Mechanism Of SIRT3 Antagonizing ECRS Progression By Inhibiting Eosinophil Differentiation

Background:Eosinophilic chronic rhinosinusitis（ECRS）is a recognized refractory rhinosinusitis,which was prone to relapse after treatment according to the guidelines,with a three-year recurrence rate of 98.5%.Eosinophils mature in bone marrow and enter the peripheral blood.It is one of the main effector cells in the inflammatory response and is recruited into the local tissue in the inflammatory state.Abnormal differentiation of eosinophils plays an important role in the development of ECRS.How physiological or pathological signals induce endometabolic remodeling of eosinophilic granulocyte,thus determining its differentiation fate and functional status,is a hot topic in the field of life science and medicine.SIRT3 has a strong deacetylation effect and plays an important role in mediating metabolic remodeling and regulating inflammatory response.However,the regulation of SIRT3 on eosinophil differentiation,especially in ECRS,remains unclear.Objective:Firstly,based on clinical samples,this study explored the internal correlation between SIRT3,eosinophils and ECRS.SIRT3 knockout mice were further introduced to construct ECRS model and explore the mechanism of the regulatory effect of SIRT3 on disease.Bone marrow primary eosinophils were cultured in vitro to study the effect of SIRT3 on eosinophile differentiation,so as to provide a new target for early clinical intervention and treatment of ECRS.Methods:1.Clinical sample analysis:1)SIRT3 expression levels in nasal mucosa of normal control group,nasal polyps of ECRS patients and non-Eosinophilic chronic rhinosinusitis（non-ECRS）patients were detected.2)To study the correlation between the expression level of SIRT3 in nasal polyp tissue of ECRS patients and Th2 inflammatory response in nasal cavity and sinuses:To compare the correlation between the expression level of SIRT3 in nasal polyps of ECRS patients and the number of eosinophils and the expression level of Major basic protein（MBP）.3)To investigate the correlation between the expression level of SIRT3 in nasal polyps of ECRS patients and clinical disease severity.2.Analysis of animal models:1)Ovalbumin（OVA）and Aspergillus protease（Ap）were used to sensitize the nasal cavity,and the ECRS model of mice was successfully constructed.2)In the OVA and Ap induced ECRS mouse model,the ECRS symptoms,nasal mucosal tissue swelling and expression of eosinophil related important proteins of Wild-Type（WT）mice and SIRT3 knockout mice were compared.3)In the OVA and Ap induced ECRS mouse model,the expression of Th2 cytokines（IL-4,IL-5,IL-13）in serum,nasal lavage fluid and nasal mucosal tissue homogenate of WT and SIRT3 gene knockout mice were compared.4)In the OVA and Ap induced ECRS mouse model,the number of eosinophils in nasal mucosa tissue and peripheral blood of WT and SIRT3 knockout mice was compared.3.Function analysis of cell experiment:Bone marrow cells from 4-5 week old mice were extracted and induced into eosinophils by cytokines in vitro.For the first 4 days,the medium was supplemented with 100ng/ml recombinant Mouse stem cell factor（m SCF）and Fms-related tyrosine kinase 3 ligand（Flt3-L）.From day 4 to day 12,only 10ng/ml IL-5 was added to the medium.Bone marrow cells from WT and SIRT3 knockout mice were extracted and the following experiments were performed.1)Compare the growth state and number of cells in the process of induction differentiation.2)The maturity of eosinophils was detected after the induction differentiation.3)After the induction of differentiation,the differential expression of eosinophilic surface molecules,receptors,chemokines and important transcription factors was detected.4)After the induction differentiation,Western blot was used to detect whether STAT5and STAT5 phosphorylation levels were differentially expressed under the stimulation of IL-5.5)After the induction of differentiation,transcriptome sequencing（RNA-Seq）was performed to screen differential genes and complete KEGG analysis;The important genes related to eosinophils were further analyzed for differential expression.Results:1.Compared with normal control group and non-ECRS patients,SIRT3 expression level in nasal polyp tissues of ECRS patients was significantly increased;The expression level of SIRT3 in nasal polyp tissues of ECRS patients was positively correlated with the number of eosinophil infiltration in tissues,the expression level of MBP and Lund-Mackay CT score.2.After OVA and Ap induced ECRS model,the frequency of scratching and sneezing increased,nasal mucosal tissue swelling was more obvious,inflammatory cell infiltration increased significantly;Moreover,the expression of SIRT3 in nasal mucosa of ECRS model mice was increased.3.In the OVA and Ap treatment group,compared with WT mice,ECRS symptoms of SIRT3^-/-mice were more serious,including mucosal swelling in the respiratory area,respiratory epithelial structure disorder,partial abruption,and increased inflammatory cell infiltration in lamina propria.The expressions of Eosinophile cationic protein（ECP）,Eosinophil Peroxidase（EPX）,CD69 and CCL5 in nasal mucosa of SIRT3^-/-mice were significantly increased.The expression levels of Th2 cytokines（IL-4,IL-5 and IL-13）in serum,nasal mucosal tissue homogenate and nasal lavage solution of SIRT3^-/-mice were significantly increased.In addition,the relative number of eosinophils in nasal mucosa and peripheral blood of SIRT3^-/-mice was significantly increased in the OVA and Ap treatment group compared with WT mice.4.Mouse bone marrow cells were extracted to induce differentiation of primary eosinophils.Compared with WT group,the number of eosinophils in SIRT3^-/-group was significantly increased,and the proportion of mature eosinophils was increased.The expressions of eosinophils surface molecules（CD69）,receptors（CCR3）,chemokines（CCL5）,and important transcription factors（GATA1,、C/ebpα、C/ebpε）were increased.In addition,SIRT3 gene knockout significantly promoted STAT5 phosphorylation in eosinophils.5.Bone marrow cells of WT and SIRT3^-/-mice were extracted and induced to be primary eosinophilic granulocytes.Differential genes were found according to RNA-Seq data and KEGG analysis was performed to enrich the pathways related to cell differentiation and proliferation.The expression of important eosinophil related genes increased in SIRT3^-/-group,such as EPX,GATA1,CCR3,Prg2,Ear2,Ear6,etc.,which was verified by q RT-PCR,which was consistent with the sequencing results.Conclusion:Our study found that the expression level of SIRT3 in nasal polyp tissues of ECRS patients was significantly increased.The expression level of SIRT3 in nasal polyp tissues of ECRS patients was positively correlated with the number of eosinophil infiltration in tissues,the expression level of MBP and Lund-Mackay CT score.SIRT3 gene knockout can promote Th2 type inflammatory response and aggravate nasal eosinophilic inflammation in mice.SIRT3 gene knockout can promote the increase of the number of primary eosinophils,promote the differentiation of eosinophils,and promote the expression of important transcription factors and numerous proteins of eosinophils.KEGG analysis was performed on the basis of RNA-Seq data,and the expression of important genes related to eosinophils was studied and verified.This study confirmed that SIRT3 gene knockout can promote the differentiation of eosinophils and promote the progression of ECRS disease,indicating that SIRT3 plays an important protective role in the nasal inflammatory microenvironment.In the course of chronic nasal inflammation,due to the negative feedback of the body,the expression of SIRT3 is increased to inhibit the inflammatory response.A new intervention target is proposed for the prevention and treatment of clinical ECRS.