胃蛋白酶激活巨噬细胞促进舌扁桃体肥大机制的研究

Mechanism of pepsin promoting lingual tonsil hypertrophy by stimulating macrophage

目的探讨咽喉反流(LPR)在舌扁桃体肥大(lingual tonsil hypertrophy,LTH)发生发展中可能的病理生理机制。方法回顾性收集2019年10月至2020年12月就诊于南方医科大学南方医院耳鼻咽喉头颈外科的因咽喉相关疾病接受手术的73例患者[男48例,女25例,年龄24~76(52.86±12.04)岁]的舌扁桃体组织,并评估其舌扁桃体分级(lingual tonsil grade,LTG)、反流症状指数(RSI)和反流体征评分(RFS)。免疫组织化学检测舌扁桃体组织中胃蛋白酶表达,免疫组织双荧光检测胃蛋白酶和巨噬细胞的共表达情况。在体外,对胃蛋白酶刺激后的体外巨噬细胞进行多种细胞学实验和通路检测。免疫组织双荧光检测胃蛋白酶阳性的高分级LTH中巨噬细胞的通路改变。采用SPSS 20.0软件进行统计分析。结果具有临床意义的LTG 3和4级的LPRD患者44例,其胃蛋白酶阳性率为88.6%(39/44),而LTG 1和2级阳性率为48.3%(14/29)。LTG与RFS/RSI阳性率(χ^(2)=23.01/19.62,P<0.001/0.001;r=0.54/0.51,P<0.001/0.001)、胃蛋白酶组织染色强度(H=21.58,P<0.001;r=0.53,P<0.001)分别呈显著正相关性。胃蛋白酶和巨噬细胞在高分级LTH中明显共定位。在体外实验中,胃蛋白酶促进巨噬细胞增殖(P值均<0.05)和促炎因子IL-6/IL-8产生(P值均<0.05)。胃蛋白酶显著上调巨噬细胞中的p38/JNK MAPK通路(P值均<0.05),并通过激活p38 MAPK通路上调巨噬细胞表达IL-6和IL-8(P值均<0.05),激活JNK通路上调IL-8(P<0.05)。p38/JNK MAPK通路在胃蛋白酶阳性LTH组织的巨噬细胞中高表达(P值均<0.05)。结论LPR是LTH的重要致病因素,巨噬细胞可能介导了胃蛋白酶诱导的炎症反应和LTH的重要发病过程。

Objective To investigate the possible pathophysiological mechanism of laryngopharyngeal reflux(LPR)in the development of lingual tonsil hypertrophy(LTH).Methods The lingual tonsil tissues were collected from 73 patients[48 males and 25 females,aged from 24 to 76(52.86±12.04)years]who underwent surgery for laryngopharyngeal diseases at the Department of Otolaryngology and Head and Neck Surgery,Southern Hospital of Southern Medical University from October 2019 to December 2020,and the lingual tonsil grade(LTG),reflux symptom index(RSI)and reflux finding score(RFS)were assessed.The expression of pepsin in LTH was detected by immunohistochemistry.The coexpression of pepsin and macrophages were detected by immunohistofluorescence.In vitro,cytological experiments and pathway assays were performed on macrophages stimulated by pepsin.Pathway alterations of macrophages in pepsin-positive high-grade LTH were detected by double-fluorescence immunohistochemistry.Data were analyzed by SPSS 20.0 software.Results There were 44 clinically significant LPRD patients with LTG 3 and 4,and the pepsin positive rate was 88.6%(39/44).While,the pepsin positive rate of LTG 1 and 2 was 48.3%(14/29).LTG was significantly positively correlated with RFS/RSI positive rate(χ^(2)=23.01/19.62,P<0.001/0.001;r=0.54/0.51,P<0.001/0.001)and pepsin tissue staining intensity(H=21.58,P<0.001;r=0.53,P<0.001),respectively.Pepsin and macrophages were clearly colocalized in high grade LTH.In vitro,pepsin promoted macrophage proliferation(P<0.05)and production of IL-6/IL-8(P<0.05).Pepsin significantly up-regulated the p38/JNK MAPK pathway in macrophages(P<0.05).Pepsin up-regulated the expression of IL-6 and IL-8 of macrophages by activating the p38 MAPK pathway(P<0.05),and up-regulated the expression of IL-8 by activating the JNK pathway(P<0.05).The p38/JNK MAPK pathways were highly expressed in macrophages of pepsin-positive LTH(P<0.05).Conclusions LPR is an important pathogenic factor in LTH.Macrophages may mediate pepsin-induced inflammation and the pathogenesis of LTH.