| PART ?Objective:To investigate the effect of erythromycin on glucocorticoid resistance of peripheral blood mononuclear cells?PBMCs?in patients with COPD.Methods:Eight healthy nonsmoking control subjects,eight healthy smokers were recruited in this study.and eight patients with stable COPD were enrolled at The First Affiliated Hospital of Guangxi Medical University.Peripheral venous blood were collected from study subject,and PBMCs were obtained under sterile condition by ficoll hypaque density gradient centrifugation.The cells were divide four group??1?the control group,PBMCs from healthy nonsmoking?2?smoking group:PBMCs from healthy smokers?3?COPD group:PBMCs from patients with stable COPD?4?COPD+EM group:PBMCs from patients with stable COPD and treatment with erythromycin?.The cells in COPD+EM group were prtreated with EM for 2 hours prior to treat with Dex(10-12-10-6M)for 2 hours,and then stimulate with TNF-??5ng/ml?for 16 hours.The level of IL-8 in supernatant were measured by ELISA.The concentration inhibition response curve was obtained,and the half inhibitory concentration?IC50-Dex?of dexamethasone in each group was calculated.The higher the IC50-Dex,the worse the sensitivity of glucocorticoid and the higher the glucocorticoid resistance.Results:1.There is no significant difference in the IC50-Dex of PBMCs between healthy people and healthy smokers.2.However,the inhibition rate of IL-8 in PBMCs from patients with COPD was significantly lower than healthy nonsmoking and healthy smokers,the IC50-Dex was significantly increased?P<0.05?.With erythromycin intervention,the inhibition rate of IL-8 was increased and IC50-Dex decreased significantly,but it was still lower than that of healthy people.Conclusion:1.Compare with healthy nonsmoking volunteer and healthy smokers,there was a significant glucocorticoid resistance in the PBMCs from patients with COPD.2.Erythromycin could reverse corticosteroid insensitivity in the PBMCs from patient with COPD.PART ?Objective:1.To explore glucocorticoid resistance in mononuclear cells exposure to cigarette smoke extract?CSE?and the effect of erythromycin intervention.2.To investigated effects of erythromycin on AP-1 expression in mononuclear cells exposure to CSE.Methods:In this part,the U937 cells were divided into three groups:?1?the control group?2?CSE group?3?CSE+ EM group.1.The cells in CSE+EM group were prtreated with EM for 2 hours while the other two groups were not pretreated,then CSE group and CSE+ EM group treat with 0.25% CSE 2 hours while control group not given.Next three group treat with Dex?10-12-10-6M?for 2 hours,and then stimulate with TNF-??5ng/ml?for 16 hours.The levels of IL-8 in supernatant were measured by ELISA.IL-8 inhibition rate and IC50–Dex were calculated by Microsoft Excel2003.2.CSE+EM group were pretreated with EM for 2 hours while the other two groups were not used,then CSE group and CSE+ EM group treat with 0.25%CSE 8 hours while control not given.Then cells were collected and protein and RNA were extracted.The AP-1?C-FOS and C-JUN?m RNA and protein were measured by PCR and WB,respectively.Results:1.Compare with control group,the IL-8 inhibition rate significantly decreased and the IC50-Dex significantly increased in the CSE group?P < 0.05?.The IC50-Dex of CSE +EM group were significantly lower than that of the CSE groups.2.The expression of C-FOS m RNA and C-JUN m RNA was significantly increased after exposure to CSE,which inhibited by erythromycin.3.The levels of C-JUN protein increased significantly after exposed to CSE.While C-FOS protein was no changed.Meanwhile,the expression of C-FOS had no changed by pretreat with erythromycin,while the expression of C-JUN protein were significantly decreased?P < 0.05?.Conclusion:1.The glucocorticoid sensitivity of human mononuclear cells were decreased exposure to CSE,Erythromycin reversing corticosteroid insensitivity of human mononuclear cells under tobacco smoke exposure,enhanced glucocorticoid anti-inflammatory effects.2.The expression of C-FOS m RNA in mononuclear cells was increased after CSE stimulation,There was no change in protein level,and erythromycin had no effect on the expression of C-FOS protein.The expression of C-JUN protein and m RNA in mononuclear cells was increased after CSE stimulation,and Erythromycin can inhibit the expression of C-JUN protein and m RNA.PART ?Objective:1.To investigate the role of C-JUN in the glucocorticoid resistance of mononuclear cells exposure to CSE.2.To investigate the pathway of erythromycin in reversing corticosteroid insensitivity in mononuclear cells exposure to CSE.Methods:1.In order to observe the role of C-JUN in glucocorticoid resistance of mononuclear cells,C-JUN SiRNA was transfected into the mononuclear cells to interference C-JUN m RNA expression.And the cells were divided into four groups:?1?Control group?2?CSE group,?3?C-JUN SiRNA group.?4?CSE +C-JUN SiRNA group.2.To observe the effect of erythromycin on the upstream pathway MAPK of C-JUN.The U937 cells were divided into six groups:?1?Control group,?2?CSE group,?3?CSE +EM group?4?CSE +SCH772984 group,?5?CSE +SP600125group,?6?CSE + SB203580 group.The levels of IL-8 in supernatant were measured by ELISA.IL-8 inhibition rate and IC50-Dex were calculated by Microsoft Excel 2003.The gene expression levels of C-JUN?P38MAPK?ERK and JNK were detected by real-time fluorescence quantitative polymerase chain reaction?RT-PCR?.The protein expression levels of C-JUN?P38MAPK?ERK and JNK were analyzed by western blotting.Results:1.Knock-down of C-JUN in mononuclear cells exposed to CSE failed to develop corticosteroid insensitivity,IC50-Dex was significantly reduced,and the difference was statistically significant?P < 0.05?.2.Corticosteroid insensitivity and increased levels of C-JUN protein and m RNA after exposed to CSE were reversed by ERK,JNK,P38 MAPK inhibitor?SCH772984,SP600125,SB203580?and low concentrations of erythromycin.3.Erythromycin has no effect on the activity of P38 MAPK,ERK,however,erythromycin suppresses activity of P-JNK in mononuclear cells exposure to CSE.Conclusion : 1.C-JUN knockdown reversing corticosteroid insensitivity in mononuclear cells exposure to CSE,It is conclude that C-JUN plays an important role in glucocorticoid resistance.2.Inhibition activity of JNK,P38 MAPK and ERK could decreased levels of C-JUN in mononuclear cells and reduce glucocorticoid resistance exposed to CSE.3.Erythromycin decreased the expression of C-JUN by inhibiting the activity of JNK,thus reversing corticosteroid resistance of human mononuclear cellss exposure to CSE. |
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