Perrone Pasquale, Notariale Rosaria, Lettieri Gennaro, Mele Luigi, La Pietra Valeria, Piscopo Marina, Manna Caterina
Department of Precision Medicine, School of Medicine, University of Campania "Luigi Vanvitelli", 80138, Naples, Italy.
Department of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, Villa Comunale, 80121, Naples, Italy.
Free Radic Biol Med. 2025 Feb 1;227:42-51. doi: 10.1016/j.freeradbiomed.2024.11.047. Epub 2024 Nov 27.
In several physiopathological processes, phosphatidylserine (PS), normally sequestered to the inner leaflet of the plasma membrane, becomes exposed to the cell surface. In erythrocytes (RBC), PS externalization is a crucial event for the removal of aged/damaged cells but can also be associated with increased prothrombotic activity. Structurally related olive oil antioxidants, including hydroxytyrosol (HT), are able to significantly reduce the percentage of PS-exposing RBC, when cells are exposed to toxic compounds such as the heavy metal mercury (Hg). The aim of the present study was to identify the molecular mechanisms underlying the protective effect, with a focus on two different phospholipid translocases, the ATP-dependent flippase ATP11C and the calcium-dependent scramblase PLSCR1, which are responsible for PS internalization and exposure, respectively. In addition to HT, its monophenol analogue, tyrosol, and its in vivo metabolite, homovanillic alcohol, were also tested. Our investigation revealed that exposure of human intact RBC to HgCl induced a decrease in flippase activity and an increase in scramblase activity, and that all the selected phenols restored the control activity, regardless of their different scavenging properties. Interestingly, all phenols restored the ATP level of control cells, which were significantly reduced by HgCl treatment. Conversely, no variation in intracellular calcium was observed under our experimental conditions. Additionally, all phenols restored the glutathione levels, significantly reduced in the presence of HgCl. In line with the data on the enzymatic activity, Western blotting analysis indicated changes in the membrane expression of the two enzymes, alterations prevented by antioxidant pre-treatment. Finally, molecular docking analysis suggests that the tested antioxidants may be able to directly interact with ATP11C. Our findings provide an experimental basis for the use of olive oil bioactive compounds in nutritional/nutraceutical strategies for the prevention of Hg-related toxicity, particularly in relation to the cardiovascular tissues.
在多种生理病理过程中，通常隔离于质膜内小叶的磷脂酰丝氨酸（PS）会暴露于细胞表面。在红细胞（RBC）中，PS外化是清除衰老/受损细胞的关键事件，但也可能与血栓形成活性增加有关。结构相关的橄榄油抗氧化剂，包括羟基酪醇（HT），当细胞暴露于重金属汞（Hg）等有毒化合物时，能够显著降低暴露PS的RBC百分比。本研究的目的是确定这种保护作用的分子机制，重点关注两种不同的磷脂转位酶，即ATP依赖的翻转酶ATP11C和钙依赖的磷脂翻转酶PLSCR1，它们分别负责PS的内化和暴露。除了HT，还测试了其单酚类似物酪醇及其体内代谢产物高香草醇。我们的研究表明，将完整的人RBC暴露于HgCl会导致翻转酶活性降低和磷脂翻转酶活性增加，并且所有选定的酚类都恢复了对照活性，无论它们的清除特性如何。有趣的是，所有酚类都恢复了对照细胞的ATP水平，HgCl处理使其显著降低。相反，在我们的实验条件下未观察到细胞内钙的变化。此外，所有酚类都恢复了谷胱甘肽水平，在存在HgCl的情况下其显著降低。与酶活性数据一致，蛋白质印迹分析表明两种酶的膜表达发生了变化，抗氧化剂预处理可防止这种变化。最后，分子对接分析表明，测试的抗氧化剂可能能够直接与ATP11C相互作用。我们的研究结果为在预防Hg相关毒性的营养/营养保健策略中使用橄榄油生物活性化合物提供了实验依据，特别是与心血管组织相关的毒性。
