Duran-Güell Marta, Garrabou Glòria, Flores-Costa Roger, Casulleras Mireia, López-Vicario Cristina, Zhang Ingrid W, Cantó-Santos Judith, Contreras Bryan J, Sánchez-Rodríguez María B, Romero-Grimaldo Berta, Horrillo Raquel, Costa Montserrat, Arroyo Vicente, Clària Joan
Biochemistry and Molecular Genetics Service, Hospital Clínic-IDIBAPS, Barcelona, Spain.
Grifols Chair, European Foundation for the Study of Chronic Liver Failure (EF CLIF), Barcelona, Spain.
FASEB J. 2023 Mar;37(3):e22817. doi: 10.1096/fj.202201526R.
Cytokine-induced inflammation and mitochondrial oxidative stress are key drivers of liver tissue injury. Here, we describe experiments modeling hepatic inflammatory conditions in which plasma leakage leads to large amounts of albumin to reach the interstitium and parenchymal surfaces to explore whether this protein plays a role in preserving hepatocyte mitochondria against the damaging actions of the cytotoxic cytokine tumor necrosis factor alpha (TNFα). Hepatocytes and precision-cut liver slices were cultured in the absence or presence of albumin in the cell media and then exposed to mitochondrial injury with the cytokine TNFα. The homeostatic role of albumin was also investigated in a mouse model of TNFα-mediated liver injury induced by lipopolysaccharide and D-galactosamine (LPS/D-gal). Mitochondrial ultrastructure, oxygen consumption, ATP and reactive oxygen species (ROS) generation, fatty acid β-oxidation (FAO), and metabolic fluxes were assessed by transmission electron microscopy (TEM), high-resolution respirometry, luminescence-fluorimetric-colorimetric assays and NADH/FADH production from various substrates, respectively. TEM analysis revealed that in the absence of albumin, hepatocytes were more susceptible to the damaging actions of TNFα and showed more round-shaped mitochondria with less intact cristae than hepatocytes cultured with albumin. In the presence of albumin in the cell media, hepatocytes also showed reduced mitochondrial ROS generation and FAO. The mitochondria protective actions of albumin against TNFα damage were associated with the restoration of a breakpoint between isocitrate and α-ketoglutarate in the tricarboxylic acid cycle and the upregulation of the antioxidant activating transcription factor 3 (ATF3). The involvement of ATF3 and its downstream targets was confirmed in vivo in mice with LPS/D-gal-induced liver injury, which showed increased hepatic glutathione levels, indicating a reduction in oxidative stress after albumin administration. These findings reveal that the albumin molecule is required for the effective protection of liver cells from mitochondrial oxidative stress induced by TNFα. These findings emphasize the importance of maintaining the albumin levels in the interstitial fluid within the normal range to protect the tissues against inflammatory injury in patients with recurrent hypoalbuminemia.
细胞因子诱导的炎症和线粒体氧化应激是肝组织损伤的关键驱动因素。在此，我们描述了模拟肝脏炎症状态的实验，其中血浆渗漏导致大量白蛋白到达间质和实质表面，以探究这种蛋白质是否在保护肝细胞线粒体免受细胞毒性细胞因子肿瘤坏死因子α（TNFα）的损伤作用方面发挥作用。肝细胞和精密肝切片在细胞培养基中于有无白蛋白的情况下培养，然后用细胞因子TNFα诱导线粒体损伤。还在脂多糖和D - 半乳糖胺（LPS/D - gal）诱导的TNFα介导的肝损伤小鼠模型中研究了白蛋白的稳态作用。分别通过透射电子显微镜（TEM）、高分辨率呼吸测定法、发光 - 荧光 - 比色测定法以及各种底物产生的NADH/FADH来评估线粒体超微结构、氧气消耗、ATP和活性氧（ROS）生成、脂肪酸β - 氧化（FAO）以及代谢通量。TEM分析显示，在无白蛋白的情况下，肝细胞更容易受到TNFα的损伤作用，与用白蛋白培养的肝细胞相比，呈现出更多圆形线粒体且嵴完整性更低。在细胞培养基中有白蛋白存在时，肝细胞还显示出线粒体ROS生成和FAO减少。白蛋白对TNFα损伤的线粒体保护作用与三羧酸循环中异柠檬酸和α - 酮戊二酸之间断点的恢复以及抗氧化剂激活转录因子3（ATF3）的上调有关。在LPS/D - gal诱导的肝损伤小鼠体内证实了ATF3及其下游靶点的参与，这些小鼠显示肝谷胱甘肽水平升高，表明白蛋白给药后氧化应激降低。这些发现揭示，白蛋白分子对于有效保护肝细胞免受TNFα诱导的线粒体氧化应激是必需的。这些发现强调了将间质液中白蛋白水平维持在正常范围内对于保护组织免受反复低白蛋白血症患者炎症损伤的重要性。
