The Mechanism And Intervention Syudies Of Glucocorticoids Inhibiting Airway Epithelial Cells Repair

PART ONETHE EXPESSION OF GLUCOCORTICOID-INDUCEDLEUCINE ZIPPER(GILZ) IN HUMAN AIRWAY EPITHELIALCELLS9HTEObjective: To explore the expression of glucocorticoid-induced leucinezipper in human airway epithelial cells9HTE treated with glucocorticoiddexamethasone.Methods: RT-PCR and western Blot detected the expressions of GILZmRNA and protein treated with DEX for6h,12h and24h.Immunofluorescence assay located GILZ protein.Results: The expressions of GILZ mRNA and protein were low in9HTEcells under normal circumstances, but were significantly changed. Itshowed that the expressions of GILZ mRNA and protein increasedobviously after DEX treated for6h, and continued to maintain a high level until24h. We also observed GILZ protein was located in the cytoplasm of9HTE cells.Conclusion: DEX quickly and significantly induced the expressions ofGILZ mRNA and protein in airway epithelial cells9HTE. PART TWOTHE SCREENING AND IDENTIFICATION OF GILZSILENCING BY SMALL INTERFERING RNA(si-RNA)Objective: To screen the best si-RNA for silencing GILZ from three GILZsiRNAs that were designed and synthesized by si-RNA technology, and usefor subsequent experiments.Methods: We designed and synthesized three GILZ siRNAs: GILZ1si-RNA, GILZ2si-RNA, GILZ3si-RNA. Then they were transfected intohuman airway epithelial cells9HTE via lipofectamine2000.9HTE cellswere collected after were silenced for48h, and we screened and identifiedthe GILZ si-RNA that had the best silencing effect by Realtime-PCR,Western Blot and immunofluorescence assays.Results: We screened that GILZ3si-RNA was the best GILZ si-RNA after GILZ1si-RNA, GILZ2si-RNA, GILZ3si-RNA were transfected into9HTEcells. The average silencing efficiency of GILZ gene was up to55.8%byRealtime-PCR, and the silencing efficiency of GILZ protein was significantby Western Blot and immunofluorescence.Conclusion: We successfully synthesized and identified to obtain a GILZsi-RNA for best silencing effect by si-RNA technology, and it was the keyof subsequent experiments. PART THREETHE STUDY OF GLUCOCORTICOIDS INHIBITING humanAIRWAY EPITHELIAL CELLS REPAIR WAS MEDIARED BYGILZObjective: To explore the influence of GCs mediated by GILZ onMAPK-ERK signaling pathway, proliferation and migration, and clarify theinhibitory effect on airway epithelial cells repair.Methods: We collected cells after non-specific si-RNA and GILZ si-RNAwere transfected into human airway epithelial cells9HTE. Then wedetected the expressions of phosphorylated and total proteins of Raf-1, Mek1/2, Erk1/2(components of the MAPK-ERK signaling pathway) in9HTE cells. MTT and CFSE assays were used to detect the cellproliferation. The cell migration was tested by wound-healing and transwellassays.Results: DEX inhibited phosphorylated protein expressions of Raf-1,Mek1/2, Erk1/2of the MAPK-ERK signaling pathway in human airwayepithelial cells9HTE, and had no significant effect on total proteinexpression. It showed that DEX inhibited the activation of MAPK-ERKsignaling pathway. We also observed that DEX inhibited the cellproliferation and migration. After GILZ si-RNA was transfected into9HTEcells to silence the expression of GILZ, the inhibitory effect onMAPK-ERK signaling pathway, proliferation and migration weresignificantly reduced.Conclusion: DEX could inhibit the activation of MAPK-ERK signalingpathway, proliferation and migration, thereby suppressing the repair ofairway epithelial cells. The inhibitory effect of DEX was mainly mediatedby GILZ. PART FOURTHE INTERVENTION STUDY OF VITAMIN A ONGLUCOCORTICOIDS INHIBITING AIRWAY EPITHELIALCELLS REPAIRObjective: To explore the effect of VitA on GCs inhibiting human airwayepithelial cells repair.Methods: ELISA assay was used to detect the EGF expression of culturesupernatant in human airway epithelialcells9HTE that were treated byDEX and ATRA for24h, and immunofluorescence and Western Blot assaystested the EGFR expressions of phosphorylated and total proteins. Then wedetected the expressions of phosphorylated and total proteins of Raf-1,Mek1/2, Erk1/2(components of the MAPK-ERK signaling pathway) in9HTE cells. MTT assay was used to detect the cell proliferation. The cellmigration was tested by wound-healing and transwell assays.Results: ATRA had no significant effect on the secretion of EGF in9HTE,but promoted the EGFR expressions of phosphorylated and total proteins.ATRA also increased the phosphorylation of Raf-1, Mek1/2, Erk1/2toreduce the inhibitory effect of DEX on MAPK-ERK signaling pathway. Inthe early stage ATRA did not influence the cell proliferation, butsignificantly increased the cell migration.Conclusion: ATRA could induce the phosphorylated protein expression ofEGFR to activate EGFR signaling pathway, then activated the downstream MAPK-ERK signaling pathway, and promoted the cell migration, therebyreducing the side-effect of DEX on inhibiting airway epithelial cells repair.