| Chapter 1 The expression and clinical significance of NOS2 in patients with allergic rhinitisObjective: The disturbance of energy metabolism,accumulation of reactive oxygen species(ROS)and secretion of pro-inflammatory cytokines are the important histopathological changes of allergic rhinitis(AR).We previously showed that the serum inducible nitric oxide synthase(i NOS/NOS2)and nasal nitric oxide concentration(n NO)were closely associated with the efficacy of subcutaneous immunotherapy in AR patients.As an upstream rate-limiting enzyme of NO synthesis,NOS2 is involved in many inflammatory and allergic diseases.This chapter aims to explore the expression of NOS2 in AR patients and its association with ROS and epithelial cell-derived cytokines in nasal mucosa.Methods: Clinicopathological specimens were collected,and quantitative real-time polymerasechain reaction(q RT-PCR)was performed to detect the expression levels of NOS1,NOS2,NOS3 and interleukin(IL)-33,thymic stromal lymphopoietin(TSLP)and IL-25.Western blotting(WB)and immunofluorescence(IF)were conducted to evaluate the protein expressions,and ROS levels in tissues were detected by dihydroethidium.The concentrations of NOS1,NOS2 and NOS3 in serum were determined by enzyme-linked immunosorbent assay(ELISA).Results: AR patients exhibited significantly higher levels of n NO and ROS in nasal mucosa than healthy controls.The m RNA and protein levels of NOS2,IL-33 and TLSP were increased in AR group than control group.Correlation analysis results showed that tissue NOS2 expression levels were positively correlated with IL-33,TLSP,IL-25 and ROS levels and n NO concentrations in AR patients.IF results indicated that the expressions of NOS2,IL-33,TLSP and IL-25 in AR nasal mucosa were enhanced and concentrated in the nasal epithelial cell region.ELISA results showed that the serum concentrations of NOS2 in AR patients were significantly higher than that in healthy controls.Conclusion: NOS2 was up-regulated in nasal mucosa and peripheral blood samples of AR patients,and ROS,IL-33,TSLP and IL-25 levels were enhanced in nasal mucosa of AR.The elevated tissue NOS2 levels were correlated with the levels of n NO,ROS,IL-33,TSLP and IL-25.These results suggest that NOS2 may be involved in the occurrence and development of AR mucosal inflammation by regulating the production of n NO,ROS and epithelial-derived cytokines.Chapter 2 NOS2 regulates HDM-induced ROS and epithelial-derived cytokines expressions in human nasal epithelial cellsObjective: We previously confirmed that NOS2 was enhanced in nasal mucosa of AR patients and its levels were associated with ROS and epithelial cell-derived cytokines levels in tissues.Prior studies reported that ROS could regulate the expression of epithelial cell-derived cytokines and participated in the occurrence and development of airway inflammation.Therefore,this chapter aims to explore the expression of NOS2 in human nasal epithelial cell(HNEp C)model induced by house dust mite(HDM),and confirm that whether NOS2 can promote epidermal cytokine expression and cellular inflammation by regulating ROS production.Methods: HNEp C was stimulated by HDM to construct cell model,and the expression levels of NOS1,NOS2,NOS3,IL-33,TSLP and IL-25 in cells were detected by WB and IF.The ROS in cell and NO levels in cell supernatant were detected.Subsequently,siRNA was applied to regulate the expression of NOS2 and N-acetyl-L-cysteine(NAC)was used to inhibit the expression of ROS,and their effects on the expression of ROS,NO and epithelial cell-derived cytokines were evaluated.Finally,the NOS2 overexpression plasmid and NAC was used to intervene HNEp C,and the expression levels of epithelial cell-derived cytokines were detected.Results: HDM could enhance the expression of NOS2 in HNEp C,and promote the expression of ROS,IL-33,TSLP and IL-25,and the release of NO,but exhibited no significant effect on the expression of NOS1 and NOS3.NOS2 knockdown could inhibit the HDM-induced NO release and intracellular ROS,IL-33,TSLP and IL-25 production in HNEp C.In HDM induced HNEp C,NOS2 overexpression promoted NO release and intracellular ROS,IL-33,TSLP and IL-25 production,and NAC inhibited NOS2-mediated IL-33,TSLP and IL-25 overexpression.Conclusion: HDM enhance NOS2 expression and promote NO release and ROS,IL-33,TSLP and IL-25 expression in HNEp C.NOS2 may promote the expressions of epithelial cell-derived cytokines and is involved in the development of cell inflammation in HNEp C induced by HDM via increasing ROS production.Chapter 3 The circRNA profiles in nasal mucosa of patients with allergic rhinitis and their clinical significanceObjective: Circular RNA(circRNA)is a new non-coding RNA,and its expression profiles exhibit obvious disease specificity.This chapter aims to explore the expression profile of circRNA in nasal mucosa of AR patients,then screen and verify circRNA with obvious differences,and analyze their clinical significances.Methods: Transcriptome sequencing was performed in nasal mucosa samples collected from 4 AR patients and 4 healthy controls,and differential m RNA and circRNA were screened for function enrichment analysis and pathway analysis.The top five differentially up-regulated and down-regulated circRNA were selected and verified by q RT-PCR in30 pairs of nasal mucosa samples,and the associations between the verified circRNA expression levels and clinical indicators of AR patients and previous research results were analyzed.Results: Sequencing results showed that m RNA and circRNA expression profiles in the nasal mucosa of AR patients exhibited significant tissue specificity.Differentially expressed circRNA were associated with transcriptional regulation,signal transduction and other biological functions,and were involved in protein degradation and absorption,m TOR and other signaling pathways.q RT-PCR verification results showed that the levels of circNOS2,circPRKAR1 B,circVASP and circMSL1 were significantly higher in the AR group than the healthy control group,and circNOS2,circVASP and circMSL1 levels were correlated with the clinical indicators of AR patients.The levels of circNOS2 were positively correlated with the levels of NOS2,ROS,IL-33 and TSLP in nasal mucosa.In addition,bioinformatic analysis showed that competitive endogenous RNA(ce RNA)regulatory network can be formed between circNOS2 and NOS2 through multiple miRNAs,and miR-513a-5p was demonstrated to be down-regulated in AR nasal mucosa.Conclusion: circRNA profiles in nasal mucosa of AR patients were tissue-specific,and the levels of circNOS2 were demonstrated to be upregulated and correlated with the expression levels of NOS2,ROS,IL-33 and TSLP in nasal mucosa of AR patients.Circ NOS2 may competitively bind miR-513a-5p to regulate NOS2 expression and promote the productions of ROS and epithelial cell-derived cytokines in nasal mucosal epithelial cells through ce RNA mechanism.Chapter 4 circNOS2/miR-513a-5p/NOS2 axis regulates the expressions of ROS and epithelial cell-derived cytokines in nasal epithelial cellsObjective: In the previous chapter,we found that circRNA profiles in nasal mucosa of AR patients were tissue-specific,and circNOS2 might regulate the expression of NOS2 and promote the productions of ROS and epithelial cell-derived cytokines in nasal mucosal epithelial cells through binding miR-513a-5p.In this chapter,the regulation and mechanism of circNOS2/ miR-513a-5p /NOS2 signaling axis on ROS and epithelial cell-derived cytokines expression in nasal mucosal epithelial cells will be explored at the cellular level.Methods: HNEp C was stimulated with HDM to construct cell models,and q RT-PCR and fluorescence in situ hybridization(FISH)were used to detect the expression levels and locations of circNOS2 and miR-513a-5p in cells.We regulate the expressions of circNOS2 and miR-513a-5p in HNEp C and stimulate it with HDM,then evaluate the expressions of NOS2,IL-33,TSLP and IL-25 by WB and IF.The ROS in cells and NO levels in cell supernatant were detected.The dual-luciferase reporter assay and the recovery assay verified whether circNOS2 and NOS2 could directly bind to mir-513a-5p.Results: HDM could induce the up-regulation of circNOS2 and down-regulation of miR-513a-5p in HNEp C.FISH confirmed that both circNOS2 and miR-513a-5p were mainly expressed in the cytoplasm.Upregulation of circNOS2 or downregulation of miR-513a-5p can induce NOS2 activation and further enhance NO release,ROS and epithelial cell-derived cytokine expression in HDM induced HNEp C.Downregulation of circNOS2 or up-regulation of miR-513a-5p could inhibit the expression of NOS2 and alleviate the NO release and ROS and epithelial cell-derived cytokines productions induced by HDM in HNEp C.The recovery assay and dual-luciferase reporter assay confirmed that circNOS2 could competitively bind to miR-513a-5p to promote NO release,ROS and epithelial-derived cytokines productions in HDMinduced HNEp CConclusion: HDM can induce the intracellular circNOS2 expression and inhibite the expression of miR-513a-5p.Circ NOS2 / miR-513a-5p/NOS2 axis may regulate the productions of ROS and epithelial cellderived cytokines in nasal epithelial cells,which is expected to be a new target for AR treatment. |
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