Caspase-9蛋白在亚慢性砷染毒大鼠睾丸间质细胞中的表达研究

doi:10.12160/j.issn.1672-5190.2023.04.003

摘要/Abstract

摘要：

[目的]探讨亚慢性砷染毒对大鼠睾丸间质细胞Caspase-9蛋白表达的影响。[方法]将40只健康成年清洁级SD雄性大鼠随机分为4组，分别为对照组以及低、中、高剂量染毒组，每组10只。采用自由饮水方式进行染毒，低、中、高剂量染毒组亚砷酸钠的染毒剂量分别为2.4、12、60 mg/L，对照组给予生理盐水，染毒试验持续14周。染毒试验结束后，检测大鼠外周血白细胞数量；采用ELISA法检测大鼠血清TNF-α及IL-1的浓度；利用透射电镜观察大鼠精子的超微结构；应用免疫组化法检测大鼠睾丸间质细胞Caspase-9蛋白的表达定位及表达水平。[结果]与对照组相比，中、高剂量染毒组大鼠血清中TNF-α、IL-1浓度及白细胞计数均显著（P<0.05）升高。透射电镜下，对照组精子顶体结构完整，厚度及染色均匀；低剂量染毒组可见精子胞膜肿胀，核染色质较均匀；中剂量染毒组精子顶体与核分离，胞膜肿胀；高剂量染毒组精子尾部中断，轴丝排列紊乱。免疫组化法分析表明，Caspase-9蛋白主要表达于睾丸间质细胞的细胞质，表达信号呈棕褐色；与对照组相比，各剂量染毒组大鼠睾丸组织内Caspase-9蛋白的表达水平均显著（P<0.05）升高。[结论]睾丸间质细胞Caspase-9蛋白表达水平升高可能是亚砷酸钠致雄性大鼠慢性生殖毒性的机制之一。

关键词: 亚慢性砷中毒, 睾丸间质细胞, 超微结构, Caspase-9蛋白, 大鼠

Abstract:

[Objective] This study was conducted to investigate the effects of sub-chronic arsenic poisoning on the expression of Caspase-9 protein in leydig cells of rats. [Method] A total of 40 healthy adult clean-grade male SD rats were randomly assigned into the following 4 groups：a control group and the low-, medium-, and high-dose arsenic exposure groups, with 10 rats in each group. The rats in the low-, medium-, and high-dose arsenic exposure groups were given sodium arsenate by drinking water at concentrations of 2.4, 12 and 60 mg/L, respectively, and those in the control group was given physiological saline. The exposure test lasted for 14 weeks. Following the exposure test, the number of peripheral white blood cells of the rats was counted; the serum levels of TNF-α and IL-1 were measured by ELISA assay; the ultrastructure of sperm was observed using transmission electron microscopy; the expression level and localization of Caspase-9 protein in leydig cells were assessed by immunohistochemical method. [Result] Compared with the control group, the serum levels of TNF-α and IL-1 as well as the number of peripheral white blood cells in the medium- and high-dose arsenic exposure groups significantly （P<0.05） increased. Under transmission electron microscopy, the sperm acrosome structure in the control group was intact, with uniform thickness and staining. In the low-dose arsenic exposure group, the sperm membrane swelled and the nuclear chromatin was uniform. In the medium-dose arsenic exposure group, the sperm acrosome and nucleus split, and the membrane swelled. In the high-dose arsenic exposure group, the sperm tail disrupted and the axial filament arrangement disordered. Immunohistochemical analysis showed that Caspase-9 protein mainly expressed in the cytoplasm of leydig cells, and the expression signal was dark brown. Compared with the control group, the expression level of Caspase-9 protein in the testicular tissue in all the arsenic exposure groups significantly （P<0.05） increased. [Conclusion] The increased expression level of Caspase-9 protein in laydig cells may be one of the mechanisms underlying chronic reproductive toxicity induced by sodium arsenite in male rats.

Key words: sub-chronic arsenic poisoning, leydig cell, ultrastructure, Caspase-9 protein, rat

中图分类号:

S859.87
R114

秦海霞, 韩菲, 戴研平, 刘改萍. Caspase-9蛋白在亚慢性砷染毒大鼠睾丸间质细胞中的表达研究[J]. 畜牧与饲料科学, 2023, 44(4): 17-21.

QIN Haixia, HAN Fei, DAI Yanping, LIU Gaiping. Expression of Caspase-9 Protein in Leydig Cells of Rats with Sub-chronic Arsenic Poisoning[J]. Animal Husbandry and Feed Science, 2023, 44(4): 17-21.

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参考文献 20

[1]	JALALUDEEN A M, RAN L, LEE W Y, et al. Ameliorating effect of selenium against arsenic induced male reproductive toxicity in rats[J]. Reproductive and Developmental Biology, 2014, 38(3):107-114. doi: 10.12749/RDB.
[2]	王平, 戴研平, 杨薇, 等. 不育患者凋亡生精细胞与ACP超微结构的关系[J]. 检验医学, 2018, 33(6):472-475.
[3]	王随心, 张玉华, 李向阳, 等. 重金属生精细胞毒性研究进展[J]. 环境与健康杂志, 2011, 28(4):367-372.
[4]	秦海霞, 高晓勤, 刘智, 等. 亚慢性砷中毒对大鼠睾丸间质细胞caspase-3表达及血清中睾酮含量的影响[J]. 中华地方病学杂志, 2016(1):14-17.
[5]	许小凤, 谈勇, 陈宣伊, 等. 补肾活血中药对雷公藤致卵巢储备下降大鼠颗粒细胞凋亡的影响[J]. 医学研究杂志, 2009, 38(12):123-125.
[6]	马惠芳, 任秀君, 图娅, 等. 针刺对高血脂合并脑缺血大鼠海马区Caspase-9蛋白表达及神经行为学的影响[J]. 针刺研究, 2008, 33(6):382-386.
[7]	赵茹, 华亚军, 万俊. 杜仲水提液对幼鼠睾丸间质细胞睾酮合成和caspase-9的影响[J]. 儿科药学杂志, 2012, 18(6):11-14.
[8]	郭泳梅, 姚红, 童娟. 慢性阻塞性肺疾病膈肌形态学改变的机制探讨[J]. 临床肺科杂志, 2012, 17(9):1675-1676.
[9]	KONG D, ZHENG T, ZHANG M, et al. Static mechanical stress induces apoptosis in rat endplate chondrocytes through MAPK and mitochondria-dependent caspase activation signaling pathways[J]. PLoS ONE, 2013, 8(7):e69403. doi: 10.1371/journal.pone.0069403
[10]	朱筑霞, 潘玮炜, 易必达, 等. 砷致大鼠海马神经细胞凋亡对学习记忆功能的影响[J]. 贵州医药, 2010, 34(9):777-780.
[11]	李玉飞, 康朝胜, 臧贵勇, 等. SYNⅠ在慢性砷中毒大鼠海马CA3区的表达[J]. 当代医学, 2009(34):3-5.
[12]	范兴君, 马子媛, 杜宗花, 等. 低剂量亚慢性砷染毒对雄性大鼠生殖功能及子代的影响[J]. 中国生育健康杂志, 2012, 23(5):361-364.
[13]	李解生, 李纯颖, 李勇, 等. 甲醛对小鼠胚胎的发育毒性[J]. 实用预防医学, 2009, 16(4):1242-1244.
[14]	吴勇. 精索静脉曲张大鼠血清抑制素B、性激素及睾丸肿瘤坏死因子的变化[D]. 佳木斯: 佳木斯大学, 2007.
[15]	曹兴午, 林凯, 李翠英, 等. 睾丸生精小管界膜和管周肌样细胞调控睾丸功能[J]. 中国男科学杂志, 2011, 25(12):59-62.
[16]	杨春林, 王明, 邓远忠, 等. 雄性大鼠脊髓损伤对生殖系细菌感染及IL-1、TNF-α、SOD表达的影响[J]. 重庆医科大学学报, 2011, 36(11):1296-1299.
[17]	戴研平, 高晓勤, 丁贤胜. 盐酸利多卡因体外对精子功能及超微结构的影响[J]. 甘肃医药, 2020, 39(9):799-801.
[18]	胡京美, 许成岩, 李媛, 等. 不同冷冻保护剂对人类精子冷冻复苏后活动能力的影响[J]. 中华男科学杂志, 2008(1):23-25.
[19]	郭海, 宋爽, 王国秀, 等. 染锰大鼠生精细胞凋亡中caspase-3 mRNA、凋亡蛋白酶活化因子-1及多聚ADP核糖聚合酶的表达变化[J]. 解剖学杂志, 2017, 40(3):254-257.
[20]	白彩军, 刘丹, 李冰. 砷的氧化应激分子机制研究进展[J]. 实用预防医学, 2012, 19(2):314-316.