高艳荣, 史雪敏, 贾玉巧, 王丽, 邰代鹏, 孙瑶, 常翔宇, 张晗, 田江, 刘璐敏, 李东颖. 黄河包头段水源水有机提取物对雄性小鼠生殖细胞凋亡及睾丸组织3-NT/Caspase-3表达的影响[J]. 环境与职业医学, 2019, 36(12): 1137-1142. DOI: 10.13213/j.cnki.jeom.2019.19272
引用本文: 高艳荣, 史雪敏, 贾玉巧, 王丽, 邰代鹏, 孙瑶, 常翔宇, 张晗, 田江, 刘璐敏, 李东颖. 黄河包头段水源水有机提取物对雄性小鼠生殖细胞凋亡及睾丸组织3-NT/Caspase-3表达的影响[J]. 环境与职业医学, 2019, 36(12): 1137-1142. DOI: 10.13213/j.cnki.jeom.2019.19272
GAO Yan-rong, SHI Xue-min, JIA Yu-qiao, WANG Li, TAI Dai-peng, SUN Yao, CHANG Xiang-yu, ZHANG Han, TIAN Jiang, LIU Lu-min, LI Dong-ying. Effects of organic extracts in source water in Baotou section of the Yellow River on germ cell apoptosis and 3-NT/Caspase-3 expressions in testis of male mice[J]. Journal of Environmental and Occupational Medicine, 2019, 36(12): 1137-1142. DOI: 10.13213/j.cnki.jeom.2019.19272
Citation: GAO Yan-rong, SHI Xue-min, JIA Yu-qiao, WANG Li, TAI Dai-peng, SUN Yao, CHANG Xiang-yu, ZHANG Han, TIAN Jiang, LIU Lu-min, LI Dong-ying. Effects of organic extracts in source water in Baotou section of the Yellow River on germ cell apoptosis and 3-NT/Caspase-3 expressions in testis of male mice[J]. Journal of Environmental and Occupational Medicine, 2019, 36(12): 1137-1142. DOI: 10.13213/j.cnki.jeom.2019.19272

黄河包头段水源水有机提取物对雄性小鼠生殖细胞凋亡及睾丸组织3-NT/Caspase-3表达的影响

Effects of organic extracts in source water in Baotou section of the Yellow River on germ cell apoptosis and 3-NT/Caspase-3 expressions in testis of male mice

  • 摘要: 背景 水体有机物污染对生殖健康的危害已成为目前重点关注的问题。我国水环境污染主要是河流有机物污染。

    目的 探讨黄河包头段水源水有机提取物对小鼠生殖细胞凋亡及睾丸组织3-硝基酪氨酸(3-nitrotyrosine,3-NT)和天冬氨酸半胱氨酸特异性蛋白3(cysteinyl aspartate specificproteinase-3,Caspase-3)表达的影响,并初步探讨其生殖毒性作用机制。

    方法 60只SPF级雄性ICR小鼠随机分为6组:对照组、黄河水有机提取物染毒组(YRW-OE组)、黄河水有机提取物+N-乙酰半胱酸(NAC)处理组(YRW-OE+NAC组)、自来水有机提取物染毒组(TW-OE组)、自来水有机提取物+NAC处理组(TW-OE+NAC组)、NAC组,每组10只。经口灌胃染毒,隔日1次,每次染毒前1 h YRW-OE+NAC组、TW-OE+NAC组及NAC组小鼠经腹腔以0.02 mL/g(以体重计)体积注射浓度为10g/L的NAC。染毒4周后,取小鼠附睾及睾丸组织,透射电镜检测生殖细胞凋亡,采用细胞计数板计数精子数量和观察精子畸形,ELISA法检测睾丸组织3-NT的表达,Western blotting法检测睾丸组织Caspase-3表达。

    结果 与对照组(4.15±0.06)×107/g,(4.84±0.65)%比较,YRW-OE组小鼠精子数量(2.86±0.06)×107/g减少,精子畸形率(19.59±1.49)%增加(P < 0.05),TW-OE组仅精子畸形率(7.89±1.16)%增加(P < 0.05);YRW-OE+NAC组小鼠精子数量(3.55±0.06)×107/g高于YRW-OE组(P < 0.05),精子畸形率(11.44±1.22)%低于YRW-OE组(P < 0.05);TWOE+NAC组小鼠精子畸形率(5.62±0.91)%低于TW-OE组(P < 0.05)。透射电镜显示,YRWOE组细胞核碎裂,核糖体和溶酶体等细胞器减少,细胞器结构不完整,凋亡细胞多见;YRW-OE+NAC组偶见核染色质固缩,核糖体、内质网和溶酶体等细胞器未见减少。与对照组比较,各染毒组小鼠睾丸组织3-NT和Caspase-3蛋白表达量明显增加,YRW-OE+NAC组3-NT (8.46±0.40)mg/L和Caspase-3蛋白(0.38±0.02)表达均低于YRW-OE组(12.28±0.78)mg/L、0.48±0.02,均P < 0.05;TW-OE+NAC组仅3-NT表达低于TW-OE组(7.23±0.47)、(9.34±0.81)mg/L,P < 0.05。

    结论 黄河水有机提取物能够引起实验小鼠睾丸生殖细胞凋亡增加,3-NT和Caspase-3蛋白表达量增加;氧化应激激活Caspase-3信号通路介导生殖细胞凋亡可能是其生殖毒性的重要作用机制之一。

     

    Abstract: Background Reproductive health hazards sourced from organic pollutants in river water have become a major concern at present. The pollution of river water in China is mainly caused by organic pollutants.

    Objective This experiment investigates the effects of organic pollutants in source water in Baotou section of the Yellow River on the apoptosis of germ cells and the expressions of 3-nitrotyrosine (3-NT) and cysteinyl aspartate specific proteinase-3 (Caspase-3) in testis of male mice, and preliminarily explores the relevant mechanism of action.

    Methods Sixty SPF ICR male mice were randomly divided into six groups:control group, Yellow River water organic extracts treatment group (YRE-OE group), Yellow River water organic extracts+N-acetylcysteine (NAC) treatment group (YRE-OE+NAC group), tap water organic extracts treatment group (TW-OE group), tap water organic extracts+NAC treatment group (TW-OE+NAC group), and NAC group, with 10 mice in each group. The YRE-OE were administered to all mice by gavage once every other day, and the mice in the YRE-OE+NAC group, TW-OE+NAC group, and NAC group were intraperitoneally injected with 10 g/L NAC in a volume of 0.02 mL/g (in body weight) 1 h before the YRE-OE administration. After four weeks of the exposure, epididymis and testis tissues were isolated to observe germ cell apoptosis under transmission electron microscope; germ cells were counted and sperm malformation was observed; 3-NT expression in testis was detected by ELISA; and Caspase-3 protein expression was detected by Western blotting.

    Results Compared with the control group(4.15±0.06)×107/g, (4.84±0.65)%, the YRE-OE group showed decreased germ cells(2.86±0.06)×107/g and increased sperm malformation rate(19.59±1.49)% (P < 0.05), and the TW-OE group only showed increased sperm malformation rate(7.89±1.16)% (P < 0.05); compared with the YRE-OE group, the YRE-OE+NAC group showed increased germ cells(3.55±0.06)×107/g and decreased sperm malformation rate(11.44±1.22)% (P < 0.05); compared with the TW-OE group, the TWOE+NAC group showed decreased sperm malformation rate(5.62±0.91)% (P < 0.05). Under transmission electron microscope, nuclear fragmentation, reduced cell organelles such as ribosomes and lysosomes, destroyed mitochondrial structure, and abundant apoptosis germ cells were observed in the YRE-OE group; however, such phenomenon was rarely observed in the YRE-OE+NAC group. Compared with the control group, the 3-NT and Caspase-3 expressions were increased in the exposure groups; the YRE-OE+NAC group showed lower 3-NT(8.64±0.40) mg/L and Caspase-3 (0.38±0.02) expressions than the YRE-OE group(12.28±0.78) mg/L, 0.48±0.02, P < 0.05; the TW-OE+NAC group only showed lower 3-NT expression(7.23±0.47) vs. (9.34±0.81) mg/L, P < 0.05 than the TW-OE group.

    Conclusion The organic matters extracted from the Yellow River could cause germ cell apoptosis and increased expressions of 3-NT and Caspase-3 in testis of mice; the germ cell apoptosis mediated by Caspase-3 signaling pathway activated by oxidative stress may be one of the mechanisms of their reproductive toxicity.

     

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