Mo Fan, Zhou Xu, Yang Mengting, Chen Leyi, Tang Zhining, Wang Chong, Cui Yanjun
Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, China-Australian Joint Laboratory for Animal Health Big Data Analytics, Zhejiang Provincial Engineering Laboratory for Animal Health Inspection & Internet Technology, College of Animal Science and Technology & College of Veterinary Medicine of Zhejiang A&F University, Zhejiang A&F University, Lin'an, Hangzhou 311300, China.
Animals (Basel). 2022 Aug 16;12(16):2093. doi: 10.3390/ani12162093.
This study was carried out to investigate the effects of trehalose (Tre) on antioxidant capacity, endoplasmic reticulum stress (ERS) response and apoptosis of heat-stressed intestinal porcine epithelial cells (IPEC-J2). IPEC-J2 cells were cultured at 37 °C until the end of the experiment (control, CON); exposed to heat stress for 2 h (43 °C, HS); or pretreated with 0.1, 1, 5, 10, and 15 mM trehalose at 37 °C for 4 h prior to heat stress exposure for 2 h. The optimum level of trehalose for protecting against HS-induced cell injuries was determined to be 10 mM, as evidenced by the highest cellular viability and lowest malondialdehyde (MDA) content and lactate dehydrogenase (LDH) activity. Based on these, IPEC-J2 cells were divided into three groups: the first group was cultured at 37 °C until the end of the experiment (control, CON); the second group was exposed to heat stress for 2 h (43 °C, HS); the third group was pretreated with 10 mM trehalose for 4 h at 37 °C prior to heat stress exposure for 2 h (Tre + HS). The reactive oxygen species (ROS) content, superoxide dismutase (SOD) activity, mitochondrial membrane potential (MMP) changes, and expressions of the manganese superoxide dismutase (SOD2), ERS and apoptosis-related proteins were determined. Compared to the CON group, HS significantly increased ROS generation (p < 0.01), decreased SOD activity (p < 0.05), and downregulated protein expression of SOD2 (p < 0.01). Compared to the HS group, Tre supplementation reduced ROS levels and increased SOD activity and SOD2 expression to the levels that were comparable to the control (p < 0.05). The HS-induced ERS response was evidenced by the increased protein expressions of glucose-regulated protein 78 (GRP78) (p < 0.01), eukaryotic translation initiation factor 2α (p-eif2α) (p < 0.01), transcription activator 4 (ATF4) (p < 0.01), and the protein expression of C/EBP homologous protein (CHOP) (p < 0.01), which were the four hallmarks of ERS. The Tre + HS group showed lower expressions of GRP78 (p < 0.01), p-eif2α (p < 0.01), ATF4 (p < 0.01), and CHOP (p < 0.01) than that of the HS group. Tre pretreatment attenuated HS-induced mitochondrial apoptosis in IPEC-J2 cells, demonstrated by the increased MMP and decreased proapoptotic proteins active caspase 3, Bax, and cytochrome c (Cyt c). Taken together, trehalose can protect against HS-induced oxidative damage and endoplasmic reticulum stress-mediated apoptosis in IPEC-J2 cells. These data may provide a nutritional strategy for alleviating heat stress in pig production.
本研究旨在探讨海藻糖（Tre）对热应激猪肠上皮细胞（IPEC-J2）抗氧化能力、内质网应激（ERS）反应及细胞凋亡的影响。IPEC-J2细胞在37℃培养至实验结束（对照组，CON）；暴露于热应激2小时（43℃，HS）；或在热应激暴露2小时前，于37℃用0.1、1、5、10和15 mM海藻糖预处理4小时。确定保护细胞免受热应激诱导损伤的海藻糖最佳水平为10 mM，这由最高的细胞活力、最低的丙二醛（MDA）含量和乳酸脱氢酶（LDH）活性得以证明。基于此，将IPEC-J2细胞分为三组：第一组在37℃培养至实验结束（对照组，CON）；第二组暴露于热应激2小时（43℃，HS）；第三组在热应激暴露2小时前，于37℃用10 mM海藻糖预处理4小时（Tre + HS）。测定活性氧（ROS）含量、超氧化物歧化酶（SOD）活性、线粒体膜电位（MMP）变化以及锰超氧化物歧化酶（SOD2）、ERS和凋亡相关蛋白的表达。与CON组相比，HS显著增加ROS生成（p < 0.01），降低SOD活性（p < 0.05），并下调SOD2蛋白表达（p < 0.01）。与HS组相比，补充海藻糖降低了ROS水平，增加了SOD活性和SOD2表达，使其水平与对照组相当（p < 0.05）。HS诱导的ERS反应表现为葡萄糖调节蛋白78（GRP78）（p < 0.01）、真核翻译起始因子2α（p-eif2α）（p < 0.01）、转录激活因子4（ATF4）（p < 0.01）以及C/EBP同源蛋白（CHOP）（p < 0.01）的蛋白表达增加，这是ERS的四个标志。Tre + HS组的GRP78（p < 0.01）、p-eif2α（p < 0.01）、ATF4（p < 0.01）和CHOP（p < 0.01）表达低于HS组。海藻糖预处理减轻了热应激诱导的IPEC-J2细胞线粒体凋亡，表现为MMP增加以及促凋亡蛋白活性半胱天冬酶3、Bax和细胞色素c（Cyt c）减少。综上所述，海藻糖可保护IPEC-J2细胞免受热应激诱导的氧化损伤和内质网应激介导的细胞凋亡。这些数据可能为缓解生猪生产中的热应激提供一种营养策略。
