one:Establishment and observation lung injury in pseudomonas aeruginosa pneumonia hTG SP-B mice model1.PCR genotype showed that hTG mice expressed either human SP-B C or T allele,but not mouse SP-B gene,and WT mice only expressed mice SP-B gene.Western blot and immunofluorescence were used to observe the expression of SP-B in the lung tissues of normal hTG SP-B-C/T mice and WT mice.The results showed that both hTG SP-B-C/T mice and WT mice had normal expression of SP-B.In addition,western blot semi-quantitative analysis showed that the SP-B levels in hTG SP-B-C/T mice and WT mice were similar.2.After modeling,the control groups of the three types of mice had good mental status,normal diet and activity,while the pneumonia group showed low spirits,significantly reduced diet and slow action.Bioluminescence levels of IVIS-200 imaging system showed that a large number of bacteria grew after mice infection.Bacteria in all three types of mice increased sharply 12 hours after infection.SP-B-C mice had the highest bacterial loads at 24,36 and 48 hours after infection,compared with SP-B-T and WT mice.The mortality rate of SP-B-C mice at 48 hours after infection was higher than that of SP-B-T and WT mice,and none of the control mice died,indicating that the three types of mice had different sensitivity to pseudomonas aeruginosa.3.The total cell number in BALF increased significantly in infected mice compared with control mice at 24 hours after infection,and the SP-B-C mice was the highest in the three types of infected mice.Only macrophages were observed in the BALF of control mice,while more than 90%of cells in the BALF of infected mice were neutrophils.Macrophages increased after infection,but there were no differences among three types of mice.After infection,BALF neutrophils increased significantly,and SP-B-C mice had the highest number of neutrophils among three types of mice.The BALF of infected mice showed a large amount of bacterial growth,while the BALF of control mice showed no bacterial growth.The bacterial CFU in SP-B-C mice was the highest among three types of mice.4.HE staining of lung tissue sections showed that,compared with control mice,the infected mice showed significant lung damage,a large number of inflammatory cells in the alveoli and the interstitials,a large amount of proteinaceous debris accumulation and alveolar wall thickening.Lung injury scores showed that the scores of all three types of infected mice were significantly higher than those of each control mice,and the injury scores of SP-B-C mice were higher than those of SP-B-T and WT mice.5.Ultrastructural analysis by transmission electron microscopy showed that many normal lamellar bodies and mitochondrial structures in the type ? alveolar epithelial cells of each control group.After infection,increased number of autophagosomes,damaged lamellar bodies,abnormal mitochondria,and decreased microvilli were obserbed in type ? alveolar cells.6.Immunofluorescence results showed that SP-B expression decreased after infection,and the number of SP-B positive expression in SP-B-C mice was significantly lower than that of SP-B-T and WT mice.Western blot results showed a similar SP-B expression trend in lung tissues and BALF of the three mice.Western semi-quantitative analysis showed that there were significant differences in SP-B expression in lung tissues and BALF among all kinds of infected mice,and SP-B-C mice had the lowest SP-B expression among the three types of infected mice.7.The determination of surface tension of lung surfactant(LAs)in BALF of mice showed that the minimum surface tension(?min)of mice in the pneumonia group was significantly higher than that in the control group,suggesting that lung surface activity was significantly impaired after infection.There was no difference in ?min in the control group of the three types of mice,while there was a significant difference in ?min in the pneumonia group,SP-B-C>SP-B-T>WT,indicating that there was a difference in the regulation of alveolar surface tension by the polymorphism of hSP-B gene under infection conditions.Part two:Susceptibility and potential mechanism of human SP-B 1580C/T gene polymorphism to pneumonia model8.Immunofluorescence and western blot analysis were used to examine the expression of activated pNF-?B protein in lung tissues.Immunofluorescence showed no expression of pNF-?B in the control group,but significantly increased in the pneumonia group.Quantitative analysis of pNF-?B positive cells showed pNF-?B expression was significantly increased in hTG SP-B-C/T mice and WT mice compared with the control group.The levels of pNF-?B in the infected SP-B-C mice were the highest compared with those in the infected SP-B-T and WT mice.Western Blot results showed the same trend,the expression of pNF-?B was significantly increased in hTG SP-B-C/T mice and WT mice after infection.The proportion of pNF-?B/NF-?B in infected SP-B-C mice was the highest,and the difference was statistically significant compared with infected SP-B-T and WT mice.9.ELISA results showed that the levels of inflammatory cytokines TNF-?,IL-6,IL-1? in serum,lung tissue homogenate and BALF of the three types of mice were significantly higher than those in control group after infection,and the levels of cytokines in SP-B-C mice after infection were significantly higher than those in SP-B-T and WT mice10.TUNEL method was used to analyze apoptosis of lung cells in infected mice.24 hours after infection,a large number of apoptotic cells were observed in all three types of mice,while no apoptotic cells were observed in control mice.Quantitative analysis of TUNEL positive cells showed that the number of apoptotic cells in infected SP-B-C mice was higher than that in infected SP-B-T and WT mice.Western blot results showed that Bcl-2 levels of the three types of infected mice were lower than those of control mice,and Bcl-2 level of SP-B-C mice was lower than that of SP-B-T and WT mice.After infection,the levels of cleaved caspase-3 in three types of mice were higher than those of control mice,and the levels of cleaved caspase-3 in SP-B-C mice were higher than those in SP-B-T and WT mice.These results indicated that apoptosis occurred in mice after infection,and apoptosis of SP-B-C mice was more severe.11.Immunofluorescence showed high expression of pMLKL in the lungs of infected mice,while no expression was found in control mice.Quantitative analysis of pMLKL positive cells showed that the number of pMLKL positive cells in SP-B-C infected mice was higher than that in SP-B-T and WT mice.Western blot results showed that lung RIP3 levels of three types of infected mice were lower than those of control mice,and RIP3 level of SP-B-C mice was lower than that of WT mice.The expression of MLKL and pMLKL increased after infection,the MLKL and pMLKL levels of SP-B-C mice were higher than those of SP-B-C and WT mice.These results indicated that necroptosis of lung cells occurred in mice after infection,and necroptosis of SP-B-C mice was more severe12.Immunofluorescence and western blot analysis were used to examine the expression of NLRP3 and pyroptosis-related proteins in lung tissues.Immunofluorescence showed significant activation of NLRP3 in infected mice,while no activation was seen in control mice.Quantitative analysis of NLRP3 positive cells showed that the number of NLRP3 positive cells in infected SP-B-C mice was higher than that in infected SP-B-T and WT mice.Western blot results showed that the levels of NLRP3 in the lungs of the three types of infected mice were higher than those of control mice,and level of NLRP3 in SP-B-C mice was higher than that of SP-B-T and WT mice.Expression of ASC and cleaved caspase-1 increased after infection,and the levels of SP-B-C mice were higher than those of SP-B-T mice.Among the three types of mice,the GSDMD level of pneumonia group was higher than that of control group,and the GSDMD level of SP-B-C mice was higher than that of SP-B-T mice and WT mice.These results indicated that pyroptosis occurred in the lungs of mice after infection,and the pyroptosis was more severe in SP-B-C mice.ConclusionIn pseudomonas aeruginosa pneumonia hTG SP-B 1580C/T mice model,infected mice exhibited much bacterial growth,severe lung injury,a large number of inflammatory cells and high mortality,reduced SP-B level,increased lung surface tension of LAs,increased inflammatory cytokines levels,NF-?B and NLRP3 signal pathway activation,as well as cell death types of apoptosis,necroptosis and pyroptosis.SP-B-C mice showed more susceptibility and more cell death compared with SP-B-T and WT mice,indicating differential regulation of SP-B gene polymorphism to pneumonia induced ARDS by regulating NF-?B and NLRP3 pathways and cell death. |
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