| Part ?:Research on regulation mechanism of Brgland ?-catenin on human nasal epithelial cells during chronic rhinosinusitisBackground:Chronic rhinosinusitis(CRS)is one of the most common diseases in otolaryngology.It is a chronic inflammatory lesion of the nasal mucosa,and its development is a complex inflammatory process.According to the European EPOS guidelines,it is divided into chronic rhinosinusitis with nasal polyps(CRSwNP)and chronic rhinosinusitis without nasal polyps(CRSsNP).In the nasal-sinus mucosa,mucociliary system,epithelial cells,neutrophils and other important components of innate immunity,in addition to the role of mechanical barriers,epithelial cells are still an immune barrier,the body's first stimulus to the outside world Road defense line.Studies have found that most of the CRSwNP patients have str:ctural and functional abnormalities in the epithelium,chronic inflammation of the mucosa and abnormal epithelial tissue remodeling are its main histological features.Nasal epithelial cells(NEPCs)are a group of adult stem cells with self-renewal and pluriPotency differentiation in the basal layer of the nasal mucosa.Their function is to maintain the stability of epithelial cells and the orderly regeneration after injury.Under normal circumstances,NEpCs initiate a normal repair process after the damage of the nasal mucosa epithelium,migrate to the injury site,and induce self-differentiation into epithelial cells to reconstruct the integrity of the epithelial structure and function.Under the pathological conditions of CRS,NEpCs are reduced in dryness after excessive or repeated injury/inflammatory signal stimulation,and the proliferative ability is decreased,and the cells are in an abnormally differentiated state,thereby initiating the abnormal damage repair process of the nasal mucosa epithelium,that is,the epithelial tissue.Hyperplasia,differentiation and remodeling.The chromatin remodeling complex(Brgl)is the core hydrolase subunit of the chromatin recombination complex BAF(Brgl/Brm associated factor)in mammals and plays an important role in mammalian embryonic development and postnatal growth.effect.Brgl can activate or inhibit the expression of thousands of genes by specifically binding to different promoters,affect cell proliferation and differentiation through regulation of transcription,and act extensively on biological tissues,and participate in the pathophysiological processes of various diseases.Stem cell proliferation and differentiation,tumorigenesis,neuronal development,heart development and inner ear development play an important role.Based on the confirmed regulation and characteristics of Brgl on cell dry maintenance and induced differentiation,we have reason to speculate that Brgl in CRS may lead to abnormal proliferation and differentiation of NEpCs,and participate in the process of epithelial remodeling of CRS.?--eatenin is a member of the catenin family.In cells,the physiological activities of cells are regulated mainly by binding specific related proteins.Among them,two functions are widely studied.One is the signal transduction factor of the classical Wnt signaling pathway to coordinately regulate the T/LEF geneo It is involved in the adhesion between cells in combination with the cadherin family(Cadherin).Studies have found that the distribution of ?-catenin in the membrane,plasma,and nucleus and the changes in spatial sites determine the changes in function and effect:When ?-catenin and Cadherin form a complex located in the cell membrane,it can maintain the dryness of stem cellso If p-catenin dissociates from the complex and is released from the cytosol,?-catenin will gradually accumulate in the cytoplasm and enter the nucleus to activate the Wnt/?-catenin pathway,thereby promoting the differentiation of stem cells.It can be seen that the membrane-nuclear translocation state of ?-catenin determines the transition state of stem cell stem and differentiation.The study found that Brgl can directly regulate P-catenin,thereby affecting Wnt signaling pathway:under normal conditions,Brgl can bind to ?-catenin,thereby enhancing the transcriptional activity of ?-catenin-TCF complex on the target gene and maintaining normal stem cells.Dryness and differentiationo whereas after Brgl down-regulation,Brgl,which has lost ATPase activity,exhibits a dominant mutational inhibition of the Wnt signaling pathway,affecting the transformation of the stem and differentiation states of the cell,and subsequently mediates the cell.Abnormal proliferation and differentiation destroy the normal development of the organism.These studies suggest that Brgl and ?-catenin have similar effects on Wnt signaling pathways in terms of stem cell dryness and fine regulation of differentiation.In summary,we speculate that Brgl affects wnt signaling by regulating p-catenin and participates in the proliferation and differentiation of NEpCs.Therefore,we use the drug to stimulate human nasal epithelial stem cells to simulate the mucosal state of chronic sinusitis from the key point of Brgl regulation of proliferation and differentiation of NEpCs,and to apply immunofluorescence to nasal polyps and normal inferior turbinate mucosa.Western and other technologies to achieve the following research purposes:? to determine the expression of Brg1 and?-catenin in NEpCs from different clinical types of CRS and its correlation with abnormal proliferation and differentiation of NEpCs??to clear the expression levels of Brgl and ?-catenin The effects of alteration and cell translocation on the prolif'eration and differentiation of NEpCs,and elucidation of the mechanism by which Brgl regulates the involvement of nasal epithelial stem cells in the pathogenesis of CRS.The implementation of this study will help to clarity the mechanism of CRS epithelial remodeling,and provide new molecular targets and ideas for the prevention and treatment of CRS from the application prospects.Material and Methods:1.Research about of tissue mucosaIn this study,20 patients with chronic rhinosinusitis(CRSwNP)with nasal polyps were used as the experimental group,and 14 patients with nasal septum deviation and no other nasal diseases.As a normal control group.During the operation,the nasal polyps of CRSwNP patients were taken,and the inferior turbinate mucosa of patients with nasal septum deviation correction was performed.The total RNA and protein in nasal polyps and inferior turbinate mucosa were extracted,and the expression levels of Brgl and ?-catenin in two types of samples were detected by RT-PCR and Western blot,and observed by irnmunohistochemistry and immunofluorescence.Expression patterms and expression localization of Brgl and ?-catenin on two types of nasal mucosa.2.Research about of the human nasal epithelial cellsHuman nasal epithelial cells(HNEpCs)were purchased and cultured.HNEpCs were stimulated with TNF-? at a concentration of 2.5 ?/ml to simulate the chronic inflammatory state of nasal epithelial stem cells,and intracellular Brgl was detected at 0h,6h,12h,24h,48h and 72h after TNF-a stimulation.The expression level of ?-catenin was detected by immunofluorescence and immunohistochemistry to detect the localization of Brgl and ?-catenin on normal HNEpCs,and the stimulation of Brgl and ?-catenin on HNEpCs after stimulation with TNF-?.Expression patterns and expression localization.Results:1.In this study,Brgl and p-catenin were expressed in the nasal polyps of CRSwNP and the inferior turbinate mucosa of the control group,and were mainly located in the nasal epithelial cells.The expression levels of Brgl and ?-catenin in the nasal mucosa of patients with CRSwNP were significantly lower than those of Brgl and ?-catenin in the control group.In the normal control group,?-catenin was expressed in the cell membrane and cytoplasm.In the CRSwNP group,?-catenin was transferred into the nucleus?however,there was no significant difference in the localized expression of Brgl between CRSwNP and the normal control group,both in the cytoplasm and nucleus.2.The study showed:the expression of Brgl and ?-catenin decreased with the prolongation of drug-stimulated cells after the TNF-? stimulated HNEpCs.From 24h after drug stimulation,the downward trend began to become apparent.The changes of Brg1 and ?-catenin expression patterns are shown in the following:?-catenin changes from cell membrane and qualitative localization to nuclear localization.With the prolongation of TNF-? stimulation time,Brgl is transferred from the nuclear part to the cytoplasm,which is betore us.The observed tissue staining results were similar.Conclusion:This study showed that both Brgl and ?-catenin were expressed in nasal epithelial cells,which were localized in the basal layer of nasal mucosa.The growth and proliferation of nasal epithelial cells derived from CRSwNP decreased significantly compared with the normal control group.In comparison,the expression of Brgl and?-catenin was significantly reduced in abnormally proliferating nasal polyps,which was further confirmed in HNEpCs in which we stimulated the inflammatory state with TNF-?.This essential difference in growth and proliferation performance may be a major cause of persistent and abnormal remodeling of nasal polyps.Part ?:The bacterial extract OM-85 BV protects mice against experimental chronic rhinosinusitisBackground:Chronic rhinosinusitis(CRS)has long been considered an important public health problem.According to international and domestic guidelines,CRS can be divided into chronic rhinosinusitis with nasal polys(CRSwNP)and Chronic rhinosinusitis without nasal polys(CRSsNP).The symptoms of chronic rhinosinusitis are complex and diverse,with symptoms such as nasal congestion,runny nose,headache and olfactory disorders.The nasal mucosa of patients with CRS is severely damaged and has a continuous inflammatory state with a Iong treatment period.The comprehensive treatment of CRS with nasal polyps is even worse,and the recurrence rate is higher.Patients with CRS are deeply troubled by it,and severe cases can even cause psychological diseases.OM-85 BV is a lyophilized lysate extracted from 8 Gram-positive and negative bacteria,which are closely related to the respiratory tract,including:Staphylococcus aureus,Streptococcus mutans,Streptococcus pyogenes,Haemophilus influenzae,Klebsiella pneumoniae,Pneumococcal pneumonia,K.catarrhahs and Stinky gram Thaliana.OM-85 BV is mainly composed of peptides,acidic proteins and amino acids.OM-85 BV is a non-pathogenic bacterial product of the human body and has immunological activity.As an immunostimulant,it can enhance the activity of immune cells and promote the production of immune factors.OM-85 BV has been widely used to treat recurrent respiratory infections in adults and children,asthma,chronic obstructive pulmonary disease and other respiratory diseases,but its role in chronic sinusitis is poorly studied.The nasal cavity and the trachea and bronchus together constitute the respiratory tract.From the perspective of tissue origin and composition,the tissue of the nasal cavity and respiratory tract is derived from the same starting point of embryonic development.The pathogenesis and treatment are similar,so we believe that the bacterial extract products treat chronic nasal-sinus The inflammation is feasible,and the following experiments were designed to investigate the therapeutic effect of OM-85 BV on chronic sinusitis.Material and Methods:1.40 female BALB/c mice aged 8-12 weeks were randomly divided into 4 groups,10 in each group.One group of mice(group A)served as a normal control group,and no drugs were used for them.The other 3 groups of mice(groups B,C,and D)were intraperitoneally injected with Aspergillus fumigatUs extract(AF),and after 1 week,AF was used to irritate the nasal cavity of mice,3 times a week.12 weeks to establish a model of chronic sinusitis.2.Three groups of mice were randomly divided into 3 groups after modeling,10 in each group,and participated in experiments as mice with chronic sinusitis.Forty mice were finally divided into 4 groups:group A,blank control group o group B,sinusitis model groupo group C,OM-85 BV plus amoxicillin combination treatment group?group D,amoxicillin treatment group.3.Group C was given oral OM-85 BV and amoxicillin.After taking 10 days,the mice was stopped for 20 days.One month was a course of treatment and three courses of treatment were given.Group D was given oral amoxicillin alone,and the frequency and time of administration were the same as group C,group A and group B were treated with sterile water instead of drugs.4.The sinus mucosa of the eligible mice was taken out,and the histological changes of the nasal mucosa of mice with CRS were determined by HE stainingo the RNA and protein of the mouse sinus mucosa were extracted,and the RT-PCR technique and Western blotting were used.Method(WB)to determine the expression of cytokine signal inhibitors:SOCS1,SOCS3,tumor necrosis factor TNF-a and interferon IFN-?in the sinus mucosa.Results:1.Compared with the normal control group,In the sinusitis model group mast cells and eosinophils infiltrated in the nasal mucosa epithelium,and the cilia in the ciliated epithelial tissue were disordered,missing,and the basement membrane tissue was thickened.2.The mRNA and protein expressions of SOCS1,SOCS3,TNF-? and IFN-?appeared in the combination of OM-85 BV and amoxicillin and the amoxicillin treatment group and the chronic sinusitis model group.Significantly reduced(0.807 vs 0.214/0.367;0.787 vs 0.345/0.428;0.841 vs 0.320/0.456;0.769 vs 0.402/0.487).3.The mRNA and protein expressions of SOCS1,SOCS3,TNF-? and IFN-? were not significantly different between OM-85 BY and amoxicillin in the combination group compared with the normal control group.4.Compared with the amoxicillin-treated group,the mRNA and protein expressions of SOCS1,SOCS3,TNF-? and IFN-? were significantly lower in the OM-85 Bv and amoxicillin combination group.Conclusion:The bacterial extract products OM-85 BV and amoxicillin are effective for CRS.OM-85 BV is combined with amoxicillin,which has obvious therapeutic effect on CRS.OM-85 BV can be used as a treatment for chronic rhinosinusitis.The application of new drugs in clinical practice has the advantages of low cost,relatively safe and convenient use,and is a treatment worthy of promotion. |
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