The Effect Of Cyclic Tensile Strain On Smoke-elicited Inflammation In Vocal Fold And The Possible Mechanisms Of Reinke’s Edema

Part ⅠAnalysis of smoking status and Eysenck Personality between the patientswith Reinke’s edema and vocal fold squamous cell carcinomaObjectives:Cigarette smoke stimulation is one of the most common and important causes of Reinke’s edema and vocal fold squamous cell carcinoma. It is interesting and of significance to know that both under cigarette smoke exposure, why did the Reinke’s edemasamples exhibit a chemoprotective reaction, and why did the two diseases face such different outcomes? Another essential factor of Reinke’s edema is excessive speaking, significant character difference can be found clearly in clinic, with vocal misuse in most Reinke’s edema patients, but not in neoplasm. Therefore, it is interesting to know whether a certain degree of vocal fold vibration is the main reason responsible for the different progression of both diseases. The present study aims to assessthe smoking statusand level of vocal use among Reinke’s edema, vocal fold squamous cell carcinoma and control groups via clinical investigation and Eysenck Personality Questionnaire (EPQ) survey.Methods:A total of122patients (55cases of vocal fold squamous cell carcinoma and67cases of Reinke’s edema), hospitalizedfrom September,2009to December,2011,and 60healthy control adults were recruited for the status investigation on smoking and EPQ survey in this study. The assessment of smoking status was based on the total smoke exposure(pack-years, PKY), which was calculated by multiplying the number ofpacks/day (1pack=20cigarettes) and the number of years during which that quantity wassmoked. The obtained PKY was then compared between Reinke’s edema and carcinoma patients, and the E-value in EPQ are statistical analyzed to assessthelevel of vocal use.Results:In assessment of the smoking status, the mean total smoke exposures, expressed as PKY, in Reinke’s edema (45.69±11.15) and vocal fold carcinoma(46.38±10.50) groups showed no significant differences under independent sample T test (t=0.35, P=0.7257).The EPQ survey indicated that the mean scores for E-value in Reinke’s edema, vocal fold carcinoma,and control groups were62.61±7.02,41.35±7.01, and49.2±5.55, respectively. The differences in extraversion among three groups were statistically significant (F=165.02,P<0.0001), while pairwise comparisons showed that there were significant differences between each group pair (P<0.05). Additionally, the constituent ratio among three groups shown to be significantly different (χ2=158.7225, P<0.0001); most Reinke’s edema patients were prone to extraversion, and most of vocal fold carcinoma patients were prone to introversion.Conclusions:There is no significant difference between Reinke’s edema and vocal fold carcinoma group in smoking status, but most Reinke’s edema patients were prone to extraversion, characterized bytalkative personality, the level of vocal use is much higher than vocal fold carcinoma group. The results suggest that the combinatorial effect of cigarette smoke-induced inflammation and vocal fold vibration-mediated anti-inflammation in Reinke’s edema mayhave eventually delayed or inhibitedits malignant transformation seen in carcinoma patients under prolonged exposure to cigarette smoke alone. Part IIComparison of CD44, COX-2and MMP-9expression between Reinke’s edema and paracancerous tissueObjectives:The mean total smoke exposures in Reinke’s edema and vocal fold carcinoma groups showed no significant differences, Cigarette smoke-induced chronic inflammation is the common pathologic feature in respiratory tissue, but most Reinke’s edema patients were prone to extraversion, characterized bytalkative personality, the level of vocal use in Reinke’s edema group is much higher than vocal fold carcinoma group. Cigarette smoke-induced chronic inflammation is the common pathologic feature in respiratory tissue. The present study aims to compareCD44, COX-2and MMP-9expression in Reink space betweenReinke’s edema and vocal fold squamous cell carcinoma, and investigate the differences in inflammation under same cigarette smoke exposure but different level of vocal fold vibration.Methods:20paracancerous and20RE samples were collected from the122patients during routine surgical resection. Meanwhile,20normal human vocal folds were collected from patients who had undergone total laryngectomyas initial treatment for other primary tumors, withoutimplicating the laryngeal cavity; they were hypopharyngeal andesophageal carcinoma patients who had no smoking history and no history of vocal abuse. Inflammatory factors of CD44, COX-2, and MMP-9in Reinke’s edema, paracancerous and normaltissues were detected by RT-PCR and Western blot.Results:After designation of the expression levels innormal tissues as100%, CD44expressed much more in Reinke’s edema group than in carcinoma group, while COX-2and MMP-9were down-regulated in Reinke’s edema compared to those in paracancerous tissues (P<0.05).Conclusions:The expression of CD44was significantly induced, whereas COX-2and MMP-9 were detected decreased in Reinke’s edema tissues, compared with paracancerous tissues. Reinke’s edema samples were influenced by smoking, which may indicate a chemoprotective mechanism ofantioxidant response in thevocal fold tissue, the study suggests that a certain degree of vocal fold vibration may be the main reason responsible for the mechanism of anti-inflammation. Part ⅢCyclic Tensile Strain upon Vocal Fold Attenuates Cigarette Smoke-elicited Inflammation in human vocal fold fibroblastsObjectives:The mean total smoke exposures in Reinke’s edema and vocal fold carcinoma groups showed no significant differences, but the level of vocal use in Reinke’s edema group is much higher than vocal fold carcinoma group. Cigarette smoke-induced chronic inflammation is the common pathologic feature in respiratory tissue, The study have founded that CD44expressed much more in Reinke’s edema group than in carcinoma group, while COX-2and MMP-9were down-regulated in Reinke’s edema compared to those in paracancerous tissues. The mechanism of chemoprotective reactionin Reinke’s edemasamples is unclear.Duringphonation, vocal fold fibroblasts experience varioustypes of mechanical forces, with tensile asthe dominant form.lt has been shown that tensile strain altersare beneficial to various injuries. Fibroblastsare the main functional population ofvocal fold during phonation, and may play a majorrole in the production of extracellular matrix molecules. We hypothesize that cyclic tensile strain upon vocal fold during phonation involves in Reinke’s edema and can attenuate the cigarette smoke-elicited inflammatory reaction in human vocal fold fibroblasts.Methods:The primary culture technique is used to culture human vocal fold fibroblasts, and then the fibroblastsare identified by immunofluorescent staining. The viabilities of cultured human vocal fold fibroblasts under cigarette smoke condensate (CSC) and/or cyclic tensile strain (CTS) were assessed by MTT assay. Cells are divided into four groups:group1, control cells, no treatment; group2, CTS treated cells, treated only with CTS; group3, CSC treated cells, treated only withCSC; group4, combined treatment, treated withboth CSC and CTS for24h. The inflammation-relatedmolecules of CD44, COX-2, and MMP-9in HVFFunder different conditions were detected by RT-PCR and Western blot. Results:These fibroblasts were characterized by immunofluorescent staining, the results showed that almost100%cells after4-6passages were fibroblasts. The viabilities of cells treated with CSC significantly reduced compared to that of untreated cells. Cells in combined treatment group with both CSC (400μg/mL,24h)andCTS (8%,0.5Hz,24h)showedclear protection, with viabilitysignificantly higher than that of CSC alone group. Treatment of human vocal fold fibroblasts with CSC led to the decrease incell viability, the reduction in CD44expression, but, the increase in COX-2and MMP-9expressions and, moreover, administration of CTS inhibitedsuch effects of CSCon HVFF (P<0.05).Conclusions:The results suggest that CTS-mediated CD44interactions in CTS intervention group are critically involved in the inhibition of cigarette smoke-induced inflammation and may have protected the cells from cigarette smoke-induced damages. The combinatorial effect of cigarette smoke-induced inflammation and CTS-mediated anti-inflammation on vocal folds in Reinke’s Edema mayhave eventually delayed or inhibitedthe progression to malignant transformation seen in carcinoma patients under prolonged cigarette smoke alone. The results of this study hold premise for the clinical therapy and suggestthat certain frequency of vocal training may be used to attenuate the CS-induced inflammatory response.