Protective Actions of α-Tocopherol on Cell Membrane Lipids of Paraquat-Stressed Human Astrocytes Using Microarray Technology, MALDI-MS and Lipidomic Analysis

Abstract

Cellular senescence is one of the main contributors to some neurodegenerative disorders. The early detection of senescent cells or their related effects is a key aspect in treating disease progression. In this functional deterioration, oxidative stress and lipid peroxidation play an important role. Endogenous antioxidant compounds, such as α-tocopherol (vitamin E), can mitigate these undesirable effects, particularly lipid peroxidation, by blocking the reaction between free radicals and unsaturated fatty acid. While the antioxidant actions of α-tocopherol have been studied in various systems, monitoring the specific effects on cell membrane lipids at scales compatible with large screenings has not yet been accomplished. Understanding the changes responsible for this protection against one of the consequences of senescence is therefore necessary. Thus, the goal of this study was to determinate the changes in the lipid environment of a Paraquat-treated human astrocytic cell line, as a cellular oxidative stress model, and the specific actions of the antioxidant, α-tocopherol, using cell membrane microarray technology, MALDI-MS and lipidomic analysis. The stress induced by Paraquat exposure significantly decreased cell viability and triggered membrane lipid changes, such as an increase in certain species of ceramides that are lipid mediators of apoptotic pathways. The pre-treatment of cells with α-tocopherol mitigated these effects, enhancing cell viability and modulating the lipid profile in Paraquat-treated astrocytes. These results demonstrate the lipid modulation effects of α-tocopherol against Paraquat-promoted oxidative stress and validate a novel analytical high-throughput method combining cell cultures, microarray technology, MALDI-MS and multivariate analysis to study antioxidant compounds against cellular senescence.

Keywords: astrocytes; human cell line; lipid peroxidation; membrane microarrays; oxidative stress.

Figure 1

Effects of Paraquat on 1321N1 cells viability with or without pre-treatment with α-tocopherol. (A) Time course and design of treatments for the evaluation of cell culture viability. (B) Time course of cell viability in control conditions and after treatment with Paraquat (500 µM) with or without a pre-treatment with α-tocopherol (1 µM). Measurements were performed every 24 h using trypan blue cell viability assay. Asterisks symbols (*) refer to post-hoc comparisons with respect to control; hash symbols (#) refer to comparisons with respect to Paraquat. Colour of symbols allude to the condition been compared. (C) Cell viability results at 24 h expressed as percentage of live cells. Shapiro-Wilk test was performed to check the normality of distributions. One-way ANOVA analysis (two-tailed) and Tukey post-hoc were conducted, α set to 0.05. p-value < 0.05 (*), p-value < 0.01 (**), p-value < 0.0001 (****), moreover, p-value <0.05 (#), p-value < 0.01 (##), p-value < 0.0001 (####).

Figure 2

Relative intensity change in different lipid adducts when comparing Paraquat treated membranes with control samples. Data are shown as percentage of control values. (A) Changes in sphingolipids (Cer, HexCer and SM). (B,C)) Changes in glycerophospholipids. PA, PG, PE, and PE O- shown in (B). LPC, PC, and PI shown in (C). Abbreviations: Ceramides (Cer), hexosylceramides (HexCer), sphingomyelins (SM), glycerophosphates (PA), glycerophosphoethanolamines (PE and its ether form PE O-), glycerophosphoglycerols (PG), glycerophosphocholines and lisophosphocholines (PC and LPC), glycerophosphoinositols and lisofosfoinositols (PI and LPI), and ether forms of glycerophosphocholines and lisophosphocholines (PC O- and LPC O-). Oxygen number inside the lipid head and fatty acids in Ceramides and Sphingomyelins are indicated by O with a suffix. Bars with red arrows are only present in Paraquat-treated samples.

Figure 3

Relative intensity change in lipid adducts when comparing Paraquat-treated membranes preceded by an α-tocopherol pre-treatment with samples treated with Paraquat only. Data are shown as percentage of control values. (A) Change in sphingolipids (Cer, HexCer and SM). (B,C)) Changes in glycerophospholipids. PA, PG, PE, and PE O shown in (B). PC, PC O-, LPC, LPC O-, PI, and LPI shown in (C). Abbreviations: Ceramides (Cer), hexosylceramides (HexCer), spingomyelins (SM), glycerophosphatidic acid (PA), glycerophosphoethanolamine (PE), glycerophosphoglycerols (PG), glycerophosphocholines and lysophosphocholines (PC and LPC), glycerophosphoinositols and lysophosphoinositols (PI and LPI). Ether forms indicated by the O-suffix. Oxygen number inside lipid head and fatty acids in Ceramides and Sphingomyelins is indicated by O with a suffix.

Figure 4

Graphic summary of lipid changes observed in CMMAs upon oxidative stress preceded or not by antioxidant treatment.