背景:蛋氨酸(Met)是动物必需氨基酸中唯一的含硫氨基酸,甲硫氨酸缺乏(MD)会导致动物组织损伤和细胞死亡。细胞死亡的常见模式包括凋亡,自噬,焦亡,坏死。然而,关于由MD引起的细胞死亡的主要模式的研究尚未报道,值得进一步研究。
方法:分离草鱼原代肝细胞,用不同剂量的Met(0、0.5、1、1.5、2、2.5mmol/L)处理,检测细胞凋亡的表达。焦亡,自噬和坏死相关蛋白。基于此,随后,我们使用脂多糖和尼日利亚霉素钠盐对焦亡进行建模,然后自噬抑制剂氯喹(CQ),AMP激活的蛋白激酶(AMPK)抑制剂化合物C(CC)和活性氧(ROS)清除剂N-乙酰基-L-半胱氨酸(NAC)进一步用于检查与焦亡相关的蛋白质的表达,自噬和AMPK途径分别在MD处理的细胞。
结果:MD上调B细胞淋巴瘤蛋白2(Bax),微管相关蛋白1轻链3II(LC3II),下调B细胞淋巴瘤-2(Bcl-2)的蛋白表达水平,隔离体1(P62),裂解的胱天蛋白酶-1,裂解的白细胞介素(IL)-1β,和肝细胞中的受体相互作用蛋白激酶(RIP)1,但对RIP3无明显影响。此外,MD显著增加肝激酶B1(LKB1)的蛋白表达,p-AMPK,和Unc-51样激酶1(ULK1)对雷帕霉素的p-靶无明显影响。随后,使用CQ增加NOD样受体热蛋白结构域相关蛋白3(NLRP3)的蛋白表达,MD抑制了cleaved-caspase-1和cleaved-IL-1β;CC的使用显着降低了MD诱导的LC3II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。
结论:MD促进自噬和凋亡,但是抑制了焦亡和坏死性凋亡.MD抑制焦亡可能与促进自噬有关。MD通过诱导ROS产生而激活AMPK,ROS产生进而促进自噬。这些结果为Met在确保动物器官正常结构和功能方面的可能机制提供了部分理论依据。此外,铁死亡与氧化还原状态密切相关,值得研究MD是否影响肝细胞的铁凋亡。
BACKGROUND: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.