瞬时受体电位锚蛋白1通道调节慢性鼻窦炎上皮细胞自噬及细胞间黏附分子表达的作用探究  

The role of transient receptor potential ankyrin1(TRPA1)channel in regulating autophagy and interadhesion molecule expression in epithelial cells of chronic sinusitis

作　　者：王丽[1] 黄永阳 张义 綦彬 万龙[1] 陈才盛[1] WANG Li;HUANG Yongyang;ZHANG Yi;QI Bin;WAN Long;CHEN Caisheng(Department of Otolaryngology Head and Neck Surgery,Huanggang Central Hospital,Huanggang,Hubei,438000,China)

机构地区：[1]黄冈市中心医院耳鼻咽喉头颈外科,湖北黄冈438000

出　　处：《中国耳鼻咽喉头颈外科》2022年第12期780-786,共7页Chinese Archives of Otolaryngology-Head and Neck Surgery

基　　金：湖北省卫生和计划生育委员会科研项目(WJ2017F087)。

摘　　要：目的 探究瞬时受体电位锚蛋白1(transient receptor potential ankyrin1,TRPA1)对慢性鼻窦炎(CRS)上皮细胞自噬与细胞间黏附分子(intercellular adhesion moleclar-1,ICAM-1)表达的影响。方法 通过免疫组织化学染色、实时荧光定量PCR及Western blot检测TRPA1在对照组、慢性鼻窦炎不伴鼻息肉(chronic nasal-sinusitis without nasal polyps,CRSsNP)及慢性鼻窦炎伴鼻息肉(chronic rhinosinusitis with nasal polyps,CRSwNP)患者鼻黏膜组织中的表达水平,并采用免疫组织化学染色和Western blot观察各组患者鼻黏膜组织内自噬水平;分离培养鼻黏膜上皮细胞,利用细胞角蛋白AE1进行免疫荧光染色鉴定上皮细胞;将鼻黏膜上皮细胞随机分为对照组、脂多糖(LPS)组、LPS+TRPA1抑制剂组,实时荧光定量PCR和Western blot检测各组细胞TRPA1在mRNA和蛋白上的表达水平,以自噬双标腺病毒mRFP-GFP-LC3感染细胞并通过荧光显微镜观察各组细胞自噬情况,并使用透射电镜观测各组细胞自噬小体形成,实时荧光定量PCR和Western blot检测各组细胞ICAM-1在mRNA和蛋白上的表达水平。结果 相较于对照组,CRSsNP与CRSwNP患者鼻黏膜上TRPA1平均光密度值升高,其mRNA相对表达量和蛋白相对表达量均上调(P<0.05),且LC3-Ⅱ平均光密度值升高,LC3-Ⅱ/LC3-Ⅰ比值升高,Beclin-1蛋白相对表达量上调而p62蛋白相对表达量下调(P<0.05),组织内自噬水平升高。成功分离到生长密集、形状为梭形或多边形以及AE1呈阳性表达的鼻黏膜上皮细胞;与LPS组比较,经过LPS与TRPA1抑制剂共处理的鼻黏膜上皮细胞TRPA1 mRNA相对表达量与蛋白相对表达量均下调(P<0.05),自噬溶酶体红色荧光减弱,自噬小体也明显减少,同时,ICAM-1mRNA相对表达量与蛋白相对表达量均下调(P<0.05)。结论CRS中TRPA1表达异常增加,自噬水平也升高,靶向抑制TRPA1能够抑制CRS上皮细胞自噬以及ICAM-1表达的升高。OBJECTIVE To explore the effect of transient receptor potential ankyrin1(TRPA1) on autophagy and the expression of intercellular adhesion molecule(ICAM-1) in epithelial cells of chronic rhinosinusitis(CRS). METHODS The expression level of TRPA1 in normal, CRSsNP and CRSwNP patients’ nasal mucosal tissues was detected by immunohistochemical staining, real-time fluorescent quantitative PCR and Western blot. immunohistochemical staining and Western blot were used to observe the autophagy level in the nasal mucosa of the groups respectively. Nasal mucosal epithelial cells were isolated and cultured, and the epithelial cells were identified by immunofluorescence staining with cytokeratin AE1.Nasal mucosal epithelial cells were randomly divided into control group, LPS group and LPS+TRPA1 inhibitor group.After grouping, real-time fluorescent quantitative PCR and Western blot were used to detect the expression of TRPA1in each group of cells on mRNA and protein level. The cells were infected with the autophagy double-labeled adenovirus mRFP-GFP-LC3 and the autophagy of each group was observed by fluorescence microscope, and a transmission electron microscope was used to observe the formation of autophagosomes in each group of cells. Real-time fluorescent quantitative PCR and Western blot were used to detect the expression levels of ICAM-1 in each group of cells on mRNA and protein. RESULTS Compared with the control group,the average optical density of TRPA1 on the nasal mucosa of CRSsNP and CRSwNP patients increased, and the relative expression of mRNA and protein were up-regulated(P<0.05).The average optical density of LC3-Ⅱ and the ratio of LC3-Ⅱ/LC3-Ⅰ increased. The relative expression of Beclin-1 protein was up-regulated and the relative expression of p62 protein was down-regulated(P <0.05), and the level of autophagy in tissues increased. Isolated nasal mucosal epithelial cells with dense growth, fusiform or polygonal shape and positive expression of AE1. Compared with the LPS group, the relative expression o

关 键 词：鼻窦炎 锚蛋白类 瞬时受体电位通道 鼻黏膜 上皮细胞 自噬 细胞粘附分子 

分 类 号：R765.41[医药卫生—耳鼻咽喉科]