Study On The Damage Mechanism Of Pepsin On Laryngeal Mucosa Epithelium

Purpose and significanceLarynghopharyngeal reflux disease(LPRD)caused by reflux of gastric contents to pharynx and larynx has received extensive attention in the otorhinolaryngological community.Due to the unclear pathogenesis of pharyngolaryngeal reflux,there is no specific method and diagnostic gold standard for the diagnosis of LPRD,and so far there is a lack of specific methods and diagnostic gold standard for the diagnosis of Otolaryngology.Nearly 50%of the patients were treated with proton pump inhibitors for acid inhibition.It has been proved that pepsin(pepsin)is the main damage factor of LPRD,but the mechanism of how pepsin injures throat mucosa epithelium is not clear,and there are few reports on it.In this study,we established the primary cell line of human laryngeal mucosa,studied the mechanism of pepsin damage to laryngeal epithelium,provided a new theory for the pathogenesis of larynx reflux disease,and explored new therapeutic strategies.MethodSubglottic,ventricular zone,epiglottis and posterior ring were taken from normal human laryngeal tissues during operation,and the primary cells of laryngeal epithelial cells were cultured by two-step enzyme and tissue grinding method.The expression of BMI-1,hTERT,p53 and pRB pathway proteins were detected by immortalized,Western blot induced by lentivirus infection in laryngeal epithelial cells,and the molecular biological changes caused by BMI-1 infection in laryngeal epithelial cells were investigated.The expression of pLVTHM-BMI-1 plasmid was constructed and induced by lentivirus infection in laryngeal epithelial cells.Cell senescence ?-galactosidase staining,EdU cell proliferation test and flow cytometry were used to verify the functional characteristics of immortalized cell line.In pH=7 environment,laryngeal epithelial immortalized cells were stimulated with different concentrations of pepsin(Omg/ml,0.1mg/ml and lmg/ml).Oxidative stress,mitochondrial membrane potential and comet assay were used to detect ROS production.Mitochondrial damage and nuclear DNA damage.The expression of inflammatory cytokines(IL1 ?,IL1 ?,IL2,IL4,IL5,IL6,IL8,IL10,IL18)was detected by fluorescence quantitative PCR.The effects of pretreatment with NADPH oxidation inhibitor(DPI and Apocynin)or ROS scavenger(Tempol)on mitochondrial damage,inflammatory cytokine expression and nuclear DNA damage in pepsin were observed.Furthermore,the expression of DNA damage marker p-HOAX and ROS pathway protein was detected by Western blot,so as to explore the possible mechanism of DNA damage mediated by Pepsin in laryngeal epithelial cells through ROS.P-H2AX and 8-OHdG immunohistochemical staining was performed in 32 specimens of vocal cord polyps associated with larynx reflux and 16 specimens of laryngeal mucosa in healthy volunteers.The data of multi-channel impedance PH in the specimen library were used to detect polyp of vocal cord(n = 32)and laryngeal mucosa specimens(n = 16)of larynx and larynx regurgitation.To investigate the relationship between pepsin and DNA damage in vocal cord polyps.Result.1.Primary cell culture of larynx and construction of immortalized cell line(1)Successful culture of primary laryngeal epithelial cells.The primary laryngeal epithelial cells were cultured by enzyme digestion and tissue grinding.the morphology of the cells was round under light microscope,the refraction was good under light microscope,the cells grew like paving stone,there was contact inhibition,and the cells could be successfully subcultured and CK staining was positive.It was proved that the cell came from epithelium.The primary cells in different regions of laryngeal cavity(including posterior ring,epiglottis,subglottic,ventricular zone)were successfully cultured,and Westernblotting was completed to prove the origin of epithelial cells.(2)Establishment of subglottic and ventiricular zone cells stably expressing BMI-1.Two immortalized laryngeal epithelial cells(subglottic BMI-1 cells and ventricular zone BMI-1 cells)were established and verified by BMI-1 mediated by our group.the overexpression of BMI-1 was detected by qPCR and Wb,and the level of hTERT protein was increased by telomerase detection.The increase of telomerase activity,western blotting suggested that the expression of p16 and p21 decreased gradually,while the expression of phosphorylated RB increased after overexpression of BMI-1.(3)Study on the function of subportal BMI-1 and ventricular zone BMI-1 cells.The results of P-galactosidase senescence analysis,Edu proliferation test and cell cycle flow assay showed that after transfection of BMI-1 into laryngeal epithelial cells,the number of senescent cells decreased significantly,the cell activity increased,and the number of cells in S phase increased.The proliferation of cells was accelerated.Tumor formation was not found in subglot-tic BMI-1 and subglottic BMI-1 cells,but not in subglottic cells and subglottic cells in nude mice.Hep-2 cells were used as positive control.2.Damage of laryngeal epithelial cells induced by PepsinBy stimulating subglottic BMI-1 and ventricular zone BMI-1 cells with dif-ferent concentrations of pepsin(Omg/ml,0.1mg/ml,1mg/ml)in pH7 environment,the results suggest that ROS production increases with the increase of pepsin concentration.Mitochondrial membrane potential increased,The secretion of inflammatory cytokines(IL1 ?,IL1?,IL6,IL8)was up-regulated,and the damage of DNA was aggravated.3.Mechanism of laryngeal epithelial injury mediated by Pepsin through ROS.After adding NADPH oxidation inhibitors(Apocynin and DPI)and ROS scavenger(Tempol),ROS production,mitochondrial damage,inflammatory cytokine secretion and DNA damage were significantly decreased.The expression of p-ERK,p-JNK and c-Jun protein was up-regulated after pepsin stimulation,but significantly down-regulated after pretreatment with apocynin.4.Relationship between Pepsin and oxidative stress DNA damage in vocal cord polyps.The intensity of immunohistochemical staining of 8-OHdG and P-H2AX in vocal cord polyp group was higher than that in normal group(P<0.01).The expression of Pepsin was positively correlated with the expression of 8-OHdG and P-H2AX in vocal cord polyps(Correlation Coefficient=-0.348,P=0.008 and Correlation Coefficient=-0.254,P=0.041).The P-H2AX score was basically similar to the 8-OHdG score.It was positively correlated with the four parameters of pharyngeal acid reflux(including acid reflux times,acid reflux time percentage,average acid clearance time)(P<0.05),and with esophageal acid reflux parameters,neutral position and total acid reflux parameters(P<0.05),and positively correlated with the four parameters of pharyngeal acid reflux(including acid reflux times,acid reflux time percentage,average acid clearance time)(P<0.05).There was a significant positive correlation between pH<4 time and the longest reflux time(P<0.05).Conclusion.1.The primary cells of posterior ring,epiglottis,subglottic and ventricular zone were successfully cultured,and the immortalized cellines of subglottic and ventricular zone stably expressing BMI-1 were established.2.Pepsin stimulation promoted the damage of DNA and mitochondria,the increase of ROS production and the secretion of inflammatory factors in laryngeal epithelial cells.3.DNA damage of laryngeal epithelial cells induced by pepsin through ROS/MAPK/inflammatory signaling pathway4.Pepsin was closely related to DNA oxidative damage in vocal cord polyps.