| Allergic rhinitis(AR)refers to the non-infectious chronic inflammatory reaction mainly mediated by immunoglobulin E(Ig E)after the nasal mucosa of atopic individuals is exposed to allergens.The symptoms mainly include sneezing,nasal itching,nasal congestion and runny nose.Epidemiological surveys have shown that the incidence of AR has gradually increased in the past few decades,and currently affects 10% to 40% of the world’s population.AR not only affects the quality of life,but can also cause a huge social and economic burden,and it has become a global health problem.According to the different types of allergen,AR is divided into seasonal AR and perennial AR.The common allergens of seasonal AR are seasonal inhalants such as pollen and fungi;while the common allergens of perennial AR are house dust mites(HDM),animal dander,cockroaches and other indoor perennial inhalants.Pollen is the main allergen of seasonal AR.Due to geographical conditions,climatic factors and human factors,the distribution of plants is obviously regional,which results in the regional characteristics of pollen dispersal.For example,Artemisia pollen is the most common allergen in summer and autumn in northern China.HDM is the primary allergen of perennial AR.HDM secretions,fecal pellets,molting,and mite corpses are all strong allergens.Exposure or inhalation of HDM can induce allergic reactions in atopic people and cause AR.However,the specific biological mechanism of AR caused by Artemisia pollen and HDM is still unclear.Therefore,exploring the molecular mechanisms of Artemisia pollen and HDM in the occurrence of AR has important scientific significance and clinical value.As the first line of defense,the epithelial barrier is essential to protect the host’s immune system from harmful pathogens.Nasal epithelial cells,as the physical barrier,chemical barrier and immune barrier inside the nasal cavity,play an important role in resisting allergens,pathogens and other foreign particles.Studies on different allergens show that allergens can damage the nasal epithelial barrier.For example,Artemisia pollen and HDM can destroy the tight junctions between nasal epithelial cells and promote the release of related inflammatory factors.Studies have shown that birch pollen allergens can enter the epithelial cells of the nasal mucosa.This process may be mediated by the caveolin-related endocytic pathway,and the allergens entered the epithelial cells may induce epithelial-related changes in signal pathways.In addition,studies have shown that the allergens in HDM can trigger epithelial cell-related immune responses,mediate the release of related inflammatory factors,and trigger allergen-related cell apoptosis.However,whether the allergenic proteins Art v 1 and Der p 1 in Artemisia pollen and HDM can enter the human nasal epithelial cell line(HNEp C),and which proteins can the allergens interact with and regulate the epithelial cells changes in specific signaling pathways after entering the epithelial cells have not yet been reported.Therefore,exploring whether Art v 1 and Der p 1 can enter nasal epithelial cells,and clarifying the changes in the corresponding signaling pathways in epithelial cells after the entry of allergens will lay the foundation for elucidating the pathogenesis of AR.Therefore,in order to explore the biological effects of Art v 1 and Der p 1,which are the main allergenic proteins of Artemisia pollen and HDM,on nasal epithelial cells,this study firstly performed transcriptome sequencing on seasonal and perennial AR nasal epithelial cells.The differentially expressed genes and the enrichment of gene signaling pathways are analyzed.In addition,we expressed and purified Art v 1 and Der p 1 proteins in vitro,studied the proteins that interact with Art v 1 and Der p 1 in nasal epithelial cells.At the same time,we explored the changes in gene transcription levels in nasal epithelial cells stimulated by Art v 1 and Der p 1 proteins,analyzed the signal pathways that allergens Art v 1 and Der p 1may regulate,and clarified the influence of allergens on nasal epithelium and its specific molecular mechanism.The main results are as follows.1.Bioinformatics analysis of nasal epithelial cell gene expression in seasonal and perennial AR.The inflammatory response,TNFα/NF-κB signaling pathway,and IL2/STAT5 signaling pathway are significantly up-regulated in AR patients allergic to Artemisia pollen,indicating the promotion of inflammatory response.AR patients allergic to HDM have significant down-regulation of G2 M,E2F,and MYC,indicating the inhibition of cell proliferation.TNF and CDK1 are the most interacting proteins.It is inferred that TNF and CDK1 may play important roles in Artemisia pollen and HDM-sensitized AR,respectively.2.Analysis of protein interaction between HNEp C and Art v 1 and Der p 1.In order to subsequently explore the interaction protein of Art v 1 and Der p 1 protein in nasal epithelial cells and analyze the changes in transcriptomics in cells after the protein stimulates HNEp C,we successfully constructed a prokaryotic expression vector containing both GST tag and His tag.The proteins Art v 1 and Der p 1 are induced and expressed in E.coli,and the proteins are purified in vitro using Ni-NTA-Agarose,which lay the foundation for subsequent experiments.In addition,we constructed,expressed and purified the Art v 1 and Der p 1 fusion protein containing green fluorescent protein(EGFP)in vitro,and found that Art v 1 and Der p 1 can enter HNEp C.We co-incubated the in vitro expressed and purified GST-Art v 1 and GST-Der p 1 proteins with the whole protein of HNEp C,and enriched the proteins interacting with Art v 1 and Der p 1 in HNEp C through GST pull-down technology.The interacting proteins were identified by mass spectrometry technology,and enrichment analysis of these proteins revealed that the functions and signal pathways of these proteins are mainly enriched in the tight junctions,cytoskeleton,membrane,endocytosis,cell cycle and apoptosis.3.The differentially expressed gene analysis of HNEp C after exposure to Art v1 and Der p 1 recombinant proteins.In order to explore the effects of allergens Art v1 and Der p 1 on HNEp C cells,HNEp C cells were treated with in vitro purified allergens Art v 1 and Der p 1,and RNA-seq analysis was performed on the treated cells.We analyzed the genes that HNEp C changed after allergen treatment by high-throughput sequencing technology and performed gene enrichment analysis on these genes.Through the functional enrichment analysis of differentially expressed genes,we found that,similar to the protein functional enrichment after mass spectrometry,we also enriched the signal pathways related to cell adhesion and tight junctions,indicating allergens may cleave related proteins by interacting with adhesion proteins on the one hand,and may also disrupt tight junctions and epithelial cell barrier function by regulating the expression of cell adhesion and tight junction-related proteins at the transcriptional level.In addition,we have also observed that differentially expressed genes are enriched in the positive regulation of cell apoptosis,the negative regulation of cell proliferation,and programmed necrosis.4.Art v 1 and Der p 1 participate in the occurrence and development of AR by affecting the pyrolysis of HNEp C.As a manifestation of programmed cell necrosis,pyrolysis plays an important role in epithelial cell-related inflammation.Based on the above studies,we found that the allergens Art v 1 and Der p 1 can mediate the positive activation of apoptosis-related signaling pathways and programmed necrosis in HNEp C,while the pyrolysis-related signaling pathways cannot be enriched due to the lack of gene annotations in KEGG and GO.In the analysis of the transcriptomics data of HNEp C treated with Art v 1 and Der p 1,we found that the IL-1β gene transcription level increased.As a key protein in the pyrolysis signal,the increase in IL-1β gene transcription level implies the pyrolysis pathway activation.Through experiments,we further found that HNEp C swelled and had many bubble-like protrusions after treatment with allergens Art v 1 and Der p 1.At the same time,Caspase-1 in the cells was activated and the release of LDH and IL-1β increased,indicating that Art v 1 and Der p 1 mediated the pyrolysis of HNEp C cells.In summary,through the above studies,we analyzed the difference in gene expression in nasal epithelial cells of patients with seasonal and perennial AR,and revealed the possible differences in biological mechanisms.Subsequently,the interacting proteins of Art v 1 and Der p 1 in HNEp C were identified and enriched,and the differentially expressed genes of HNEp C caused by sensitized proteins were analyzed,and the pyroptosis pathway was verified to clarify that Art v 1 and Der p 1participates in the occurrence and development of AR by affecting the pyroptosis of HNEp C,laying a foundation for the in-depth exploration of the pathogenesis of AR mediated by Artemisia pollen and HDM allergen and precise treatment. |
PDF Full Text Request