活性羰基化合物诱导的氧化应激致细胞能量代谢障碍和毒性的机制

发布时间：2018-04-04 03:00

本文选题：RCS　切入点：氧化应激　出处：《西北农林科技大学》2015年硕士论文

【摘要】：活性羰基化合物(Reactive carbonyl species,RCS),为一类体内糖代谢,脂质过氧化(Lipid peroxidation,LPO)和食物加工过程中不断产生的,包含一个或多个羰基基团的高活性亲电化合物。RCS易与细胞蛋白质,脂质和核酸发生反应,导致细胞损伤。甲基乙二醛(Methylglyoxal,MG)和乙二醛(Glyoxal,GO)为RCS中活性很高的两种二羰基化合物。MG和GO通过诱导蛋白羰基化、改变蛋白质结构和功能、增加氧化应激和羰基应激,而导致炎症、糖尿病和非酒精性脂肪性肝炎(Non-alcoholic steatohepatitis,NASH)等疾病。目前认为RCS的细胞毒性机制包括诱导氧化应激,羰基应激,蛋白与蛋白或核酸交联,基因突变,细胞凋亡等。RCS可通过线粒体途径导致细胞凋亡,但是其造成线粒体损伤的具体机制仍需要进一步的探索。因此本试验从氧化应激,线粒体膜电位(Mitochondrial membrane potential,MMP)变化,线粒体脂肪酸β-氧化(Fatty acidβ-oxidation,FAO)等方面探讨RCS的毒性机制。1.通过细胞毒性试验检测和活性氧类(Reactive oxygen species,ROS),LPO,H2O2浓度等氧化应激指标及MMP的检测,研究RCS细胞毒性的氧化损伤机制。结果表明,随着RCS浓度的增加,大鼠肝细胞(BRL 3A)和胚胎成纤维细胞(3T3-L1)活力下降,细胞毒性增强,细胞ROS上升,MMP下降。而RCS捕获剂氨基胍(Aminoguanidine,AG),超氧阴离子清除剂Trolox,过氧化氢酶,羟基清除剂甘露醇均可显著抑制GO的这种作用。2.通过细胞ATP含量,蛋白表达和酶活性检测,研究RCS诱导的氧化应激致细胞能量代谢障碍的机制。结果表明,GO显著降低BRL 3A细胞ATP含量,而AG、过氧化氢酶和Trolox能使细胞ATP恢复正常水平。随着RCS浓度的增加,BRL 3A和3T3-L1细胞脂肪酸结合蛋白(Fatty acid-binding protein,FABP)、脂肪酸转运蛋白(Cluster of differentiation 36/fatty acid translocase,CD36)表达显著下降。这些可能是RCS诱导NASH的分子机制。RCS时间选择性的抑制肉毒碱棕榈酰转移酶-1(Carnitine palmitoyltransferase-1,CPT1)活性。证实线粒体脂肪酸摄入障碍可能是造成FAO功能障碍的机制之一。GO诱导BRL 3A和3T3-L1细胞胰岛素受体底物-1(Insulin receptor substrates,IRS-1)表达降低,MG使3T3-L1细胞IRS-1表达降低,IRS-1丝氨酸磷酸化(IRS-1 Ser307 phosphorylation,p-IRS-1)表达增加,导致胰岛素抵抗(Insulin resistance,IR),进一步证实线粒体发生FAO障碍。综上所述,两种RCS可通过诱导氧化应激导致细胞线粒体能量代谢障碍。能量代谢障碍的可能机制是:⑴通过抑制脂肪酸转运蛋白表达;⑵通过某个时间段抑制CPT1活性。抑制脂肪酸转运蛋白而导致脂质累积,可能为RCS诱导NASH的分子机制。因此,两种RCS导致细胞毒性的机制是:⑴氧化应激诱导的细胞能量代谢障碍;⑵线粒体膜电位降低;⑶氧化应激。
[Abstract]:Reactive carbonyl compounds (Reactive carbonyl, species, RCS), as a kind of in vivo glucose metabolism, lipid peroxidation (Lipid, peroxidation, LPO) continuous production and food processing process, contains one or more carbonyl groups of highly active electrophilic compounds.RCS and cellular proteins, lipids and nucleic acid reaction. Leading to cell damage. Two methyl ethyl aldehyde (Methylglyoxal, MG) and B two (Glyoxal, GO) for aldehyde activity is very high in RCS two.MG and GO two carbonyl compounds by induced protein carbonylation, changes in protein structure and function, increased oxidative stress and carbonyl stress, and cause inflammation, steatohepatitis diabetes mellitus and non alcoholic hepatitis (Non-alcoholic, steatohepatitis, NASH) and other diseases. The mechanism of RCS induced cell toxicity including oxidative stress, carbonyl stress, protein and protein or nucleic acid crosslinking, gene mutation, cell apoptosis through mitochondrial.RCS Pathways leading to apoptosis, but the specific mechanism of mitochondrial injury still needs further exploration. Therefore this experiment from oxidative stress, mitochondrial membrane potential (Mitochondrial membrane, potential, MMP), mitochondrial fatty acid oxidation (Fatty acid beta -oxidation, FAO) and other aspects of.1. toxicity mechanism of RCS by cell toxicity test and reactive oxygen species (Reactive oxygen, species, ROS), LPO, H2O2 and MMP stress index detection concentration of oxidation, oxidative damage mechanism of cytotoxicity in RCS cells. The results showed that with the increase of the concentration of RCS in rat liver cells (BRL 3A) and embryonic fibroblasts (3T3-L1) activity decreased the cytotoxicity of ROS cells increased, MMP decreased and RCS increased. The capture agent aminoguanidine (Aminoguanidine, AG) Trolox, superoxide anion scavenger, catalase, hydroxyl scavenger mannitol could significantly inhibit the GO A role of.2. through the cell ATP content, protein expression and enzyme activity, the mechanism of cell energy metabolism disorder caused by oxidative stress induced by RCS. The results showed that GO significantly decreased the content of ATP and BRL 3A cells, AG, catalase and Trolox can make ATP cells back to normal levels. With the increase of RCS concentration, BRL 3A 3T3-L1 cells and fatty acid binding protein (Fatty acid-binding, protein, FABP), fatty acid transport protein (Cluster of differentiation 36/fatty acid translocase, CD36) was significantly decreased. These may be the molecular mechanism of.RCS induced by RCS NASH time selective inhibition of carnitine palmitoyl transferase -1 (Carnitine palmitoyltransferase-1 CPT1) confirmed the activity. Mitochondrial fatty acid intake disorders may be one of the mechanisms leading to dysfunction of FAO induced by.GO BRL 3A and 3T3-L1 cell insulin receptor substrate -1 (Insulin receptor substr Ates, IRS-1) MG expression decreased, 3T3-L1 cells decreased expression of IRS-1, serine phosphorylation of IRS-1 (IRS-1 Ser307 phosphorylation, p-IRS-1) expression increased, leading to insulin resistance (Insulin resistance, IR, FAO) further confirmed mitochondrial disorders. To sum up, two kinds of RCS cells through mitochondrial energy metabolism disorder induced oxidative stress. The mechanism of energy metabolism disorder is as follows: through inhibition of fatty acid transport protein expression; the inhibition of CPT1 activity by a certain period of time. The inhibition of fatty acid transport protein and lead to lipid accumulation, the possible molecular mechanism of RCS induced by NASH. Therefore, two kinds of mechanism of the RCS induced cytotoxicity is as follows: cell energy metabolism and oxidative stress induction; the decreased mitochondrial membrane potential; the oxidative stress.

【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S856

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