Chongqing Key Laboratory of Forage & Herbivore, College of Animal Science and Technology, Southwest University, Chongqing, 400716, PR China; Institute of Ecological Research, Key Laboratory of Southwest China Wildlife Resources Conservation (Ministry of Education), China West Normal University, Nanchong, Sichuan, 637002, PR China.
Geomathematics Key Laboratory of Sichuan Province, Chengdu University of Technology, Chengdu, 610059, PR China.
Theriogenology. 2020 Jan 15;142:104-113. doi: 10.1016/j.theriogenology.2019.09.031. Epub 2019 Sep 19.
Heat stress causes reversible changes in tight junction proteins in immature Sertoli cells via inhibition of the AMPK signaling pathway; these effects are accompanied by an increase in the early apoptotic rate and decrease in the cell viability of Sertoli cells. Since heat stress is known to also cause oxidative damage, in the present study, we investigated whether the earlier mentioned effects of heat stress were brought about via the induction of oxidative stress in boar Sertoli cells. Immature Sertoli cells obtained from 3-week-old piglets were subjected to heat treatment (43 °C, 30 min), and the percentage of ROS-positive cells, the malonaldehyde (MDA) concentration, and the activity of the antioxidases, including superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) were measured. Next, the Sertoli cells were treated with N-acetyl-l-cysteine (NAC) (1 mmol/L, 2 h), an antioxidant agent, before they were exposed to heat stress. The effects of NAC on ROS accumulation, MDA levels, antioxidase activity, the CaMKKβ-AMPK signaling pathway and expression of tight junction proteins were assessed. The results showed that heat stress reversibly increased the percentage of ROS-positive cells and MDA levels, and decreased the activity of SOD, GSH-Px, and CAT. Pretreatment with NAC abrogated these effects of heat stress. Additionally, NAC reversed the heat stress-induced decrease in the expression of CaMKKβ and dephosphorylation of AMPK. NAC also obviously rescued the heat stress-induced downregulation of tight junction proteins (claudin-11, JAM-A, occludin, and ZO-1) both at the mRNA and protein level. In conclusion, the findings indicate that oxidative damage participates in heat stress-induced downregulation of tight junction proteins in Sertoli cells by inhibiting the CaMKKβ-AMPK axis. Further, NAC reversed the effects of heat stress on tight junction proteins; this means that it has potential as a protective agent that can prevent reproductive dysfunction in boars under conditions of heat stress.
热应激通过抑制 AMPK 信号通路引起未成熟支持细胞中紧密连接蛋白的可逆变化；这些作用伴随着支持细胞早期凋亡率的增加和细胞活力的降低。由于已知热应激也会引起氧化损伤，因此在本研究中，我们研究了热应激对猪支持细胞的上述作用是否是通过诱导氧化应激引起的。从 3 周龄仔猪中获得未成熟的支持细胞，进行热处理（43°C，30min），并测量 ROS 阳性细胞的百分比、丙二醛（MDA）浓度以及抗氧化酶的活性，包括超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）和过氧化氢酶（CAT）。然后，在暴露于热应激之前，用抗氧化剂 N-乙酰-L-半胱氨酸（NAC）（1mmol/L，2h）处理支持细胞。评估 NAC 对 ROS 积累、MDA 水平、抗氧化酶活性、CaMKKβ-AMPK 信号通路和紧密连接蛋白表达的影响。结果表明，热应激可逆地增加了 ROS 阳性细胞的百分比和 MDA 水平，降低了 SOD、GSH-Px 和 CAT 的活性。NAC 预处理消除了这些热应激的作用。此外，NAC 逆转了热应激诱导的 CaMKKβ 表达降低和 AMPK 去磷酸化。NAC 还明显挽救了热应激诱导的紧密连接蛋白（claudin-11、JAM-A、occludin 和 ZO-1）在 mRNA 和蛋白质水平的下调。总之，这些发现表明，氧化损伤通过抑制 CaMKKβ-AMPK 轴参与热应激诱导的支持细胞中紧密连接蛋白的下调。此外，NAC 逆转了热应激对紧密连接蛋白的作用；这意味着它具有作为保护性剂的潜力，可以防止热应激条件下公猪的生殖功能障碍。
