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目的 研究氧化应激在邻苯二甲酸单(2-乙基己基)酯(mono-2-ethylhexyl-phthalate, MEHP)诱导小鼠初级精母细胞(GC-2spd)毒性中的作用及机制。方法 将培养的GC-2spd细胞分为对照组(含0.1%二甲基亚砜的无血清培养基)、MEHP染毒组(1、10、100μmol/L MEHP 3个不同剂量组)、N-乙酰半胱氨酸(N-acetylcysteine, NAC)预处理组(100μmol/L NAC+100μmol/L MEHP),MEHP染毒24 h, NAC预处理2 h, NAC的预处理浓度(100μmol/L)通过CCK-8实验确定。采用DCFH-DA探针法检测细胞内活性氧(reactive oxide species, ROS)水平，JC-1法检测线粒体膜电位，Western blot法检测细胞内线粒体凋亡通路相关蛋白和p-STAT3Tyr705、P53蛋白表达水平。结果 随着MEHP染毒剂量升高，细胞内代表活性氧水平的绿色荧光信号增多；不同剂量MEHP染毒组的线粒体膜电位均下降，表现为红绿荧光细胞比例比值减小，10和100μmol/L MEHP染毒组的红绿荧光细胞比例比值分别为5.15±1.68和4.09±1.72,明显低于对照组(7.91±1.24)(P<0.05);促凋亡蛋白Bax与抗凋亡蛋白Bcl-2表达水平比值(Bax/Bcl-2)在1、10、100μmol/L MEHP染毒组分别为1.23±0.17、2.64±0.43和4.75±0.73,与对照组(0.52±0.11)相比明显增加(P<0.05);cytochrome C蛋白表达水平在100μmol/L MEHP染毒组升高为0.83±0.09,cleaved caspase-9蛋白表达水平在10和100μmol/L MEHP染毒组升高为0.41±0.03和0.52±0.09,cleaved caspase-3蛋白表达水平在10和100μmol/L MEHP染毒组升高为0.60±0.12和0.84±0.17,与对照组相比差异均具有统计学意义(P<0.05);STAT3/p53通路中，p-STAT3Tyr705蛋白表达水平在10和100μmol/L MEHP染毒组分别下降为0.70±0.14和0.41±0.04,P53蛋白表达水平在10和100μmol/L MEHP染毒组分别升高为1.32±0.05和1.66±0.22,与对照组相比差异均具有统计学意义(P<0.05);NAC预处理后，与100μmol/L MEHP组相比，NAC预处理组ROS生成减少，线粒体膜电位升高为5.92±1.64,Bax/Bcl-2下降为1.92±0.06, cytochrome C、cleaved caspase-9和cleaved caspase-3蛋白表达水平分别下降为0.57±0.07、0.35±0.04和0.53±0.06,p-STAT3Tyr705蛋白表达水平升高为0.86±0.07,P53蛋白表达水平下降为1.01±0.06,差异均有统计学意义(P<0.05)。结论 MEHP诱导GC-2spd细胞凋亡可能与ROS介导的STAT3/p53通路调控线粒体凋亡通路有关。
Abstract：OBJECTIVE To investigate the role and possible mechanism of oxidative stress on mouse primary spermatocyte(GC-2 spd) toxicity induced by mono-2-ethylhexyl-phthalate(MEHP). METHODS GC-2 spd cells were cultured in vitro and divided into control group(serum free medium containing 0.1% DMSO), MEHP-treated group(including 1, 10, 100 μmol/L MEHP three different dose groups), N-acetylcysteine(NAC) pretreatment group(100 μmol/L NAC+100 μmol/L MEHP). GC-2 spd cells were treated with MEHP 24 h, and pretreated with NAC 2 h. The concentration of NAC(100 μmol/L) was determined by CCK-8 experiment. The levels of intracellular reactive oxide species(ROS) were detected by DCFH-DA probe, mitochondrial membrane potential was detected by JC-1 method, and the expression levels of proteins in mitochondrial apoptosis pathway, p-STAT3Tyr705 and P53 proteins were detected by Western blot. RESULTS With the increase of MEHP dose, the level of intracellular green fluorescence signal representing ROS increased. The mitochondrial membrane potential in cells exposed to different doses of MEHP decreased, which showed that the ratio of red-green fluorescent cells decreased, and the ratio of red-green fluorescent cells in 10 and 100 μmol/L groups were 5.15±1.68 and 4.09±1.72, respectively, which were significantly lower than that in the control group(7.91±1.24)(P<0.05). The ratio of expression levels of pro-apoptotic protein Bax to anti-apoptotic protein Bcl-2(Bax/Bcl-2) in 1, 10 and 100 μmol/L MEHP groups were 1.23±0.17, 2.64±0.43 and 4.75±0.73, respectively, which were significantly increased compared with the control group(0.52±0.11)(P<0.05). The expression levels of cytochrome C protein increased to 0.83±0.09 in the 100 μmol/L group, the cleaved caspase-9 protein increased to 0.41±0.03 and 0.52±0.09 in the 10 and 100 μmol/L groups, the cleaved caspase-3 protein increased to 0.60±0.12 and 0.84±0.17 in the 10 and 100 μmol/L groups, and the differences were statistically significant compared with the control group(P<0.05). In STAT3/p53 pathway, the expression levels of p-STAT3Tyr705 protein decreased to 0.70±0.14 and 0.41±0.04 in 10 and 100 μmol/L groups, the P53 increased to 1.32±0.05 and 1.66±0.22 in 10 and 100 μmol/L groups, and the differences were statistically significant compared with the control group(P<0.05). Compared with 100 μmol/L MEHP group, ROS production decreased, mitochondrial membrane potential increased to 5.92±1.64, Bax/Bcl-2 decreased to 1.92±0.06, the expression levels of cytochrome C, cleaved caspase-9, and cleaved caspase-3 proteins decreased to 0.57±0.07, 0.35±0.04 and 0.53±0.06, respectively, the expression level of p-STAT3Tyr705 protein increased to 0.86±0.07, the P53 protein decreased to 1.01±0.06 in NAC pretreatment group, and the differences were statistically significant(P<0.05). CONCLUSION MEHP induces apoptosis of GC-2 spd cells, which may be related to the mitochondrial apoptosis pathway regulated by the ROS-mediated STAT3/p53 pathway.
[1] LATINI G,FERRI M,CHIELLINI F.Materials degradation in PVC medical devices,DEHP leaching and neonatal outcomes [J].Curr Med Chem,2010,17(26):2979-2789.
[2] WANG L X,GONG M Y,XU Y,et al.Phthalates in dust collected from various indoor environments in Beijing,China and resulting non-dietary human exposure [J].Building Environ,2017,124 (2017):315-322.
[3] 黄盼盼，王晨晨，邱春生，等.水环境中PAEs的赋存、环境风险及水质标准[J].环境工程，2020,38(5):23-29.
[4] NIU L L,XU Y,XU C,et al.Status of phthalate esters contamination in agricultural soils across China and associated health risks [J].Environ Pollut,2014,195:16-23.
[5] 王力强，厉曙光.化妆品中酞酸酯类化合物含量及人群暴露研究[J].卫生研究，2016,45(2):331-337.
[6] FU G Q,DAI J,LI Z,et al.The role of STAT3/p53 and PI3K-Akt-mTOR signaling pathway on DEHP induced reproductive toxicity in pubertal male rat [J].Toxicol Appl Pharmacol,2020,404:115151.
[7] YANG L C,YANG B,LU D L,et al.The dynamic assessment of toxicity and pathological process of DEHP in germ cells of male Sprague Dawley rats [J].Roprod Biol,2020,20(4):465-473.
[8] YANG G T,ZHANG W J,QIN Q Z,et al.Mono(2-ethylhexyl) phthalate induces apoptosis in p53-silenced L02 cells via activation of both mitochondrial and death receptor pathways [J].Environ Toxicol,2015,30(10):1178-1191.
[9] WANG J K,ZHAO T X,CHEN J D,et al.Multiple transcriptomic profiling:p53 signaling pathway is involved in DEHP-induced prepubertal testicular injury via promoting cell apoptosis and inhibiting cell proliferation of Leydig cells [J].J Hazard Mater,2021,406:124316.
[10] SEKARAN S,BALAGANAPATHY P,PARSANATHAN R,et al.Lactational exposure of phthalate causes long-term disruption in testicular architecture by altering tight junctional and apoptotic protein expression in Sertoli cells of first filial generation pubertal Wistar rats [J].Hum Exp Toxicol,2015,34(6):575-590.
[11] THOMAS M O,PETRICE W B,PATRICIA L M.Mono-(2-ethylhexyl) phthalate increases spermatocyte mitochondrial peroxiredoxin 3 and cyclooxygenase 2 [J].J Androl,2008,29(3):293-303.
[12] 李桢，易玲娜，王文文，等.MEHP对小鼠生精细胞凋亡及Bcl-2 mRNA表达的影响[J].现代预防医学，2019,46(5):884-887.
[13] SOBARZO C M,ROSANA NDE M,LIVIA L,et al.Mono-(2-ethylhexyl) phthalate (MEHP) affects intercellular junctions of Sertoli cell:a potential role of oxidative stress [J].Reprod Toxicol,2015,58:203-212.
[14] EWELINA W G,PATRYCJA C W,MARTA L S,et al.P53 protein in proliferation,repair and apoptosis of cells [J].Protoplasma,2014,251(3):525-533.
[15] XU D J,LIU L B,ZHAO Y J,et al.Melatonin protects mouse testes from palmitic acid-induced lipotoxicity by attenuating oxidative stress and DNA damage in a SIRT1-dependent manner [J].J Pineal Res,2020,69(4):e12690.
[16] NIU G L,WRIGHT K L,MA Y H,et al.Role of Stat3 in regulating p53 expression and function [J].Mol Cell Biol,2005,25(17):7432-7440.
[17] PHAM T H,PARK H M,KIM J,et al.STAT3 and p53:dual target for cancer therapy [J].Biomedicines,2020,8(12):637.
[18] 陈田.发育期PFOS暴露的肺损伤效应及机制研究[D].武汉：华中科技大学，2011.
[19] 刘迪，于子翔，张寒雪，等.矢车菊素-3-O-葡糖苷干预核因子-κB通路抑制过氧化氢诱导的细胞凋亡[J].卫生研究，2020,49(5):775-779.
[20] CHANG W H,WU M H,PAN H A,et al.Semen quality and insulin-like factor 3:associations with,nary and seminal levels of phthalate metabolites in adult males [J].Chemosphere,2017,173:594-602.
[21] CAMACHO L,LATENDRESSE J R,MUSKHELISHVILI L,et al.Effects of intravenous and oral di(2-ethylhexyl) phthalate (DEHP) and 20% Intralipid vehicle on neonatal rat testis,lung,liver,and kidney [J].Food Chem Toxicol,2020,144:111497.
[22] WANG Y B,BAI L,ZHANG J Y,et al.Lepidium draba L.leaves extract ameliorated cyclophosphamide-induced testicular toxicity by modulation of ROS-dependent Keap1/Nrf2/HO1,Bax/Bcl2/p53/caspase-3,and inflammatory signaling pathways [J].J Food Biochem,2021:e13987.
[23] 王军可.线粒体-内质网结构偶联介导线粒体钙离子超载在DEHP致未成熟睾丸发育损伤中的作用及机制研究[D].重庆：重庆医科大学，2021.
[24] YANG J B,MOITREYEE C K,SUSAN M S,et al.Novel roles of unphosphorylated STAT3 in oncogenesis and transcriptional regulation [J].Cancer Res,2005,65(3):939-947.
[25] GELAIN A,MORI M,MENEGHETTI F,et al.Signal transducer and activator of transcription protein 3 (STAT3):an update on its direct inhibitors as promising anticancer agents [J].Curr Med Chem,2019,26(27):5165-5206.
[26] 黄意湘，马晓晓，郝欣蕊，等.邻苯二甲酸单乙基己基酯对神经干细胞增殖和迁移的作用[J].中国药理学与毒理学杂志，2016,30(5):8.
[27] WU J,YANG C L,LIU J,et al.Betulinic acid attenuates T-2-toxin-induced testis oxidative damage through regulation of the JAK2/STAT3 signaling pathway in mice [J].Biomolecules,2019,9(12):787-799.
[28] LIN X M,LIU S B,LUO Y H,et al.10-HDA induces ROS-mediated apoptosis in A549 human lung cancer cells by regulating the MAPK,STAT3,NF-kappaB,and TGF-beta1 signaling pathways [J].Biomed Res Int,2020:3042636.
基本信息:
DOI：10.19813/j.cnki.weishengyanjiu.2022.02.017
中图分类号:R114
引用信息:
[1]付国庆,代娟,张浩,等.氧化应激介导STAT3/p53通路调控邻苯二甲酸单(2-乙基己基)酯诱导的小鼠初级精母细胞凋亡[J].卫生研究,2022,51(02):278-285.DOI:10.19813/j.cnki.weishengyanjiu.2022.02.017.
基金信息:
湖北省教育厅科学研究计划指导项目(No.B2020014); 职业危害识别与控制湖北省重点实验室开放基金项目(No.OHIC2021Y04)
2022-03-28
2022-03-28