The Inflammatory Mechanism Of Pepsin Damaging Laryngeal Mucosa Through The ROS-TXNIP-NLRP3 Pathway

Background and ObjectiveStomach contents contain gastric acid,pepsin,bile acid and chyme,etc.,which caused mucosal damage when they reflux to the pharynx and larynx.Pepsin is the main damage medium of the contents,but the damage mechanism is still not clear.The increased intracellular reactive oxygen species(ROS)level and the activation of Nod-like receptor family protein-3(NLRP3)inflammasome may be related to it.ROS are oxygenated compounds with high-energy electrons in cells,which participate in cellular activities such as energy metabolism and immune response under physiological conditions.Under pathological conditions,abnormal ROS content can damage cell structure and mediate chronic inflammation.NLRP3 inflammasome is an intracellular protein polymer that can be activated by intracellular and extracellular stimuli such as ROS to activate caspasel,thereby cutting the substrate precursor inflammatory cytokines IL1β and IL18 and causeing inflammation.Previous studies have shown that pepsin stimulation can mediate increased transcription of IL1 and IL8 in laryngeal epithelial cells and increase ROS levels.In this study,we proposed that pepsin mediates inflammation through the ROS-NLRP3 pathway,which is an important mechanism for LPR to damage laryngeal mucosa.Materials and methodsImmortalized cell lines were stimulated by pepsin.ROS levels were determined by dihydroethidium(DHE)staining and fluorescence quantification.The total protein of immortalized cells was extracted,and the expression levels of TXNIP,caspasel,IL1β,IL18 and corresponding splice were detected by western blot(WB).ROS inhibitors Apocynin,DPI,Tempol and NLRP3 specific inhibitor MCC950 were used for inhibitor protection experiments.The levels ROS,pepsin,caspasel,IL1β,and IL18 in the non-diseased mucosa of the postcricoid area in patients with LPR-related laryngeal lesions were detected,and their correlation was analyzed.Results1.Pepsin increased ROS and TXNIP levelsAfter Pepsin stimulated immortalized laryngeal epithelial cells for 2 days,DHE staining fluorescence quantification showed that the ROS level of immortalized laryngeal epithelial cells increased.WB detection showed increased expression of TXNIP.2.Pepsin up-regulated the expression levels of caspasel and IL1 beta-protein splicesUnder the same stimulation conditions,WB results showed that the expressions of cl-caspasel and cl-ill in mature forms of caspasesl and IL1 were increased,while there was no significant trend of IL18 and its shear bodies3.Protective effect of ROS inhibitors on pepsin stimulationThree ROS inhibitors(Apocynin,DPI and Tempol)were preincubated to inhibit pepsin to up-regulate ROS to different degrees,and Apocynin had the most obvious effect.After inhibition of ROS by Apocynin,the shear activation level of caspase 1 and IL1β decreased obviously.4.Protective effect of MCC950 on pepsin stimulusAfter pre-incubation of MCC950 specific inhibition of NLRP3 inflammasomes,the shear activation levels of caspasesl and IL1 significantly decreased,and the up-regulation of ROS levels was also inhibited.5.Pepsin of laryngeal mucosa in patients with laryngeal lesions was associated with ROS,caspasel and IL1 levelsImmunohistochemical results showed that 6 and 14 of the 20 subjects with negative pepsin expression were grouped accordingly.The expression levels of ROS,Caspasel and IL1 in the positive group were significantly higher than those in the negative group,and there was no significant difference in the level of IL18 between the two groups.ROS level,Caspasel and IL1 expression integral were significantly correlated with pepsin integral.ConclusionPepsin stimulation up-regulated ROS levels and TXINP expression and activated NLRP3 inflammaosomes in immortalized laryngeal epithelial cells,making increased expression of inflammatory cytokines IL1β.Both ROS inhibitor Apocynin and NLRP3 inhibitor MCC950 can inhibit the up-regulation of ROS and the activation of NLRP3 mediated by pepsin.The positive rate of pepsin expression in the mucosa of in laryngeal mucosa of patients with LPR related laryngeal lesions was 70%,and pepsin level was correlated with ROS,Caspasel and IL1 levels of laryngeal mucosa.Pepsin stimulation may mediate inflammatory injury of laryngeal mucosa through the ROS-NLRP3 pathway.