Research On Oxidative Stress Works In LPS-induced Autophagy In Ana-1Pulmonary Macrophage

Abstract: Objective: Nowadays there are few researches on theinteraction and mechanism about oxidative stress works in LPS-inducedautophagy.This experimental observing the impact on the injury of mice’spulmonary macrophage （Ana-1） induced by LPS-stimulated, and studythe oxidative stress works in LPS-induced mechanism and autophagymechanism of Ana-1and related molecular mechanism through the DNAinjury and autophagy of Ana-1. Methods:1. Establish LPS-inducedAna-1model and oxidative stress injury model of LPS-induced inAna-1. With Ana-1induced by LPS, Comet assay kit could be used todetect the expression of LPS-induced Ana-1’s DNA injury, ROSfluorescent probe-DHE to detect the expression of ROS in Ana-1, andtotal SOD assay kit with WST to detect the expression of total SOD inAna-1. With oxidative stress injured Ana-1induced by LPS, Comet assaykit could be used to detect the expression of LPS-induced Ana-1’s DNAinjury, ROS fluorescent probe-DHE to detect the expression of ROS inAna-1, and total SOD assay kit with WST to detect the expression of totalSOD in Ana-1. Then compare the indices’ changes of Ana-1under twodifferent situations mentioned above.2. Autophagy of LPS-inducedAna-1and that of LPS-induced Ana-1which is injured by oxidative stress.Four hours after using1ug/ml LPS to induce Ana-1（LPS group）, use reverse transcription-polymerase chain reaction （RT-PCR） to detect themRNA expression changes of autophagy-related LC3-Ⅱ, autophagy ofAns-1under the effect of LPS through Lipofectamine2000GFP-LC3（green fluorescent protein-microtubule associated protein light chain3）and RFP-LC3（red fluorescent protein-microtubule associated proteinlight chain3）, and the expression of BECN1and LC3Ⅰ/Ⅱ （WesternBlot）. As for LPS-induced Ana-1under oxidative stress （LPS+H₂O₂group, two hours after being induced by12.5uM exogenous H₂O₂, fourhours with1ug/ml LPS）, use RT-PCR to detect the mRNA expressionchanges of autophagy-related LC3-Ⅱ, autophagy under the effect of LPSthrough Lipofectamine2000GFP-LC3and RFP-LC3, and the expressionof BECN1and LC3Ⅰ/Ⅱ （Western Blot）. After treatment of Vitamin Cunder oxidative stress （one hour after inducing with2000ug/ml Vitamin C,add12.5uM H₂O₂, two hours later add1ug/ml LPS for four hours）,observe the autophagy of LPS-induced Ana-1, and the expression ofLC3Ⅰ/Ⅱ （Western Blot）. Compare with the control group by PBS.Compare the indices changes and differences between each two of thecontrol group, LPS group, LPS+H₂O₂group, and Vitamin C group.Result:1. Compared with the control group, the expression of ROS inLPS-induced Ana-1rises up, P<0.01; total SOD decreases, P<0.01;Comet assay kit detected DNA injury of Ana-1, P<0.01, indicatingoxidative stress injury of Ana-1could be induced by LPS. The expressionof ROS in LPS-induced Ana-1with oxidative stress is dramatically higher, P<0.01, total SOD decreased, P<0.01; Comet assay kit detected DNAinjury of Ana-1, P<0.01, with clear cell injury.2. Observing thefluorescence intensity and numbers of the control group, LPS group, andLPS+H₂O₂group under confocal microscope, the results are as follows:compared with the control group, the fluorescence intensity and numberof the LPS increases, P<0.01; the fluorescence intensity and number ofLPS+H₂O₂group obviously higher than that of LPS group, P<0.01.3.The expression changes of LC3-Ⅱ m RNA detected throughRT-PCRshows that the expression of LC3-Ⅱ mRNA in LPS+H₂O₂group is morethan that of LPS group.4. Observing the GFP-LC3and RFP-LC3transfection of the control group, LPS group, LPS+H₂O₂group andVitamin C group under confocal microscope, the results are as follows:autophagosome in LPS group goes up compared with the control group;the expression of autophagosome in cytoplasm of LPS+H₂O₂group ishigher than that of the control group; autophagosome in Vitamin C groupgoes down compared with the control group.5. The detection of BECN1protein through Western Bolt demonstrates that the expression of BECN1in LPS+H₂O₂group is more than that of LPS group, P<0.01; theexpression of BECN1in LPS group increases compared with the controlgroup, P<0.01. The detection of LC3Ⅰ/Ⅱprotein through Western Boltshows that the expression of LC3of Vitamin C group is less than that ofLPS+H₂O₂group, P<0.01; the expression of LC3of LPS+H₂O₂group ismore than that of LPS group, P<0.01; the expression of LC3of LPS group is more than that of the control group, P<0.01. Conclusion:1.LPScould induce the oxidative stress of mice’s Ana-1,which leads to cellinjury. oxidative stress could aggravate such stress and intensify DNAinjury of the cell.2. oxidative stress with LPS could accelerate theautophagy of mice’s Ana-1, increasing BECN1and LC3Ⅰ/Ⅱprotein.The autophagy of Ana-1under oxidative stress is relevant tooxidative stress DNA injury of the cell.3.With treatment of Vitamin C theantioxidant, it manifests that Vitamin C could decrease the expression ofLC3Ⅰ/Ⅱ protein, GFP-LC3and RFP-LC3of Ana-1under LPS-inducedoxidative stress. It also proves, once more, LPS could intensify cell injuryof Ana-1and then accelerate cell autophagy.