The Effects Of Erythromycin On PPARγ Of Macrophges Induced By Cigarette Smoke

OBJECTIVE:1.To investigate the effects of erythromycin on inflammation of macrophages by inhibiting the oxidative stress.2.To investigate the effects of erythromycin on the expression of PPARγ in macrophages induced by cigarette smoke.METHODS: Human monocyte U937 cells were used as the observing object of the study,and they were induced to human macrophages incubated 24 h with 200ng/ml PMA.CCK8 was used to detect the appropriate concentration of cigarette smoke extract and erythromycin on cells proliferation and toxicity.Groups: control group、 the CSE group(incubation with 1% CSE for 24 h)、CSE + Erythromycin group(incubation with 1 ug/ml erythromycin for 24 h before expose to 1% CSE stimulation for 24h),GW9662 group(10 umol/ml GW9662 incubation 24 h).The ROS release level was detected by Fluorescence microplate reader;The expression of inflammatory factors IL-6 and IL-8 were detected by enzyme-linked immunosorbent assay;Western blot methods was used to detect the expression of PPARγ and NF-κB and levels in the total protein of each group;The relative expression of PPARγ and NF-κB p65 mRNA were detected by real-time fluorescence quantitative PCR.The interrelationship between PPARγ and NF-κB p65 was detected by immunocoprecipitation(Co-IP).RESULTS: 1.Compared with the control group,the final concentration of CSE was less than 1% and the final concentration of EM was less than 10ug/ml,which had no significant effect on cell proliferation,and the cell proliferation was inhibited significantly when the final concentration were 2% and 3% of CSE or 100ug/ml EM(P < 0.05).2.1%CSE could increase the release of ROS,IL-6 and IL-8 of human macrophages(p <0.05).1ug/ml erythromycin preincubated for 24 h was able to inhibit the up-regulation of ROS、IL-6 and IL-8 in human macrophages induced by 1%CSE(p <0.05).3.1% CSE could obviously inhibit the expression of PPAR γ protein and mRNA in human macrophages(p < 0.05)and promoted the expression of NF-κB p65 protein and mRNA in human macrophages(p < 0.05),and preincubation of 1ug/ml erythromycin for 24 h restored the expression of PPAR γ protein and PPARγ mRNA expression(p < 0.05),and inhibited the NF-κB p65 protein and NF-κB p65 mRNA expression(p < 0.05).Furthermore,we could not investigate that there was time-dependent effect on the expression of PPARγ when erythromycin was used.4.Compared with the control group,PPAR γ inhibitor GW9662 significantly inhibited PPARγ and mRNA expression(p <0.05)and promoted the expression of NF-κB p65 protein and the release of IL-6、IL-8 and ROS(p <0.05).5.After immunoprecipitation and Western blot joint detection,PPARγ and NF-κB p65 were correlated with each other;Compared with the control group,1%CSE could inhibit the up-regulation of PPARγ protein expression and promote the expression of NF-κB p65(P<0.05);Compared with CSE group,erythromycin in CSE+EM group was able to up-regulate the expression of PPAR γand inhibit the expression of NF-κB p65(P<0.05);Compared with the control group,The expression of PPARγ in the GW9662 group was inhibited while the expression of NF-κB p65 was up-regulated(P<0.05).CONCLUTIONS: 1.Oxidative stress exposed by cigarette smoke may inhibite the expression of PPARγ,which in turn leads to increased expression of NF-κB p65,thereby promoting the release of inflammatory cytokines IL-6 and IL-8 and causing inflammation.2.Erythromycin may increase the expression of PPARγ by inhibiting the ROS release,which in turn leads to inhibite the expression of NF-κB p65 and inhibiting the release of inflammatory cytokines IL-6 and IL-8 and shows an anti inflammatory effect.