| Objectives:Acute exacerbation is a major event in the natural course of chronic obstructive pulmonary disease(COPD),and recurrent episodes of acute exacerbations can lead to worse clinical consequences and a heavier financial burden.Frequent acute exacerbation phenotype is a special phenotype of chronic obstructive pulmonary disease(COPD),which has the characteristics of poor prognosis,high mortality,and heavy economic burden.In addition,we understand the frequency and mechanism of frequent acute exacerbations.Still not complete.There is currently no effective method to improve the prognosis,which requires special clinical attention.This study was designed to detect the differential expression of proteins in patients with frequent exacerbations of COPD,to assess whether these expression profiles are unique in COPD patients,and may reveal the molecular pathogenesis of frequent acute exacerbations of COPD.Methods:Part ?: Screening and verification of subtype-specific proteins of chronic obstructive pulmonary frequent exacerbators based on TMT-LC-MS/MS1)Detection of differentially expressed proteins in lung tissue of patients with frequent acute aggravation(FE)and infrequent acute aggravation(IE)using TMT-LC-MS/MS technique,and performing a series of activities such as Cluster,GO and KEGG Information analysis,screening to select differential proteins associated with frequent acute exacerbations of COPD.2)Western Blot technique was used to detect the expression of differential proteins at the protein level to verify the accuracy of proteomic results.Part ?: Screening of serum biomarker of chronic obstructive pulmonary frequent exacerbators based on TMT-LC-MS/MS1)Based on proteomic screening results and literature reports,whether ELISA is used to verify whether Biglycan is highly expressed in the serum of patients with FE can be used as a biomarker for frequent acute exacerbations.2)The accuracy,specificity and sensitivity of the protein model were analyzed by ROC curve method.Part ?: Role of Biglycan molecule in CSE+LPS-induced airway inflammation1)Westen Blot was used to detect the expression levels of NLRP3,ASC,IL-1? and caspase-1 in lung tissues of frequent exacerbaotors and infrequent exacerbators;2)Location of Biglycan expression in lung tissues;3)Acute exacerbation model of COPD in vitro was established by CSE and LPS stimulation;4)Exogenously addition of human recombinant protein Biglycan and CSE+LPS,the secretion of inflammatory factors IL-1? and IL-6 were detected by ELISA,and Western Blot was used to detect the expression levels of NLRP3,caspase-1,IL-1? and ASC,to evaluate whether there were differences in the degree of inflammatory response.5)NLRP3 inhibitors were given to investigate whether Biglycan is involved in regulating inflammatory responses by activating NLRP3 inflammasomes.Results:Part ?: Screening and verification of subtype-specific proteins of chronic obstructive pulmonary frequent exacerbators based on TMT-LC-MS/MS1)Through this experimental study,we identified 4506 proteins and screened 23 differentially expressed proteins in the lung tissues of patients with FE and IE.Among them,9 differentially expressed proteins and 14 differentially expressed proteins were frequently expressed in frequent acute recombination.Compared with non-frequent acute plus recombination;2)Three differential proteins(PIGR,Biglycan and MHC II)were validated by Western blot analysis in the FE and IE groups of the cohort and validation cohort,respectively,and the reliability of the proteomic results was verified at the protein level.Part ?: Screening of serum biomarker of chronic obstructive pulmonary frequent exacerbators based on TMT-LC-MS/MSThe results of ELISA showed that the expression of Biglycan in serum of FE group was significantly higher than that of IE group(P < 0.05)1)The serum Biglycan level of frequent exacerbators was significantly higher than that of infrequent exacerbators and normal controls(P < 0.05);2)Serum Biglycan is more accurate in identifying frequent exacerbators of COPD.The model differentially diagnosed patients with frequent exacerbators and infrequent exacerbators with an AUC value of 0.899,with a sensitivity of 81.9% and a specificity of 68.9%.Part ?: Role of Biglycan molecule in CSE+LPS-induced airway inflammation1)NLRP3,ASC,IL-1? and Caspase-1 were significantly higher in lung tissue of patients with frequent acute exacerbations than those with infrequent acute exacerbations;2)Biglycan protein is mainly expressed in alveolar macrophages and extracellular matrix;3)Biglycan can aggravate the inflammatory response induced by LPS treatment in 16 HBE and A549 cells.4)Biglycan participates in the acute exacerbation of COPD by activating NLRP3 inflammasomes to regulate the expression of Caspase-1 and IL-1? and affecting airway inflammation and pyroptosis.Conclusions:1.A total of 4506 proteins were identified,and 23 differentially expressed proteins(DEPs)were screened in lung tissues of patients with FE and IE.Among them,9 DEPs were highly regulated and 14 DEPs were down regulated in FE group compared with IE group.DEPs(PIGR,Biglycan and MHC II)were verified to be consistent with proteomic results by Western Blot analysis.2.The serum level of Biglycan in the FE group was significantly higher than that in the IE group.The ROC curve analysis showed that the accuracy of Biglycan in identifying frequent exacebators from infrequent exacebators of COPD was higher,with a sensitivity of 81.8% and a specificity of 68.9%.3.Biglycan participates in the acute exacerbation of COPD by activating NLRP3 inflammasomes to regulate the expression of Caspase-1 and IL-1?,thereby affecting airway inflammation,high expression of Biglycan can make 16 HBE and A549 more prone to acute exacerbations. |
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