Differential Expression Of Proteins In Pseudomonas Aeruginosa-Induced Acute Lung Injury

BackgroundAcute lung injury (ALI) and its presentation with more severe hypoxemia, the acute respiratory distress syndrome (ARDS), are critical illness in the respiratory system and common causes of respiratory failure. Even if the oxygen therapy, antibiotics, mechanical ventilation and liquid management support technologies have made substantial progress in recent years, the mortality rate remains as high as 40% -60%. Some studies have referred that the high mortality with ALI / ARDS has a lot references to the diagnostic criteria, because current clinical diagnostic criteria in used are the non-specific standards and lacking of dependablity with the prospective study of risk factors. This means that we can not rely on the current distinction to make the diagnosis of ALI / ARDS which are caused by severe pneumonia, sepsis, mechanical ventilation, trauma and so on.(According to the intensive care unit, the investigation of ALI / ARDS etiology shows that the proportion of severe pneumonia and sepsis were 50% and 26%, subsequent are trauma mechanical ventilation). It is precisely these risk factors of ALI / ARDS which could obtain the early diagnosis and treatment are the key to reducing mortality.Protein is the direct executor of gene function which has become the most major biological research object in post-genome era, the associated technology called proteomics.Proteomics are new techniques that provide powerful tools for studying the holoprotein of the cell or tissue. By studying the diseased tissues or cells, Proteomics can improve elucidate molecular classification of diseases and discover sensitive biomarkers that are useful for early diagnosis,treatment, and prediction of deseases. Because of the proteomics can provide new clues to explore the pathogenesis of the diseases, it is considered to have important clinical value and prospects.ObjectiveProteomics we performed to analyze and identify the differentially expressed proteins in ALI which was induced by P. aeruginosa infection and even to provide potential specific biomarker and establish further targeted therapy.Methods1. Sixty adult Specific-pathogen-free male Sprague–Dawley rats were randomly separated into four groups: P. aeruginosa group, Oleic acid group, mechanical Ventilation group and control group.Lung tissues and BAL were obtained at the set times.Then the lung tissues were fixed and taken for histopathology, were homogenized and calculated bacterial colonization and were measured wet-to-dry weigh ratios (W/D).The levels of BAL TNF-a,IL-1βwere detected by ELISA.2. Two-dimensional electrophoresis(2-DE)was used to separate the total proteins, Images of CBB R520-stained 2D gel were alanyzed and then established matchset gel. The differential expression proteins were identified by peptide mass fingerprint(PMF)based on matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS)and searched in database of proteins by Mascot software.3. The methods of QPCR,Western blot and Immunohistochemistry were used to verificate the differentially expressed proteins which were associated with P. aeruginosa.Results1. Compared to the control group, TNF-a,IL-1β,W/D and lung injury score had significantly elevated in other three groups.2. 22 differential protein spots were found and 11 of them were up expressed in PAE group.18 proteins were analyzed and identified by MALDI-TOF-MS. Two proteins were related to the pathogenesis of P. aeruginosa,such as Peroxiredoxin1 and Calreticulin.3. In order to validate the reliability of the identified results,the expression of Peroxiredoxin1 and Calreticulin were detected by QPCR,Western blot and Immunohistochemistry .The results displayed that two of them were up-regualted in ALI of P. aeruginosa,whereas they were down-regualted in the other groups,which were consistent with our 2-DE analysis results.ConclusionsIn the experiment , we successfully established three acute lung injury models and identified 18 differential proteins expression by comparative proteomics. This findings indicated that Peroxiredoxin1 and Calreticulin may play an important role in ALI process that influence immunity, inflammation, and antioxidize regulation. Our study will provides a useful clue to elucidate the infection-induced ALI pathogenesis.