| ObjectiveTo investigate the protective effect of lycium barbarum polysaccharide on endothelial dysfunction induced by oxidative stress and its potential molecular mechanism by establishing the oxidative stress model of rat thoracic aortic endothelial cells in vitro.MethodsThe cell relative viability of RTAEC was analyzed by CCK-8 assay when cells were treated with Ang?at different final concentrations(0,10-9,10-8,10-7,10-6,10-5mol/L)for different time?3,6,12,24,48 h?,and incubated in medium containing LBP at 400,800,1600,3200,6400?g/mL for 2h.The effects of Ang?(10-6mol/L,24h)and LBP?3200?g/mL,2h?on the level of reactive oxygen species?ROS?in RTAECs was measured using 2?,7?-dichlorofluorescein diacetate?DCF-DA?assay and the fluorescence intensity was monitored via flow cytometry and microscope.The effects of Ang?(10-6mol/L,24h)and LBP?400,3200?g/mL,2h?on protein expression of ZonulaOccludens-1?ZO-1?,occludin,endothelial growth factor receptor?EGFR?,phosphorylated endothelial growth factor receptor?p-EGFR?,human epidermal growth factor receptor-2?HER2/ErbB2?,phosphorylated human epidermal growth factor receptor-2?p-HER2/p-ErbB2?,phosphatidylinositol-3-kinase?PI3K?,protein kinase B?PKB/AKT?,phosphorylated protein kinase B?p-PKB/p-AKT?,endothelial nitric oxide synthase?e-NOS?and phosphorylated endothelial nitric oxide synthase?p-e-NOS?were tested via western blot.The expressive changes of occludin,ZO-1,EGFR,ErbB2 were observed by immunofluorescence.The effects of Ang?(10-6mol/L,24h),LBP?3200?g/mL,2h?,AC480?2?mol/L,26h?,WM?5?mol/L,26h?on gene expression of EGFR,ErbB2,AKT,e-NOS were analyzed by RT-qPCR.Data were analyzed using one-way ANOVA for multiple groups and unpaired Student's t-test for double groups via SPSS for Windows software?version 22.0,SPSS Inc.,IL?.The measurement data were expressed by mean±standard deviation?x?s?,and the enumeration data were expressed by median±quartile spacing?M?Q?in our study.ResultsCompared with the control group,the cell relative viability of RTAEC treated with Ang?decreased with the increase of Ang?concentration and cultural time and reached lowest with 10-6mol/L Ang?for 24h?P<0.05?,and the level of reactive oxygen species?ROS?in cells significantly increased in Ang?(10-6mol/L,24h)group?P<0.05?.Moreover,the expression of occludin and ZO-1,tight junction protein that can maintain the integrity of blood vessel endothelium,could be decreased by Ang?(10-6mol/L,24h)?P<0.05?.But if cells were incubated with LBP before the cells treated with Ang?,the changes caused by Ang?could be converted.That is to say,vs Ang?group,the relative viability of cells could be increased?P<0.05?,the level of ROS in cells?P<0.05?could be abated,and the expression of occludin and ZO-1 could be up-regulated when cells were incubated with LBP before the cells treated with Ang?.Protein expression of EGFR,p-EGFR could up-regulate when cells were treated with Ang?but down-regulate when cells were pretreated with LBP before the cells were treated with Ang?.However,cells incubated in medium containing Ang?could decrease the protein expression of ErbB2,p-ErbB2,PI3K,p-e-NOS?P<0.05?,p-AKT?P<0.05?,but the expression of them could be added when cells were incubated with LBP before the cells treated with Ang?.Effects of Ang?and LBP on gene expression of EGFR and ErbB2 was the same as effects of Ang?and LBP on protein expression of EGFR and ErbB2?P<0.01?.The effects of Ang?and LBP on p-e-NOS,p-AKT can be blocked by WM,specific inhibitor of PI3K,but not AC480,selective inhibitors of EGFR and ErbB2.AC480 only blocked the effect of Ang?and LBP on p-ErbB2.It should be noted in particular that the gene and protein expression of e-NOS,AKT were almost unaffected by Ang?,LBP,WM and AC480.Conclusion Lycium barbarum polysaccharide?LBP?can ameliorate endothelial dysfunction induced by oxidative stress via regulating EGFR,ErbB2,and PI3K/AKT/e-NOS in the rat thoracic aortic endothelial cells. |
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