新型干细胞iPSC-MSCs缓解ox-LDL诱导的HUVEC氧化应激损伤的研究
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摘要
目的探讨诱导性多能干细胞源性间充质干细胞(iPSC-MSCs)对氧化型低密度脂蛋白(ox-LDL)诱导的人脐静脉内皮细胞(HUVEC)氧化应激损伤的缓解作用及其机制。方法建立人脐静脉内皮细胞(HUVEC)的ox-LDL损伤模型,采取条件培养基预孵育和Transwell装置共培养2种方式发挥iPSC-MSCs的作用;细胞增殖-毒性检测(CCK8)法测定细胞活力,DCFH-ROS荧光探针法检测细胞内活性氧(ROS)水平,Western Blot法测定细胞内烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶4型(NOX4)的蛋白表达,荧光定量PCR检测核因子相关因子2(Nrf2)和血红素氧合酶1(HO-1)的mRNA表达。结果与iPSC-MSCs条件培养基(CM)共孵育后,ox-LDL-HUVEC模型产生的ROS水平明显下降(P<0.05),且下降幅度与iPSC-MSC-CM提取过程中的共孵育时间有关,在共孵育24 h CM(24 hcm)组达到最大(P<0.05);与iPSC-MSCs进行Transwell共培养后,ox-LDL-HUVEC模型细胞活力显著提高(P<0.05),NOX4蛋白表达显著下降(P<0.05),Nrf2和HO-1 mRNA表达明显增加(P<0.05);iPSC-MSCs-Transwell共培养对单HUVEC的NOX4表达无明显影响,但可显著上调其Nrf2和HO-1的mRNA表达水平(P<0.05)。结论 iPSCMSCs可缓解ox-LDL引起的HUVEC细胞活力损害,该过程可能与抑制ROS水平增高,下调NOX4表达及促进Nrf2和HO-1转录增多有关,提示iPSC-MSCs在动脉粥样硬化及氧化应激性疾病方面具有应用前景。
Objective To investigate the alleviative effect of mesenchymal stem cells derived from induced pluripotent stem cell(iPSC-MSCs)on oxidized low density lipoprotein(ox-LDL)induced oxidative stress injury in human umbilical vein endothelial cells(HUVEC)and its probable mechanism. Methods The ox-LDL-HUVEC injury model was developed,and iPSC-MSCs were applied to observe its effect on the model. The application of iPSC-MSCs to HUVECs was developed in two ways:conditioned medium(CM)pre-incubation and Transwell co-culture. Cell viability was measured using cell counting kit 8(CCK8). Intracellular reactive oxygen species(ROS)level was measured using DCFH-ROS probing method. The expression of nicotinamide adenine dinucleotide phosphate(NADPH) oxidase type 4(NOX4) protein was detected by Western Blot. Quantitive real-time PCR was applied to detect the mRNA expression of Nuclear Factor Erythroid 2-Related Factor 2(Nrf2)and heme oxygenase-1(HO-1). Results After pre-incubation with iPSC-MSC-CM,the ROS level produced by the ox-LDL-HUVEC model was significantly lowered(P<0.05). The extent of this effect was related to the coincubation time-length during the iPSC-MSC-CM extraction process and reached its maximum in the 24-hour co-incubation group(24 hcm,P<0.05). After the Transwell co-culture applied with iPSC-MSCs,the model demonstrated a significant higher level of cell viability(P<0.05),down regulation of NOX4 protein expression(P<0.05)and up regulation of Nrf2 and HO-1 transcription(P<0.05),compared to the model group that wasn't applied with co-culture. And the iPSC-MSCsTranswell co-culture demonstrated no effect on the expression of NOX4 but a significant up regulation of Nrf2 and HO-1 mRNA expression(P<0.05)in HUVEC. Conclusion iPSC-MSCs were capable of alleviating the damage of cell viability induced by ox-LDL in HUVEC,which was probably related to the inhibition of intracellular ROS increase,the down regulation of NOX4 protein expression and up regulation of Nrf2 and HO-1 transcription. The iPSC-MSCs have a great prospect of application to atherosclerosis and oxidative stress injury-related diseases.
Objective To investigate the alleviative effect of mesenchymal stem cells derived from induced pluripotent stem cell(iPSC-MSCs)on oxidized low density lipoprotein(ox-LDL)induced oxidative stress injury in human umbilical vein endothelial cells(HUVEC)and its probable mechanism. Methods The ox-LDL-HUVEC injury model was developed,and iPSC-MSCs were applied to observe its effect on the model. The application of iPSC-MSCs to HUVECs was developed in two ways:conditioned medium(CM)pre-incubation and Transwell co-culture. Cell viability was measured using cell counting kit 8(CCK8). Intracellular reactive oxygen species(ROS)level was measured using DCFH-ROS probing method. The expression of nicotinamide adenine dinucleotide phosphate(NADPH) oxidase type 4(NOX4) protein was detected by Western Blot. Quantitive real-time PCR was applied to detect the mRNA expression of Nuclear Factor Erythroid 2-Related Factor 2(Nrf2)and heme oxygenase-1(HO-1). Results After pre-incubation with iPSC-MSC-CM,the ROS level produced by the ox-LDL-HUVEC model was significantly lowered(P<0.05). The extent of this effect was related to the coincubation time-length during the iPSC-MSC-CM extraction process and reached its maximum in the 24-hour co-incubation group(24 hcm,P<0.05). After the Transwell co-culture applied with iPSC-MSCs,the model demonstrated a significant higher level of cell viability(P<0.05),down regulation of NOX4 protein expression(P<0.05)and up regulation of Nrf2 and HO-1 transcription(P<0.05),compared to the model group that wasn't applied with co-culture. And the iPSC-MSCsTranswell co-culture demonstrated no effect on the expression of NOX4 but a significant up regulation of Nrf2 and HO-1 mRNA expression(P<0.05)in HUVEC. Conclusion iPSC-MSCs were capable of alleviating the damage of cell viability induced by ox-LDL in HUVEC,which was probably related to the inhibition of intracellular ROS increase,the down regulation of NOX4 protein expression and up regulation of Nrf2 and HO-1 transcription. The iPSC-MSCs have a great prospect of application to atherosclerosis and oxidative stress injury-related diseases.
引文
[1]Zambon A,Pauletto P,Crepaldi G.Review article:the metabolic syndrome-a chronic cardiovascular inflammatory condition[J].Aliment Pharmacol Ther,2005,22 Suppl 2:20-23.
[2]Badimon L,Storey RF,Vilahur G.Update on lipids,inflammation and atherothrombosis[J].Thromb Haemost,2011,105 Suppl 1:S34-S42.
[3]Dong Y,Fernandes C,Liu Y,et al.Role of endoplasmic reticulum stress signalling in diabetic endothelial dysfunction and atherosclerosis[J].Diab Vasc Dis Res,2017,14(1):14-23.
[4]Yamanaka S.Induced pluripotent stem cells:past,present,and future[J].Cell Stem Cell,2012,10(6):678-684.
[5]Wang ZX,Wang CQ,Li XY,et al.Mesenchymal stem cells alleviate atherosclerosis by elevating number and function of CD4(+)CD25(+)FOXP3(+)regulatory T-cells and inhibiting macrophage foam cell formation[J].Mol Cell Biochem,2015,400(1-2):163-172.
[6]石卉,梁美玲,杨至圣,等.iPSC-MSCs抑制ApoE(-/-)小鼠动脉粥样硬化斑块形成及其机制[J].热带医学杂志,2017,17(4):452-455,499.
[7]Zhang G,Zou X,Huang Y,et al.Mesenchymal stromal cellderived extracellular vesicles protect against acute kidney injury through anti-oxidation by enhancing Nrf2/ARE activation in rats[J].Kidney Blood Press Res,2016,41(2):119-128.
[8]Kretlow JD,Jin YQ,Liu W,et al.Donor age and cell passage affects differentiation potential of murine bone marrow-derived stem cells[J].BMC Cell Biol,2008,9:60.
[9]Lian Q,Zhang Y,Zhang J,et al.Functional mesenchymal stem cells derived from human induced pluripotent stem cells attenuate limb ischemia in mice[J].Circulation,2010,121(9):1113-1123.
[10]梁美玲,曾武涛,陈伟燕,等.新型干细胞iPSC-MSCs对泡沫细胞胆固醇含量的影响及调节机制研究[J].热带医学杂志,2015,15(8):1010-1013,1056.
[11]Shi H,Liang M,Chen W,et al.Human induced pluripotent stem cell-derived mesenchymal stem cells alleviate atherosclerosis by modulating inflammatory responses[J].Mol Med Rep,2018,17(1):1461-1468.
[12]董曦,孙桂波,罗云,等.间充质干细胞条件培养基激活Nrf2/ARE通路对抗H2O2致H9C2细胞的氧化应激损伤[J].中国病理生理杂志,2015,31(6):961-966.
[13]Gamkrelidze M,Mamamtavrishvili N,Bejitashvili N,et al.Role of oxidative stress in pathogenesis of atherosclerosis[J].Georgian Med News,2008,(163):54-57.
[14]王梦龙,万军.NADPH氧化酶在常见心血管疾病中的表达与作用[J].中国医药导报,2015,12(26):34-38.
[15]Konior A,Schramm A,Czesnikiewicz-Guzik M,et al.NADPHoxidases in vascular pathology[J].Antioxid Redox Signal,2014,20(17):2794-2814.
[16]陈瑗,周玫.自由基-炎症与衰老性疾病[M].北京:科学出版社,2007.
[17]Guo S,Chen X.The human Nox4:gene,structure,physiological function and pathological significance[J].J Drug Target,2015,23(10):888-896.
[18]鞠传霞,侯琳,丛培阳,等.扇贝糖胺聚糖对OX-LDL诱导血管内皮细胞氧化应激损伤的抑制作用机制研究[J].中国药房,2015,26(28):3938-3941.
[19]Yamamoto M,Kensler TW,Motohashi H.The KEAP1-NRF2System:a thiol-based sensor-effector apparatus for maintaining redox homeostasis[J].Physiol Rev,2018,98(3):1169-1203.
[20]Dai Y,Zhang H,Zhang J,et al.Isoquercetin attenuates558oxidative stress and neuronal apoptosis after ischemia/reperfusion injury via Nrf2-mediated inhibition of the NOX4/ROS/NF-kappaBpathway[J].Chem Biol Interact,2018,284:32-40.
[21]Ding Y,Zhang B,Zhou K,et al.Dietary ellagic acid improves oxidant-induced endothelial dysfunction and atherosclerosis:role of Nrf2 activation[J].Int J Cardiol,2014,175(3):508-514.
[22]Zrelli H,Kusunoki M,Miyazaki H.Role of hydroxytyrosoldependent regulation of HO-1 expression in promoting wound healing of vascular endothelial cells via Nrf2 De Novo synthesis and stabilization[J].Phytother Res,2015,29(7):1011-1018.
[23]Huang CS,Lin AH,Liu CT,et al.Isothiocyanates protect against oxidized LDL-induced endothelial dysfunction by upregulating Nrf2-dependent antioxidation and suppressing NFκB activation[J].Mol Nutr Food Res,2013,57(11):1918-1930.
[2]Badimon L,Storey RF,Vilahur G.Update on lipids,inflammation and atherothrombosis[J].Thromb Haemost,2011,105 Suppl 1:S34-S42.
[3]Dong Y,Fernandes C,Liu Y,et al.Role of endoplasmic reticulum stress signalling in diabetic endothelial dysfunction and atherosclerosis[J].Diab Vasc Dis Res,2017,14(1):14-23.
[4]Yamanaka S.Induced pluripotent stem cells:past,present,and future[J].Cell Stem Cell,2012,10(6):678-684.
[5]Wang ZX,Wang CQ,Li XY,et al.Mesenchymal stem cells alleviate atherosclerosis by elevating number and function of CD4(+)CD25(+)FOXP3(+)regulatory T-cells and inhibiting macrophage foam cell formation[J].Mol Cell Biochem,2015,400(1-2):163-172.
[6]石卉,梁美玲,杨至圣,等.iPSC-MSCs抑制ApoE(-/-)小鼠动脉粥样硬化斑块形成及其机制[J].热带医学杂志,2017,17(4):452-455,499.
[7]Zhang G,Zou X,Huang Y,et al.Mesenchymal stromal cellderived extracellular vesicles protect against acute kidney injury through anti-oxidation by enhancing Nrf2/ARE activation in rats[J].Kidney Blood Press Res,2016,41(2):119-128.
[8]Kretlow JD,Jin YQ,Liu W,et al.Donor age and cell passage affects differentiation potential of murine bone marrow-derived stem cells[J].BMC Cell Biol,2008,9:60.
[9]Lian Q,Zhang Y,Zhang J,et al.Functional mesenchymal stem cells derived from human induced pluripotent stem cells attenuate limb ischemia in mice[J].Circulation,2010,121(9):1113-1123.
[10]梁美玲,曾武涛,陈伟燕,等.新型干细胞iPSC-MSCs对泡沫细胞胆固醇含量的影响及调节机制研究[J].热带医学杂志,2015,15(8):1010-1013,1056.
[11]Shi H,Liang M,Chen W,et al.Human induced pluripotent stem cell-derived mesenchymal stem cells alleviate atherosclerosis by modulating inflammatory responses[J].Mol Med Rep,2018,17(1):1461-1468.
[12]董曦,孙桂波,罗云,等.间充质干细胞条件培养基激活Nrf2/ARE通路对抗H2O2致H9C2细胞的氧化应激损伤[J].中国病理生理杂志,2015,31(6):961-966.
[13]Gamkrelidze M,Mamamtavrishvili N,Bejitashvili N,et al.Role of oxidative stress in pathogenesis of atherosclerosis[J].Georgian Med News,2008,(163):54-57.
[14]王梦龙,万军.NADPH氧化酶在常见心血管疾病中的表达与作用[J].中国医药导报,2015,12(26):34-38.
[15]Konior A,Schramm A,Czesnikiewicz-Guzik M,et al.NADPHoxidases in vascular pathology[J].Antioxid Redox Signal,2014,20(17):2794-2814.
[16]陈瑗,周玫.自由基-炎症与衰老性疾病[M].北京:科学出版社,2007.
[17]Guo S,Chen X.The human Nox4:gene,structure,physiological function and pathological significance[J].J Drug Target,2015,23(10):888-896.
[18]鞠传霞,侯琳,丛培阳,等.扇贝糖胺聚糖对OX-LDL诱导血管内皮细胞氧化应激损伤的抑制作用机制研究[J].中国药房,2015,26(28):3938-3941.
[19]Yamamoto M,Kensler TW,Motohashi H.The KEAP1-NRF2System:a thiol-based sensor-effector apparatus for maintaining redox homeostasis[J].Physiol Rev,2018,98(3):1169-1203.
[20]Dai Y,Zhang H,Zhang J,et al.Isoquercetin attenuates558oxidative stress and neuronal apoptosis after ischemia/reperfusion injury via Nrf2-mediated inhibition of the NOX4/ROS/NF-kappaBpathway[J].Chem Biol Interact,2018,284:32-40.
[21]Ding Y,Zhang B,Zhou K,et al.Dietary ellagic acid improves oxidant-induced endothelial dysfunction and atherosclerosis:role of Nrf2 activation[J].Int J Cardiol,2014,175(3):508-514.
[22]Zrelli H,Kusunoki M,Miyazaki H.Role of hydroxytyrosoldependent regulation of HO-1 expression in promoting wound healing of vascular endothelial cells via Nrf2 De Novo synthesis and stabilization[J].Phytother Res,2015,29(7):1011-1018.
[23]Huang CS,Lin AH,Liu CT,et al.Isothiocyanates protect against oxidized LDL-induced endothelial dysfunction by upregulating Nrf2-dependent antioxidation and suppressing NFκB activation[J].Mol Nutr Food Res,2013,57(11):1918-1930.