The Role And Mechanism Of Pulmonary Surfactant On Acute Lung Injury Induced By Sepsis

BackgroudSepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection.Acute lung injury(ALI)is a common complication of sepsis.The manifestation in the lung is mainly due to the destruction of the lung epithelium and vascular endothelial barrier,the possible mechanism is that the uncontrolled inflammation involved with activation of lung epithelial cell apoptosis and mitochondrial dysfunction,so the recovery of mitochondrial function are essential for recovery.Pulmonary surfactant(PS)can improve the respiratory function and reduce lung injury in lipopolysaccharide(LPS)-induced acute lung injury rat model,but its specific mechanism remains unknown.ObjectiveI.Isolate rat primary alveolar type Ⅱ epithelial cells(AEC Ⅱ)and use lipopolysaccharide(LPS)to establish lipopolysaccharide-induced alveolar type Ⅱ epithelial cell injury model to explore the effects of pulmonary surfactant intervention.2.Transcriptome detection of rat model of acute lung injury induced by lipopolysaccharide,through cluster analysis,differential gene analysis,or based on data prediction system results,explore changes in ALI-related genes.3.Use lipopolysaccharide-induced alveolar type Ⅱ epithelial cells model to perform qPCR verification to screen for highly relevant differential genes and pathways.4.Establish a rat model of lipopolysaccharide-induced acute lung injury,and give lung surfactant intervention to detect lung histology,serum inflammatory factors and related signaling pathway protein levels,and explore the role and possible mechanism of lung surfactant on acute lung injury.MethodsPart Ⅰ.Extract and culture of primary AEC Ⅱ of SD rats immunohistochemical staining by cell morphology,selective markers SPA,SPB,and SPC,identification by immunofluorescence,and determination of the cultured by CCK8 cell proliferation experiment the activity of primary alveolar epithelial cells.Part Ⅱ.Establishe the model of LPS-induced AEC Ⅱ injury,and establishes different concentrations of LPS(0μg/ml,25 μg/ml,50 μg/ml,75 μg/ml,100 μg/ml,150 μg/ml concentration gradient)and stimulation time(Oh,2h,4h,6h,8h),observe cell morphology,CCK8 to detect cell viability,select LPS optimal stimulation concentration time,and PS intervention,observe cell morphology,CCK8 to detect normal group before and after intervention LPS group In the LPS PS group,cell viability,metabolic factors(IL-6,IL-8,TNF-α)and AEC Ⅱ cells occurred at 2h 4h and 6h after intervention.Part Ⅲ.Inject the LPS intraperitoneally in SD rats to establish the rat model of sepsis.The normal group and the lung tissue of the LPS group are used to extract total RNA.RNA-seq sequencing is performerd,and the differential genes are obtained according to the analysis of gene expression.ClusterProfiler is applied The software performs GO function enrichment analysis on the collected differential gene sets,and KEGG pathway enrichment analysis on LPS-induced ALI-related genes and their pathways.Part Ⅳ.Establish the LPS-induced AEC Ⅱ injury model,and qPCR verifies the expression level of mitochondrial function-related genes.Construct LPS-induced acute lung injury rat model and give PS intervention to compare the survival rate,blood gas analysis,inflammatory factors(IL-6,IL-8,TNF-α),lung tissue in normal group,LPS group,LPS+PS group Pathway protein,lung tissue ATP content.ResultsPart Ⅰ.Successfully extracted,cultured,and identified the primary AEC Ⅱ of SD rats.CCK8 test showed that it reached the best growth state at 72h.Part Ⅱ.The model of alveolar type II epithelial cell injury induced by lipopolysaccharide was successfully constructed.The cell morphology and CCK8 results suggested that the optimal LPS stimulation concentration was 75 μg/ml and the optimal stimulation time was 2h.At 72 hours after culturing AEC Ⅱ,cells were divided into two groups:LPS group and LPS+PS group according to the random number table method.After 2 hours of cell culture,no obvious change in cell morphology was observed in the normal group;75 g/ml LPS treatment Cells in the group began to appear deformed,with blurred edges and burrs.Cytoplasmic particles decreased,nuclei contracted,and the number of cells decreased.They gradually became vacuoles.The nucleus was lost,and no particles were seen in the cytoplasm;75 g/ml In the LPS+80 g/ml PS group,the edges of the cells showed that the pseudopodia extended outward the cytoplasmic particles increased and the nucleus did not shrink significantly.The CCK8 drug toxicity test showed that within 24 hours of cell culture,the viability of cells in the normal control group was the best,the cell viability in the LPS+PS group was second,and the cell viability in the LPS group was the worst The percentage of apoptosis in the LPS group was the highest,and there was no significant difference in the percentage of apoptosis between the LPS+PS group and the normal control group.The levels of IL-6,IL-8,and TNF-α in the cell supernatant of the LPS group were the highest at each time point;after the addition of PS,the expression levels of inflammatory factors decreased at each time point,and the difference was statistically significantPart Ⅲ.The rat model of sepsis was successfully established Total RNA was extracted from lung tissues of normal group and LPS group,and RNA-seq sequencing was performed After analysis and comparison,there were 8820 genes differentially expressed in NC group and LPS group.4197 genes were down-regulated in LPS(relative to NC group),while 4624 genes were up-regulated in LPS group.Screen the genes with significantly different expressions in the NC group and the LPS group,use clusterProfiler software to perform gene enrichment analysis of GO(Gene Ontology)function and KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway enrichment analysis on the differential gene sets.The results show that lipopolysaccharide can The genes related to the induction of mitochondrial apoptosis in alveolar cells include the expression of Ndufs2,Scol,Sirt2,and inhibit the expression of anti-apoptosis related genes Pinkl and Bcl2.Part Ⅳ.After establishement of LPS-induced AEC Ⅱ injury model,qPCR verification results showed that the expression of anti-apoptosis-related gane Bcl2 was suppressed,and the mitochondrial anti-apoptosis-related gene Bcl-2 and Bim-Bax-Bcl-2-Bak signaling pathway were selected for animal verification.Established LPS-induced acute lung injury rat model and gave bovine lung surfactant(PS)intervention,survival time of LPS+PS group,blood gas analysis index,pathological changes of lung tissue,lung edema score,serum inflammatory factors concentration were significantly improved compared with the LPS group,and the expression of HMGB1 protein in the lung tissue of the LPS+PS group was decreased,and the activity of ATPase was increased.The mitochondrial pro-apoptotic proteins Bax,Bim,Bak and the apoptosis proteins Cleaved-Caspase3,Cleaved-Parpl decereased.The differences were statistically significant.ConclusionsIn this study,by extracting primary alveolar type Ⅱ epithelial cells(AEC Ⅱ)from SD rats,a model of alveolar type Ⅱ epithelial cell injury induced by lipopolysaccharide was established,and it was found that pulmonary surfactant can inhibit the primary alveolar typeⅡ induced by lipopolysaccharide LPS Damage to epithelial cells.The transcriptome changes in the rat model of acute lung injury induced by lipopolysaccharide indicated that acute lung injury activates mitochondrial function-related signaling pathways,the expression of antiapoptosis gene Bcl-2 decrease.After in vitro experiments,the results show that bovine lung surfactants could effectively improve the acute lung injury induced by lipopolysaccharide.The mechanism may be by changing the mitochondrial anti-apoptosis gene Bcl-2 and Bim-Bax-Bcl-2-Bak signaling pathway to improve the mitochondrial function of alveolar epithelial cells,reduces apoptosis,reduces lung tissue injury and improves survival of rats.