Effect And Mechanism Of Acid Suppression Therapy On Postoperative Recurrence Of Vocal Leukoplakia

As research continues,it becomes consensus that laryngopharyngeal reflux(LPR),as a co-factor,participates in the pathogenesis and development of vocal leukoplakia.Postoperative recurrence of vocal leukoplakia is frequently encountered in clinic,and is difficult to deal with it.Therefore,searching for therapeutic methods to decrease postoperative recurrence rate of vocal leukoplakia has become the focus and hot spot in clinical research.The mucosa of vocal cord is stratified squamous epithelium.The integrity of vocal mucosal barrier is essential to maintaining the shape and function of vocal cord.Structural and functional abnormalities of mucosal barrier in vocal leukoplakia maybe the major causes leading to recurrence and progress of this disease.LPR may result in or aggravate the damage of mucosal barrier.Yet there is little clear evidence confirms this hypothesis.Chronic inflammation plays a vital role in the occurrence and development of many diseases.In the development of laryngeal carcinoma,multiple signal transduction pathways are activated.NF-?B pathway,which mediates inflammatory reaction in the body,is of great importance.Activation of NF-?B and signal transduction pathway regulated by NF-?B maybe one of the underlying mechanisms for the occurrenceandprogressofvocalleukoplakia.This study aims to clarify the significance of acid suppression therapy for preventing recurrence of vocal leukoplakia,and to discuss the underlying mechanism for impact of LPR on mucosal barrier,thereforetoprovide a newtheoryandtherapyideafor furtherclinicaltreatment ofvocalleukoplakia.The paper contains two parts,including three clinical and basic researches on effect of acidsuppressiontherapyonpostoperativerecurrenceofvocalleukoplakia.Part I: Effect of acid suppression therapy on postoperative recurrence of vocal leukoplakiaObjective : Via observing the influence of laryngopharyngeal reflux(LPR)on outcome of vocal leukoplakia,the roles of LPR in the pathogenesis and pathological process of vocal leukoplakia were discussed,and the significance of acid suppression in the treatment of this disease was elucidated.Methods : According to the results of ambulatory 24-hour double-probe(simultaneous esophageal and pharyngeal)pH monitoring(pHmetry),the patients suffering from vocal leukoplakia were divided into LPR group and non-LPR group.Meanwhile,the patients of LPR group were divided into subgroups with and without oral administration of proton-pump inhibitor(PPI)based on informed consent.All patients received evaluation by electrolaryngendoscope,voice handicap index-10(VHI-10),reflux symptom index(RSI)and reflux finding score(RFS)before operation and 8 months after operation.They were asked to have follow-up visits monthly with a minimal follow-up duration of 12 months.For those with recurrence,the times of relapse were recorded,biopsies were performed and progressions of vocal leukoplakia were evaluated based on their initial pathological grades.Results:The differences of the average values of preoperative RSI,RFS and VHI-10 between the LPR group and non-LPR group were all statistically significant(all P<0.05).After operation,the differences of the mean values of RSI and RFS between the two groups were statistically significant(both P<0.05),while that of VHI-10 was insignificant(P>0.05).In the LPR group,both differences between the preoperative and postoperative mean values of RSI,RFS were statistically significant(both P<0.05),but that of VHI-10 was insignificant(P>0.05).In the non-LPR group,statistical analyses showed similar results with those of the LPR group.The recurrence rates of vocal leukoplakia in the LPR group and non-LPR group were 61.5% and 26.8% respectively,and the difference was statistically significant(P<0.05).In the PPI treatment group,the differences of the mean values of RSI,RFS and VHI-10 between the before treatment group and after treatment group were statistically significant(both P<0.05).In the LPR group,the cumulative recurrence rates of subgroups with and without administration of PPI were 58.3% and100%.However,there was no significant difference between the two groups.Conclusion:Standard PPI administration to patients suffering from vocal leukoplakia accompanied with LPR can alleviate the symptoms of LPR,relieve hoarseness,and decrease postoperative recurrence rate of vocal leukoplakia.Part II: Protective effect of acid suppression therapy on postoperative vocal mucosal barrier in vocal leukoplakiaObjective:Via observing the influence of acid suppression therapy on the structure and function of postoperative vocal mucosal barrier in patients suffering from vocal leukoplakia accompanied with LPR,the protective effect of this therapeutic strategy on the vocal mucosal barrier was discussed.Methods:The patients suffering from vocal leukoplakia accompanied with LPR were divided into groups with and without oral administration of PPI based on informed consent.Mucosal specimens of vocal cord were collected from all the patients during before PPI treatment and mucosal specimens of vocal cord were collected from the patients with suspected recurrence at 8weeks after PPI treatment.HE staining was used to observe the histopathological changes of the mucosa.Transmission electron microscopy(TEM)was utilized to detect morphology of mucosa epithelium tight junction.Western blot was used to detect the expression of the tight junction protein claudin-1 and ZO-1.Immunofluorescence method was adopted to detect position changes of the tight junction proteins,and apoptosis was determined with TUNELmethod.Results:HE staining showed decrease of inflammatory cell infiltration and obvious improvement of structure integrality in the specimens of after PPI-treated group.TEM found obvious increase of electron-densed substance around the mucosa epithelium tight junction and obvious improvement in the morphology of tight junction.Compared with the group before PPI treatment,the expression of protein claudin-1 and ZO-1 got decreased significantly(P<0.05).Immunofluorescence detection revealed increased expression of protein claudin-1 and ZO-1 in cytoplasm(P<0.05)and TUNNEL examination showed obvious increased amount of apoptotic cells in the specimens from the group before PPI treatment(P<0.05).Conclusion : Refluxed material will lead to structural damage of tight junction,abnormalities in function and expression of tight junction-related proteins.Damage of intracellular connection may result in expansion of intracellular space and therefore increased permeability of vocal mucosa epithelium.Acid suppression therapy can protectvocal mucosal barrier via elimination of this factor.Part ? Improvement of postoperative mucosal inflammation in vocal leukoplakia by acid suppression therapyObjective To study the mechanism of vocal mucosal barrier damage mediated by NF-?B and NF-?B-regulated signaling pathway via probing the expression of inflammatory factors and essential proteins for node of NF-?B signaling pathway.Methods The patients suffering from vocal leukoplakia accompanied with LPR were treated with oral administration of PPI.Mucosal specimens of vocal cord were collected from all the patients during before PPI treatment and mucosal specimens of vocal cord were collected from the patients with suspected recurrence at 8 weeks after PPI treatment.HE staining was used to score the pathological changes of the mucosa.ELISA was utilized to detect the levels of inflammatory factors including TNF-?,IL-1 and IL-6.Western blot was used to detect the expression of p-p65,p-IKK and p-I?B.Immunofluorescence method was adopted to detect the entrance of p65 to cell nucleus.RESULTS Inflammation scores evaluated with HE staining in the specimens of PPI-treated group were significantly lower than those of PPI pre-treatment group(P<0.05).In PPI pre-treatment,the expressions of IL-1?,TNF-?and IL-6 in the specimens of 8 weeks after operation were insignificantly different from those obtained during operation.But in the PPI-treated group,the expressions were down-regulated.The difference of p65 expression between PPI-treated group and PPI pre-treatment group was statistically insignificant(P>0.05).The expression of p-p65 in the PPI-treated group was lower than that of the PPI pre-treatment group(P<0.05).The expressions of IL-1?,TNF-?and IL-6were positively related with that of NF-?B-P65,but negatively with that of p-I?B.Immun of luorescence method revealed the entrance of p65 to cell nucleus in PPI pre-treatment group,which meant that NF-?B was activated.In the PPI-treated group,no activated p65 could be observed in the cell nuclei.Concusions The possible mechanism of vocal mucosal barrier damage in vocal leukoplakia accompanied with LPR maybe the vocal mucosal inflammationmediated by NF-?B and NF-?B-regulated signaling pathway activated with refluxed materials.