镉对胎盘绒毛外滋养层HTR-8/SVneo细胞内抗氧化酶活性的影响
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摘要
目的:探究氯化镉(CdCl_2)对胎盘绒毛外滋养层HTR-8/SVneo细胞内活性氧(ROS)水平和抗氧化酶活性的影响。方法:用不同浓度的CdCl_2(0、3、6、12μmol/L)处理HTR-8/SVneo细胞24 h,或者用12μmol/L CdCl_2处理HTR-8/SVneo细胞不同时间(0、6、12、24 h)后,CCK-8检测细胞活性;采用时间依赖模型,显微镜下观察细胞形态变化;流式细胞术检测细胞内ROS含量变化;试剂盒法检测细胞内超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GPx)活性及丙二醛(MDA)水平的变化;用12μmol/L CdCl_2与500μmol/L ROS清除剂NAC共处理HTR-8/SVneo细胞24 h,观察NAC对细胞的保护效应。结果:CdCl_2可以显著抑制HTR-8/SVneo细胞活性,且呈剂量和时间依赖性(P<0.01);随着CdCl_2处理时间的延长,HTR-8/SVneo细胞逐渐皱缩、变圆;细胞内ROS水平和MDA含量呈时间依赖性升高,而SOD、CAT和GPx活性呈时间依赖性降低;NAC可以显著抑制CdCl_2引起的ROS及MDA含量升高,缓解CdCl_2引起的形态损伤和抗氧化酶活性降低(P<0.05)。结论:镉可以引起HTR-8/SVneo细胞内ROS升高,并导致SOD、CAT、GPx活性降低和脂质过氧化。
Objective:To investigating the effects of CdCl_2 on reactive oxygen species(ROS) generation and antioxidant system in extravillous trophoblast HTR-8/Svneo cells.Methods:HTR-8/SVneo cells were treated with different concentrations of CdCl_2(0、3、6、12 μmol/L) for 24 h, or treated with 12 μmol/L CdCl_2 for different times(0,6, 12, 24 h). The cell viability was detected by CCK-8 assay and morphological changes were observed by microscope. The intracellular ROS level was detected by flow cytometry. The treated cells were collected for the assessments of the activities of antioxidant enzymes such as superoxide dismutase(SOD), catalase(CAT), and glutathione peroxidase(GPx) as well as the contents of malonyldialdehyde(MDA). After HTR-8/SVneo cells were treated with12 μmol/L CdCl_2 and 500 μmol/L NAC for 24 h, we detected the morphological changes, ROS generation, MDA content and antioxidant enzyme activities.Results:CdCl_2 significantly reduced the viability of HTR-8/SVneo cells in a dose-and time-dependent manner(P<0.01). With the increase of treated time, the cell morphology became shrinkage and turned round. CdCl_2 significantly increased the ROS and MDA content(P<0.05, P<0.01), however,activities of SOD, CAT, and GPx were reduced(P<0.05, P<0.01). In addition, NAC significantly alleviated Cd induced morphological changes, ROS generation, MDA content and depletion of antioxidant enzyme in HTR-8/SVneo cells(P<0.05, P<0.01).Conclusion:Our results indicate that ROS generation play an important role in Cd induced depletion of antioxidant enzyme and lipid peroxidation.
Objective:To investigating the effects of CdCl_2 on reactive oxygen species(ROS) generation and antioxidant system in extravillous trophoblast HTR-8/Svneo cells.Methods:HTR-8/SVneo cells were treated with different concentrations of CdCl_2(0、3、6、12 μmol/L) for 24 h, or treated with 12 μmol/L CdCl_2 for different times(0,6, 12, 24 h). The cell viability was detected by CCK-8 assay and morphological changes were observed by microscope. The intracellular ROS level was detected by flow cytometry. The treated cells were collected for the assessments of the activities of antioxidant enzymes such as superoxide dismutase(SOD), catalase(CAT), and glutathione peroxidase(GPx) as well as the contents of malonyldialdehyde(MDA). After HTR-8/SVneo cells were treated with12 μmol/L CdCl_2 and 500 μmol/L NAC for 24 h, we detected the morphological changes, ROS generation, MDA content and antioxidant enzyme activities.Results:CdCl_2 significantly reduced the viability of HTR-8/SVneo cells in a dose-and time-dependent manner(P<0.01). With the increase of treated time, the cell morphology became shrinkage and turned round. CdCl_2 significantly increased the ROS and MDA content(P<0.05, P<0.01), however,activities of SOD, CAT, and GPx were reduced(P<0.05, P<0.01). In addition, NAC significantly alleviated Cd induced morphological changes, ROS generation, MDA content and depletion of antioxidant enzyme in HTR-8/SVneo cells(P<0.05, P<0.01).Conclusion:Our results indicate that ROS generation play an important role in Cd induced depletion of antioxidant enzyme and lipid peroxidation.
引文
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[28]Gawe?S,Wardas M,Niedworok E,et al.Malondialdehyde(MDA)as a lipid peroxidation marker[J].Wiad Lek,2004,57(9-10):453-455.
[2]Jacobo-Estrada T,Cardenas-Gonzalez M,Santoyo-Sanchez M,et al.Evaluation of kidney injury biomarkers in rat amniotic fluid after gestational exposure to cadmium[J].J Appl Toxicol,2016,36(9):1183-1193.
[3]Mikolic A,Piasek M,Sulimanec Grgec A,et al.Oral cadmium exposure during rat pregnancy:assessment of transplacental micronutrient transport and steroidogenesis at term[J].J Appl Toxicol,2015,35(5):508-519.
[4]Blum J L,Xiong J Q,Hoffman C,et al.Cadmium associated with inhaled cadmium oxide nanoparticles impacts fetal and neonatal development and growth[J].Toxicol Sci,2012,126(2):478-486.
[5]Nakamura Y,Ohba K I,Ohta H.Participation of metal transporters in cadmium transport from mother rat to fetus[J].J Toxico Sci,2012,37(5):1035-1044.
[6]Rafati Rahimzadeh M,Rafati Rahimzadeh M,Kazemi S,et al.Cadmium toxicity and treatment:an update[J].Caspian J Intern Med,2017,8(3):135-145.
[7]Blundell C,Tess E R,Schanzer A S,et al.A microphysiological model of the human placental barrier[J].Lab Chip,2016,16(16):3065-3073.
[8]Lager S,Powell T L.Regulation of nutrient transport across the placenta[J].J Pregnancy,2012,2012:7153-7167.
[9]Levy R,Smith S D,Yusuf K,et al.Trophoblast apoptosis from pregnancies complicated by fetal growth restriction is associated with enhanced p53 expression[J].Am J Obstet Gynecol,2002,186(5):1056-1061.
[10]Burton G J,Jauniaux E.What is the placenta[J].Am J Obstet Gynecol,2015,213(4):S6.e1-S6.e4.
[11]Forbes K,Westwood M.The IGF axis and placental function.a mini review[J].Horm Res,2008,69(3):129-137.
[12]Carter A M,Enders A C,Pijnenborg R.The role of invasive trophoblast in implantation and placentation of primates[J].Philos Trans R Soc Lond B Biol Sci,2015,370(1663):20140070.
[13]Zhang W,Yang L,Kuang H,et al.Acute toxicity of quantum dots on late pregnancy mice:Effects of nanoscale size and surface coating[J].J Hazard Mater,2016,318:61-69.
[14]Wang Z,Wang H,Xu Z M,et al.Cadmium-induced teratogenicity:association with ROS-mediated endoplasmic reticulum stress in placenta[J].Toxicol Appl Pharmacol,2012,259(2):236-247.
[15]Adebambo O A,Shea D,Fry R C.Cadmium disrupts signaling of the hypoxia-inducible(HIF)and transforming growth factor(TGF-β)pathways in placental JEG-3 trophoblast cells via reactive oxygen species[J].Toxicol Appl Pharm,2018,342:108-115.
[16]Kantola M,Purkunen R,Kroger P,et al.Accumulation of cadmium,zinc,and copper in maternal blood and developmental placental tissue:differences between Finland,Estonia,and St.Petersburg[J].Environ Res,2000,83(1):54-66.
[17]Chen J,Shaikh Z A.Activation of Nrf2 by cadmium and its role in protection against cadmium-induced apoptosis in rat kidney cells[J].Toxicol Appl Pharm,2009,241(1):81-89.
[18]Zheng J L,Yuan S S,Wu C W,et al.Acute exposure to waterborne cadmium induced oxidative stress and immunotoxicity in the brain,ovary and liver of zebrafish(Danio rerio)[J].Aquat Toxicol,2016,180:36-44.
[19]汪纪仓.镉致大鼠肝细胞毒性机理研究[D].扬州大学,2010.
[20]Lei X G,Zhu J H,Cheng W H,et al.Paradoxical roles of antioxidant enzymes:basic mechanisms and health implications[J].Physiol Rev,2016,96(1):307-364.
[21]Fujii J,Homma T.Application of glutathione as antioxidative and anti-aging drugs[J].Curr Drug Metab,2015,16(7):560-571.
[22]Claustrat B,Leston J.Melatonin:physiological effects in humans[J].Neurochirurgie,2015,61(2-3):77-84.
[23]Straaten O V,Man S D,Waard M C D.Vitamin Crevisited[J].Crit Care,2014,18(4):1-13.
[24]Singh U,Devaraj S,Jialal I.Vitamin E,oxidative stress,and inflammation.[J].Annu Rev Nutr,2005,25(25):151-174.
[25]Dringen R.Metabolism and functions of glutathione in brain.[J].Prog Neurobiol,2000,62(6):649-671.
[26]Kiran Kumar K M,Naveen Kumar M,Patil R H,et al.Cadmium induces oxidative stress and apoptosis in lung epithelial cells[J].Toxicol Mech Methods,2016,26(9):658-666.
[27]Athmouni K,Belhaj D,Gammoudi S,et al.Nano-encapsulation using macrocyclic carbohydrate polymers(β-cyclodextrins)of Periploca angustifolia extract:physical stability and protective effect against cadmium-induced alterations in HepG2 cells[J].Int J Biol Macroml,2019,125:711-720.
[28]Gawe?S,Wardas M,Niedworok E,et al.Malondialdehyde(MDA)as a lipid peroxidation marker[J].Wiad Lek,2004,57(9-10):453-455.