| BackgroundOtitis media with effusion(OME)is a non-purulent inflammatory disease of the middle ear characterized by fluid accumulation in the tympanic cavity,and is also known as exudative otitis media,tympanic effusion,catarrhal otitis media,and serous otitis media.OME may be accompanied by conductive hearing loss in many patients at the same time.OME can be divided into acute OME and chronic OME,which is one of the common diseases in otolaryngology,general medicine and pediatrics.OME can develop in both adults and children,but is more common in children.Its prevalence is only slightly lower than that of upper respiratory tract infections in pediatric patients and it is one of the most common diseases in children.Previous overseas studies have found that between 15%and 40%of children aged 1 to 5 years have asymptomatic OME through routine tympanic membrane screening in healthy children and 91%of these children under the age of 2 years have had at least one acute episode of OME.Untimely or inappropriate treatment of patients with OME may lead to pathological changes in the tympanic membrane and middle ear,such as adhesive otitis media,tympanosclerosis,and middle ear cholesteatoma,which can put patients at risk for severe hearing loss and may in turn delay the speech development of affected children.The current situation of OME urgently requires us to conduct indepth studies on the pathogenesis of OME,explore its possible therapeutic modalities,and search for new therapeutic targets.The development of OME is closely related to a variety of factors and its pathogenesis has not been fully elucidated to date.Mechanical obstruction or dysfunction of the eustachian tube is thought to be an important cause of OME,and infections and immune responses to OME have gradually attraction from researchers.The eustachian tube is a narrow channel connecting the tympanum to the nasopharynx,and its dysfunction(ETD)causes the air in the nasopharynx to be unable to enter the tympanic cavity,causing the middle ear to be in a state of negative pressure.Prolonged negative pressure will affect gaseous exchange in the tympanic mucosa,resulting in mucosal ischemia and increased vascular permeability,thereby causing middle ear exudation.Surface tension may be one of the important factors affecting the function of the eustachian tube.Surface tension is present in the gas-liquid interface of the liquid surface due to the attraction between liquid molecules.This causes extremely small tension to be produced on the liquid surface,which is known as surface tension.Surface tension is ubiquitous in nature and is present in the human body,in human body fluids such as gastric juice,urine,digestive juices,liquid films in the middle ear and alveolar lining layer.Surface tension has an extremely important role in the human body.Current studies suggests that OME development and progression is intimately associated with the normal opening of the eustachian tube(ET)and changes in surface tension are likely to be closely related to the opening of the ET.Immune factors are another research hotspot in the pathogenesis of OME.Allergic reactions and their associated disorders are considered to be one of the most important causes of OME.The prevalence of allergic rhinitis in children with OME is between 82%and 93%.The immune mechanisms of OME are complex and diverse,with different changes in inflammatory mediators in different middle ear effusions(e.g.,serous effusions,mucoid effusions)and different corresponding immune mechanisms.Surface tension is a dynamic process that is precisely regulated by the surfactant.Surfactants in the human body are complex mixtures of phospholipids and proteins that have the property of reducing surface tension at the gas-liquid interface.Surfactant protein A(SPA)is the main protein component of surfactants and the most abundant non-serum protein in lung surfactants.SPA can influence the dynamic equilibrium of surfactants,promote changes in the physical structure of lipids,and regulate the function and metabolism of surfactants.In addition to binding and modulating pathogens,SPA binds to the surface of host defense cells and promotes or inhibits immune cell activity through a variety of cellular pathways.SPA plays a vital role in many bodily functions,but its role in OME has received little attention.The aim of this study was to investigate the role of SPA in OME development and progression and the related molecular mechanisms.By constructing an experimental OME mouse model,we tested the changes in SPA expression in the eustachian tube tissue and secretions in OME,and also explored the changes of surface tension of the eustachian tube and the effect of SPA on macrophage migration.An in-depth study of the relationship between SPA and OME is important to clarify the role of SPA in the human body,which helps us to further understand the mechanism of OME development and progression and provides a new theoretical basis for the prevention and treatment of OME in clinical practiceStudy protocol and resultsChapter 1:Constructing an OME mouse model using inactivated Streptococcus pneumoniae(S.pn)and validating the reliability of the model.Study protocol:Healthy BALB/c m ice weighing approximately 15-20 g were used to construct the OME model by tympanic injection of inactivatedS.pn(5 μL/ear)via the external ear canal.The control group was injected with phosphate-buffered saline(PBS,5 μl/ear).The morphology of the tympanic membrane was observed by microscopy or fiber optic endoscopy,and the changes of hearing level and tympanum pressure in the model mice were assessed by using auditory brainstem responses(ABR)and tympanometry to further validate the successful model construction.HE staining was used to detect the S.pn induced biological characteristics of the OME model.Study results:1.The OME model induced by S.pn was successfully constructed and tympanic membrane congestion and effusion could be observed under the microscope.HE staining showed thickening of the tympanic mucosa in the OME group.There was no such change in the tympanic membrane and tympanum in the control group(PBS group).2.The results of the hearing function test suggested that the ABR detection threshold was increased in mice in the OME group,accompanied by a negative tympanum pressure and the differences were statistically significant.Conclusion:The OME model of BALB/c mice was induced by inactivated Streptococcus pneumoniae.The observation of tympanic effusion,ABR changes,tympanogram differences,HE staining,etc.showed that the model was successfully constructed.Chapter 2:Exploring the changes in surface tension and SPA expression in mouse eustachian tube.Study protocol.The grouping and modeling methods were the same as in Part Ⅰ.The surface tension of the rinse fluid was measured using the TACLIS interfacial rheometer in the tympanum and eustachian of each group of successfully constructed model mice(on the third day of model construction).The changes of SPA expression in the eustachian tube tissues of OME mice and control mice were also observed using ELISA,real-time PCR,Western blotting,and immunofluorescence confocal microscopy.The correlation between the two were analyzed.Study results:1.The results of the interfacial rheometer test showed that the surface tension of the tympanum and eustachian tube rinse fluid in the OME group of mice was significantly higher than that in the control group,and the difference in the surface tension values between the two groups was statistically significant(P=0.014).2.ELISA,real-time PCR,Western blot,and immunofluorescence experiments showed that the expression of SPA was significantly reduced in the eustachian tube tissue of OME mice,and the difference was statistically significant(P<0.001).3.Surface tension was negatively correlated with SPA expression(r=-0.525,P=0.037)Conclusion:1.The expression of SPA in OME mouse eustachian tube flushing fluid decreased with the increase of surface tension,which indicated that SPA in eustachian tube may play a key role in the change of eustachian tube surface tension,thus affecting the opening process of eustachian tube.2.The decrease of SPA expression in the eustachian tube of OME mice may be a potential therapeutic target of OMEChapter 3:Determination of the effects of SPA on macrophage migration and possible mechanisms of actionStudy protocol.It has been shown that macrophages have an important role in the degradation and synthesis of surfactants.Transwell assay was used to measure the effect of SPA on macrophage migration.The RNA-seq assay predicted the changes of macrophage-related gene expression after SPA treatment,and the predicted results of RNA-seq assay were further validated by Western blot to study the molecular mechanism of SPA on macrophage migration.Study results:1.The migration capacity of macrophages was increased by treatment of macrophages with different concentrations of SPA.The change in migration capacity is closely related to the concentration of SPA.2.A total of 13,973 genes were detected by RNA-seq,and a total of 5,772 differentially expressed genes(DEGs)between the study and control groups were analyzed and selected,of which 2,952 were upregulated and 2,770 were downregulated.Among them,three protein subfamilies of Rho GTPase,which are intimately associated with macrophage migration were DEGs(P<0.05).3.Western blot was used for further validation of the changes in the three protein subfamilies of Rho GTPase and showed that SPA may affect macrophage migration through Rac1/2/3 and RhoA.The expression of Cdc42 did not change significantly in macrophages after treatment with different concentrations of SPA.Conclusion:1.SPA has chemotaxis on macrophages.When the concentration of SPA exceeds a certain threshold,the chemotaxis on macrophages is inhibited.2.After the co culture of SPA and macrophages,the expression of Rac 1/2/3 and RhoA changed,and the expression of Cdc42 had no significant difference.Therefore,SPA may affect the migration of macrophages in ET of OME mice through Rac 1/2/3 and RhoA. |
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