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分子氢对雄性小鼠生殖系统高水平小剂量电离辐射损伤的防护作用
郭佳铭^1△,和生辉^2△,熊泽森³,刘哲¹,赵海男¹,刘鹏飞⁴,颜宏利⁵,高福¹,李百龙^1*
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(1. 海军军医大学(第二军医大学)海军医学系舰船辐射医学防护教研室, 上海 200433; 2. 解放军总医院第六医学中心医务部, 北京 100048; 3. 北部战区陆军参谋部门诊部, 济南 250002; 4. 解放军 91126 部队医院, 大连 116041; 5. 海军军医大学(第二军医大学)长海医院生殖医学中心, 上海 200433 ^△共同第一作者 ^*通信作者)

摘要:

目的探讨分子氢对雄性小鼠生殖系统高水平小剂量电离辐射损伤的防护作用及机制。方法将小鼠精原细胞系GC-1 spg细胞分为对照组、加氢组、4 Gy照射组、4 Gy照射加氢组，在各组细胞处理后24 h时采用流式细胞术检测细胞凋亡率。将72只雄性BALB/c小鼠分为对照组、加氢组、0.25 Gy照射组、0.25 Gy照射加氢组、0.5 Gy照射组、0.5 Gy照射加氢组，每组12只。小鼠加氢处理采用饮用富氢水联合呼吸高浓度氢气法。各组小鼠经相应处理后24 h分离睾丸组织进行H-E染色，并于内眦静脉取血，采用ELISA试剂盒检测促性腺激素释放激素（GnRH）、血清卵泡刺激素（FSH）、黄体生成素（LH）、睾酮含量。照射后4周分离小鼠双侧附睾制备精子悬液，以精子彗星电泳实验检测精子DNA损伤情况。结果 4 Gy照射加氢组GC-1 spg细胞的24 h凋亡率低于4 Gy照射组，差异有统计学意义（t=7.186，P<0.01）。加氢处理缓解了0.5 Gy照射导致的小鼠睾丸组织损伤，纠正了0.25 Gy、0.5 Gy照射诱发的FSH水平升高（t_{0.25 Gy}=3.195 8，P_{0.25 Gy}=0.019；t_{0.5 Gy}=10.723 4，P_{0.5 Gy}<0.05），并减轻了0.25 Gy、0.5 Gy照射后4周小鼠精子DNA彗星拖尾程度和DNA损伤情况（尾部面积：t_{0.25 Gy}=16.592 3，t_{0.5 Gy}=15.891 5；尾部DNA含量：t_{0.25 Gy}=11.296 5，t_{0.5 Gy}=13.785 0；尾部DNA百分含量：t_{0.25 Gy}=26.834 0，t_{0.5 Gy}=10.325 7；尾长：t_{0.25 Gy}=16.865 4，t_{0.5 Gy}=15.441 2；尾矩：t_{0.25 Gy}=26.979 4，t_{0.5 Gy}=13.174 2；Olive尾矩：t_{0.25 Gy}=24.752 4，t_{0.5 Gy}=6.896 1；P均<0.05）。结论分子氢通过降低精原细胞凋亡、调节激素紊乱、减轻精子DNA损伤等方式对雄性小鼠生殖系统高水平小剂量辐射损伤发挥防护作用。

关键词: 分子氢辐射防护精原细胞细胞凋亡雄激素类

DOI：10.16781/j.0258-879x.2018.11.1188

投稿时间：2018-06-19修订日期：2018-10-09

基金项目:

Hydrogen alleviates high-level low-dose irradiation-induced injury of male reproductive system in mice

GUO Jia-ming^1△,HE Sheng-hui^2△,XIONG Ze-sen³,LIU Zhe¹,ZHAO Hai-nan¹,LIU Peng-fei⁴,YAN Hong-li⁵,GAO Fu¹,LI Bai-long^1*

(1. Department of Marine Radiation Medicine, Faculty of Naval Medicine, Navy Medical University(Second Military Medical University), Shanghai 200433, China;
2. Department of Medical Service, Sixth Medical Center, General Hospital of PLA, Beijing 100048, China;
3. Department of Outpatient, Army Staff of PLA Northern Theater Command, Jinan 250002, Shandong, China;
4. No. 91126 Hospital of PLA, Dalian 116041, Liaoning, China;
5. Department of Reproductive Medicine, Changhai Hospital, Navy Medical University(Second Military Medical University), Shanghai 200433, China
^△Co-first authors.
^* Corresponding author)

Abstract:

Objective To explore the protective effects of molecular hydrogen on high-level low-dose irradiation-induced male reproductive system injury in mice and the underlying mechanism. Methods Cultured spermatogonia-derived cell line GC-1 spg was randomized into control group, hydrogen group, 4 Gy radiation group and 4 Gy radiation+hydrogen group. The apoptotic rate of the cells was detected by flow cytometry assay at 24 h after treatment in each group. Seventy-two male BALB/c mice were randomized into control group, hydrogen group, 0.25 Gy radiation group, 0.25 Gy radiation+hydrogen group, 0.5 Gy radiation group and 0.5 Gy radiation+hydrogen group, with 12 mice in each group. The hydrogen treatment was conducted by hydrogen-rich water administration and high-concentration hydrogen gas inhalation. At 24 h after treatment, the testes were dissected and sectioned for H-E staining, and blood samples from the internal canthus vein were collected to determine the levels of gonadotropin-releasing hormone (GnRH), follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone using ELISA. At 4 weeks after radiation, the bilateral epididymides were isolated to prepare sperm suspensions, and the DNA damage of the spermatozoa was examined using the neutral single cell gel electrophoresis. Results The 24 h apoptosis rate of GC-1 spg cells was significantly decreased in the 4 Gy radiation+hydrogen group compared with the 4 Gy radiation group (t=7.186, P<0.01). Hydrogen obviously reverted the histological damage of the testes induced by 0.5 Gy irradiation, significantly inhibited 0.25 Gy and 0.5 Gy radiation-caused surge of FSH (t=3.195 8, P=0.019; t=10.723 4, P<0.05), and significantly ameliorated comet tailing and damage of the sperm DNA at 4 weeks after radiation (tail area t_{0.25 Gy}=16.592 3, t_{0.5 Gy}=15.891 5; tail DNA t_{0.25 Gy}=11.296 5, t_{0.5 Gy}=13.785 0; tail DNA% t_{0.25 Gy}=26.834 0, t_{0.5 Gy}=10.325 7; tail length t_{0.25 Gy}=16.865 4, t_{0.5 Gy}=15.441 2; tail moment t_{0.25 Gy}=26.979 4, t_{0.5 Gy}=13.174 2; Olive tail moment t_{0.25 Gy}=24.752 4,t_{0.5 Gy}=6.896 1; all P<0.05). Conclusion Molecular hydrogen protects male mouse reproductive system from high-level low-dose radiation through reducing spermatogonium apoptosis, adjusting hormone disturbance and ameliorating sperm DNA damage.

Key words: molecular hydrogen radiation protection spermatogonia apoptosis androgens