| Background:Olfactory disorder(OD),a common symptom in otolaryngology,has an incidence which as high as 12% in people all over the world.Along with course of an illness extension,Physical and mental health as well as quality of life will be seriously affected.Chronic sinusitis(CRS)is one of the main causes of olfactory dysfunction in clinic.About 65-83% of CRS patients are accompanied by olfactory dysfunction to varying degrees.The important role of immune response in the pathogenesis and development of CRS was emphasized in EPOS 2020.It has been reported that the olfactory score(TDI)of patients with CRS may be related to the expression levels of inflammatory factors such as IL-2,IL-5,IL-6,IL-10 and IL-13 in nasal mucus.After functional endoscopic sinus surgery,68% of patients with CRSw OD had no improvement in olfactory function,and9% had aggravation of olfactory disorder,suggesting that in addition to anatomical factors,the immune imbalance of nasal cavity and paranasal sinus may play a vital role in the progression of CRS-induced olfactory disorder.Macrophages have strong plasticity and heterogeneity,which are a key component of the innate immune system.They can respond to the growth factors and cytokines in the mucosal tissue microenvironment of nasal olfactory area,polarize into M1 and M2 phenotypes,and maintain a balance between tissue inflammatory damage and repair(M1 macrophages are inflammatory phenotypes,while M2 macrophages are repair phenotypes).However,the drivers of the relationship between CRSw OD and macrophage mediated regulation of tissue damage repair balance have not yet been found.Recent studies have shown that a close link between inflammation and metabolism,may with mediating by sirtuins.SIRT3 is a deacetylase localized in mitochondria of Sirtuins protein family.Some researchers proposed that SIRT3 expression in macrophages of diabetic mice decreased during inflammatory phenotype transforming to repair phenotype,leading to inflammation imbalance and aggravating inflammatory reaction,which suggests that SIRT3 plays an important regulatory role in the balance of inflammation injury repair.However,in the disease progression of CRSw OD,SIRT3 and macrophage phenotypic transformation and immune dysregulation have rarely been reported.Therefore,the following research is of great significance for the targeted diagnosis and treatment of CRSw OD: the investigation of the change process of inflammatory damage and repair balance in the local tissue immune microenvironment during the occurrence and development of CRSw OD in a bid to looking for corresponding potential targets.Objective:Starting with the effect of SIRT3 in CRSw OD regulating macrophage polarization and function affecting disease progression,this study will further explore the possible potential molecular mechanism through multi-omics sequencing,screen the important gene or metabolite targets that affect the balance of inflammatory damage and repair in the occurrence and development of CRSw OD,and provide a theoretical basis for the further study of the potential drug therapeutic targets of CRSw OD.Methods:1.Clinical sample analysis: exploring the correlation between SIRT3 expression and olfactory score in patients with CRS,collecting the clinical samples of nasal mucosa in olfactory region,detecting the overall expression level of SIRT3 in nasal mucosa of olfactory region and analyzing relationship between the expression of SIRT3 in repair macrophages and olfactory function.2.Construction and analysis of animal model.1)Constructing mouse CRSw OD model(1mg / ml ova,100 ul,sensitized by intraperitoneal injection for 3 times;100mg /ml ova,10 ul / side,nasal drip for 8 weeks),detecting the progress of CRS inflammation in mice by behavior and imaging,and evaluating the olfactory function of mice through the experiment of buried food.The overall expression level of SIRT3 in nasal mucosa of mouse olfactory region and its expression in repair macrophages,as well as the expression of inflammatory injury and repair related cytokines were detected by histology.2)Cultivating SIRT3-knockout mice,constructing CRSw OD model on this basis,and studying the effect of SIRT3 on the occurrence and development of CRSw OD disease in vivo through correlation detection and comparative analysis.3)Constructing the CRSw OD model of mice with local macrophage deletion in nasal mucosa with the related detection and comparative analysis,and study the effect of SIRT3 on the balance of inflammatory damage and repair in the disease progression of CRSw OD by regulating macrophage polarization in vivo.3.Study on cellular and molecular mechanisms.1)verifying the cytological effect of SIRT3 on the polarization and function of reparative macrophages by culturing macrophages derived from bone marrow of mice with different genotypes in vitro.2)Through the combined analysis of bone marrow-derived macrophage transcriptome sequencing and TM widely targeted metabolome sequencing,the differential genes and metabolites were screened,and the possible related signal pathways were explored,which were simply verified by RT-q PCR.Clinical sample analysis: To explore the correlation between SIRT3 expression and clinical characteristics of CRS.To collect clinical specimens and analyze the correlation between SIRT3,M2 macrophage content and clinical score in CRS nasal mucosa.Results:1.The expression of SIRT3 macrophages in the repaired nasal mucosa decreased compared with that in the repaired nasal mucosa of patients with SIRT3;2.The mouse CRSw OD model induced by ova was stable.In OVA group,the expression of SIRT3 and the surface molecule CD206 of repair macrophages in nasal mucosa of olfactory area decreased,the expression of injury inflammatory factor m RNA increased,while the expression of repair cytokine m RNA decreased;3.SIRT3-knockout mice accelerated the progress of olfactory dysfunction,with decreased expression of repair macrophage marker proteins in the nasal mucosa of the olfactory region.There was no significant difference in the expression of IL-5 and IFN-γ,the expression of Th2 effector molecules(IL-4,IL-13)decreased,and the expression of Th1 and Th17 inflammatory factors(IL-1β,IL-17)increased.Macrophage-associated inflammatory effector molecules(IL-1β,TNF-α)were both elevated and repair effector molecules(EGF,TGF-β)were all lower.4.Removal of mouse nasal macrophages can significantly aggravate the inflammatory damage of nasal mucosa in olfactory area of OVA-induced CRSw OD mouse model,with accelerating the progress of olfactory disorder.Expression of IL-4,IL-5,IL-13,EGF,TGF-β in the nasal mucosa of the olfactory region of mice with excluded nasal macrophages decreased,and the expression of IL-1β,IFN-γ,TNF-α,IL-17,TNF-α increased.However,SIRT3 deletion could reverse the related effect of macrophage elimination on aggravating the inflammatory injury of nasal mucosa in the olfactory region of OVA-induced CRSw OD mouse model.5.Mouse bone marrow hematopoietic stem cells were induced to differentiate into reparative macrophages in vitro.The experimental system was stable.The transcriptome and TM widely targeted metabolome were sequenced and analyzed together to screen the interaction between potential differential genes and differential metabolites and focus on FOXO and HIF-1 signal pathways.RT-q PCR verified that the m RNA expression of relevant targets was consistent with the sequencing results.Conclusion:Both the samples of CRSw OD patients and OVA-induced mouse model showed that the total expression of SIRT3 in nasal mucosa of olfactory area decreased,the number of repair macrophages decreased,and the expression of SIRT3 in repair macrophages also decreased.In vivo studies found that both SIRT3 deletion and local macrophage deletion in nasal cavity would accelerate the occurrence and development of CRSw OD,and tilt the balance between inflammatory injury and repair in nasal mucosa of olfactory region to injury,while the loss of both would tilt the balance to repair.After SIRT3 deficient macrophages were further stimulated to polarize into repair macrophages by IL-4 and IL-13,transcriptome and TM widely targeted metabolome sequencing and joint analysis screened out the interaction between potential differential genes and differential metabolites.RT-q PCR verification suggested that SIRT3 may promote the polarization and function of repair macrophages and inhibit the disease progression of CRSw OD through FOXO or HIF-1 signaling pathway.The completion of this study provides a theoretical reference for further screening potential drug targets of CRSw OD. |
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