| Purpose and SignificanceLingual Tonsillar Hypertrophy(LTH)is caused by lymphoid tissue hyperplasia at the root of the tongue,and is generally ignored by the clinic because of its clinical symptoms.In recent years,more than 70%of adult patients with obstructive sleep apnea hypopnea syndrome(OSAHS)have glossopharyngeal plane obstruction caused by hypertrophy of tongue tonsil,which is considered to be one of the important reasons affecting surgical efficacy.Therefore,the etiology of hypertrophy of tongue tonsil has been studied gradually.Recently,emerging evidence of Laryngopharyngeal Reflux(LPR)is becoming one of the important pathogenic factors of hypertrophy of tongue tonsil.Pepsin is considered to be one of the main factors of laryngopharyngeal mucosa injury in gastric reflux.The detection of pepsin in saliva or tissue is an objective index for the diagnosis of laryngopharyngeal reflux.In previous articles published by our research team,it was found that pepsin was significantly overexpressed in hypertrophy tongue tonsil tissue and hypertrophy adenoid tissue in children.However,the main effectant cells and molecular mechanism of pepsin causing local inflammatory reaction and pathological hypertrophy of tongue tonsil are unknown.This study will carry out three aspects of research:1.Immunohistochemical detection of the correlation between pepsin expression in tongue tonsil tissue and hypertrophy grade of tongue tonsil,and explore the relationship between pepsin and macrophages;2.In vitro experiments on the role of pepsin in activating macrophage proliferation and inflammatory factor expression;3.Finally,to explore whether pepsin can activate the inflammatory pathway of macrophages and promote hypertrophy of tongue tonsils.Objects and methods1.Research objects1.1 Tongue tonsil tissue(N=73)from patients admitted to the Department of Otolaryngology,Nanfang Hospital,Southern Medical University from October 2019 to August 2020 were collected.These patients were hospitalized for surgical treatment due to obstructive sleep apnea hypopnea syndrome(26 cases),chronic tonsillitis(11 cases),vocal cord polyp(23 cases),and vocal cord leukoplakia(13 cases).2.Research methods2.1 Before surgery,the patients were asked and recorded their personal life and medical history related to the study,and a Reflux Finding Score(RFS)(RFS>7:Positive;RFS≤7:Negative)and Reflux Symptom Index(RSI)(RFS>13:Positive;RFS≤13:Negative)score;lingual tonsil tissue was finally obtained during general anesthesia.for follow-up experiments.2.2 Lingual tonsillar hypertrophy was graded on a laryngoscope scale from 0 to 4 according to Friedman’s criteria.The expression of pepsin in lingual tonsil tissue was detected by immunohistochemistry.Colocalization of pepsin and CD68 macrophage immunofluorescence double staining was performed in lingual tonsil tissue.2.3 Recruit healthy volunteers,draw 20ml of peripheral venous blood,extract mononuclear cells in the blood,and induce and culture them into macrophages.Macrophages were stimulated with different concentrations of pepsin,the proliferation changes of macrophages were tested with CCK-8,Edu and cell cycle,and the inflammatory factors IL-6 and IL-8 and MAPK pathway markers were detected by RTPCR and Western Blotting experiments expression changes.Finally,the activation of MAPK pathway was detected in lingual tonsil tissue.2.4 Data were processed by statistical software SPSS 20.0,and normality&homogeneity of variance tests were used to determine the test methods used for different data.Chi-square test for trend was used to compare the difference in positive rate of reflux meter between different grades of lingual tonsillar hypertrophy.KruskalWallis test and Spearman correlation analysis were used to compare bilevel variable data.Differences among multiple groups of data were compared using analysis of variance.Two-tailed P<0.05 was identified as significant.Results1.Correlation between pepsin and macrophages in lingual tonsil hypertrophic tissue.The 73 lingual tonsil hypertrophy cases included in this study were classified as:16 cases(21.9%)of lingual tonsil hypertrophy grade 1,13 cases(17.8%)of lingual tonsillar hypertrophy grade 2,23 cases(31.5%)of lingual tonsillar hypertrophy grade 3,Twenty-one cases(28.8%)of lingual tonsillar hypertrophy were grade 4.Among them,grade 3 and 4 are clinically significant grading of hypertrophy of tongue tonsil.The positive rate of pepsin in grade 3 and 4 tonsils was 88.6%(39 cases/44 cases).Chi-square test for trend analysis showed that there were significant differences in the positive rates of RFS and RSI in different grades of lingual tonsillar hypertrophy(p<0.001).The results of Spearman rank correlation analysis showed that there was a positive correlation between the positive rate of lingual RFS and RSI and the grade of lingual tonsillar hypertrophy(p<0.001).Kruskal-Wallistest rank sum test found that the staining intensity of pepsin was significantly different from the grading of lingual tonsillar hypertrophy(p<0.001).Spearman correlation analysis proved that there was a positive correlation between the staining intensity of pepsin and the grading of lingual tonsillar hypertrophy(p<0.001).The co-localization of CD68-positive macrophages and pepsin in the hypertrophic lingual tonsil tissue was clearly observed by immunofluorescence double staining,but not in the low-grade tissue of tongue tonsil hypertrophy.2.pepsin stimulates macrophage proliferation and inflammatory cytokine expression.In vitro cell experiments,CCK-8 experiments proved that pepsin can promote the proliferation of macrophages(p<0.05),Edu experiments also verified that pepsin upregulates the proliferation level of macrophages(p<0.05),and Cell cycle assay based on flow cytometry showed that pepsin promoted the differentiation of macrophages from G1 phase to S phase(p<0.05);RT-PCR experiments confirmed that the mRNA levels of macrophage inflammatory factors IL-6 and IL-8 were up-regulated by pepsin stimulation(p<0.05),Western Blotting experiments confirmed that pepsin stimulated macrophage IL-6 and IL-8 protein level expression increased(p<0.05).3.Pepsin promotes lingual tonsillar hypertrophy by mediating inflammation through the MAPK pathway in macrophages.Pepsin can activate the MAPK inflammatory signaling pathway in macrophages in vitro,and up-regulate the protein expression of pathway markers P-p38 MAPK,PJNK and c-jun(p<0.05);Compared with tonsillar hypertrophy grade 3/4+pepsin negative),MAPK signaling pathway markers(P-p38 MAPK in macrophages in lingual tonsillar hypertrophy tissue of the experimental group(lingual tonsillar hypertrophy grade 3/4+pepsin positive)and P-JNK)were significantly overexpressed(p<0.05).ConclusionLaryngeal reflux is an important pathogenic factor of lingual tonsillar hypertrophy,and pepsin may be involved in the pathophysiological process of lingual tonsil hypertrophy by activating macrophage proliferation and inflammatory factor expression. |
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