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| 氢气通过减少活性氧生成抑制线粒体凋亡发挥对小鼠精原细胞的电离辐射防护作用 |
| 马龙1,蔡山林2,郝伟3,高福2,李百龙2,郭佳铭2*,颜宏利1* |
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(1. 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;
2. 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;
3. 海军军医大学(第二军医大学)长海医院内分泌科, 上海 200433
*通信作者) |
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| 摘要: |
| 目的 探索H2对小鼠精原细胞电离辐射损伤的防护效应及机制。方法 将小鼠精原细胞系GC-1细胞分为4组：对照组、H2组、照射组和照射加H2组。照射组和照射加H2组细胞予单次60Co γ射线照射，累积辐射剂量为8 Gy（剂量率为0.897 Gy/min）。H2组和照射加H2组细胞于照射前使用H2细胞培养系统（75% H2、20% O2和5% CO2）培养1 h。照射后24 h，用CCK-8和流式细胞术分别检测照射和H2处理对GC-1细胞活力和凋亡的影响。照射后2 h，用2'，7'-二氯二氢荧光素二乙酸酯（DCFH-DA）荧光探针和线粒体膜电位JC-1荧光探针分别检测照射和H2处理对GC-1细胞内活性氧（ROS）和线粒体膜电位的影响。照射后24 h，用蛋白质印迹法检测照射和H2处理对GC-1细胞线粒体凋亡通路蛋白B淋巴细胞瘤相关蛋白x（Bax）、细胞色素c（Cyt-c）和cleaved-caspase 3（caspase 3活化产物）表达的影响。结果 CCK-8检测结果显示H2提高了照射后GC-1细胞的活力（P<0.01），流式细胞术检测结果显示H2降低了照射后GC-1细胞的凋亡率（P<0.01）。特异性荧光探针染色结果显示，H2不仅抑制照射后GC-1细胞内ROS的产生，还抑制照射后GC-1细胞线粒体膜电位的降低（P<0.01或P<0.05）。蛋白质印迹法检测结果显示，H2抑制照射后GC-1细胞内线粒体凋亡蛋白Bax、Cyt-c和cleaved-caspase 3的表达（P<0.01或P<0.05）。结论 H2通过减少ROS生成保护线粒体膜电位、抑制线粒体凋亡通路，对60Co γ射线导致的小鼠精原细胞电离辐射损伤起防护作用。 |
| 关键词: 氢 电离辐射 活性氧 线粒体凋亡通路 精原细胞 |
| DOI：10.16781/j.0258-879x.2021.10.1085 |
| 投稿时间：2021-03-19修订日期：2021-07-08 |
| 基金项目:国家重点研发计划（2018YFC1004900，2018YFC1005002），上海市科学技术委员会科技英才扬帆计划（19YF1459000），军队计划生育专项（18JS003）. |
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| Hydrogen protecting spermatogonia from ionizing radiation by reducing reactive oxygen species production and inhibiting mitochondrial apoptosis |
| MA Long1,CAI Shan-lin2,HAO Wei3,GAO Fu2,LI Bai-long2,GUO Jia-ming2*,YAN Hong-li1* |
(1. Department of Reproductive Medicine, Changhai Hospital, Naval Medical University(Second Military Medical University), Shanghai 200433, China;
2. Department of Marine Radiation Medicine, Faculty of Naval Medicine, Naval Medical University(Second Military Medical University), Shanghai 200433, China;
3. Department of Endocrinology, Changhai Hospital, Naval Medical University(Second Military Medical University), Shanghai 200433, China
*Corresponding authors) |
| Abstract: |
| Objective To explore the protective effect and mechanism of hydrogen (H2) on ionizing radiation injury of mouse spermatogonia. Methods Mouse spermatogonia GC-1 cells were divided into 4 groups:control group, H2 group, irradiation group and irradiation plus H2 group. The cells in the irradiation group and the irradiation plus H2 group were given single 60Co γ ray irradiation with a cumulative dose of 8 Gy (dose rate 0.897 Gy/min). The cells in the H2 group and the irradiation plus H2 group were cultured in a H2 cell culture system (75% H2, 20% O2 and 5% CO2) for 1 h before irradiation. Cell counting kit 8 (CCK-8) and flow cytometry were used to detect the effects of irradiation and H2 treatment on viability and apoptosis of GC-1 cells 24 h after irradiation. The 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and mitochondrial membrane potential JC-1 fluorescence probes were used to detect the effects of irradiation and H2 treatment on intracellular reactive oxygen species (ROS) and mitochondrial membrane potential of GC-1 cells 2 h after irradiation. Western blotting was used to detect the effects of irradiation and H2 treatment on the expression of mitochondrial apoptosis pathway proteins (B-cell lymphoma-associated protein x[Bax], cytochrome c[Cyt-c] and cleaved caspase 3[an activation product of caspase 3]) in GC-1 cells 24 h after irradiation. Results CCK-8 results showed that H2 significantly increased the viability of GC-1 cells after irradiation (P<0.01), and flow cytometry showed that H2 significantly reduced the apoptosis rate (P<0.01). The results of specific fluorescent probe staining showed that H2 reduced the increase of intracellular ROS and inhibited the decrease of mitochondrial membrane potential after irradiation (P<0.01 or P<0.05). Western blotting results showed that H2 inhibited the expression of mitochondrial apoptotic proteins (Bax, Cyt-c and cleaved caspase 3) in GC-1 cells after irradiation (P<0.01 or P<0.05). Conclusion H2 can protect mouse spermatogonia from ionizing radiation injury of 60Co γ ray irradiation by reducing ROS production, protecting mitochondrial membrane potential, and inhibiting mitochondrial apoptotic pathway. |
| Key words: hydrogen ionizing radiation reactive oxygen species mitochondrial apoptotic pathway spermatogonium |
