Investigation Into The Role By Which Twist1 Protein Confers Eosinophil Apoptosis Resistance In The Nasal Mucosa Of Allergic Rhinitis

Objective:Allergic rhinitis(Allergic Rhinitis,AR)is a Non-infectious inflammatory disease of the nasal mucosa,which is caused by IgE-mediated immune reaction to inhaled allergens.Presently,although the research on its pathogenesis continue goes deeper,it has not been fully explained,and the clinical treatment methods are still difficult to achieve satisfactory results.Eosinophils(Eos)are typical effector cells for allergic rhinitis and many inflammatory diseases.In the airway tissue or subcutaneous tissue of allergic diseases,there is usually a large accumulation of Eos.However,the mechanism of Eos accumulation in local tissues is still not fully understood,and the resistance to apoptosis may be one reason for Eos accumulation.Twist1 protein is an apoptosis inhibitor,and Ras is associated with the sustaining apoptosis resistance in cancer cells.After reviewing the literature,their apoptosis resistance effects with Eos have not been reported.Therefore,this study aims to investigate the role of Twistl in the pathogenesis of eosinophilia in AR,further reveal the occurrence and development mechanism of allergic rhinitis,and provide scientific theoretical basis for more effective clinical treatments.Methods:1.Nasal mucosa specimens were collected from patients with allergic rhinitis and non-allergic rhinitis,evaluate the proportion of Eos and the activation level of Rat Sarcoma(Ras)protein;add cisplatin to Eos and evaluate cell apoptosis by flow cytometry.2.Total RNA of Eos were Extracted for RNAseq analysis,detect the expression of Twist1 mRNA and evaluate its correlation with the activation level of Ras.3.EoL-1 cells were to sensitize and challenged by OVA,then detect the protein level of Twistl after adding FTS(RAS inhibitor)to Eos and to detect the activity of Ras.Knock out HIF-1α in EoL-1 cells by RNA interference(RNAi)technology.Then the content of p38,phosphorylated p38 and HIF-1α,as well as Twistl protein and mRNA level,were detected and evaluated the role of MAPK-HIF-1α signaling pathway mediated Ras up-regulation of Twistl in eosinophils.4.Isolate Eos from the nasal mucosal tissues of patients with AR and extract the protein;then perform immunoprecipitation to identify the interaction between Twistl and GTPase-activating protein(GAP);knock out Twistl of EoL-1 cells and detect the competitive binding of Ras and Twistl to GAP by competitive ELISA method.5.Cisplatin was added to activated Eos to induce apoptosis,and FTS was added to verify the role of Ras activation in Eos apoptosis resistance.Result:1.Compared with nAR group,significantly higher Eos counts,Ras.GTP expression,K-Ras(Ras,in short)activation and less Ras.GDP expression was detected in Eos of the AR group in nasal mucosal tissue;after Eos exposure to cisplatin,Apoptotic Eos in AR patients is significantly less than nAR patients,this effect is eliminated after adding FTS,and the protein levels of caspase7,caspase3 and p53 in Eos of AR patients are lower than nAR patients;2.AR Eos expresses high levels of Twistl,and the level of Twistl is positively correlated with the activation of Ras;3.After challenged by OVA,the Twist level increased significantly and Ras activation increased in EoL-1s,while inhibition of Ras abolished the sensitizationinduced Twist expression in EoL-1s.Ras activation promotes the expression of Twist1 through the P38MAPK-HIF-1α pathway;4.Ras activation was still found in sensitized WT EoL-1 cells but not in Twistdeficient(KO)EoL-1s;The results demonstrate that Twist1 physically contacts GAP and prevents GAP to bind Ras,that sustains Ras activation in Eos;while the activation of Ras was not found in EoL-1s after Twist1 knockout.5.Depletion of Twistl or in the presence of FTS effectively blocked Eos’s apoptosis resistance.Conclusion:High Ras activation and Twistl was detected in the AR nasal mucosal tissueisolated Eos,their interaction conferred Eos the apoptosis resistance.