Effects Of Erythromycin On Oxidative Stress Pathway Of Human Macrophages Stimulated By Cigarette Smoke

Part Ⅰ Antioxidative effect of erythromycin on human macrophage SIRT1 stimulated by cigarette smokeObjective: To observe the regulation of erythromycin on oxidative stress involving human macrophages SIRT1 stimulated by cigarette smoke,and to explore the mechanism of antioxidative stress of erythromycin.Methods: Human histiocytic lymphoma cells(U937)were selected for culture preincubated with PMA at a final concentration of 200ng/ml for 24 h to induce macrophages,and the induced macrophages were taken as experimental objects.Group macrophages: control group,CSE group,CSE+ erythromycin(EM)group,CSE+NAM group and CSE+NAM+EM group.According to the experimental results,1%CSE,10 g/ml EM and 0.02 mol/LNAM were used for the intervention.DCFH-DA method was used to detect the release of ROS in each group of cells.Western blot(WB)was used to detect SIRT1 protein expression in each group.Fluorescence quantitative polymerase chain reaction(RT-PCR)was used to detect SIRT1 m RNA expression.Enzyme-linked immunosorbent assay(ELISA)was used to detect the expression of oxidative stress factors superoxide dismutase(SOD)and malondialdehyde(MDA)in the cell supernatant of each group.Results: 1.The expression of ROS in each group: compared with the control group,the release of ROS was increased after CSE stimulation(P <0.05);EM pre-incubation for 24 h can inhibit the release of ROS from macrophages(P < 0.05).Compared with the control group,the release of ROS in the CSE+NAM group was increased(P<0.05),and the release of ROS after the pre-incubation of EM was reduced(P<0.05).2.SIRT1 protein expression:Compared with the control group,SIRT1 protein expression in CSE group and CSE+NAM group was down-regulated(P<0.05),erythromycin pre-incubation for 24 h can up-regulate the expression of SIRT1(P<0.05).3.SIRT1 m RNA expression: Compared with the control group,SIRT1 m RNA expression levels in the CSE and CSE+NAM groups were decreased(P<0.05).erythromycin pre-incubation for 24 h can up-regulate the expression of SIRT1 m RNA(P<0.05).4.SOD expression: compared with the control group,the expression of SOD was down-regulated under CSE stimulation.Compared with CSE group,The expression of SOD in CSE+NAM group cells decreased significantly(P<0.05),the expression of SOD could increase after erythromycin preincubated for 24h(P<0.05).5.MDA expression: Compared with the control group,the expression of MDA was increased stimulated by CSE.Compared with CSE group,the expression of MDA in the CSE+NAM group increased significantly(P<0.05),the expression level of MDA decreased after erythromycin preincubated for 24h(P < 0.05).Conclusion: 1.Cigarette smoke stimulation caused a decrease in the expression of SIRT1 in human macrophages,which leads to the increase of ROS release,which in turn caused a decrease in SOD expression and an increased level of MDA expression,resulting in an increase in oxidative stress response in cells.2.The mechanism of erythromycin anti-oxidative stress may be through up-regulating the expression of SIRT1,thereby inhibiting the release of ROS,and then causing the up-regulation of SOD expression and inhibiting the expression of MDA,inhibiting the oxidative stress response of cells.Part Ⅱ Antioxidative effect of erythromycin on human macrophage PPARγstimulated by cigarette smokeObjective: To observe the regulation of erythromycin on oxidative stress involving human macrophages PPARγ stimulated by cigarette smoke,and to explore the mechanism of antioxidative stress of erythromycin.Methods: Human monocyte cell line U937 cells were selected and induced into macrophages by PMA.Experimental groups: control group,CSE group,CSE+EM group,CSE+GW9662 group,CSE+GW9662+EM group.According to the experimental results,1%CSE,10 g/ml EM,and 10 mol/ml GW9662 were used for the intervention.DCFH-DA method was used to detect the release level of ROS.WB was used to detect the expression of PPAR protein,and RT-PCR was used to detect the expression of PPAR m RNA.The expression of SOD and MDA in supernatant was detected by ELISA.Results: 1.Compared with the control group,the release level of ROS in the CSE group was increased(P<0.05),and the expression of ROS decreased after erythromycin pre-incubation for 24 h.Compared with the control group,the release of ROS in the CSE+GW9662 group was increase(P<0.05),erythromycin pre-incubation for 24 h can reduce the expression of ROS(P<0.05).2.PPARγ protein expression: Compared with the control group,the expression of PPARγ protein in the CSE group and CSE+GW9662 group was decreased(P<0.05),and the expression level of PPARγ protein increased after erythromycin pre-incubation(P<0.05).3.PPARγ m RNA expression: Compared with the control group,the expression of PPARγ m RNA in CSE group and CSE+GW9662 group was down-regulated(P<0.05),and the expression level of PPARγ m RNA was up-regulated after pre-incubation with erythromycin(P<0.05).4.SOD expression: Compared with the control group,the expression of SOD in the CSE,CSE+GW9662 group was decreased(P<0.05),compared with the CSE group,the expression of SOD in the CSE+GW9662 group decreased significantly(P<0.05),erythromycin can upregulate the expression of SOD(P<0.05).5.Expression of MDA: Compared with the control group,the expression of MDA in the CSE,CSE+GW9662 group was increased(P<0.05),compared with the CSE group,the expression of MDA in the CSE+GW9662group increased significantly(P<0.05),MDA expression decreased after erythromycin pre-incubation(P<0.05).Conclusion: 1.Cigarette smoke stimulation caused a decrease in the expression of PPARγ in human macrophages,which leads to the increase of ROS expression,and then leads to the decrease of SOD expression and the increase of MDA expression,and finally induces the oxidative stress response.2.Erythromycin can upregulate the expression of PPARγ,thereby inhibiting the expression of ROS,further promoting the expression of SOD,and reducing the expression of MDA,thereby inhibiting the oxidative stress caused by cigarette smoke.Part Ⅲ The relationship between SIRT1 and PPARγ in human macrophages stimulated by cigarette smoke and the role of erythromycinObjective: To investigate the effect of PPARγ inhibitor GW9662 on the expression of SIRT1 in human macrophages stimulated by cigarette smoke,to explore the relationship between SIRT1 and PPARγ,and the possible mechanism of erythromycin’s anti-oxidative stress.Methods: Select macrophages induced by PMA as the test object.Experimental grouping: control group,CSE group,CSE+EM group,CSE+GW9662 group,CSE+GW9662+EM group,the intervention conditions are the same as the second part.WB detected the expression of SIRT1 protein.RT-PCR was used to detect the expression of SIRT1 m RNA.Spearman correlation analysis was used to analyze the relationship between SIRT1 and PPARγ.Results: 1.Expression of SIRT1 protein: Compared with the control group,the expression of SIRT1 protein in human macrophages was reduced under CSE stimulation,and the expression of SIRT1 protein was upregulated after erythromycin pre-incubation for 24h(P<0.05).Compared with the control group,the expression of SIRT1 protein in the CSE+GW9662 group was decreased(P<0.05).the expression of SIRT1 protein was upregulated after erythromycin pre-incubation for 24h(P<0.05).2.The expression of SIRT1 m RNA: Compared with the control group,the expression of SIRT1 m RNA in human macrophages was reduced under CSE stimulation.the expression of SIRT1 m RNA was upregulated after erythromycin pre-incubation for 24h(P<0.05).Compared with the control group,the expression of SIRT1 m RNA in the CSE+GW9662 group was decreased(P<0.05),the expression of SIRT1 m RNA was upregulated after erythromycin pre-incubation for 24h(P<0.05).3.Correlation analysis: PPARγwas positively correlated with SIRT1 protein and m RNA(r=0.5920,p=0.006;r=0.8689,p<0.0001).Conclusion: 1.GW9662 inhibitor inhibits the expression of PPARγ,SIRT1 expression increases,SIRT1 may be a downstream factor of PPARγ.2.Oxidative stress induced by cigarette smoke may be achieved through the PPARγ-SIRT1 signaling pathway.3.Erythromycin inhibition of oxidative stress induced by tobacco smoke may be achieved by regulating the PPARγ-SIRT1 pathway.