龟鳖类动物以其较强的对低氧、缺氧、食物缺乏等胁迫的耐受能力及抗氧化防御能力而受到广泛关注。在温度剧烈变化时，生物体内有氧代谢受到干扰，会产生大量ROS进而造成组织氧化损伤，而在龟鳖类动物中这种情况发生较少。中华草龟具有较中华鳖、拟鳄龟等更强的组织抗氧化防御能力，探究其在急性冷应激及冬眠过程中抗氧化防御系统在生理及分子水平上的变化，可为全面了解龟鳖类动物的应激响应机理提供基础数据，并为中华草龟养殖提供参考性数据。因此，本研究提出科学问题：中华草龟体内的抗氧化防御系统如何响应急性冷应激和冬眠两种低温胁迫？

为了回答这一问题，本研究以中华草龟（Chinemys reevesii）为研究对象，分别进行急性冷应激实验和冬眠实验。前者实验处理为：将实验动物于实验室28℃下驯化4周后，禁食48h，分别取样记为对照组（28℃禁食后取样）、冷应激组（禁食后水温迅速降至8 °C保持12h，取样）、恢复组（禁食后水温迅速降至8 °C保持12h，恢复至28℃保持24h，取样）；后者实验处理为：实验动物于实验室28℃下驯化4周后，逐渐降低室温至自然温度，分别取样记为冬眠前组（超过90%动物停止进食后取样）、冬眠中组（平均水温最低点取样）、出眠后组（超过90%动物出眠后取样）。每个处理组取样10只（n=10），取其脑、肝、肾、脾脏组织及血浆，称重并测定其以细胞转录因子Nrf2介导的抗氧化酶系统（SOD、CAT、GPx）基因的表达量、抗氧化酶活力、小分子抗氧化剂VC系统中GLO基因表达量、GLO酶活力及VC含量、与总抗氧化能力及MDA含量。探究中华草龟的抗氧化防御系统对急性冷应激和冬眠这两种低温胁迫的响应。

主要结果和结论如下：

1.急性冷应激过程中，中华草龟肝脏组织Nrf2转录因子与Cu/Zn SOD、GPx4的表达量出现同步上调，脑组织中各相关基因表达量相对稳定。各组织中GPx4表达量均显著上调。各组织抗氧化酶（CAT、GPx、SOD）活力变化存在组织特异性，大部分酶活力在冷应激与其后恢复期上升。

2.急性冷应激实验期间，中华草龟肾脏中GLO基因表达量未发生显著变化，其酶活力显示出温度依赖性。脾、脑组织中VC水平相对稳定，中华草龟经历急性冷应激后肾组织中VC含量下降，肝组织VC含量上升。

3.急性冷应激过程中，中华草龟肝组织总抗氧化能力显著升高，脾组织抗氧化能力在恢复期下降，肾、脑组织总抗氧化能力在实验期间没有发生显著改变。在实验期间，中华草龟各组织均没有受到氧化损伤。

4.冬眠期间，中华草龟肝组织中Nrf2转录因子与Cu/Zn SOD表达量同步上调，脾组织中Nrf2转录因子与Cu/Zn SOD、GPx1表达量均呈先升高后恢复的模式。各组织抗氧化酶活力变化存在组织特异性，中华草龟肝、脾组织中SOD酶活力显著上升。冬眠期间肝组织中CAT酶活力先升高后降低，肾、脾组织中CAT酶活力分别在出眠后、冬眠中显著降低。GPx酶活力在各组织中均有所降低。

5.出眠后中华草龟肾脏中GLO基因表达量显著升高。中华草龟肾组织中VC含量在冬眠中降低，出眠后肝、肾、脑组织中VC含量增加，血浆中VC含量在冬眠期间下降。冬眠期间脑组织中VC系统发挥重要的抗氧化作用。

6.冬眠期间中华草龟肾组织中总抗氧化能力没有发生显著变化，肝组织中总抗氧化能力先升高后恢复，脑组织中总抗氧化能力在冬眠中显著下降，出眠后上升但未达到原始水平，脾脏中总抗氧化能力在出眠后显著上升，血浆中总抗氧化能力先升高后降低。在实验期间，中华草龟各组织均未受到明显氧化损伤。

Turtles have been widely concerned for their strong antioxidant defense capability and tolerance to stress, such as hypoxia, anoxia and food deficiency. When temperature changes drastically, the aerobic metabolism in lots of species will be affected, a large amount of ROS being produced, leading to oxidative damage of the body, which is lessly occured in turtles. The pond turtle Chinemys reevesii has been reported to have stronger antioxidant defense capabilities than the soft shelled turtle Pelodiscus sinensis and the snapping turtle Chelydra serpentina. From this perspective, exploring the physiological and molecular response of the antioxidant defense system of the pond turtle during acute cold exposure and hibernation may provide basic data for a comprehensive understanding of the stress response mechanism of turtles and also contribute reference data for C. Reevesii breeding. Therefore, the present study was designed to explore the following scientific question: how does the antioxidant defense system of C. Reevesii response to acute cold exposure and during long winter hibernation?

For the above purpose, pond turtles bought from a turtle farm in Jiangsu province were subjected to acute cold exposure experiments and hibernation experiments. For the former experiment, animals were acclimated in the lab for 4 weeks at 28℃. Then, after fasted for 48hs, samples were taken and recorded as control group (sampling after the turtles were fasted at 28℃), cold exposure group (turtles were exposed at 8℃ for 12h, then sampling), and recovery group (turtles were exposed at 8℃ for 12h, transferred to 28℃ for 24h, then sampling). For the latter experiment, after animals were acclimated in the lab for 4 weeks at 28℃, the room temperature was gradually lowered to the natural temperature, and samples were taken and recorded as pre-hibernation group (sampling after more than 90% of animals stopped eating), hibernation group (sampling at the lowest point of average water temperature), and arousal group (sampling after more than 90% of animals arousal).Ten samples (n = 10) were taken in each treatment group. The brain, liver, kidney, spleen tissues and plasma were sampled for measuring gene expressions and enzyme activities in the antioxidant enzyme system (SOD, CAT and GPx), mediated by cellular transcription factor Nrf2; GLO gene expression, GLO enzyme activity and VC content in small molecule antioxidant VC system; along with total antioxidant capacity (T-AOC) and content of MDA.

The following are the main results and conclusions:

1. During acute cold exposure, the expressions of Nrf2, Cu/Zn SOD, and GPx4 in liver were simultaneously up-regulated, however, the expressions of related genes in brain were relatively stable. GPx4 expression was significantly up-regulated in all tissues. The activity of antioxidant enzymes (CAT, GPx, SOD) varied from tissue to tissue. Most enzyme activities increased during cold exposure and subsequent recovery periods.

2. During the acute cold exposure, GLO gene expression in kidney did not change significantly. GLO enzyme activity showed temperature dependence. The contents of VC in spleen and brain tissues were relatively stable. Vitamin C content decreased in kidney, but increased in liver after cold exposure.

3. In the process of acute cold exposure, hepatic total antioxidant capacity (T-AOC) increased significantly, then decreased during recovery period; while T-AOC kept stable in kidney and brain. No oxidative damage occurred during the experimental process.

4. During hibernation, the expressions of Nrf2 and Cu/Zn SOD genes in liver up-regulated simultaneously, while the expressions of Nrf2, Cu/Zn SOD, and GPx1 in spleen increased firstly and then recovered. The antioxidant enzyme activity of each tissue showed tissue specificity during hibernation. The activity of SOD enzyme in liver and spleen tissues increased significantly, while the activity of CAT enzyme in liver increased firstly and then decreased. Renal and splenic CAT enzyme activities decreased after arousal and during hibernation, respectively. The GPx enzyme activity was reduced in all tissues.

5. The GLO gene expression in kidney increased significantly after arousal. The content of VC in kidney decreased during hibernation, and it increased in liver, kidney and brain after arousal. The content of VC in plasma decreased during hibernation.  Results indicated that the VC system may play an important antioxidant role in brain during hibernation.

6. Renal T-AOC kept stable during hibernation, while T-AOC increased firstly and then recovered in liver. Hepatic T-AOC declined during hibernation, rised again after arousal, but did not recover to the original level. Splenic T-AOC increased significantly after arousal, while it increased firstly and then decreased in plasma. During the experiment, all tissues were efficiently protected from oxidative damage by the antioxidant defense system.