| Invasive fungal rhinosinusitis (IFRS) is a serious disease with a high mortalityrate that has increased over the last decade due to the growing number ofimmunocompromised patients.To explore the pathogenesis of IFRS, there sections ofexperiments were constructed.Section1A preliminary study on aspergillus fumigates-induced IKKαcontroled maspin protein expression in respiratory epithelial cellsObjective To study if the expression of maspin protein in respiratory epithelialcells was downregulated through IκB kinase-α (IKKα)-controlled mechanism in anaspergillus fumigates-induced model in rat.Method Inactivated Aspergillus fumigatus hyphae (AFH) suspension was usedto induce respiratory epithelial cells (REC) cultured in vitro in rat, with PBS buffer ascontrol. By RT-PCR, the mRNA expression of maspin was quantified, and byimmunocytochemistry the expressions of maspin and IKKα in REC were observed.Furthermore AFH (from level1to level3) suspension was prepared to induce REC.Then Western blot hybridization techniques were used to detect the expression ofmaspin and IKKα protein. All data were processed by analysis of variance andtwo-variable correlation analysis.Result By RT-PCR, a statistically significant (t=2.463, P <0.05) expression ofmaspin mRNA was found (0.128±0.059in AFH group vs2.972±0.353in controlgroup). By Immunocytochemistry, the difference of maspin protein color in differentgoups was shown statistically significant in integrated scoring (t=3.721, P <0.05)(weak positive in AFH group, moderately positive in control group). While in IKKαcolor study, the difference between the two groups was also statistically significant (t =6.825, P <0.05) in integrated scoring, with a moderate positive in AFH group andweak positive in control group. By Western Blot hybridization, Grayscale ratio ofmaspin and β-actin was0.912±0.023,in control group，0.607±0.030,0.476±0.019,0.416±0.017in AFH1~3groups，respectively, with a statistically significantdifference within the four groups (F=281.91, P <0.05); While Grayscale ratio ofIKKα and β-actin was0.624±0.012in control group,0.739±0.020,0.778±0.010,0.927±0.017, respectively, in AFH1~3groups; with a statistically significantdifference within the four groups (F=200.91，P＜0.05). Moreover, the differencebetween any two groups from both AFH group (including supgroup1,2and3) andcontrol group was statistically significant. Two-variable correlation analysis indicateda negative correlation between expression of maspin and IKKα (r=-0.911, P <0.05).Conclusion Induced by aspergillus fumigates, the rat respiratory epithelia mightupregulate the expression of IKKα with a downregulated expression of maspinprotein.Section2A novel model of invasive fungal rhinosinusitis in ratsObjective Invasive fungal rhinosinusitis (IFRS) is a life-threateninginflammatory disease that affects immunocompromised patients, but animal modelsof the disease are scarce. This study aimed to develop an IFRS model in neutropenicrats.Method The model was established in three consecutive steps: unilateral nasalobstruction with a Merocel sponge, followed by the administration ofcyclophosphamide (CPA), and finally nasal inoculation with Aspergillus fumigatus.Fifty healthy Wistar rats were randomly divided into five groups, with group I as thecontrols, group II undergoing unilateral nasal obstruction alone, group III nasalobstruction with fungal inoculation, group IV nasal obstruction with theadministration of CPA, and group V nasal obstruction with the administration of CPAand fungal inoculation. Hematology, histology, and mycology investigations wereperformed. Result The changes in the rat absolute neutrophil counts (ANCs) werestatistically different across the groups. The administration of CPA decreased theANCs, whereas nasal obstruction with fungal inoculation increased the ANCs, andnasal obstruction did not change them. Histological examination of the rats in groupV revealed the hyphal invasion of sinus mucosa and bone, thrombosis, and tissueinfarction. No pathology indicative of IFRS was observed in the remaining groups.Positive rates of fungal culture in tissue homogenates from the maxillary sinus(62.5%) and lung (25%) were found in group V, whereas groups I, II, III and IVdemonstrated no fungal culture in the homogenates.Conclusion A rat IFRS model was successfully developed through nasalobstruction, CPA-induced neutropenia, and fungal inoculation. The disease modelclosely mimics the pathophysiology of anthropic IFRS.Section3Experimental Study on IKKα regulation of Maspin Expression inRats Invasive Fungal RhinosinusitisObjective The early study showed that induced by aspergillus fumigates, the ratrespiratory epithelia might upregulate the expression of IKKα with a downregulatedexpression of maspin protein.This study was aimed to explore the expression ofmaspin and IKKα in rats model of invasive fungal rhinosinusitis.Method The rats model of invasive fungal rhinosinusitis were developed. Fiftyhealthy Wistar rats were randomly divided into five groups, with group I as thecontrols, group II undergoing unilateral nasal obstruction alone, group III nasalobstruction with fungal inoculation, group IV nasal obstruction with theadministration of CPA, and group V nasal obstruction with the administration of CPAand fungal inoculation. By RT-PCR, the mRNA expression of maspin was quantified,and by immunohistochemical study the expressions of maspin and IKKα in thegroups were observed. Then Western blot hybridization techniques were used todetect the expression of maspin and IKKα protein. All data were processed byanalysis of variance.Result By immunohistochemical study, the difference of maspin protein color indifferent goups was shown statistically significant in integrated scoring (F=14.58 P<0.05)(weak positive in group V, moderately positive in the remaining groups).While in IKKα color study, the difference between the five groups was alsostatistically significant (F=18.80P<0.05) in integrated scoring, with a moderatepositive in groups III and V, weak positive in the remaining groups.By RT-PCR, a statistically significant (F=13.678P<0.05) expression of maspinmRNA was found and the expression in group V was lower than that in the remaininggroups (P<0.05).By Western Blot hybridization, there was a statistically significant differencewithin the five groups in the grayscale ratio of maspin and β-actin (F=6.976P<0.05).The ratio in group V was lower than that in the remaining groups (P<0.05); WhileGrayscale ratio of IKKα and β-actin was a statistically significant difference in thefive groups (F=18.156P<0.05).The ratio in groups III and V were higher than that inthe remaining groups (P<0.05).The results of invasive fungal sinusitis IKKα protein up expression, anddownregulation of maspin; stuffy nose with fungal group, IKKα protein proteinenhanced expression without maspin downregulated; the blank control group, simplystuffy nose group, stuffy nose with immunosuppression group did not see IKKαprotein enhanced expression and downregulation of maspin.Conclusion IKKα activation does not cause maspin down in normal immunefunction, while in the case of the immunodeficiency, the activation of IKKα promoteddown of the maspin.ConclusionThe innovation of this study was the discussion on relationship between IFRSand IKKα regulation of maspin expression.1. Induced by aspergillus fumigates, the rat respiratory epithelia mightupregulate the expression of IKKα with a downregulated expression of maspinprotein.2. A rat IFRS model was successfully developed through nasal obstruction,CPA-induced neutropenia, and fungal inoculation. The disease model closely mimics the pathophysiology of anthropic IFRS.3. There is not simple linear relationship between upregulated IKKα anddownregulated maspin in vivo,which is different from it in vitro study. IKKαactivation does not cause maspin down in normal immune function, while in the caseof the immunodeficiency, the activation of IKKα promoted down of the maspin. Itmight be one of the molecular mechanisms of invasion mechanism in IFRS. |
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