苯所致再生障碍性贫血小鼠骨髓细胞活性氧和细胞周期的改变
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摘要
目的探讨骨髓细胞活性氧(ROS)和细胞周期在苯所致小鼠再生障碍性贫血中的作用。方法取无特定病原体级雄性CD1小鼠随机分为对照组和染毒组,每组10只。染毒组小鼠予皮下注射剂量为2 mL/kg体质量的苯和玉米油(1∶1,V/V)混合物,对照组小鼠皮下注射等体积玉米油;每周注射3次,累计注射25次。染毒结束后,检测小鼠周围血血常规和网织红细胞百分比,对小鼠股骨进行组织病理学检查,以固相萃取-气相色谱-质谱法检测小鼠血液、肝脏和骨髓中苯及其代谢产物氢醌、苯酚的水平,以流式细胞术检测骨髓单个核细胞(BMMNCs)中ROS水平与细胞周期,以免疫印迹法检测BMMNCs中细胞周期蛋白D1(Cyclin D1)和周期蛋白依赖性激酶-4(CDK4)的表达。结果自第10次染毒起,染毒组小鼠体质量低于同时间点对照组(P<0.05)。染毒结束后,染毒组小鼠周围血中白细胞、红细胞、血小板的计数和血红蛋白水平、网织红细胞百分比均低于对照组(P<0.05);骨髓组织病理学检查示,染毒组小鼠骨髓造血细胞减少,非造血细胞增多。本研究成功建立了苯所致再生障碍性贫血小鼠模型。与对照组比较,染毒组小鼠血液、肝脏及骨髓中苯、氢醌、苯酚的水平均升高(P<0.05)。对于染毒组小鼠,苯水平由高至低依次为血液、肝脏和骨髓;氢醌和苯酚主要蓄积在血液和骨髓中。与对照组比较,染毒组小鼠BMMNCs中ROS水平、S期细胞比例和CyclinD1、CDK4的蛋白相对表达水平均升高(P<0.01),G1期细胞比例降低(P<0.01)。结论苯及其代谢物引起的骨髓细胞ROS水平升高及S期细胞阻滞在苯所致小鼠再生障碍性贫血发生发展中具有重要作用。
Objective To explore the effect of reactive oxygen species(ROS) and cell cycle in bone marrow cells in benzene-induced aplastic anemia(AA) mouse model. Methods Specific pathogens free male CD1 mice were randomly divided into control group and exposure group(n=10, each group). The mice in exposure group were subcutaneously injected with benzene at a dose of 2 mL/kg body weigh diluted 1 ∶1 in corn oil, while the mice in control group were treated with equal volume of corn oil, 3 times a week for a total of 25 times. After exposure, the blood routine and reticulocyte percentage of peripheral blood of mice were examined. The femur histopathology was performed. The levels of benzene and its metabolites hydroquinone, and phenol in blood, liver and bone marrow were tested by solid-phase extraction gas chromatography mass spectrometry. The level of ROS and the changes of cell cycle in bone marrow mononuclear cells(BMMNCs) were determined by flow cytometry. The protein expression of Cyclin D1 and cyclin-dependent kinase 4(CDK4) in BMMNCs was detected by Western blot. Results Since the 10 th benzene exposure, the body mass of mice in the exposure group was lower than that in the control group at the same time point(P<0.05). After the benzene exposure, all the counts of white blood cell, red blood cell, platelet, and hemoglobin level and reticulocyte percentage in peripheral blood of mice in the exposure group were decreased when compared with the control group(P<0.05). Bone marrow histopathological examination showed that bone marrow hematopoietic cells were decreased and non-hematopoietic cells were increased in the exposure group. In this study, a mouse model of AA induced by benzene was successfully established. The levels of benzene, hydroquinone and phenol in exposure group increased in blood, liver, and bone marrow compared to control group(P<0.05). Furthermore, the level of benzene from high to low were blood, liver and bone marrow, while the levels of hydroquinone and phenol were mainly stored in the blood and bone marrow in exposure group. Compared with the control group, the level of ROS, S phase fraction, and the relative protein expression of Cyclin D1 and CDK4 in BMMNCs increased, while the G1 phase fraction decreased in exposure group(P<0.01). Conclusion The results suggest that benzene and its metabolites induce an increase of ROS level and S phase cell arrest, that play an important role in the pathogenesis and development of benzene-induced AA.
Objective To explore the effect of reactive oxygen species(ROS) and cell cycle in bone marrow cells in benzene-induced aplastic anemia(AA) mouse model. Methods Specific pathogens free male CD1 mice were randomly divided into control group and exposure group(n=10, each group). The mice in exposure group were subcutaneously injected with benzene at a dose of 2 mL/kg body weigh diluted 1 ∶1 in corn oil, while the mice in control group were treated with equal volume of corn oil, 3 times a week for a total of 25 times. After exposure, the blood routine and reticulocyte percentage of peripheral blood of mice were examined. The femur histopathology was performed. The levels of benzene and its metabolites hydroquinone, and phenol in blood, liver and bone marrow were tested by solid-phase extraction gas chromatography mass spectrometry. The level of ROS and the changes of cell cycle in bone marrow mononuclear cells(BMMNCs) were determined by flow cytometry. The protein expression of Cyclin D1 and cyclin-dependent kinase 4(CDK4) in BMMNCs was detected by Western blot. Results Since the 10 th benzene exposure, the body mass of mice in the exposure group was lower than that in the control group at the same time point(P<0.05). After the benzene exposure, all the counts of white blood cell, red blood cell, platelet, and hemoglobin level and reticulocyte percentage in peripheral blood of mice in the exposure group were decreased when compared with the control group(P<0.05). Bone marrow histopathological examination showed that bone marrow hematopoietic cells were decreased and non-hematopoietic cells were increased in the exposure group. In this study, a mouse model of AA induced by benzene was successfully established. The levels of benzene, hydroquinone and phenol in exposure group increased in blood, liver, and bone marrow compared to control group(P<0.05). Furthermore, the level of benzene from high to low were blood, liver and bone marrow, while the levels of hydroquinone and phenol were mainly stored in the blood and bone marrow in exposure group. Compared with the control group, the level of ROS, S phase fraction, and the relative protein expression of Cyclin D1 and CDK4 in BMMNCs increased, while the G1 phase fraction decreased in exposure group(P<0.01). Conclusion The results suggest that benzene and its metabolites induce an increase of ROS level and S phase cell arrest, that play an important role in the pathogenesis and development of benzene-induced AA.
引文
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[22] GRIGORYAN H,EDMANDS W M B,LAN Q,et al.Adductomic signatures of benzene exposure provide insights into cancer induction[J].Carcinogenesis,2018,39(5):661-668.
[23] PENG D,JIAXING W,CHUNHUI H,et al.Study on the cytogenetic changes induced by benzene and hydroquinone in human lymphocytes[J].Hum Exp Toxicol,2012,31(4):322-335.
[24] 韦海燕.苯中毒的骨髓造血细胞microRNA表达谱与miR-34a-5p对苯毒性的调控研究[D].南京:东南大学,2016.
[25] 陈玉婷,云林,徐龙妹,等.MicroRNA-7在DNA损伤修复中研究进展,[J].中国职业医学,2015,42(5):575-578.
[26] 董锁花,王芳,包金风.DNA甲基化对细胞周期的调控[J].中国细胞生物学学报,2018,40(12):2083-2089.
[27] 付小娟.生物钟基因Per1对Cyclins-CDKs-CKIs细胞周期网络的调控作用及抑癌机理[D].重庆:重庆医科大学,2016.
[28] 袁丹.雌激素及其受体拮抗剂对人子宫内膜样癌Ishikawa细胞形态变化及p57kip2、Cyclin D1、CDK4表达影响的研究[D].遵义:遵义医学院,2018.
[29] HE S,YANG S,NIU M,et al.HMG-box transcription factor 1:a positive regulator of the G1/S transition through the Cyclin-CDK-CDKI molecular network in nasopharyngeal carcinoma[J].Cell Death Dis,2018,9(2):100.
[30] PEDRAZA N,CEMELI T,MONSERRAT M V,et al.Regulation of small GTPase activity by G1 cyclins[J].Small GTPases,2019,10(1):47-53.
[31] 建方方,刘华,冯炜炜,等.CXCL5通过上调Cyclin D1促进卵巢癌细胞增殖[J].现代妇产科进展,2018,27(4):255-258.
[32] PREYA U H,LEE K T,KIM N J,et al.The natural terthiophene α-terthienylmethanol induces S phase cell cycle arrest of human ovarian cancer cells via the generation of ROS stress[J].Chem Biol Interact,2017,272:72-79.
[33] LONG M,WU J,HAO J,et al.Selenocystine-induced cell apoptosis and S-phase arrest inhibit human triple-negative breast cancer cell proliferation[J].In Vitro Cell Dev Biol Anim,2015,51(10):1077-1084.
[34] WANG J L,QUAN Q,JI R,et al.Isorhamnetin suppresses PANC-1 pancreatic cancer cell proliferation through S phase arrest[J].Biomed Pharmacother,2018,108:925-933.
[2] 刘旻,江嘉欣,陈嘉斌.低水平职业性苯接触人群健康损害及基因多态性影响研究进展[J].中国职业医学,2018,45(4):511-516.
[3] HOSTYNEK J J,LAMEL S A,MAIBACH H I.Benzene absorption in animals and man:an overview[J].Rev Environ Health,2012,27(2/3):85-101.
[4] FENGA C,GANGEMI S,COSTA C.Benzene exposure is associated with epigenetic changes (Review)[J].Mol Med Rep,2016,13(4):3401-3405.
[5] SUN R,ZHANG J,WEI H,et al.Acetyl-l-carnitine partially prevents benzene-induced hematotoxicity and oxidative stress in C3H/He mice[J].Environ Toxicol Pharmacol,2017,51:108-113.
[6] MAN Z,MENG X,SUN F,et al.Global identification of HIF-1α target genes in benzene poisoning mouse bone marrow cells[J].Int J Environ Res Public Health,2018,15(11).doi:10.3390/ijerph15112531.
[7] SUN S,ZHANG C,GAO J,et al.Benzoquinone induces ROS-dependent mitochondria-mediated apoptosis in HL-60 cells[J].Toxicol Ind Health,2018,34(4):270-281.
[8] LUO H,LIANG H,CHEN J,et al.Hydroquinone induces TK6 cell growth arrest and apoptosis through PARP-1/p53 regulatory pathway[J].Environ Toxicol,2017,32(9):2163-2171.
[9] UZMA N,KUMAR B S,HAZARI M A.Exposure to benzene induces oxidative stress,alters the immune response and expression of p53 in gasoline filling workers[J].Am J Ind Med,2010,53(12):1264-1270.
[10] TANIA M,SHAWON J,SAIF K,et al.Cordycepin downregulates Cdk-2 to interfere with cell cycle and increases apoptosis by generating ROS in cervical cancer cells:in vitro and in silico study[J].Curr Cancer Drug Targets,2019,19(2):152-159.
[11] VELASCO LEZAMA R,BARRERA ESCORCIA E,MU?OZ TORRES A,et al.A model for the induction of aplastic anemia by subcutaneous administration of benzene in mice[J].Toxicology,2001,162(3):179-191.
[12] 赵娜,宋向荣,刘秋英,等.骨髓间充质干细胞对苯所致小鼠造血损伤修复作用[J].中国职业医学,2017,44(5):537-541.
[13] XIAO Y,WANG Y,LI L,et al.Homing of chloromethylbenzoyl ammonia-labeled bone marrow mesenchymal stem cells in an immune-mediated bone marrow failure mouse model in vivo[J].Genet Mol Res,2014,13(1):11-21.
[14] HOFFMANN M J,SINKO P J,LEE Y H,et al.Pharmacokinetic studies in Tg.AC and FVB mice administered [14C] benzene either by oral gavage or intradermal injection[J].Toxicol Appl Pharmacol,2001,174(2):139-145.
[15] SUN R,XU K,ZHANG Q,et al.Plasma metabonomics investigation reveals involvement of fatty acid oxidation in hematotoxicity in Chinese benzene-exposed workers with low white blood cell count[J].Environ Sci Pollut Res Int,2018,25(32):32506-32514.
[16] JAMEBOZORGI I,MAHJOUBI F,POURYAGHOUB G,et al.Micronucleus,nucleoplasmic bridge,and nuclear budding in peripheral blood cells of workers exposed to low level benzene[J].Int J Occup Environ Med,2016,7(4):227-233.
[17] 赵金垣.临床职业病学[M].3版.北京:北京大学医学出版社,2017:347-351.
[18] 杨文睿,武志洁,赵馨,等.骨髓残存造血评估指标:重型再生障碍性贫血患者免疫抑制治疗疗效的重要预后因素[J].中华血液学杂志,2014,35(12):1095-1099.
[19] 陈萍,宋善俊.再生障碍性贫血诊断治疗专家共识解读[J].临床内科杂志,2016,33(10):719-720.
[20] CARBONARI D,CHIARELLA P,MANSI A,et al.Biomarkers of susceptibility following benzene exposure:influence of genetic polymorphisms on benzene metabolism and health effects[J].Biomark Med,2016,10(2):145-163.
[21] BADHAM H J,RENAUD S J,WAN J,et al.Benzene-initiated oxidative stress:effects on embryonic signaling pathways[J].Chem Biol Interact,2010,184(1/2):218-221.
[22] GRIGORYAN H,EDMANDS W M B,LAN Q,et al.Adductomic signatures of benzene exposure provide insights into cancer induction[J].Carcinogenesis,2018,39(5):661-668.
[23] PENG D,JIAXING W,CHUNHUI H,et al.Study on the cytogenetic changes induced by benzene and hydroquinone in human lymphocytes[J].Hum Exp Toxicol,2012,31(4):322-335.
[24] 韦海燕.苯中毒的骨髓造血细胞microRNA表达谱与miR-34a-5p对苯毒性的调控研究[D].南京:东南大学,2016.
[25] 陈玉婷,云林,徐龙妹,等.MicroRNA-7在DNA损伤修复中研究进展,[J].中国职业医学,2015,42(5):575-578.
[26] 董锁花,王芳,包金风.DNA甲基化对细胞周期的调控[J].中国细胞生物学学报,2018,40(12):2083-2089.
[27] 付小娟.生物钟基因Per1对Cyclins-CDKs-CKIs细胞周期网络的调控作用及抑癌机理[D].重庆:重庆医科大学,2016.
[28] 袁丹.雌激素及其受体拮抗剂对人子宫内膜样癌Ishikawa细胞形态变化及p57kip2、Cyclin D1、CDK4表达影响的研究[D].遵义:遵义医学院,2018.
[29] HE S,YANG S,NIU M,et al.HMG-box transcription factor 1:a positive regulator of the G1/S transition through the Cyclin-CDK-CDKI molecular network in nasopharyngeal carcinoma[J].Cell Death Dis,2018,9(2):100.
[30] PEDRAZA N,CEMELI T,MONSERRAT M V,et al.Regulation of small GTPase activity by G1 cyclins[J].Small GTPases,2019,10(1):47-53.
[31] 建方方,刘华,冯炜炜,等.CXCL5通过上调Cyclin D1促进卵巢癌细胞增殖[J].现代妇产科进展,2018,27(4):255-258.
[32] PREYA U H,LEE K T,KIM N J,et al.The natural terthiophene α-terthienylmethanol induces S phase cell cycle arrest of human ovarian cancer cells via the generation of ROS stress[J].Chem Biol Interact,2017,272:72-79.
[33] LONG M,WU J,HAO J,et al.Selenocystine-induced cell apoptosis and S-phase arrest inhibit human triple-negative breast cancer cell proliferation[J].In Vitro Cell Dev Biol Anim,2015,51(10):1077-1084.
[34] WANG J L,QUAN Q,JI R,et al.Isorhamnetin suppresses PANC-1 pancreatic cancer cell proliferation through S phase arrest[J].Biomed Pharmacother,2018,108:925-933.