Epigallocatechin-3-gallate Protects HUVECs From Pm2.5-induced Oxidative Stress Injury By Activating Nrf2/HO-1 Pathways

BackgroudThe exposure to ambient fine particulate matter(PM2.5) is responsible for certain cardiovascular diseases(CVDs). Endothelial dysfunction and oxidative stress likely play roles in PM2.5-induced harmful effects. Epigallocatechin-3-gallate(EGCG), the major polyphenolic constituent of green tea, is a potent antioxidant that exerts protective effects on CVDs by scavenging free radicals. The aim of the present study was to investigate whether EGCG could inhibit PM2.5-induced oxidative stress by activating the transcription factor nuclear factor E2-related factor 2(Nrf2)/heme oxygenase-1(HO-1) pathway in human umbilical vein endothelial cells(HUVECs). PM2.5(200μg/ml) increased both cell death and intracellular ROS levels significantly, whereas EGCG(50~400μM) inhibited these effects in a concentration-dependent manner. Western blotting and PCR demonstrated that EGCG increased Nrf2 and HO-1 expression in HUVECs that had been exposed to PM2.5. PD98059(a selective inhibitor of extracellular signal regulated kinase [ERK]-1/2) and SB203580(a selective inhibitor of p38 MAPK), but not SP600125(a selective inhibitor of c-jun N-terminal kinase [JNK]), attenuated the EGCG-induced Nrf2 and HO-1 expression. In addition, silencing Nrf2 abolished EGCG-induced Nrf2 and HO-1 upregulation. The present study suggests that EGCG protects HUVECs from PM2.5-induced oxidative stress injury by upregulating Nrf2/HO-1 via activation of the p38 MAPK and the ERK1/2 signaling pathways.Section 1 Role of EGCG on PM2.5-indued Human umbilicalvein endothelial cells injury Objective:In order to observe the effect of EGCG on PM2.5-induced HUVEC injury human umbilical vein endothelial cells(HUVECs) were treated with soluble constituents of atmospheric fine particulate matter PM2.5. Methods: 1. Cell linesHuman umbilical vein endothelial cells lines were purchased from Shanghai Cell Bank, Chinese Academy of Sciences. 2. Methods 2.1 The extraction of soluble components of PM2.5 2.2 Culture of human umbilical vein endothelial cellsHUVECs were grown in DMEM supplemented with 10%(v/v) heat-inactivated fetal bovine serum and 1%(v/v) penicillin–streptomycin at 37°C in a humidified atmosphere containing 5% CO2 and 95% air. The cells can be passaged until reached confluence more than 80%. 2.3 Determination of indicators 2.3.1 Cell survival was detected by Cell Counting Kit-8(CCK-8) 2.3.2 Detection of intracellular levels of reactive oxygen species in HUVECs(DCFH2-DA probe labeled flow cytometry and confocal microscopy) Results: 1. The effects of EGCG on HUVECs survival.The HUVECs survival, detected after being treated with 50~200u M EGCG for 24 hours, showed that 300 and 400μM of EGCG significantly reduced cell survival. Therefore, the 50~200μM EGCG were selected for subsequent experiments.2. PM2.5 decreased the HUVECs survival.PM2.5 concentration-dependent decreased cell survival when HUVECs were treated with 50~400μg/ml PM2.5 for 24 hours, and the IC50 value was ~200μg/ml. 3. EGCG reversed PM2.5-indued HUVECs injury.HUVECs were pre-incubated with 50~400μM EGCG for 30 minutes followed by 200μg/ml PM2.5 for another 24 hours. The CCK-8 result showed that EGCG reversed PM2.5-induced decrease of cell survival concentration-dependently. 100 and 200μM EGCG almost entirely offset the cell survival decrease induced by PM2.5. 4. EGCG reduced PM2.5-induced intracellular levels of ROS in HUVECs.HUVECs were pre-incubated with 100μM EGCG for 30 minutes followed by 200μg/ml PM2.5 for another 24 hours. The intracellular levels of ROS in PM2.5 treatment groups increased significantly, and the elevation of intracellular ROS can be completely reversed by EGCG. Conclusion: 1. When HUVECs were treated with 200μg/ml PM2.5, the cell survival decreased and intracellular ROS increased significantly. 2. 100μM of EGCG completely reversed PM2.5-induced cell survival decrease and intracellular ROS increase.Section 2 Molecular mechanisms of the protective effect ofEGCG on PM2.5-indued HUVECs injury Objective:In order to study the molecular mechanisms of the protective effect of EGCG on PM2.5-indued HUVECs injury the expression of Nrf2 and HO-1 were detected when HUVECs were pre-incubated with 100μM EGCG for 30 minutes followed by 200μg/ml PM2.5 soluble components for another 24 hours. And we want to know whether MAPK inhibitor could affect the expression of Nrf2 and HO-1 in HUVECs under the treatment above. Methods: 1. Cell linesHuman umbilical vein endothelial cells lines were purchased from Shanghai Cell Bank, Chinese Academy of Sciences. 2. Methods Culture of human umbilical vein endothelial cellsHUVECs were grown in DMEM supplemented with 10%(v/v) heat-inactivated fetal bovine serum and 1%(v/v) penicillin–streptomycin at 37°C in a humidified atmosphere containing 5% CO2 and 95% air. The cells can be passaged until reached confluence more than 80%. 3. Determination of indicators 3.1 Cell survival was detected by Cell Counting Kit-8(CCK-8) 3.2 Detection of intracellular levels of reactive oxygen species in HUVECs(DCFH2-DA probe labeled flow cytometry and confocal microscopy) 3.3 Detection of the protein expression of Nrf2 and HO-1 in HUVECs(Western blot) 3.4 Detection of the m RNA expression of Nrf2 and HO-1 in HUVECs(Real-time PCR) Results: 1. PM2.5 induced decrease of intracellular protein and m RNA expression of Nrf2 and HO-1 in HUVECs.HUVECs were treated with 50~400μg/ml PM2.5 for 24 hours showed 50 and 100μg/ml PM2.5 increased intracellular expression of Nrf2 and HO-1, while 200, 300 and 400ug/ml PM2.5 decreased intracellular expression of Nrf2 and HO-1 sharply. 2. EGCG reversed PM2.5-induced intracellular expression of Nrf2 and HO-1 decrease in HUVECs.HUVECs were pre-incubated with 50~400μM EGCG for 30 minutes followed by 200μg/ml PM2.5 for another 24 hours. The decrease of intracellular expression of Nrf2 and HO-1 can be reversed by EGCG concentration-dependently. 3. The effect of MAPK inhibitors on cell viability in HUVECs which were treated with EGCG followed by PM2.5.HUVECs were pre-treated with three MAPK inhibitors PD98059, SP600125 and SB203580 for 1 hour followed by 100μM EGCG for 30 min, and then treated with 200μg/ml PM2.5 for another 24 hours. Then cell viability was detected and the results showed that that PD98059 and SB203580 completely blocked EGCG induced increase of cell viability, whereas SP600125 did not have this effect. 4. The effect of MAPK inhibitors on intracellular ROS in HUVECs which were treated with EGCG followed by PM2.5.HUVECs were pre-treated with three MAPK inhibitors PD98059, SP600125 and SB203580 for 1 hour followed by 100μM EGCG for 30 min, and then treated with 200μg/ml PM2.5 for another 24 hours. Then intracellular ROS was detected and the results showed that that PD98059 and SB203580 completely blocked EGCG-induced decrease of intracellular ROS, whereas SP600125 did not have this effect. 5. HUVECs which were treated with EGCG followed by PM2.5.HUVECs were pre-treated with three MAPK inhibitors PD98059, SP600125 and SB203580 for 1 hour followed by 100μM EGCG for 30 min, and then treated with 200μg/ml PM2.5 for another 24 hours. Then intracellular expression of Nrf2 and HO-1 were detected and the results showed that that PD98059 and SB203580 completely blocked EGCG-induced increase of intracellular expression of Nrf2 and HO-1, whereas SP600125 did not have this effect. Conclusion: 1. PM2.5 decreased intracellular expression of Nrf2 and HO-1 HUVECs, while EGCG completely reversed the effect of PM2.5. 2. The protective effect of EGCG on PM2.5-induced cell damage may be through ERK1/2 and JNK pathway but not p38 MAPK pathway.Section 3 Crucial role of Nrf2 gene in the antioxidant effect ofEGCG Objective:Expression of Nrf2 and HO-1 were detected in Nrf2 gene silencing model, when HUVECs were treated with EGCG followed by PM2.5. Methods: 1. Cell linesHuman umbilical vein endothelial cells lines were purchased from Shanghai Cell Bank, Chinese Academy of Sciences. 2. Methods 2.1 Culture of human umbilical vein endothelial cellsHUVECs were grown in DMEM supplemented with 10%(v/v) heat-inactivated fetal bovine serum and 1%(v/v) penicillin-streptomycin at 37°C in a humidified atmosphere containing 5% CO2 and 95% air. The cells can be passaged until reached confluence more than 80%. 2.2 Establishment of HUVECs transfected with plasmid that silenced Nrf2 3. Determination of indicators 3.1 Cell survival was detected by Cell Counting Kit-8(CCK-8) 3.2 Detection of intracellular levels of reactive oxygen species in HUVECs(DCFH2-DA probe labeled flow cytometry and confocal microscopy) 3.3 Detection of the protein expression of Nrf2 and HO-1 in HUVECs(Western blot). Results: 1. Nrf2-sh RNA plasmid was constructed successfully.Protein expression of Nrf2 in HUVECs transfected with constructed Nrf2-sh RNA plasmid decreased by about 50% compared with normal cells which indicate that the plasmid construction and transfected successfully. 2. After Nrf2-sh RNA construction and transfection successfully, the effect of EGCG-induced increase of cell viability was abolished in HUVECs explore to PM2.5.Nrf2-sh RNA transfected HUVECs and normal HUVECs were treated with EGCG for 30 minutes followed by 200μg/ml PM2.5 for another 24 hours. The result showed that Nrf2 silencing decreased cell viability in transfected HUVECs compare with in normal HUVECs3. After Nrf2-sh RNA construction and transfection successfully, the effect of EGCG-induced increase of the expression of Nrf2 and HO-1 were abolished in HUVECs explore to PM2.5.Nrf2-sh RNA transfected HUVECs and normal HUVECs were treated with EGCG for 30 minutes followed by 200μg/ml PM2.5 for another 24 hours. The result showed that Nrf2 silencing increased intracellular ROS in transfected HUVECs compare with in normal HUVECs.4. After Nrf2-sh RNA construction and transfection successfully, the effect of EGCG-induced increase of the expression of Nrf2 and HO-1 were abolished in HUVECs explore to PM2.5.Nrf2-sh RNA transfected HUVECs and normal HUVECs were treated with EGCG for 30 minutes followed by 200μg/ml PM2.5 for another 24 hours. The result showed that Nrf2 silencing decreased Nrf2 and HO-1 protein expression in transfected HUVECs compare with in normal HUVECs. Conclusion:The protective effect of EGCG on PM2.5-induced cell damage was achieved by increasing intracellular expression of HO-1 which was activated by Nrf2 in HUVECs.