Mechanisms Of Clara Cell 10-kDa Protein Functions As An Anti-inflammation Molecule In Airway Epithelium

PartⅠThe effect of Clara cell 10-kDa protein inairway epithelium inflammationExperimentⅠConstruction and identification the expression vector ofhuman Clara cell 10-kd protein geneBackground: Chronic sinusitis and nasal polyps are chronic inflammatory diseases innasal cavity and nasal sinus. The pathogenesis of them has not been elucidated by now. Claracell 10-kd protein (CC10) is a multifunction protein with anti-inflammatory andimmunomodulatory effects. Previous research has demonstrated that CC10 is downregulatedin chronic sinusitis and nasal polyps, but how the CC10 protein functions on these diseasesremain unclear. Gene transfection is an important means to study the function of protein. Tostudy the function of CC10 protein on airway inflammatory diseases, we construct CC10plasmid which can be expressed in eukaryotic cell, it is helpful for further research.Objective: To construct and identify the expression vector of human Clara cell 10-kdprotein gene.Methods: The cDNA fragment that contains the full coding region of CC10 gene wasacquired from human inferior turbinate tissue, and then the cDNA was ligated topcDNA3.1/V5-His TOPO TA vector. CC 10 plasmid was transfected to bronchi epithelial cellline BEAS-2B by liposome mediated gene transfer method. The expression level of CC10protein was detected by the techniques of immunofluorescence and western blot.Results: After the identification of PCR and restriction enzymes analysis, therecombinant plasmid was sequenced and confirmed which contained the correct and entirenucleotide sequence of the CC10 DNA. The CC10 protein was expressed in BEAS-2B cellsafter being transfected with reconstructive plasmid.Conclusion: The human CC10 expression plasmid was successfully constructed andacquired stably protein expression in BEAS-2B cell line. ExperimentⅡThe Clara cell 10-kDa protein functions as an anti-inflammationfactor in airway epitheliumBackground: Clara cell 10-kd protein (CC10) is a multifunction protein withanti-inflammatory and immunomodulatory effects. Previous research had demonstrated thatCC10 plays an important role in airway inflammation. Human bronchial epithelial cell lineBEAS-2B is a widespread used model for study of aiway epithelial cell, and gene transfectionis an important means to study the function of protein. To elucidate the function of CC10 inairway inflammation, in this study, we transfected the bronchial epithelial cell line BEAS-2Bwith mock or CC10 plasmid, and compared the response of them after the treatment withIL-1β.Objective: To set up a model of alway epithelium inflammation by using the cytokineIL-1βstimulate the BEAS-2B cell line, and to elucidate whether the CC10 protein plays apotential role in this model.Methods: The bronchial epithelial cell line BEAS-2B was transfected with pcDNA3.1 orCC10 plasmid for 48 hours, and then the cells were treated with 10 ng/ml IL-1βfor 6 hours,the downstream gene IL-8 mRNA and protein levels were detected by the methods ofreal-time reverse transcription polymerase chain reaction and enzyme linked immunosorbentassay.Results: The expression of IL-8 in BEAS-2B cells was low, neither pcDNA3.1 norCC10 plasmid had any effect on IL-8 expression. Compared with controls, the IL-8 mRNAwas upregulated up to 150-fold in BEAS-2B cells trasfectd with mock and stimulated withIL-1βfor 6 hours, and the protein level was also upregulated up to 62-fold after beingstimulated with IL-1βfor 24 hours. However, the CC10 protein could inhibit the IL-1βinduced IL-8 expression in BEAS-2B, the IL-8 mRNA and protein expression weresignificantly downregulated at 5-fold and 2-fold, respectively (P＜0.05).Conclusion: The human CC10 protein can inhibit the IL-1βinduced IL-8 expression inBEAS-2B and may play an anti-inflammation role in aiway epithelium. PartⅡThe mechanism of Clara cell 10-kDa protein functions as ananti-inflammation factor in airway epitheliumBackground: Clara cell 10-kd protein (CC10) is a multifunction protein withanti-inflammatory and immunomodulatory effects. Previous research had demonstrated thatCC10 protein can inhibit the IL-1βinduced IL-8 expression in BEAS-2B and may play ananti-inflammation role in aiway epithelium, but the detailed mechanism of CC10 fuctions asan anti-inflammation factor remains unknown. Because NF-κB can be activated by IL-1βinaiway inflammation and IL-8 gene expression is also regulated by NF-κB. So, we assumeCC10 fuctions as an anti-inflammation factor in aiway epithelium is through inhibiting theactivation of NF-κB.Objective: To ensure wherther CC10 can suppress the activation of NF-κB in airwayepithelium inflammation and identify the detailed mechanism.Methods: We first detected the NF-κB activity in BEAS-2B cells after transfection withmock or CC10 plasmid and stimulated with IL-1βby the means of luciferase report genetransfection and western blot. Then we detected the important proteins p-IκB-αand p-IKKα/βin NF-κB signal pathway. Eventually we determined wherther CC10 can directly interactionwith p65 by the means of co-immunoprecipitation.Results: The activity of NF-κB was up-regulated up to 6.6-fold in BEAS-2B cells aftertreatment with 10 ng/ml IL-1β, and this ascensus can be significantly inhibited by CC10, thedecrease was to 64.7% (P＜0.05). The total p65 protein expression level was not changed inIL-1βstimulated BEAS-2B cells after transfection with mock or CC10, but the nuclear p65protein in BEAS-2B was immediataly increased after IL-1βstimulation compared with controls, howerer, when tranfected with CC10 plasmid, this increase was also attenuated.Similarly with nuclear p65 protein, the p-IκB-αprotein was also immediataly increased afterIL-1βstimulation compared with controls, and can be attenuated by CC10, but the p-IKKα/βprotein after IL-1βstimulation was not changed in BEAS-2B after being transfeced withmock and CC10 plasmid. Finally, the result of co-immunoprecipitation demonstrated thatthere was no direct interaction between CC 10 and p65.Conclusion: The CC10 protein can inhibit the activation of NF-κB and fuctions as ananti-inflammation factor in alway epithelium, this effect is via inhibiting the phosphorylationof IκB-α.