聚苯乙烯纳米塑料致雄性小鼠睾丸睾酮合成障碍的作用机制研究

doi:10.16431/j.cnki.1671-7236.2025.04.004

摘要/Abstract

摘要： 【目的】探究聚苯乙烯纳米颗粒(NPs)对雄性小鼠生殖系统造成的损伤和影响睾酮合成的作用机制。【方法】将24只清洁级雄性C56小鼠随机分为4组，分别为对照组及低(0.1 mg/L)、中(1 mg/L)、高(10 mg/L)剂量聚苯乙烯NPs组，每组6只，饮水暴露，试验期90 d。试验结束后，小鼠眼球采血，脱颈处死后剥离附睾及睾丸，采集精子。睾丸称重，统计睾丸指数，利用HE染色观察睾丸组织病理变化，使用综合精液分析系统分析精子总活率及精子总活力；利用ELISA试剂盒检测睾丸组织丙二醛(MDA)含量、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GSH-Px)活性以及血清睾酮含量；利用Western blotting检测氧化应激、凋亡相关蛋白表达水平；通过实时荧光定量PCR和Western blotting分别检测睾酮合成相关基因的mRNA和蛋白表达水平。【结果】与对照组相比，中、高剂量聚苯乙烯NPs组小鼠睾丸指数显著或极显著升高(P＜0.05；P＜0.01)，小鼠精子总活力及总活率、血清睾酮含量均显著或极显著降低(P＜0.05；P＜0.01)。ELISA检测结果显示，与对照组相比，各聚苯乙烯NPs处理组小鼠睾丸组织中MDA含量均极显著升高(P＜0.01)，T-SOD、CAT和GSH-Px活性显著或极显著降低(P＜0.05；P＜0.01)。Western blotting检测结果显示，与对照组相比，中、高剂量聚苯乙烯NPs组小鼠睾丸组织中核因子E2相关因子2(Nrf2)蛋白表达量极显著升高(P＜0.01)，3β-羟类固醇脱氢酶(HSD-3β)蛋白表达量均极显著降低(P＜0.01)；各聚苯乙烯NPs处理组小鼠睾丸组织血红素加氧酶-1(HO-1)蛋白表达量均极显著降低(P＜0.01)；高剂量聚苯乙烯NPs组小鼠睾丸组织中天冬氨酸半胱氨酸特异性蛋白酶9(Caspase9)、Caspase3、Bcl2相关X蛋白(Bax)/B淋巴细胞瘤-2(Bcl-2)蛋白表达量均极显著升高(P＜0.01)，胆固醇侧链裂解酶(CYP11A)蛋白表达量显著降低(P＜0.05)。实时荧光定量PCR结果显示，与对照组相比，各聚苯乙烯NPs处理组小鼠睾丸组织中HSD-17β、细胞色素P450胆固醇侧链裂解酶(P450 scc)、类固醇激素急性调节蛋白(StAR)基因表达量均极显著下降(P＜0.05；P＜0.01)；中、高剂量聚苯乙烯NPs组小鼠睾丸组织中HSD-3β、P450c17基因表达量均极显著或显著下降(P＜0.01；P＜0.05)。【结论】聚苯乙烯NPs能诱导小鼠睾丸组织氧化应激进而引发细胞发生，最终导致睾丸损伤和睾酮合成障碍。

关键词: 聚苯乙烯; 纳米塑料(NPs); 氧化应激; 凋亡; 睾酮合成

Abstract: 【Objective】 The aim of this study was to explore the effects of polystyrene nanoparticles (NPs) on reproductive system damage and testosterone synthesis in male mice.【Method】 A total of 24 clean grade male C56 mice were randomly divided into 4 groups:Control group and low (0.1 mg/L),medium (1 mg/L) and high (10 mg/L) dose polystyrene NPs groups,with 6 mice in each group.The experiment lasted for 90 d.After the test,blood was collected from eyeballs of mice,epididymis and testicles were removed after neck removal,and sperm was collected.Testis were weighed,testicular index was counted,testicular histopathological changes were observed by HE staining,total sperm motility and total sperm viability were analyzed by comprehensive semen analysis system.The content of malondialdehyde (MDA), the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) in testicular tissue, and the content of serum testosterone were detected by ELISA kit.Western blotting was used to detect the expression of oxidative stress and apoptosis-related proteins.The mRNA and protein expression of genes related to testosterone synthesis were detected by Real-time quantitative PCR and Western blotting,respectively.【Result】 Compared with control group,the testicular index of mice in medium and high dose polystyrene NPs groups was significantly or extremely significantly increased (P＜0.05 or P＜0.01),the total motility and total viability of sperm and serum testosterone content of mice were significantly or extremely significantly decreased (P＜0.05 or P＜0.01).The ELISA results showed that compared with control group,the MDA content in testis tissue of mice in polystyrene NPs treated groups was extremely significantly increased (P＜0.01),and the activities of SOD，CAT and GSH-Px were significantly decreased or extremely significantly decreased (P＜0.05 or P＜0.01).Western blotting results showed that compared with control group,the expression of nuclear factor E2 related factor 2 (Nrf2) protein in testicular tissue of mice in medium and high dose polystyrene NPs groups was extremely significantly increased (P＜0.01),and the expression of 3β-hydroxysteroid dehydrogenase (HSD-3β) protein was extremely significantly decreased (P＜0.01).The expression of heme oxygenase-1 (HO-1) protein in testicular tissue of mice in polystyrene NPs treated groups was extremely significantly decreased (P＜0.01).The expression of aspartic cysteine-specific protease 9 (Caspase9),Caspase3,Bcl2-associated X protein (Bax)/B lymphoblastoma-2 (Bcl-2) protein in testicular tissue of mice in high dose polystyrene NPs group were extremely significantly increased (P＜0.01)，and the expression of cholesterol side chain lyase (CYP11A) protein was significantly decreased (P＜0.05).Real-time quantitative PCR results showed that compared with control group, the expression of HSD-17β,cytochrome P450 cholesterol side chain lyase (P450 scc) and steroid-hormone acute regulatory protein (StAR) genes in testicular tissue of mice in polystyrene NPs treated groups were significantly or extremely significantly decreased (P＜0.05 or P＜0.01).The expression of HSD-3β and P450c17 genes in testicular tissue of mice in medium and high dose polystyrene NPs groups were extremely significantly or significantly decreased (P＜0.01 or P＜0.05). 【Conclusion】 Polystyrene NPs could induce oxidative stress in testicular tissue of mice,which led to cytogenesis,and eventually led to testicular injury and testosterone synthesis disorder.

Key words: polystyrene; nanoplastics (NPs); oxidative stress; apoptosis; testosterone synthesis

中图分类号:

S859.8

缪心媛, 许晴雨, 夏苏干, 林天金, 蔡国栋, 邹辉, 顾建红, 袁燕, 刘宗平, 卞建春. 聚苯乙烯纳米塑料致雄性小鼠睾丸睾酮合成障碍的作用机制研究[J]. 中国畜牧兽医, 2025, 52(4): 1488-1498.

MIAO Xinyuan, XU Qingyu, XIA Sugan, LIN Tianjin, CAI Guodong, ZOU Hui, GU Jianhong, YUAN Yan, LIU Zongping, BIAN Jianchun. Mechanism of Action of Testosterone Synthesis Disorder in Male Mice Induced by Polystyrene Nanoplastics[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(4): 1488-1498.

参考文献

[1] BRADNEY L,WIJESEKARA H,PALANSOOSIYA K N,et al.Particulate plastics as a vector for toxic trace-element uptake by aquatic and terrestrial organisms and human health risk[J].Environment International,2019,131:104937.
[2] 任元毅.微塑料颗粒对男性生殖系统的影响及机制[J].中国男科学杂志,2023,37(6):127-131.REN Y Y.The effects and mechanisms of microplastic particles on the male reproductive system[J].Chinese Journal of Andrology,2023,37(6):127-131.(in Chinese)
[3] DE SOUZA M A,KLOAS W,ZARFL C,et al.Microplastics as an emerging threat to terrestrial ecosystems[J].Global Change Biology,2018,24(4):1405-1416.
[4] YU Z,FAN X,ZHAO X,et al.Polystyrene nanoplastics induce lipid metabolism disorder by activating the PERK-ATF4 signaling pathway in mice[J].Acs Applied Materials&Interfaces,2024,16(27):34524-34537.
[5] YANG Z S,BAI Y L,JIN C H,et al.Evidence on invasion of blood,adipose tissues,nervous system and reproductive system of mice after a single oral exposure:Nanoplastics versus microplastics[J].Biomedical and Environmental Sciences,2022,35(11):1025-1037.
[6] ZOU L,XU X,WANG Y,et al.Neonatal exposure to polystyrene nanoplastics impairs microglia-mediated synaptic pruning and causes social behavioral defects in adulthood[J].Environmental Science&Technology,2024,58(27):11945-11957.
[7] SUN J,TENG M,ZHU W,et al.microRNA and gut microbiota alter intergenerational effects of paternal exposure to polyethylene nanoplastics[J].ACS Nano,2024,18(27):18085-18100.
[8] 成娅,韩飞,梁月辉,等.聚苯乙烯纳米塑料致雄性小鼠生殖毒性效应及初步机制[J].陆军军医大学学报,2023,45(9):876-884.CHENG Y,HAN F,LIANG Y H,et al.Reproductive toxicity and preliminary mechanism of polystyrene nanoplastics in male mice[J].Journal of Army Medical University,2023,45(9):876-884.(in Chinese)
[9] CAI P,WANG Y,FENG N,et al.Polystyrene nanoplastics aggravate reproductive system damage in obese male mice by perturbation of the testis redox homeostasis[J].Environmental Toxicology,2023,38(12):2881-2893.
[10] LIANG Y,YANG Y,LU C,et al.Polystyrene nanoplastics exposure triggers spermatogenic cell senescence via the Sirt1/ROS axis[J].Ecotoxicology and Environmental Safety,2024,279:116461.
[11] 王洪艳,马佳羽,李泽堃,等.微塑料聚丙烯和镉联合作用对小鼠睾丸的毒性影响[J].吉林医药学院学报,2024,45(3):165-170.WANG H Y,MA J Y,LI Z K,et al.Effect of microplastic polypropylene and cadmium on testicles of mice[J].Journal of Jilin Medical College,2024,45(3):165-170.(in Chinese)
[12] 冯志强,王腾飞,赵善江,等.Nrf2/Keap1-ARE通路的生物学功能及其信号调控[J].中国畜牧兽医,2022,49(1):81-90.FENG Z Q,WANG T F,ZHAO S J,et al.Research progress on Nrf2/Keap1-ARE signaling pathway and its transcriptional regulation[J].China Animal Husbandry&Veterinary Medicine,2022,49(1):81-90.(in Chinese)
[13] 夏俭有,刘波,潘铁军.聚苯乙烯微塑料通过重塑肠道微生物和上调Claudin 1蛋白表达诱导小鼠睾丸损伤[J].生态毒理学报,2024,19(3):353-362.XIA J Y,LIU B,PAN T J.Polystyrene microplastics induce testicular injury in mice by remodeling gut microbiota and upregulating Claudin 1 protein expression[J].Asian Journal of Ecotoxicology,2024,19(3):353-362.(in Chinese)
[14] LI Z,XIAN H,YE R,et al.Gender-specific effects of polystyrene nanoplastic exposure on triclosan-induced reproductive toxicity in zebrafish (Danio rerio)[J].Science of the Total Environment,2024,932:172876.
[15] 李盼,杨玉菲,薛振,等.微纳米塑料对陆生哺乳动物毒性效应的研究进展[J].生态毒理学报,2023,18(6):168-176.LI P,YANG Y F,XUE Z,et al.Research progress of toxic effects of micro-nano plastics on terrestrial mammals[J]. Asian Journal of Ecotoxicology,2023,18(6):168-176.(in Chinese)
[16] YANG X,ZHANG X,YAO Q,et al.T-2 toxin impairs male fertility by disrupting hypothalamic-pituitary-testis axis and declining testicular function in mice[J].Chemosphere,2019,234:909-916.
[17] 朱琳,周燕,田坤明,等.微塑料暴露对雄性小鼠肠道菌群和生殖功能的影响[J].现代预防医学,2023,50(5):813-819.ZHU L,ZHOU Y,TIAN K M,et al.Effects of microplastic exposure on intestinal flora and male reproductive function in male mice[J].Modern Preventive Medicine,2023,50(5):813-819.(in Chinese)
[18] HE Q,LI X,XIE C,et al.Long-term nanoplastics exposure contributes to impaired steroidogenesis by disrupting the hypothalamic-testis axis:Evidence from integrated transcriptome and metabolome analysis[J].Journal of Applied Toxicology,2024.Doi:10.1002/jat.4696.Online ahead of print.
[19] 赵乾秀,白淼,白宇超,等.聚苯乙烯纳塑料诱导Caco-2细胞氧化应激和线粒体损伤的研究[J].生态毒理学报,2024,19(4):284-293.ZHAO Q X,BAI M,BAI Y C,et al.Polystyrene nanoplastics induce oxidative stress and mitochondrial damage in Caco-2 cells[J].Asian Journal of Ecotoxicology,2024,19(4):284-293.(in Chinese)
[20] LI W,YAO T,ZHANG X,et al.Oxylipin profiling analyses reveal that ω-3 PUFA is more susceptible to lipid oxidation in sheep testis under oxidative stress[J].Animal Reproduction Science,2024,268:107567.
[21] XIONG G,ZHANG H,PENG Y,et al.Subchronic co-exposure of polystyrene nanoplastics and 3-BHA significantly aggravated the reproductive toxicity of ovaries and uterus in female mice[J].Environmental Pollution,2024,351:124101.
[22] ZICKRI M B,MOUSTAFA M H,FASSEH A E,et al.Antioxidant and antiapoptotic paracrine effects of mesenchymal stem cells on spermatogenic arrest in oligospermia rat model[J].Annals of Anatomy,2021,237:151750.
[23] JIA T,CAI J,HE S,et al.UV-aged polystyrene nanoplastics aggravate intestinal barrier damage by overproduction of ROS[J].Environmental Toxicology and Pharmacology,2024,108:104448.
[24] 刘好,彭晓莉,韩彬,等.飞燕草素通过Nrf2/HO-1信号通路改善镉致雄性大鼠生殖损伤[J].成都医学院学报,2024,19(3):390-394.LIU H,PENG X L,HAN B,et al.Delphinidin ameliorates cadmium-induced reproductive damage in male rats via the Nrf2/HO-1 signaling pathway[J].Journal of Chengdu Medical College,2024,19(3):390-394.(in Chinese)
[25] 杨巧侠,王成果,王元欣.乌司他丁预处理对HepG2细胞氧化损伤保护作用的研究[J].医学信息,2019,32(13):72-76.YANG Q X,WANG C G,WANG Y X.Protective effect of ulinastatin pretreatment on oxidative damage in HepG2 cells[J].Journal of Medical Information,2019,32(13):72-76.(in Chinese)
[26] 罗金婷,许发芳,王磊,等.红景天多糖对低氧环境下猪睾丸间质细胞增殖及凋亡的影响[J].畜牧兽医学报,2024,55(6):2441-2450.LUO J T,XU F F,WANG L,et al.The effect of RSP on cell proliferation and apoptosis of porcine Leydig cells with hypoxia[J].Acta Veterinaria et Zootechnica Sinica,2024,55(6):2441-2450.(in Chinese)
[27] LIANG B,ZHONG Y,HUANG Y,et al.Underestimated health risks:Polystyrene micro-and nanoplastics jointly induce intestinal barrier dysfunction by ROS-mediated epithelial cell apoptosis[J]. Particle and Fibre Toxicology,2021,18(1):20.
[28] IJAZ M U,RAFI Z,HAMZA A,et al.Mitigative potential of kaempferide against polyethylene microplastics induced testicular damage by activating Nrf-2/Keap-1 pathway[J].Ecotoxicology and Environmental Safety,2024,269:115746.
[29] SUI A,YAO C,CHEN Y,et al.Polystyrene nanoplastics inhibit StAR expression by activating HIF-1alpha via ERK1/2 MAPK and AKT pathways in TM3 Leydig cells and testicular tissues of mice[J].Food and Chemical Toxicology,2023,173:113634.