红枣色素对H_2O_2诱导血管内皮细胞损伤的保护作用
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摘要
探讨红枣色素对H_2O_2诱导人血管内皮细胞(human umbilical vein endothelial cells,HUVECs)损伤的保护作用。体外培养HUVECs,随机分为6组:空白对照组、H_2O_2损伤组、红枣色素低、中、高剂量组(50、150、250 mg/L红枣色素)和阳性对照组(50μmol/L VE),采用显微镜观察细胞形态,四甲基偶氮盐法检测细胞增殖活力,试剂盒法测定乳酸脱氢酶(lactate dehydrogense,LDH)活性,流式细胞仪检测细胞凋亡率和线粒体膜电位水平,透射电子显微镜观察细胞超微结构。结果表明,红枣色素可保护H_2O_2对HUVECs的损伤作用,能明显改善受损细胞形态、超微结构,显著降低细胞增殖抑制率、LDH活性、细胞凋亡率,明显提高线粒体膜电位水平(P<0.05),且存在明显的剂量依赖关系。由此推断,红枣色素对氧化应激诱导血管内皮细胞损伤具有一定的保护作用,其机制可能与其抗氧化、抑制细胞凋亡和维持线粒体膜电位有关。
This research was designed to explore the protective effect of Chinese red jujube pigment(CRJP) on human vascular endothelial cell injury induced by oxidative hydrogen in vitro. For this purpose, human vascular endothelial cells were cultured in vitro and randomly divided into 6 groups: control group, oxidative hydrogen injury group, CRJP low-, medium-and high-dose groups(50, 150, 250 mg/L), and positive control group(50 μmol/L VE). Cellular morphology, proliferation viability, LDH activity, apoptosis rate, mitochondrial membrane potential and ultrastructure of all groups were determined by inverted microscope, MTT assay, kit assay, flow cytometry and transmission electron microscope, respectively. The results showed that CRJP treatment at various doses had a protective effect on vascular endothelial cell injury induced by oxidative hydrogen. Cellular morphology and ultrastructure of the CRJP intervention groups were improved obviously along with a significant decrease in proliferation inhibition rate, LDH activity and apoptosis rate and a significant increase in mitochondrial membrane potential(P < 0.05) in a dose-dependent manner. These results indicate that Chinese red jujube pigment has a protective effect on human vascular endothelial cell injury induced by oxidative hydrogenin vitro and the underlying mechanism may be related to its antioxidant capacity, and its ability to inhibit apoptosis and maintain mitochondrial membrane potential.
This research was designed to explore the protective effect of Chinese red jujube pigment(CRJP) on human vascular endothelial cell injury induced by oxidative hydrogen in vitro. For this purpose, human vascular endothelial cells were cultured in vitro and randomly divided into 6 groups: control group, oxidative hydrogen injury group, CRJP low-, medium-and high-dose groups(50, 150, 250 mg/L), and positive control group(50 μmol/L VE). Cellular morphology, proliferation viability, LDH activity, apoptosis rate, mitochondrial membrane potential and ultrastructure of all groups were determined by inverted microscope, MTT assay, kit assay, flow cytometry and transmission electron microscope, respectively. The results showed that CRJP treatment at various doses had a protective effect on vascular endothelial cell injury induced by oxidative hydrogen. Cellular morphology and ultrastructure of the CRJP intervention groups were improved obviously along with a significant decrease in proliferation inhibition rate, LDH activity and apoptosis rate and a significant increase in mitochondrial membrane potential(P < 0.05) in a dose-dependent manner. These results indicate that Chinese red jujube pigment has a protective effect on human vascular endothelial cell injury induced by oxidative hydrogenin vitro and the underlying mechanism may be related to its antioxidant capacity, and its ability to inhibit apoptosis and maintain mitochondrial membrane potential.
引文
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[23]杨阳,段维勋,金振晓,等.姜黄素对血管内皮细胞过氧化氢损伤的保护作用及其机制研究[J].中国体外循环杂志,2012,9(4):239-242.DOI:10.3969/j.issn.1672-1403.2011.04.015.
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[25]贾国荣,俞小瑞,李红波,等.应用流式细胞术检测17β-雌二醇对H2O2诱导的视网膜神经细胞凋亡的保护作用[J].西安交通大学学报(医学版),2008,29(1):47-50.
[26]QIANG Hui,WANG Kunzheng,ZHANG Chen,et al.Panax notoginseng saponins protect rabbit bone marrow stromal cells against hydrogen peroxide-induced apoptosis[J].Journal of Chinese Integrative Medicine,2010,8(2):131-137.DOI:10.3736/jcim20100207.
[27]BYUN C H,KOH J M,KIM D K,et al.Alpha-lipoic acid inhibits TNFalpha-induced apoptosis in human bone marrow stromal cells[J].Journal of Bone and Mineral Research:the Official Journal of the American Society for Bone and Mineral Research,2005,20(7):1125-1135.
[28]李妍,赵东海,张巍,等.柴胡皂苷d对人神经母细胞瘤SH-SY5Y细胞线粒体膜电位及细胞凋亡的影响[J].上海中医药杂志,2016,50(6):90-93.DOI:10.16305/j.1007-1334.2016.06.028.
[29]LEE Y,GUSTAFSSON A B.Role of apoptosis in cardiovascular disease[J].Apoptosis,2009,14(4):536-548.DOI:10.1007/s10495-008-0302-x.
[30]YANG J,HAN Y,CHEN C,et al.EGCG attenuates high glucoseinduced endothelial cell inflammation by suppression of PKC and NF-κB signaling in human umbilical vein endothelial cells[J].Life Sciences,2013,92(10):589-597.DOI:10.1016/j.lfs.2013.01.025.
[2]PAPEZIKOVA M,PEKAROVA H,KOLAIOVA A,et al.Uric acid modulates vascular endothelial function through the down regulation of nitric oxide production[J].Free Radical Reseach,2013,47(2):82-88.DOI:10.3109/10715762.2012.747677.
[3]HULSMANS M,DOOREN E V,HOLVOET P.Mitochondrial reactive oxygen species and risk of atherosclerosis[J].Current Atherosclerosis Reports,2012,14(3):264-276.DOI:10.1007/s11883-012-0237-0.
[4]FEDERICO A,CARDAIOLI E,POZZO P D,et al.Mitochondria,oxidative stress and neurodegeneration[J].Journal of the Neurological Sciences,2012,322(1/2):254-262.DOI:10.1016/j.jns.2012.05.030.
[5]LI H G,HORKE S,FORSTERMANN U.Oxidative stress in vascular disease and its pharmacological prevention[J].Trends in Pharmacological Sciences,2013,34(6):313-319.DOI:10.1016/j.tips.2013.03.007.
[6]马奇虎.枣皮红色素的提取、纯化及稳定性研究[D].银川:宁夏大学,2014:35-36.
[7]ZHANG Qiong,SHEN Guangning,WANG Shuzhen,et al.Identification of pigments from jujube fruit skin[J].农业科学与技术,2010,11(4):110-112;183.DOI:10.16175/j.cnki.1009-4229.2010.04.033.
[8]向延菊,王大伟.利用微波技术提取红枣色素工艺研究[J].保鲜与加工,2010,10(1):45-47.DOI:10.3969/j.issn.1009-6221.2010.01.014.
[9]鲁晓翔.黄酮类化合物抗氧化作用机制研究进展[J].食品研究与开发,2012,33(3):220-224.DOI:10.3969/j.issn.1005-6521.2012.03.064.
[10]赵文恩,李茜倩.F R A P法测定大枣枣皮红色素的总抗氧化能力[J].郑州大学学报(工学版),2011,32(3):28-30.DOI:10.3969/j.issn.1671-6833.2011.03.007.
[11]李勇.大枣枣皮红色素的分离、生物活性及稳定性的研究[D].郑州:郑州大学,2013:39-43.
[12]郑安然,邵佩兰,郭晓丹,等.枣皮红色素结构初步鉴定及抗氧化活性研究[J].食品科技,2016,41(11):230-235.DOI:10.13684/j.cnki.spkj.2016.11.050.
[13]邵佩兰,徐明,郭晓丹,等.红枣色素中活性成分含量与抗氧化活性的相关性[J].食品工业科技,2016,37(17):171-174.DOI:10.13386/j.issn1002-0306.2016.17.025.
[14]郑安然,邵佩兰,郭晓丹,等.提取方法对红枣色素抗氧化活性的影响[J].北方园艺,2016(23):129-133.DOI:10.11937/bfyy.201623029.
[15]王宇霆,周海燕,吕小翠.白藜芦醇对过氧化氢氧化应激损伤人脐静脉血管内皮细胞的保护作用[J].中华医学杂志,2013,93(15):1174-1177.DOI:10.3760/cma.j.issn.0376-2491.2013.15.015.
[16]LI Zilin,LIU Jincheng,HU Jing,et al.Protective effects of hyperoside against human umbilical vein endothelial cell damage induced by hydrogen peroxide[J].Journal of Ethnopharmacology,2012,139(2):388-394.DOI:10.1016/j.jep.2011.11.020.
[17]SHEN Yafen,WU Xin,CAO Gang,et al.Effect of 5-HMF on protection of hydrogen peroxide-induced injury in human umbilical vein endothelial cells[J].Journal of Chinese Pharmaceutical Sciences,2015,24(3):164-168.DOI:10.5246/jcps.2015.03.020.
[18]WANG L,HAO Q,WANG Y D,et al.Protective effects of dehydroepiandrosterone on atherosclerosis in ovariectomized rabbits via all eviating in flam matory injury in end othelial cells[J].Atherosclerosis,2011,214(1):47-57.DOI:10.1016/j.atherosclerosis.2010.07.043.
[19]唐香,程训民,江时森,等.替米沙坦对糖尿病大鼠心肌线粒体膜电位及心肌细胞凋亡的作用[J].解放军医学杂志,2010,35(7):792-779.
[20]RAMOS J W.The regulation of extracellular signal-regulated kinase(ERK)in mammalian cells[J].International Journal of Biochemistry&Cell Biology,2008,40(12):2707-2719.DOI:10.1016/j.biocel.2008.04.009.
[21]徐春华.MTT法检测大豆异黄酮对癌细胞的生长抑制作用[J].中央民族大学学报(自然科学版),2012,21(1):58-62.DOI:10.3969/j.issn.1005-8036.2012.01.014.
[22]宋欢欢.ICA对H2O2诱导的血管内皮细胞内质网应激损伤的拮抗作用及机制[D].西安:第四军医大学,2014:23-26.
[23]杨阳,段维勋,金振晓,等.姜黄素对血管内皮细胞过氧化氢损伤的保护作用及其机制研究[J].中国体外循环杂志,2012,9(4):239-242.DOI:10.3969/j.issn.1672-1403.2011.04.015.
[24]洪浩,傅淑平,偶晨,等.梓醇对高糖所致血管内皮细胞损伤的保护作用[J].世界科学技术(中医药现代化),2015,17(4):846-849.DOI:10.11842/wst.2015.04.016.
[25]贾国荣,俞小瑞,李红波,等.应用流式细胞术检测17β-雌二醇对H2O2诱导的视网膜神经细胞凋亡的保护作用[J].西安交通大学学报(医学版),2008,29(1):47-50.
[26]QIANG Hui,WANG Kunzheng,ZHANG Chen,et al.Panax notoginseng saponins protect rabbit bone marrow stromal cells against hydrogen peroxide-induced apoptosis[J].Journal of Chinese Integrative Medicine,2010,8(2):131-137.DOI:10.3736/jcim20100207.
[27]BYUN C H,KOH J M,KIM D K,et al.Alpha-lipoic acid inhibits TNFalpha-induced apoptosis in human bone marrow stromal cells[J].Journal of Bone and Mineral Research:the Official Journal of the American Society for Bone and Mineral Research,2005,20(7):1125-1135.
[28]李妍,赵东海,张巍,等.柴胡皂苷d对人神经母细胞瘤SH-SY5Y细胞线粒体膜电位及细胞凋亡的影响[J].上海中医药杂志,2016,50(6):90-93.DOI:10.16305/j.1007-1334.2016.06.028.
[29]LEE Y,GUSTAFSSON A B.Role of apoptosis in cardiovascular disease[J].Apoptosis,2009,14(4):536-548.DOI:10.1007/s10495-008-0302-x.
[30]YANG J,HAN Y,CHEN C,et al.EGCG attenuates high glucoseinduced endothelial cell inflammation by suppression of PKC and NF-κB signaling in human umbilical vein endothelial cells[J].Life Sciences,2013,92(10):589-597.DOI:10.1016/j.lfs.2013.01.025.