Effect And Mechanism Of Combination Of Erythromycin And Dexamethasone On Inflammation Of Monocytes Treated With Cigarette Smoke Extract

Objective To observe the effects of combination of erythromycin (EM)and dexamethasone (Dex) on inflammation of peripheral blood mononuclearcells (PBMCs) from chronic obstructive pulmonary diease (COPD) patients.Methods Ten cases of COPD patients with smoking and ten cases ofhealthy subjects with non-smoking were recruited. Peripheral blood of each casewas collected and mononuclear cells were obtained by ficoll hypaque densitygradient centrifugation. The cells were divided into three groups (control group:PBMCs from healty subjects, COPD group: PBMCs from COPD patients, EMgroup: PBMCs from COPD patients+EM, and combination treatment group:PBMCs from COPD patients+EM+Dex). The cells in EM group treated werepretreated with EM for2hours prior to treated with Dex (10-6-10-12M) for1 hour, and then stimulated with TNF-α overnight. The cells in control group andCOPD group were cultured with different concentrations of Dex for1hours andthen stimulated with TNF-α overnight. The cells in combination treatment grouptreated were pretreated with EM and Dex (10-6M) for2hours prior to treatedwith Dex (10-6-10-12M) for1hour, and then stimulated with TNF-α overnight.The levels of IL-8in supernatant were examined with enzyme-linkedimmunosorbent assay (ELISA). Draw semi-logatithmic curve according todifferent concentrations of dexamethasone and IL-8inhibition ratio. IC50-Dexwas calculated by Microsoft Excel2003. All data were analyzed by using SPSSsoftware (version16.0).Results (1) IL-8inhibition ratio of COPD group (10-6:51.10±3.02%，10-7:50.08±3.30%，10-8:50.20±2.55%，10-9:46.80±2.38%，10-10:25.50±3.00%) was significantly lower than control group（77.08±2.80%，76.51±3.52%，73.60±3.52%，68.30±3.02%，50.51±2.43%）. IC50-Dex of COPD group(54.9±2.2nmol/μL) was significantly higher than control group (0.20±0.08nmol/μL), P<0.05.(2) IL-8inhibition ratio of EM group (10-6：55.82±3.11%，10-7：52.23±3.31%，10-8：51.68±2.43%，10-9：48.92±2.29%，10-10：29.98±3.07%) and combination treatment group (70.88±4.29%，69.52±3.22%，66.18±2.60%，62.28±2.62%，42.53±2.46%) were significantly higher thanCOPD group, especially combination treatment group. P<0.05. IC50-Dex of EMgroup (5.67±1.15nmol/μL) and combination treatment group (1.03±0.06nmol/μL) were significant lower than COPD group, especially combinationtreatment group. P<0.05.Conclusion (1) IC50-Dex of COPD patients with smoking was higher,confirmed the presence of corticosteroid resistance of COPD.(2) IC50-Dex andIL-8release of EM and combination treatment group were lower than that of COPD group, which demonstrated that EM and combination treatment couldreverse corticosteriod resistance and increase anti-inflammatory effects,especially combination treatment of EM and Dex. Objective To observe the effects of and combination of erythromycin (EM)and dexamethasone (Dex) on inflammation of monocytes cigarette smokeextract (CSE).Methods U937cells were used to be research object and divided into fourgroups (control group, CSE group, EM group and combination treatment group).The cells in EM group treated with CSE were pretreated with EM (10μg/ml) for2hours. The cells were treated with dexamethasone (10-6-10-11M) for1hr andthen stimulated with TNF-α (10ng/ml) overnight. The cells in CSE group werenot pretreated with EM. The cells in control group were cultured with differentconcentrations of dexamethasone for1hours and then stimulated with TNF-αovernight. The cells in combination treatment group treated with CSE werepretreated with EM (10μg/ml) and dexamethasone (10-6M) for2hours. Thecells were treated with dexamethasone (10-6-10-11M) for1hr and thenstimulated with TNF-α (10ng/ml) overnight. The levels of IL-8in supernatantwere examined with enzyme-linked immunosorbent assay (ELISA). Drawsemi-logatithmic curve according to different concentrations of dexamethasoneand IL-8inhibition ratio. IC50-Dex was calculated by Microsoft Excel2003. Alldata were analyzed by using SPSS software (version16.0).Results (1) IL-8inhibition ratio of CSE group (10-6:44.79±3.32%，10-7:42.44±3.50%，10-8:39.25±2.60%，10-9:35.20±2.40%，10-10:14.92±3.18%)was significantly lower than control group (61.05±3.15%，60.78±3.70%，59.12±2.37%，55.20±2.52%，37.71±3.43%). IC50-Dex of CSE group (762.90±66.85nmol/μL) was significantly higher than control group (5.72±1.10nmol/μL), P<0.05.(2) IL-8inhibition ratio of EM group (10-6:55.59±3.26%，10-7:53.66±3.11%，10-8:50.85±2.55%，10-9:45.49±2.82%，10-10:29.54±2.66%) and combination treatment group (49.93±3.36%，48.26±3.51%，45.28±2.63%，40.03±2.39%，22.05±3.16%，9.08±2.75%，5.82±2.53%) were significantly higher than CSE group. IC50-Dex of EM group(112.57±17.88nmol/μL) and combination treatment group (24.36±7.28nmol/μL) were significantly lower than CSE group, especially combinationtreatment group. P<0.05.Conclusion (1) IC50-Dex of CSE group was higher, which confirmed thepresence of corticosteroid resistance of COPD.(2) IC50-Dex and IL-8release ofEM and combination treatment group were lower than that of CSE group, whichdemonstrated that EM and combination treatment could reverse corticosteriodresistance and increase anti-inflammatory effects, especially combinationtreatment of EM and Dex. Objective To study whether combination of erythromycin (EM) anddexamethasone (Dex) reverse corticosteroid resistance through inhibitingPI3K-δ/AKT pathway and increasing HDAC2protein expression of monocytestreated with cigarette smoke extract (CSE).Methods U937cells were used to be research object.(1) To observewhether PI3K-δ inhibition could reverse corticosteriod resistance, the cells weredivided into three groups (control group, CSE group inhibitor group). The cellsin inhibition group treated with CSE were pretreated with IC87114(PI3K-δinhibition,1μM) and dexamethasone (10-6M) for2hours. The cells weretreated with dexamethasone (10-6-10-11M) for1hr and then stimulated withTNF-α (10ng/ml) overnight. The cells in CSE group were not pretreated withIC87114. The cells in control group were cultured with different concentrationsof dexamethasone for1hours and then stimulated with TNF-α overnight.(2) Toobserve the mechanism of EM and combination treatment on anti-inflammatoryeffects of CSE-induced monocytes, the cells were divided into six groups(control group, CSE group, dexamethasone group, EM group, combinationgroup and inhibition group). The cells in EM group treated with CSE werepretreated with EM (10μg/ml) for2hours and then stimulated with TNF-α (10ng/ml) overnight. The cells in CSE group were not pretreated with EM. Cells ininhibition group treated with CSE were pretreated with IC87114for2hours andthen stimulated with TNF-α overnight. The cells in dexamethasone group treatedwith CSE were pretreated with dexamethasone (10-6M) for2hours and then stimulated with TNF-α overnight. Cells in combination treatment group werepretreated with EM (10μg/ml) and dexamethasone (10-6M) for2hours and thenstimulated with TNF-α overnight. The cells in control group were stimulatedwith TNF-α overnight. The levels of IL-8in supernatant were examined withenzyme-linked immunosorbent assay (ELISA). IC50-Dex was calculated byMicrosoft Excel2003. HDAC2, PAkt and Akt protein expressions were detectedby western blotting. Quantificational real-time PCR (qRT-PCR) was used tomeasure HDAC2RNA expression. HDAC activity was measured on nuclear cellprotein by Fluo-Lys HDAC activity assay kit. All data were analyzed by usingSPSS software (version16.0).Results (1) IL-8inhibition ratio of CSE group (10-6:44.79±3.32%，10-7:42.44±3.50%，10-8:39.25±2.60%，10-9:35.20±2.40%，10-10:14.92±3.18%)was significantly lower than control group (61.05±3.15%，60.78±3.70%，59.12±2.37%，55.20±2.52%，37.71±3.43%) and inhibitor group (56.70±2.82%，54.45±2.67%，52.96±2.33%，46.82±3.02%，30.14±2.86%). P<0.05.(2) IC50-Dex of CSE group (762.9±66.8nmol/μL) was significantly higherthan control group (5.7±1.1nmol/μL) and inhibition group (14.1±3.8nmol/μL). P<0.05.(3) HDAC2protein expression in CSE group wassignificantly lower than control group, EM group and combination treatmentgroup, P<0.05. HDAC2protein in combination treatment group wassignificantly higher than EM group, P<0.05. As positive control, IC87114alsoreversed the reduction of HDAC2protein induced by CSE, P<0.05. There wasno significant difference between CSE group and Dex group，P>0.05.(4)HDAC2mRNA expression in CSE group was significantly lower than controlgroup, EM group and combination treatment group, P<0.05. HDAC2mRNA incombination treatment group was significantly higher than EM group, P<0.05. As positive control, IC87114also reversed the reduction of HDAC2mRNAinduced by CSE, P<0.05. There was no significant difference between CSEgroup and Dex group，P>0.05.(5) HDAC activity in CSE group group weresignificantly lower than control group, EM group and combination treatmentP<0.05. As positive control, IC87114also reversed the reduction of HDACactivity induced by CSE, P<0.05. There was no significant difference betweenCSE group and Dex group.(6) PAkt protein expression in CSE group wassignificantly lower than control group, EM group and combination treatmentgroup, P<0.05. As positive control, IC87114also reversed the increment of PAktexpression induced by CSE, P<0.05. There was no significant difference of PAktbetween CSE group and Dex group. P>0.05.(7) There were no significantdifference of Akt protein expression among the groups. P>0.05.Conclusion (1) IC50-Dex of inhibition group was higher, which confirmedthe presence of corticosteroid resistance of CSE-induced monocytes.(2)Combination treatment could increase HDAC2protein expression and reducePAkt expression, which was especially combination treatment. Therefore, EMand combination of EM and Dex reverse corticosteroid resistance throughinhibiting PI3K-δ/AKT pathway and increasing HDAC2protein expression ofcigarette smoke extract (CSE)-induced monocytes, especially combinationtreatment.