| Background:Chronic rhinosinusitis with nasal polyps was a common rhinosinusitis diseases in Otolaryngology department.The morbidity of this disease was about 1%-4%.Current recommended therapeutic regimen usually calls for surgery and glucocorticoid treatment.Even though the development of functional endoscopic sinusitis surgery(FESS),the recurrence rate of CRSw NP is still very high according to a multicenter clinical researches.Repeated surgeries are needed which cause serious economic burden to both the patients and society.Conservative treatment usually relies on glucocorticoid because the pathogenesis of CRSw NP is unclear.Thus investigating pathogenesis of CRSw NP and figuring out the therapeutic targets which lead to accurate treatment become a hot topic in research.Previous researches illustrated that pathological changes include :thickening and destroying of epithelial layer,glandular hyperplasia,thickening of basal lamina,tissue edema and infiltration of inflammatory cells[1].Research on nasal middle meatus illustrted that blood flow of middle meatus was siginifcantly less than inferior meatus,hemorheology of tissue was positive related with oxygen content,thus they thought middle meatus was an hypoxia enviroment [2]..Mucosa around sinus ostium swelled which caused by inflammation stimulation,inhaled normal oxygen tension air could not insert into ostium which leads to low oxygen tension in the sinus[3],this maybe one of the reasons why hypoxia was found in the middle meatus.Hypoxia inducible factor-1α(HIF-1α)and osteopontin,which only expressed after hypoxia stimulation,were found elevated expression in nasal polyps tissues [4,5].This were another proof for hypoxia existing in middle meatus.However the mechanism that hypoxia leads to CRSw NP was not clear.HIF1α and HIF2α continuous express and transcript in normal cells[6].The two proteins undergo proteasomal degradation via the ubiquitin-dependent pathway under aerobic conditions.HIF-1α/HIF-2α escape ubiquitination and proteasomal degradation under hypoxia condition[7].Thus HIF1αexpression was closely related with hypoxia condition.HIF1α and HIF2α both bind to the same hypoxia responsive element(HRE)at the target gene loci and change its activity of transcription,more than 100 genes could be regulated by HIF1α[8].HIF1αexpression elevated in nasal polyps tissues [5],and its downstream factors-VEGF were also found up regulation [9].This research will discuss the different immune reaction of different parts of nasal epithelial cells,figure out the expression of HIF1α,HIF2α and inflammatory cytokines,and related regulatory mechanism.Object: Hypoxia creates a microenvironment conducive to polypogenesis by regulating the immune responses of the nasal polyp epithelium.To explore the differences in immunocompetence of nasal polyp and control epithelial cells responds to hypoxia and identify a potential relationship with polypogenesis.Method: Seventeen inferior turbinate(IT)samples from healthy control subjects and 40 IT or nasal polyp(NP)samples from patients with chronic rhinosinusitis with nasal polyps(CRSw NP)were obtained.All samples were divided into three groups: IT from control(IT(N)),IT from CRSw NP(IT(NP)),and NPs.What’s more,NP(CRS)group were further separated into eosinophilic NP(CRS)group and non-eosinophilic NP(CRS)group according to EPOS 2012.The biopsy were divided into two part: for immunohistochemistry experiment and primary cells culturing.Immunohistochemistry was performed on 30 samples to test for the presence of interleukin(IL)-5,thymic stromal lymphopoietin(TSLP),IL-17 A,interferon(IFN)-γ,IL-17 A receptor(IL-17AR),hypoxia inducible factor(HIF)1α,and HIF2α in NP tissues and IT tissues from both CRSw NP and normal healthy subjects.Primary human epithelial cells from CRSw NP and normal mucosa were cultured under hypoxic or normoxic conditions.Changes in cytokine expression,including IL-5,TSLP,IL-17 A,and IFN-γ,in the supernatants of cultured cells were analyzed by performing enzyme-linked immunosorbent assays(ELISAs).Western blot were analyzed with the Perkin Elmer Operetta high-content/high-throughput imaging system utilizing Columbus image analysis software to quantify HIF1α and HIF2α expression in epithelial cells.The correlation analysis were done between clinical characteristic and expression of HIF1α,HIF-2α,inflammatory cytokines.Results: Immunohistochemistry revealed strong epithelial cells of the NP and IT(N)group strongly expressed HIF2α,and HIF1α,epithelial cells of the IT(N)group strongly expressed IL-17 A and IL-17 AR.,while epithelial expression of IL-17 A was lowest in the NP group compared with the other groups.There was a positive relationship between IL-17 A and IL-17 AR.Eosinophilic CRSw NP epithelial cells expressed more IL-5 and TSLP than non-eosinophilic CRSw NP epithelial cells.Non-eosinophilic CRSw NP epithelial cells expressed more IL-17 A and IL-17 AR than eosinophilic CRSw NP epithelial cells.There was no significant differences between eosinophilic CRSw NP and non-eosinophilic CRSw NP on IFN-γ expression.Immunohistochemistry illustrated HIF1α,HIF2α protein expression in epithelial cells were positive related with endoscope score of NP(CRS)group.Negative relation were observed between HIF1α and eosinophil infiltration degree.ELISA results illustrated there were no marked changes on the secretion of IL-5,IFN-γ,TSLP,IL-17 A in each groups.However IL-17 A secretion showed differences as time prolonged in three groups: IL-17 A expression,which decreased upon prolonged exposure to hypoxia in IT(N)group,was increased in both the NP and IT(NP)groups;IL-5,TSLP and IFN-γ secretion were not different under hypoxic conditions in whole groups.Non-eosinophilic CRSw NP epithelial cells and eosinophilic CRSw NP epithelial cells showed similar trend and degree in cytokines secretion changes under hypoxia condition.Western blot and high-content/high-throughput analysis revealed prominent increases in HIF1α and HIF2α expression under hypoxic conditions for 48 hours in NP epithelial cells compared with normoxic cells.Our study illustrated the time-dependency and negative relation of HIF1α,HIF2α and IL-17 A expression : increased expression of HIF1α,HIF2α and decreased secretion of IL-17 A was observed in NP epithelial cells and after prolonged exposure to hypoxia.However,IT(N)epithelial cells also indicated time-dependency but positive relation between HIF1α,HIF2α and IL-17 A expression.Non-eosinophilic CRSw NP epithelial cells and eosinophilic CRSw NP epithelial cells showed similar trend and degree in HIF1α and HIF1α expression and IL-17 A secretion changes under hypoxia condition.Conclusion: Hypoxia plays a key role in the immunocompetence of nasal polyp and normal nasal epithelial cells.Hypoxia induced expression of HIF1α,HIF2α in nasal epithelial cells,consistenceand time-dependency were found between HIF1α,and IL-17 A expression in NP and IT(CONTROL)group.But it was opposite tendency in NP and IT(CONTROL)group.Hypoxia involved in pathogenesis by regulating IL-17 A secretion and HIF1α and HIF2α expression,start the downstream inflammatory response in the nasal polyps epithelium.Otherwise,NP epithelial cells and IT(CRS)epithelial cells showed the similar tendency on cytokines changes under hypoxia in vitro,but different degree of expression in vivo.This may illustrate environment may play an important role in pathogenesis of CRSw NP.These conclusion provide the researchers new target for exploring the pathogenesis of CRSw NP and new clinical therapy... |
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