Role Of P2X7R/NLRP3 Signaling Pathway In Chronic Rhinosinusitis With Nasal Polyps And Murine Acute Rhinosinusitis

Part? Role of P2X7R/NLRP3 Signaling Pathway in Chronic Rhinosinusitis with Nasal PolypsObjective: To explore the role of purine 2X7 receptor(P2X7R)and P2X7R-mediated NOD-like receptor pyrin domain containing 3(NLRP3)inflammasome activation in chronic rhinosinusitis with nasal polyps.Methods: Thirty-two specimens of chronic rhinosinusitis with nasal polyps were collected as the experimental group,and 16 normal middle turbinate specimens were collected as the control group.The expression of P2X7 R in nasal mucosa was detected by Western blot(WB),immunofluorescence(IF)and Real-Time polymerase chain reaction(RT-PCR).Seven specimens of chronic rhinosinusitis with nasal polyps were used for primary human nasal epithelial cell culture.Lipopolysaccharide(LPS)combined with2'(3')-O-(4-benzylbenzoyl)ATP(Bz ATP)stimulated for 24 hours,the expression of interleukin 1b(IL-1b)detecting by WB was used to determine the combined stimulation effect of LPS and Bz ATP on primary human nasal epithelial cells.The selective P2X7 R antagonist,A740003,was used to antagonize the stimulation of LPS combined with Bz ATP on primary human nasal epithelial cells.WB and RT-PCR were used to detect the expression of P2X7 R and NLRP3 after A740003 treatment,and ELISA to detect the change of IL-1b in the cell culture supernatant.Results: 1.WB,IF and RT-PCR results showed that the expression of P2X7 R,NLRP3and IL-1b in chronic rhinosinusitis with nasal polyps is higher than that in the control group,especially in eosinophilic chronic rhinosinusitis with nasal polyps;2.The stimulating effect of LPS combined with Bz ATP on primary human nasal epithelial cells is stronger than that of LPS alone,and the expression of IL-Ib is significantly increased;3.The expression of P2X7 R,NLRP3 and IL-1b were up-regulated in primary human nasal epithelial cells induced by LPS combined with Bz ATP.After adding A740003,the expression of P2X7 R did not change significantly,but the expression of NLRP3 and IL-1?were down regulated.Conclusions: 1.The expression of P2X7 R,NLRP3 and IL-1b in chronic rhinosinusitis with nasal polyps was higher than that in control group;2.P2X7R/NLRP3 inflammasome signaling pathway can be augmented by LPS combined with Bz ATP,but suppressed by A740003.It is speculated that P2X7R/NLRP3 signaling pathway may be involved in the pathogenesis of chronic rhinosinusitis with nasal polyps.Part? Role of P2X7R/NLRP3 Signaling Pathway in Murine Acute Rhinosinusitis Objective: To establish a stable mouse model of acute rhinosinusitis.To explore the role of P2X7 R and P2X7R-mediated NLRP3 inflammasome activation in murine acute rhinosinusitisMethods: Sixty C57 BL/6 mice feeding in Specific Pathogen Free(SPF)Laboratory Animal Room were recruited for experiment.The mice were divided into two groups in this randomized and controlled study.Twelve animals were randomly selected as the control group,and no treatment was done.The remaining 48 were used as the experimental group.After the mice in the experimental group were anesthetized by intraperitoneal injection of chloral hydrate,a swelling sponge was inserted into the right nasal cavity of the mice under a microscope.After the mice were awake,20 ?l of 1 mg/ml LPS was dripped into the right nasal cavity.7 days later,the mice were sacrificed under anesthesia.4 mice in the control group+12 mice in the experimental group were perfused with PBS-paraformaldehyde and frozen sections were made;4 mice in the control group+12 mice in the experimental group were used for protein extraction;4 mice in the control group+12 mice in the experimental group were used to extract m RNA from the right nasa l cavity mucosa;and 4 mice in the control group+12 mice in the experimental group were used to collect 1 ml of nasal cavity lavage after the right nasal cavity was lavaged.HE staining was used to observe the infiltration of inflammatory cells in the nasal cavity and paranasal sinuses and the morphological changes of the nasal cavity mucosa was used to determine whether the acute rhinosinusitis mouse model was successful;WB and IF were used to detect the expression of P2X7 R and NLRP3;RT-PCR was used to detect P2X7 R and IL-1b m RNA expression;enzyme-linked immunosorbent assay(ELISA)detects IL-1b expression in nasal lavage fluid.Results: 1.The results of HE showed that a large number of neutrophils exuded in the nasal cavity and sinuses of the experimental group mice,and the cilia were severely damaged,while the control group mice had no neutrophil exudation in the nasal cavity and sinuses,and the cilia were not damaged;2.WB and IF results showed that the expression of P2X7 R and NLRP3 in the experimental group was significantly higher than that in the control group;3.RT-PCR results showed that the m RNA expression of P2X7 R and IL-1b increased significantly in the experimental group;4.ELISA results showed that the expression of IL-1b in the nasal lavage fluid of the experimental group mice was significantly increased.Conclusions: 1.A murine acute rhinosinusitis model can be successfully established by inserting the swelling sponge+dripping LPS into the mouse nasal cavity;2.The expression of P2X7 R,NLRP3 and IL-1b in the nasal mucosa of mice with acute rhinosinusitis is up-regulated.The P2X7R/NLRP3 signaling pathway may be involved in the occurrence and development of murine acute rhinosinusitis.