| Bronchial asthma,a common chronic airway inflammation disease with complex pathogenesis and pathomechanism,is dually influenced by genetic and environmental factors and involvs various kinds of cells,cell components and biochemical processes.Though the pathogenesis of asthma has been widely studied,a number of controversies persist..Interleukin-13(IL-13)is a type-2 helper T cell(Th2)cytokine and has been shown to play important roles in the pathogenesis of asthma,but the corresponding mechanisms have remained unclear.Since so far most of the studies have been focused on specific molecules and pathways,we attempted in this work to study the roles of IL-13 in asthma at the proteomic level in order to achieve comprehensive understanding of the pathomechanism.The work consists of three parts,namely,1)the establishiment of an IL-13 induced asthma model on human bronchial smooth muscle cells(HBSMC),2)differential proteomic analysis on the changes caused by the treatment of IL-13 with or without further treatment with R-terbutaline(a β2-adrenergic receptor agonist),and 3)function and pathway analysis of the differentially regulated proteins.Firstly,we established a cellular asthma model by stimulating HBSMC with 50 ng/m L IL-13.Further,the celles were treated with R-Ter at concentrations of 0.05 μM,1 μM and 10 μM Twenty-four or 48 hours after IL-13 or R-Ter treatment,the HBSMC from the ten groups(control group included)were subjected for cell size and proliferation measurements.The results showed that compared with the control group,the IL-13 induction,both after 24 and 48 hours,led to significant increase of the cell size,while compared with the IL-13-induced group,R-Ter administrated groups showed obvious decease of the cell size,suggesting hypertrophy caused by IL-13 stimulation and recovery by the treatment of the agonist.Different from the cell size changes,the proliferation of HBSMC was not observed to be altered by either IL-13 stimulation or IL-13 stimulation plus R-Ter treatment.Soluble proteins were extracted from the cells of the ten groups by ultrasonication and consecutively submitted to proteomic analysis consisting nondenaturing two-dimensional gel electrophoresis(2-DE),picking of differential spots and nano ultra-performance liquid chromatography-tandem mass spectrometry(nano-UPLC-MS/MS).A total of 220 spots showing different staining intensitites were excised from the 10 gels(one representivie gel for each group)and subjected to standardized procedures of destaining,reduction and alkylation,in-gel digestion and peptide extraction.The peptide mixtures from each spots were analyzed by quantitative nano-UPLC-MS/MS.Due to the high sensitivity of the employed MS instrument,each spot were assigned to contain 2-107 proteins while 792 kinds of proteins were detected in all.Following the logic that the staining intensity of a spot is more likely to be caused by the changes of the relatively abundant proteins,five most abundant proteins in each spot were extracted and their quantites in different gels were compared with the thresholds of fold change ≥1.5 for upregulation and ≤0.67 for downregulation.Totally 89 proteins showed up-or down-regulation in the comparison.Protein classification and gene ontology analysis showed that these proteins were mainly involved in metabolism,stress response,transcription and translation,cell structure and cell physiological activities.In-depth examination of the differentially regulated enzymes of metabolism suggested the induction of IL-13 lead to enhanced glycolysis and suppressed pentose phosphate pathway,possibly as a result of faster energy production.Administration of different concentrations of R-Ter appeared to help the IL-13 stimulated cells to recover the levels of these metabolic processes.Also,the IL-13 treated cells showed upregulation of proteins responsible for inhibition of aggregation of misfolded proteins,protein sorting,processing,transporting,etc.,while administration of R-Ter downregulated them to a certain degree.The changes of stress-responsive proteins suggested that IL-13 induction may cause an abnormal environment for the cells and lead to the rise of cellular inflammation response.IL-13 induction also appeared to result in unstable binding of the actin filament,which might cause instablility of cytoskeleton structures.Moreover,downregulation of caldesmon and fibronectin caused by IL-13 were observed,which might be related to the decreased contraction of the bronchia smooth muscle in asthma.Likewise,the administration of R-Ter improved the situation.Cellular translation were found to be enhanced after the IL-13 induction and R-Ter also reversed the changes.Interestingly,POTE ankyrin domain family member I was found to be expressed in relatively high level only in the group of 48 hours after IL-13 induction but totally undetected in other groups.Reference searching showed there were few studies on the function of this protein but only suggested by gene analysis in other diseases to be pro apoptotic.Probably,the long term asthmatic status might cause apoptosis while the agonist drug helped to improve the situation.Another work I have been working on during the undergraduate years was related to the methodology of nondenaturing proteomics.We established a nondenaturing proteomic strategy and applied for global mapping of rat plasma proteins.Plasma from adult male rats were separated by nondenaturing micro 2DE and 136 CBB-stained spots were numbered and subjected to in-gel digestion and label-free quantitative LC-MS/MS.Totally 199 protein were identified,but the results revealed that each gel spot contained multiple proteins and meantime each protein was assigned in multiple spots.Since such results could not be appropriately presented as a conventional 2DE map,i.e.a list or a gel pattern with one or a few proteins annotated to each spot.Therefore,the LC-MS/MS quantity data was used to reconstruct the gel distribution of each protein and a library containing 199 native protein maps was established for rat plasma.Futher,since proteins that formed a complex would migrate together during the nondenaturing 2DE and thus show similar gel distributions,correlation analysis was attempted for similarity comparison between the maps.The protein pairs showing high correlation coefficients included some well-known complexes,suggesting the promising application of native protein mapping for interaction analysis.With the importance of rat as the most commonly used laboratory animal in biomedical research,we expect this work would facilitate relevant studies by providing not only a reference library of rat plasma protein maps but a means for functional and interaction analysis. |
