CAJ | 학술논문

目的观察白藜芦醇对小鼠脑微血管内皮细胞(bEnd.3)过氧化氢(H2O2)氧化应激损伤的保护作用.方法使用不同浓度的H2O2(100、300、500、800、1 000 μmol/L),作用不同时间(6、12、24 h)处理bEnd.3细胞,建立氧化应激模型;然后采用不同浓度的白藜芦醇(RSV) (0.1、1.0、5.0、10.0、20.0、40.0、60.0μmol/L)与H2O2共处理bEnd.3细胞.实验为对照组、H2 O2组、RSV+H2O2组.用细胞增殖检测试剂盒(CCK-8法)检测细胞存活率;用乳酸脱氢酶(LDH)和超氧化物歧化酶(SOD)检测试剂盒检测LDH和SOD的活力;用Hoechst 33342以及流式细胞术检测细胞凋亡;用Western blot法检测β-连环蛋白(β-catenin)、蛋白激酶B(Akt)的磷酸化水平、B细胞淋巴瘤/白血病-2 (bcl-2)、活化酪半胱氨酰天冬氨酸特异性蛋白酶(cleaved Caspase-3)的表达水平.结果 500 μmol/L H2O2刺激bEnd.3细胞24h,细胞的存活率降至50％左右(P＜0.05);与H2O2组比较,bEnd.3细胞经实验浓度的RSV(10 μmol/L)处理后存活率由(46.5±5.0)％升高至(70.4±3.4)％(P＜0.05),LDH活力由(161.0±2.9)％降至(108.0±3.3)％(P＜0.05),SOD活力由(23.0±5.5)％升高至(50.7±14.7)％(P＜0.05),同时细胞凋亡率由(7.12±0.59)％降至(2.24±0.37)％(P＜0.05);Western blot结果显示:RSV+H2 O2组β-catenin、磷酸化Akt (p-Akt)、bcl-2蛋白表达增强,cleaved Caspase-3蛋白表达减弱(P＜0.05).结论实验剂量的RSV对bEnd.3细胞氧化应激损伤具有保护作用,其机制可能通过介导磷酸肌醇3激酶(PI3K)/Akt信号通路和稳定细胞骨架而实现.
목적 관찰백려호순대소서뇌미혈관내피세포(bEnd.3)과양화경(H2O2)양화응격손상적보호작용.방법 사용불동농도적H2O2(100、300、500、800、1 000 μmol/L),작용불동시간(6、12、24 h)처리bEnd.3세포,건립양화응격모형;연후채용불동농도적백려호순(RSV) (0.1、1.0、5.0、10.0、20.0、40.0、60.0μmol/L)여H2O2공처리bEnd.3세포.실험위대조조、H2 O2조、RSV+H2O2조.용세포증식검측시제합(CCK-8법)검측세포존활솔;용유산탈경매(LDH)화초양화물기화매(SOD)검측시제합검측LDH화SOD적활력;용Hoechst 33342이급류식세포술검측세포조망;용Western blot법검측β-련배단백(β-catenin)、단백격매B(Akt)적린산화수평、B세포림파류/백혈병-2 (bcl-2)、활화락반광안선천동안산특이성단백매(cleaved Caspase-3)적표체수평.결과 500 μmol/L H2O2자격bEnd.3세포24h,세포적존활솔강지50％좌우(P＜0.05);여H2O2조비교,bEnd.3세포경실험농도적RSV(10 μmol/L)처리후존활솔유(46.5±5.0)％승고지(70.4±3.4)％(P＜0.05),LDH활력유(161.0±2.9)％강지(108.0±3.3)％(P＜0.05),SOD활력유(23.0±5.5)％승고지(50.7±14.7)％(P＜0.05),동시세포조망솔유(7.12±0.59)％강지(2.24±0.37)％(P＜0.05);Western blot결과현시:RSV+H2 O2조β-catenin、린산화Akt (p-Akt)、bcl-2단백표체증강,cleaved Caspase-3단백표체감약(P＜0.05).결론 실험제량적RSV대bEnd.3세포양화응격손상구유보호작용,기궤제가능통과개도린산기순3격매(PI3K)/Akt신호통로화은정세포골가이실현.
Objective To investigate the effect of resveratrol on hydrogen peroxide-treated mouse brain microvascular endothelal cells (bEnd.3).Methods bEnd.3 cells were treated with hydrogen peroxide (H2O2) of different concentrations (100,300,500,800 and 1 000 μmol/L) for differennt durations (6,12,24 h) to establish the hydrogen peroxide-treated bEnd.3 model.The effect of different concentrations of resveratrol (0.1,1.0,5.0,10.0,20.0,40.0,and 60.0 μmol/L) on the model was investigated.Cells were divided into three groups:the control group,H2O2 group and resveratrol (RSV) + H2O2 group.The cell viability was measured by cell counting kit-8 (CCK-8) assay.Lactate dehydrogenase (LDH) and superoxide dismutase (SOD) were measured by commercially available kits.Cell apoptosis was detected by Hoechst 33342 and flow cytometer.The expression of β-catenin,p-protein kinase B (Akt),B-cell lymphoma/leukemia-2 (bcl-2) and cleaved Caspase-3 was detected by Western blotting.Results The cell viability in the H2O2 groups was significantly lower than that in the control group (P ＜ 0.05),and the growth inhibition ratio for 24 h groups was 50％ inhibitory concentration (IC50) when the concentration of H2O2 was 500 μmol/L.As compared with the H2O2 group,the cell viability of the RSV group treated with 10 μmol/L RSV was increased from (46.5 ±5.0)％ to (70.4 ±3.4)％ (P ＜ 0.05),the activity of LDH decreased from (161.0 ± 2.9) ％ to (108.0 ± 3.3) ％ (P ＜ 0.05),the activity of SOD increased from (23.0 ±5.5)％ to (50.7 ± 14.7)％ (P＜0.05),and the apoptosis rate decreased from (7.12 ± 0.59) ％ to (2.24 ± 0.37) ％ (P ＜ 0.05).Western blotting revealed that the expression of β-catenin,p-Akt and bcl-2 was increased,and the expression of cleaved Caspase-3 was decreased in RSV + H2O2 group (P ＜ 0.05).Conclusion The present study suggests the PI3K/Akt pathway and the regulation of the cytoskeleton stability might be involved in the protective effect of the experimental dose of RSV on hydrogen peroxide-treated bEnd.3 cells.