脂多糖诱导的肝衰竭中巨噬细胞能量代谢变化

摘要/Abstract

摘要： 目的探讨苹果酸脱氢酶底物及产物在急性肝衰竭(acute liver failure,ALF)患者中的变化趋势,以及经脂多糖(lipopolysaccharide,LPS)诱导的小鼠ANA-1巨噬细胞能量代谢特点。方法收集16例研究对象血清,其中ALF组8例,正常体检组8例,使用液相色谱-质谱联合技术(liquid chromatograph mass spectrometer,LC-MS)检测苹果酸脱氢酶底物及产物(苹果酸和草酰乙酸)水平。体外培养小鼠ANA-1巨噬细胞,分为对照组及LPS造模组,试剂盒检测细胞上清肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)以及细胞内苹果酸脱氢酶1(malate dehydrogenase 1,MDH1)、乳酸、葡萄糖、三磷酸腺苷(adenosine-5'-triphosphate,ATP)水平,蛋白质印迹法检测细胞内MDH1蛋白含量。结果与正常组相比,ALF组血清苹果酸脱氢酶底物苹果酸增多,产物草酰乙酸减少,差异有统计学意义(P<0.05)。在体外培养的小鼠ANA-1巨噬细胞中,LPS处理组细胞上清中TNF-α水平高于对照组,为[ (1722.501±76.261)pg/mL比(255.010±16.139)pg/mL(P<0.05),说明LPS诱导巨噬细胞释放了炎症因子。同时,与对照组(15.710±0.302)ng/mL相比,LPS处理组细胞内MDH1水平降低(11.831±0.335)ng/mL,蛋白含量减少(P<0.05)。此外,LPS组ANA-1细胞内乳酸及葡萄糖水平升高[ (0.281±0.016)mmol/L比(0.081±0.012)mmol/L,(0.081±0.006)μmol/mL比(0.033±0.004)μmol/mL],ATP水平下降[ (61.766±11.982)μmol/gprot比(130.786±25.386)μmol/gprot],(P<0.05)。结论 ALF时,LPS抑制巨噬细胞内MDH1活性,诱导线粒体相关能量代谢障,乳酸和葡萄糖增多、ATP合成减少。

关键词: 急性肝衰竭, 苹果酸脱氢酶1, 脂多糖, 巨噬细胞, 能量代谢

Abstract: Objective During the process of acute liver failure (ALF), endotoxin activated macrophages to release cytokines and induced changes in cellular metabolism. The aim of this study was to investigate the change of the substrates and products of malate dehydrogenase, and to analyze the metabolic changes of mice ANA-1 macrophages in response to lipopolysaccharide (LPS). Methods A total of 16 subjects were enrolled, including 8 ALF cases and 8 were healthy controls. Liquid Chromatograph Mass Spectrometer (LC-MS) was used to analyze the level of the substrates and related metabolite (malate and oxaloacetate). The Mice ANA-1 macrophages cultured in vitro were divided into normal control group and LPS group [treated by LPS (5μg/ml)]. The levels of tumor necrosis factor-α (TNF-α), malate dehydrogenase 1 (MDH1), lactic acid, glucose, adenosine-5'-triphosphate (ATP) were tested. Western-blot was used to detect the intracellular MDH1 protein content. Results Compared with the normal group, the malate increased and oxaloacetate decreased in ALF group (P<0.05). In LPS-induced mice ANA-1 macrophages, the level of TNF-α in supernatant increased [(1722.501 ± 76.261) pg/mL vs (255.010 ± 16.139) pg/mL], P<0.05, which indicates that LPS stimulated macrophages release cytokines. Compared with the control group (15.710 ± 0.302) ng/mL, the level of MDH1(11.831 ± 0.335) ng/mL and protein content in the LPS group were reduced (P<0.05). In addition, the levels of lactate and glucose in ANA-1 cells treated by LPS increased [(0.281 ± 0.016) mmol/L vs (0.081±0.012) mmol/L, (0.081 ± 0.006) μmol/mL vs (0.033 ± 0.004) μmol/mL], and the ATP levels decreased [(61.766 ± 11.982) μmol/gprot VS (130.786 ± 25.386) μmol/gprot], P<0.05. Conclusion During the process of ALF, LPS suppressed the activity of MDH1 in macrophages, induced mitochondrial-related metabolism disorders, resulted in the increase of lactate and glucose, and decrease of ATP production.

Key words: Acute liver failure, Malate dehydrogenase 1, Lipopolysaccharide, Macrophage, Energy metabolism

郭金, 石春霞, 邓威, 张璐懿, 陈倩, 龚作炯. 脂多糖诱导的肝衰竭中巨噬细胞能量代谢变化[J]. 肝脏, 2022, 27(9): 973-977.

GUO Jin, SHI Chun-xia, DENG Wei, ZHANG Lu-yi, CHEN Qian, WANG Yao, GONG Zuo-jiong. Metabolic changes of macrophages in LPS-induced liver failure[J]. Chinese Hepatolgy, 2022, 27(9): 973-977.

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