Nrf2 / Are Pathways In Pm < Sub > 2.5 < / Sub > On Human Umbilical Vein Endothelial Cell Injury And Rg1 Reduce The Role Of The Damage

Pa`rt1Study the toxic effects of air PM_2.5on Human umbilical vein endothelial cells（HUVECs）Objective: To study the cell toxicity of the three extracts （orangic） and the water-solublecomponents （WSC）and acid-soluble components（ASC） from air PM_2.5.Method: Use the filter membrane method to collect air PM_2.5in TaiYuan area. RegardingHUVEC as target cell, explore the toxic effects of the two orangic and inorangic extracts fromPM_2.5with different concentration including0,50,200,400,800,1200μg/ml on cell viabilityand oxidantive injury respectively, use MTT to determine the cell survive ability andfluorescence microscope to detect the changes of intracellular level of ROS, MDA.Results: As concentration of the three extracts from PM_2.5increases, the cell viability levelshows decending trend, on the contrary, the level of ROS and MDA appears ascending trend.When treated the HUVECs with PM_2.550,100,200,400,800,1200μg/ml the cell viability oforangic components is97.00±1.80（P>0.05）,95.33±2.30,69.30±3.61,54.82±4.32,30.77±4.09,26.08±4.23（P<0.05）, the water-soluble components（WSC）99.00±1.00,99.02±0.57（P>0.05）,91.06±3.21,83.17±1.54,71.34±3.26,59.28±2.45（P<0.05） and acid-soluble components（ASC） is99.06±0.82,98.97±2.18,96.85±3.23（P>0.05）,90.28±2.15,85.32±1.73,79.28±2.98（P<0.05）,When treated the HUVECs with50,100,200,400,800μg/ml, the ROS level of orangiccomponents is154±11,164±10（P>0.05）,214±17,224±27,241±18（P<0.05） the water-solublecomponents（WSC）159±46,159±40,160±58（P>0.05）,220±36,228±43（P<0.05）, andacid-soluble components164±9,168±7,170±17（P>0.05）,196±18,224±11（P<0.05）, the MDAlevel of orangic components is0.88±0.09（P>0.05）,0.89±0.19,1.27±0.21,1.87±0.36,1.96±0.15（P<0.05）, the water-soluble components（WSC）0.90±0.03,0.91±0.06,0.93±0.21（P>0.05）,1.37±0.36,1.47±0.15（P<0.05）, and acid-soluble components（ASC）0.91±0.24,0.93±0.20,0.96±0.34,1.01±0.15（P>0.05）,1.50±0.15（P<0.05）. And the two trends both have siginificantstatistical significance compared with control group. The cell survive ability of orangic extractgroup is lower than the other extract group, the level of oxidantive injury and metabolic markersof other group with different PM_2.5concentration is different between the three extracts.Conclusion: The three extracts from air PM_2.5both have certain cell toxicity, moreover, thetoxicity of orangic components is higher than other components. Oxidantive stress is one of cellimpairrment way. Part2Ginsenoside Rg1protects against the particulate matter (PM_2.5)-induced cell injuryand cell death by modulating intracellular redox via Nrf2pathway in Human umbilicalvein endothelial cells （HUVECs）Objective: The purpose of this study was to explore the possible mechanism of PM_2.5-inducedendothelial cell injury, cell death and cytoprotective effects of the Rg1.Methods: After stimulation with PM_2.5and ginsenoside Rg1for the indicated concontration,the proliferation ability of HUVECs were assessed by CCK-8assay. The oxidative stress ofHUVECs were assessed by ROS and MDA assay. Furthermore, the influence of PM_2.5andginsenoside Rg1on the expression of HO-1was investigated by RT–PCR and Western blottingassays. The confocal microscopy was applied to observe the Nrf2expression and nucleitranslocation.Results: PM_2.5, at concentrations of200,400, and800μg/ml, reduced HUVECs viability by30.7±2.8%,46.2±3.9%and70.2±2.8%, respectively, and the IC50value for PM_2.5to reduceHUVEC viability by50%was526.9μg/ml. Incubation for48h with Rg12.5,10and40μg/mlconcentration-dependently antagonized the HUVECs viability decrease induced by PM_2.5.Coincubation with Rg12.5,10,40μg/ml, the IC50of PM_2.5increased to592.3,825.6and1232.1μg/ml respectively. PM_2.5dose-dependently stimulated oxidative stress generation in HUVECs.In addition to cell death, pretreatment with Rg1could also significantly increase HO-1mRNAand protein expression and decrease ROS level and MDA production. The green fluorescenceintensities in control and HUVECs incubated with PM_2.5800μg/ml were148.0±4.0and241.0±7.0（P <0.05）, respectively. PM_2.5（100-800μg/ml） increases intracellular fluorescence ina concentration dependent manner. In presence of Rg110μg/ml and40μg/ml, the maximal greenfluorescence intensity increases induced by PM_2.5800μg/ml were reduced to216.0±2.0and206.0±3.0（P <0.05） respectively. MDA concentration within control HUVECs was0.87±0.07nmol/mg protein. PM_2.5（100-800μg/ml） concentration dependently increased MDA with themaximum being1.96±0.09nmol/mg protein. Coculture with Rg1（10and40μg/ml） decreasedPM_2.5-induced MDA production by72.96%（P<0.05） and58.66%（P<0.05）,respectively.Analysis using laser confocal microscopy revealed that Rg1treatment led to adramatic increase in the level of Nrf2. The ratio of cytoplasmic Nrf2protein to nuclear Nrf2protein was markedly reduced and reached maximum at40μg/ml （0.09） compared with that ofthe PM_2.5only（0.3） and control（0.4）.Conclusions: our results suggest that Rg1could attenuate PM_2.5-induced cell damage by augmenting the cellular antioxidant defense upregulated heme oxygenase-1（HO-1） expression toprotect HUVECs against oxidative stress. In addition, Rg1also promoted nuclear translocationof nuclear factor erythroid2-related factor （Nrf2）. which put new evidence on thecardiovascular-protective mechanism of Rg1against the oxidative stress of PM_2.5in Nrf2pathway in HUVECs. Part3The study of toxicity effects of the organic component of PM_2.5on HUVECs afterthe RNA of Nrf2was interferedObjective: To study the toxicity effects of the organic component of PM_2.5on HUVECs afterthe RNA of Nrf2was interfered.Methods: Design and construct the short hairpin carrier direct against Nrf2, transfect cellswith lipofectin, select the ShRNA, take human umbilical veins endothelial cells as the targetcells, study the toxicity of different concentrations （0,400μg/ml） of PM_2.5on the cells vitalityand oxidative damages. Assay the survival ratio of cells with CCK-8, measrue the reactiveoxygen species, western-blot was used to detect the expression of HO-1in these cells.Results: After treatment HUVECs for24h with the organic component of the PM_2.5,the cellsvitality was descend to54.86%,however when the RNA of Nrf2was interfered, the survival ratioof cells was descend obviously to36.94%.With Rg140μg/ml pretreatment1h then cocultured24h with the organic component of the PM_2.5the cell vitality was increased to89.58%, when theRNA of Nrf2was interfered the cell vitality was descend again to42,14%（P<0.05）, Aftertreatment HUVECs for6h with the organic component of the PM_2.5,the ROS level was increasedto224.0,however when the RNA of Nrf2was interfered, the the ROS level was increasedobviously to302.0. With Rg140μg/ml pretreatment1h then cocultured24h with the organiccomponent of the PM_2.5the ROS level was descend to164.0, when the RNA of Nrf2wasinterfered the the ROS level was increased again to271.0（P<0.05）.The HO-1protein expressionin PM_2.5and Rg140μg/ml pretreament group were more than the contral group. When the RNAof Nrf2was interfered, the HO-1protein expression was decreased, the cell viability wasdescend by39.1%(PM_2.5400μg/ml),36.0%(PM_2.5400μg/ml with Rg1), and ROS was increased by40.2%(PM_2.5400μg/ml),38.0%(PM_2.5400μg/ml with Rg1), compared withnegative control group, and they both are of significant difference in statistics.Conclusion: Nrf2can protect the cells from cytotoxicity caused by the organic components offine particle PM_2.5and the protective effect of Rg1on HUVECs was related with anti-oxidant ofNrf2pathway.