摘要
目的:研究枸杞多糖(LBP)对过氧化氢(H_2O_2)诱导人内皮样细胞EA.hy926氧化损伤的作用及机制。方法:用H_2O_2建立EA.hy926细胞氧化损伤模型,细胞共分为5个组:正常对照(control)组、模型(damage)组(H_2O_2,50 mmol/L)、LBP(100 mg/L)组、抗损伤(anti-damage)组(50 mg/L、100 mg/L和200 mg/L LBP+50 mmol/L H_2O_2)和LY294002(20μmol/L)组。采用CCK-8法检测细胞的活力;用Annexin V/PI双染法流式细胞术检测不同处理后EA.hy926细胞的凋亡;吖啶橙(AO)/溴乙啶(EB)染色法观察细胞凋亡的形态特征;划痕法测定细胞的迁移能力;一氧化氮(NO)试剂盒检测细胞培养上清中NO的水平;用ELISA法测定血管内皮生长因子(VEGF)的水平;Western blot检测cleaved caspase-3、Bax、Bcl-2、内皮型NO合酶(eNOS)、p-eNOS和p-Akt的蛋白水平。结果:量效结果显示浓度为100 mg/L的LBP预处理EA.hy926细胞1 h,然后加入H_2O_2(50 mmol/L)处理24 h对减轻H_2O_2诱导的EA.hy926细胞损伤的作用最显著。与damage组比较,LBP预处理可提高EA.hy926细胞的活力(P<0.05),减少细胞凋亡(P<0.05),并促进细胞迁移;上清液中VEGF和NO水平升高;Bcl-2/Bax比率明显升高,下调cleaved caspase-3蛋白水平,并上调eNOS和p-eNOS蛋白水平。此外,加入PI3K抑制剂LY294002后,LBP对H_2O_2损伤的EA.hy926细胞的保护作用减弱,表现为NO水平和p-Akt蛋白水平降低。结论:LBP能减轻H_2O_2对EA.hy926细胞的损伤作用,缓解H_2O_2诱导的凋亡,其机制与激活PI3K/Akt/eNOS信号通路密切相关。
AIM： To study the effect of Lycium barbarum polysaccharides（ LBP） on oxidative stress injury of human endothelium-like EA. hy926 cells induced by hydrogen peroxide（ H2O2）. METHODS： The EA. hy926 cell model of oxidative stress injury was established by H2O2 treatment. The EA. hy926 cells were divided into 5 groups： control group,damage（ H2O2 at 50 mmol/L） group,LBP（ 100 mg/L） group,anti-damage groups（ LBP at 50 mg/L,100 mg/L or 200 mg/L ＋ 50 mol/L H2O2）,and LY294002（ 20 μmol/L） group. The effect of LBP at different concentrations on the cell viability of EA. hy926 cells was measured by CCK-8 assay,and the optimum concentration of LBP was screened out.The apoptotic of EA. hy926 cells was analyzed by flow cytometry. Acridine orange/ethidium bromide（ AO/EB） staining was used to observe the morphological characteristics of the apoptotic cells. The cell migration ability was detected by scratch method. The levels of nitric oxide（ NO） and vascular endothelial growth factor（ VEGF） in the cell culture medium were examined. The protein levels of cleaved caspase-3,Bax,Bcl-2,endothelial NO synthase（ eNOS）,p-eNOS and pAkt were determined by Western blot. RESULTS： LBP at concentration of 100 mg/L significantly attenuated the injury of EA. hy926 cells induced by H2O2,as indicated by improved cell viability（ P〈0. 05） and decreased apoptosis（ P〈0. 05）. Pretreatment with LBP elevated the levels of NO and VEGF（ P〈0. 05）,and promoted the migration ability of EA. hy926 cells. LBP also increased the Bcl-2/Bax ratio,down-regulated the protein level of cleaved caspase-3,and upregulated the protein levels of eNOS and p-eNOS. The protective effect of LBP were abolished by pretreatment of the EA. hy926 cells with the inhibitor of PI3K（ P〈0. 05）. As a result,the protein level of p-Akt was down-regulated,and the level of NO was also significantly reduced. CONCLUSION： LBP has protective effect on H2O2-induced EA. hy926 cells by attenuating apoptosis of the cells. The mechanism is closely related to the activation of PI3K/Akt/eNOS signaling pathway.
作者
王玉林
吕林林
王霞
高永清
周兆
刘革修
王林静
WANG Yu-lin;LV Lin-lin;WANG Xia;GAO Yong-qingx;ZHOU Zhao;LIU Ge-xiu;WANG Lin-jing(School of Public Health,Guangdong Pharmaceutical University,Guangzhou 510006,China;Institute of Hematology,Basic Medical College of Jinan University,Guangzhou 510632,China)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2018年第6期975-981,共7页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.81270568)
