Methionine (Met) is the only sulfur-containing amino acid among animal essential amino acids, and methionine deficiency (MD) causes tissue damage and cell death in animals. The common modes of cell death include apoptosis, autophagy, pyroptosis, necroptosis. However, the studies about the major modes of cell death caused by MD have not been reported, which worth further study. Primary hepatocytes from grass carp were isolated and treated with different doses of Met (0, 0.5, 1, 1.5, 2, 2.5 mmol/L) to examine the expression of apoptosis, pyroptosis, autophagy and necroptosis-related proteins. Based on this, we subsequently modeled pyroptosis using lipopolysaccharides and nigericin sodium salt, then autophagy inhibitors chloroquine (CQ), AMP-activated protein kinase (AMPK) inhibitors compound C (CC) and reactive oxygen species (ROS) scavengers N-acetyl-L-cysteine (NAC) were further used to examine the expression of proteins related to pyroptosis, autophagy and AMPK pathway in MD-treated cells respectively. MD up-regulated B-cell lymphoma protein 2 (Bax), microtubule-associated protein 1 light chain 3 II (LC3 II), and down-regulated the protein expression levels of B-cell lymphoma-2 (Bcl-2), sequestosome 1 (p62), cleaved-caspase-1, cleaved-interleukin (IL)-1β, and receptor-interacting protein kinase (RIP) 1 in hepatocytes, while it did not significantly affect RIP3. In addition, MD significantly increased the protein expression of liver kinase B1 (LKB1), p-AMPK, and Unc-51-like kinase 1 (ULK1) without significant effect on p-target of rapamycin. Subsequently, the use of CQ increased the protein expression of NOD-like receptor thermal protein domain associated protein 3 (NLRP3), cleaved-caspase-1, and cleaved-IL-1β inhibited by MD; the use of CC significantly decreased the protein expression of MD-induced LC3 II and increased the protein expression of MD-suppressed p62; then the use of NAC decreased the MD-induced p-AMPK protein expression. MD promoted autophagy and apoptosis, but inhibited pyroptosis and necroptosis. MD inhibited pyroptosis may be related regarding the promotion of autophagy. MD activated AMPK by inducing ROS production which in turn promoted autophagy. These results could provide partial theoretical basis for the possible mechanisms of Met in ensuring the normal structure and function of animal organs. Furthermore, ferroptosis is closely related to redox states, it is worth investigating whether MD affects ferroptosis in hepatocytes.

中文翻译：

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蛋氨酸缺乏抑制草鱼（Ctenopharyngodon idella）原代肝细胞焦亡：可能通过激活 ROS-AMPK-自噬轴


蛋氨酸（Met）是动物必需氨基酸中唯一的含硫氨基酸，蛋氨酸缺乏（MD）会导致动物组织损伤和细胞死亡。常见的细胞死亡方式包括细胞凋亡、自噬、细胞焦亡、坏死性凋亡。但有关MD引起的细胞死亡的主要方式的研究尚未见报道，值得进一步研究。分离草鱼原代肝细胞，用不同剂量的Met（0、0.5、1、1.5、2、2.5 mmol/L）处理，检测细胞凋亡、焦亡、自噬和坏死性凋亡相关蛋白的表达。在此基础上，我们随后使用脂多糖和尼日利亚素钠盐，然后使用自噬抑制剂氯喹（CQ）、AMP激活蛋白激酶（AMPK）抑制剂化合物C（CC）和活性氧（ROS）清除剂N-乙酰基-L来模拟细胞焦亡。进一步使用-半胱氨酸（NAC）分别检测MD处理的细胞中与焦亡、自噬和AMPK通路相关的蛋白的表达。 MD 上调 B 细胞淋巴瘤蛋白 2 (Bax)、微管相关蛋白 1 轻链 3 II (LC3 II)，并下调 B 细胞淋巴瘤-2 (Bcl-2)、隔离体的蛋白表达水平1 (p62)、cleaved-caspase-1、cleaved-interleukin (IL)-1β 和肝细胞中的受体相互作用蛋白激酶 (RIP) 1，但对 RIP3 没有显着影响。此外，MD 显着增加了肝激酶 B1 (LKB1)、p-AMPK 和 Unc-51 样激酶 1 (ULK1) 的蛋白表达，而对雷帕霉素的 p 靶点没有显着影响。 随后，使用 CQ 增加了 MD 抑制的 NOD 样受体热蛋白结构域相关蛋白 3 (NLRP3)、cleaved-caspase-1 和 cleaved-IL-1β 的蛋白表达； CC的使用显着降低了MD诱导的LC3 II的蛋白表达，并增加了MD抑制的p62的蛋白表达；然后使用 NAC 降低了 MD 诱导的 p-AMPK 蛋白表达。 MD促进自噬和细胞凋亡，但抑制细胞焦亡和坏死性凋亡。 MD抑制细胞焦亡可能与促进自噬有关。 MD 通过诱导 ROS 产生激活 AMPK，进而促进自噬。这些结果可为Met保证动物器官正常结构和功能的可能机制提供部分理论依据。此外，铁死亡与氧化还原状态密切相关，MD是否影响肝细胞的铁死亡值得研究。