淫羊藿苷通过AKT/P53通路抑制S-亚硝基谷胱甘肽诱导的内皮细胞凋亡
Icariin reduces S-nitrosogultathione induced endothelial cell apoptosis through modulating AKT/P53 pathway
目的 观察淫羊藿苷(ICA)对S-亚硝基谷胱甘肽(GSNO)诱导的内皮细胞凋亡的影响,并探讨其机制.方法 内皮细胞株EA.hy926由浙江大学提供,将细胞按整群随机抽样法,分成空白对照组、损伤组(GSNO组)、不同浓度(高、中和低浓度)ICA干预组(ICA组)以及蛋白激酶B(AKT)通路的蛋白酶抑制剂LY294002抑制高、中和低浓度ICA组.空白对照组为EA.hy926细胞于96孔板培养,未予任何干预;GSNO组为EA.hy926细胞于96孔板培养48 h后给予1 mmol/L GSNO处理;高、中和低浓度ICA干预组为EA.hy 926细胞于96孔板培养24 h后分别加入10、1和0.1 μmol/L的ICA预保护24 h,再给予1 mmol/L GSNO处理;LY294002抑制高、中和低浓度ICA组为EA.hy 926细胞于96孔板培养24h后分别给予高(10.μmol/L)、中(1μmol/L)和低浓度(0.1 μmol/L) ICA干预的同时加入1 μmol/L LY294002,再作用24 h后给予1 mmol/L GSNO进行干预.噻唑蓝染色(MTT)法测定各组细胞存活率.根据不同试剂盒要求,检测各组细胞乳酸脱氢酶(LDH)活性、丙二醛(MDA)含量、活性氧(ROS)活性和线粒体膜电位.Western blot法检测各组细胞AKT/磷酸化AKT(p-AKT)、人抑癌蛋白53(P53)、细胞色素C(CYC)、内皮型一氧化氮合成酶(eNOS)/磷酸化eNOS(p-eNOS)以及procaspase-3/caspase-3蛋白表达水平.结果 (1)各组EA.hy926细胞存活率:GSNO组细胞存活率明显低于空白对照组(P<0.01),而高、中和低浓度ICA组均明显高于GSNO组(P均<0.01).(2)各组EA.hy926细胞LDH活性:GSNO组的细胞LDH活性明显高于空白对照组[(142.65±5.56) U/L比(50.01±3.42) U/L,P<0.05],而高、中和低浓度ICA组则均明显低于GSNO组[分别为(98.02±3.52)、(105.29±6.89)和(117.16±4.27) U/L比(142.65±5.56) U/L,P均<0.05],且降低LDH活性的效果具有浓度依赖性.(3)各组EA.hy926细胞MDA含量:GSNO组细胞MDA含量明显高于空白对照组[(11.14±0.37)nmol/mg比(5.21±0.18) nmol/mg,P<0.05],而高、中和低浓度ICA组均明显低于GSNO组[分别为(6.60±0.41)、(6.83±0.21)和(8.29±0.07) nmol/mg比(11.14±0.37)nmol/mg,P均<0.05].(4)各组EA.hy926细胞内ROS活性:GSNO组ROS活性明显高于空白对照组[(173.15±11.12)%比100%,P<0.05],而高、中和低浓度ICA组均明显低于GSNO组[(122.56±8.09)%、(134.52±9.09)%、(149.89±9.16)%比(173.15±11.12)%,P均<0.05],其中以高浓度ICA组尤为明显.(5)各组EA.hy926细胞线粒体膜电位:GSNO组线粒体膜电位明显高于空白对照组(0.84±0.04比0.12±0.04,P<0.05),而高、中和低浓度ICA组均低于GSNO组(分别为0.57±0.08、0.63±0.02、0.66±0.04比0.84±0.04,P均<0.05).(6)各组EA.hy926细胞内AKT/p-AKT表达:GSNO组细胞p-AKT蛋白表达水平明显低于空白对照组(P<0.05),而高、中和低浓度ICA组则均明显高于GSNO组(P均<0.05),且呈现浓度依赖性的趋势.而LY294002抑制高、中和低浓度ICA组p-AKT蛋白表达水平与GSNO组比较差异则无统计学意义.(7)各组EA.hy926细胞胞浆和胞核内P53蛋白表达:GSNO组胞浆和胞核内P53蛋白表达均明显高于空白对照组(P均<0.05),而高、中和低浓度ICA组均明显低于GSNO组(P均<0.05),LY294002抑制高、中和低浓度ICA组与GSNO组比较差异则无统计学意义.(8)各组EA.hy926细胞线粒体和胞浆内CYC、procaspase-3和caspase-3蛋白表达水平:GSNO组线粒体内CYC蛋白表达水平低于空白对照组(P<0.05),而胞浆中却高于空白对照组(P<0.05).高、中浓度ICA组线粒体CYC蛋白表达水平均高于GSNO组(P<0.05),而胞浆中却低于GSNO组(P<0.05),低浓度ICA组线粒体和胞浆中CYC蛋白表达水平与GSNO组比较差异均无统计学意义.各组procaspase-3和caspase-3蛋白表达水平趋势与CYC同.(9)各组EA.hy926细胞内eNOS/p-eNOS蛋白表达:GSNO组细胞p-eNOS蛋白表达水平与空白对照组比较差异无统计学意义.而高、中和低浓度ICA组细胞中p-eNOS蛋白表达水平均明显高于GSNO组(P均<0.05),但不同浓度组间差异无统计学意义.而LY294002抑制高、中和低浓度ICA组p-eNOS蛋白表达水平与GSNO组比较差异均无统计学意义.结论 ICA可抑制GSNO诱导的内皮细胞凋亡,其机制可能与ICA激活AKT通路从而下调P53活性有关.
更多Objective To observe the effects of icariin on S-nitrosogultathione (GSNO) induced endothelial cell apoptosis,and to explore the relative mechanisms.Methods EA.hy926 cell line was provided by Zhejiang University and cells were divided into blank control group,GSNO group (1 mmol/L GSNO),icariin (ICA) intervention group (GSNO ± different concentrations (high,medium and low:10,1 and 0.1 μmol/L ICA) and 1 μmol/L LY294002 pretreatment groups (AKT protease pathway inhibitor on top of ICA groups) by cluster random sample method.After 48 hours.EA.hy 926 cell survival was detected by thiazolyl blue tetrazolium bromicle (MTr) method.Lactate dehydrogenase (LDH) activity,malonaldehyde (MDA) content,reactive oxygen species (ROS) level,mitochondrial membrane potential were also measured.The protein expression of protein kinase B (AKT)/phosphorylation protein kinase B (p-AKT),people tumor-suppressor protein (protein 53,P53),cytochrome C (CYC),endothelial nitric oxide synthetase (eNOS)/phosphorylation endothelial nitric oxide synthetase (p-eNOS),procaspase-3/ caspase-3 was detected by Western blot.Results (1) The cell survival rate was significantly lower in GSNO group than in the blank control group (P < 0.01),which was significantly higher in the high,medium and low concentration ICA groups than in the GSNO group (all P < 0.01).(2) The LDH activity was significantly higher in the GSNO group than in the blank control group ((142.65 ± 5.56) U/L vs.(50.01 ± 3.42) U/L,P < 0.05),which was significantly reduced by high,medium and low concentration ICA ((98.02 ±3.52),(105.29 ±6.89) and (117.16 ±4.27) U/L vs.(142.65 ±5.56) U/L,all P < 0.05) in a dose-dependent manner.(3) The MDA content was significantly higher in GSNO group than in the blank control group ((11.14 ±0.37) nmol/mg vs.(5.21 ±0.18) nmol/mg,P< 0.05),which could be reduced by pretreatment with high,medium and low concentration ICA ((6.60 ±0.41),(6.83 ±0.21) and (8.29 ± 0.07) nmol/mg vs.(11.14 ± 0.37) nmol/mg,all P < 0.05).(4) The ROS content was significantly higher in GSNO group than in the blank control group ((173.15 ± 11.12)% (relative ratio to the blank control group),P < 0.05),which could be significantly reduced by pretreatment with high,medium and low concentration ICA ((122.56 ± 8.09) %,(134.52 ± 9.09) %,and (149.89 ± 9.16) % (the ratio of the above are compared with the blank control group),P < 0.05).(5) The mitochondrial membrane potential was significantly higher in the GSNO group (0.84 ± 0.04) than in the blank control group (0.12 ±0.12),which could be significantly reduced by pretreatment with high,medium and low concentration ICA ((0.57 ± 0.08),(0.63 ± 0.02),(0.66 ± 0.04) vs.(0.84 ±.0.04),all P < 0.05).(6) The expression of AKT/p-AKT was significantly lower in GSNO group than in the blank control group (P < 0.05),which could be significantly upregulated by pretreatment with high,medium and low concentration ICA in a concentration-dependent manner,above effects could be blocked by LY294002 (all P < 0.05).(7) The expression of P53 was significantly higher in GSNO group than in the blank control group (P < 0.05),which could be significantly down regulated by pretreatment with high,medium and low concentration ICA in a concentration-dependent manner,above effects could be blocked by LY294002 (all P < 0.05).(8) The expression of CYC and caspase-3 was significantly reduced in the mitochondria and increased in cytoplasm post GSNO treatment compared to blank control group,which could be reversed by pretreatment with high,medium and low concentration ICA(all P < 0.05).(9) The expression of eNOS/peNOS was similar between GSNO and the blank control group,while it was significantly upregulated by pretreatment with high,medium and low concentration ICA and this effect could be blocked by LY294002 (all P < 0.05).Conclusion Icariin could reduce GSNO induced endothelial cell apoptosis through activating AKT pathway and downregulating P53 activity.
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