The Effects Of Erythromycin On Cigarette Smoke Extract-Induced SIRT1

PARTI THE EFFECTS OF ERYTHROMYCIN ON REACTIVE OXYGEN AND INFLAMMATION CYTOKINE INDUCED BY CIGARETTE SMOKE EXTRACT IN HUMAN MACROPHAGESOBJECTIVE:1. To study the effects of erythromycin on reactive oxygen species (ROS) induced by cigarette smoke extract (CSE) in human macrophages, and whether erythromycin have influenced ROS level induced by CSE; 2. To study the effects of erythromycin on CSE-induced interleukin 6 (IL-6), interleukin 8(IL-8) and tumor necrosis factor a(TNF-a), and whether erythromycin(EM) have effects on these inflammation cytokines release of human macrophages exposed to CSE.METHODS:The U937 monocytic cells were transformed to macrophages induced by phorbol 12-myristate 13-acetate (PMA) in accordance with standard procedures. Human macrophages growth curve was analyzed by MTT method, human macrophages were divided into three groups:(1) normal control group, (2) CSE group:(CSE1%:incubation with CSE1% for 24h), (3)CSE+ EM:(Pre-incubation EM for 24,48 h before adding CSE1%24h). Flow cytometry was used to measure the changes of intracellular ROS using a ROS probe (2’-7’dichloro fluorescein diacetate,DCFH-DA).IL-6, IL-8 and TNF-a Levels in supernatant were measured by Enzyme-linked immunosorbent assay (ELISA).RESULTS:The levels of ROS in CSE group were up-regulated compared to normal control group, (P<0.01); The levels of ROS released by human macrophages was down-regulated in group of pre-incubation EM for 24h before adding CSE1%24h compared with CSE group (p<0.01); Pre-incubation EM for 24h before adding CSE further depress the levels of ROS compared to CSE group (p<0.01). CSE 1% treatment significantly increased IL-6, IL-8 and TNF-arelease (P<0.01), from human macrophages cells at 24h of treatment. Compared to treatment with CSE 1%, pretreated macrophages with EM for 24 h decrease the production of IL-6 and TNF-asignificantly (P< 0.01), the production of IL-8 was decreased, but no significantly (P>0.01). Pretreated macrophages with EM for 48 h decreased the production of IL-6, IL-8 and TNF-asignificantly compared to treatment with CSE 1%(P<0.01)CONCLUSIONS:1. CSE can lead to increace in the levels of ROS and release of IL-6, IL-8 and TNF-a in hunman macrophages.2. EM can inhibit the levels of ROS and the release of IL-6, IL-8 and TNF-ain hunman macrophages which induce by CSE.PARTII THE EFFECTS OF ERYTHROMYCIN ON NF-κB AND SIRT1 PROTEIN INDUCED BY CIGARETTE SMOKE EXTRACTOBJECTIVE:1. The object of this experimentation was to research the effects of CSE-induced Sirtuins1 (SIRT1) and nuclear factor-κB (NF-κB) proteins in human macrophages. And the effects of erythromycin on CSE-induced SIRT1 and NF-κB proteins in human macrophages.2. To research the effect of erythromycin on CSE-induced interaction of SIRT1 and NF-κB proteins in human macrophages.METHODS:The U937 monocytic cells were transformed to macrophages induced by phorbol 12-myristate 13-acetate (PMA) in accordance with standard procedures. Cells were divided into six groups:(1) normal control group, (2) CSE group, (3) CSE+EM(24h):(Pre-incubation with EM for 24h before adding CSE1%24h),(4)CSE+EM(48h):(Pre-incubation with EM for 48 h before adding CSE1%48h), (5)nicotinamide (NAM):(NAM:incubation with 20mM NAM for 24h), (6)1-Pyrrolidinecarbodithioic acid ammonium salt(PDTC):(PDTC:Pre-incubation with 20nM PDTC for 24h before adding CSE1%24h). SIRT1 and NF-κB proteins were determined by Western blot.Human macrophages were divided into four groups:(1) normal control group, (2) CSE group:(CSE1%:incubation with CSE for 24h), (3) CSE+ EM(24h):(Pre-incubation with EM for 24h before adding CSE1%24h),(4)CSE+ EM(48h):(Pre-incubation with EM for 48 h before adding CSE1%48h). The effect of erythromycin on CSE-induced interaction of SIRT1 and NF-κB proteins were determined by co-immunoprecipitation(Co-IP).RESULTS:There were significantly down regulated of SIRT1 protein in group of adding CSE1% 24h compared with control group(P<0.01), and CSE1% increased the expression of NF-κB protein compared to control group(P<0.01).Pre-incubation with EM for 24h increased in the expression of SIRT1 protein compared to the group of adding CSE1% 24h (P<0.01), and pre-incubation with EM decreased the expression of NF-κB protein compared to CSE1% group(P<0.01).Pre-incubation with EM for 48h further increased in the expression of SIRT1 protein compared to the group of adding CSE1% 24h, and further decreased the expression of NF-κB protein compared to CSE1% group.SIRT1 and NF-κB protein interaction in human macrophages directly determined by Co-IP. CSE stimulation decreased the expression of SIRT1 proteins in human macrophages, therefore increased the expression of NF-κB protein. Pre-incubation with EM for 24h increased the expression of SIRT1 protein in human macrophages, therefore decreased the expression of NF-κB protein. Extend the pre-incubation with EM from 24h to 48h, increased the expression of SIRT1 protein and decreased the expression of NF-κB protein even more.CONCLUSIONS:1. CSE decreased SIRT1 proteins in human macrophages, increased NF-κB proteins, accelerate inflammation cytokine release.2. EM increased SIRT1 proteins in human macrophages,decreased NF-κB proteins,inhibit inflammation cytokine release.PARTIII THE EFFECT OF ERYTHROMYCIN ON SIRT1, NF-K B PROTEINS IN LUNG CIGARETTE SMOKE EXPOSED MICE AND LUNG TISSUE OF MICE.OBJECTIVE:1. To research the effects of erythromycin on lung tissue of mice which cigarette exposed.2.To research the effects of erythromycin on CSE-induced SIRT1 and NF-κB proteins in lung of mice.METHODS:8-week-old male Balb/C mouse were randomized into three groups::normal control group; CSE group (exposure to cigarette smoke 24h/day, last for 12 weeks); EM group (treatment with EM 100mg/kg-day before exposure to cigarette smoke, the others treatment like CSE group). After the expiration, the lung of mouse was observed by optical microscopy and HE staining techniques, and used Western blot determine SIRT1 and NF-κB proteins of mice’s lung.RESULTS:Obvious inflammation and emphysema was observed in lung of CSE group, and treatment with EM make less inflammation and emphysema in lung than CSE group.Cigarette smoke exposure significantly decreased the expression of SIRT1 protein in lung of mice, and increased the expression of NF-κB protein at the same time. Treatment with EM increased the expression of SIRT1 protein and decreased the expression of NF-κB protein induce by cigarette smoke exposure.CONCLUSIONS:1. EM relief inflammatory in lung of mice maybe connect with increased of SIRT1 protein,decreased the expression of NF-κB protein.2.EM relief emphysema maybe connect with increased of SIRT1 protein in lung of mice.