Hydrogen peroxide initiates oxidative stress and proteomic alterations in meningothelial cells

Abstract

Meningothelial cells (MECs) are fundamental cells of the sheaths covering the brain and optic nerve, where they build a brain/optic nerve-cerebral spinal fluid (CSF) barrier that prevents the free flow of CSF from the subarachnoid space, but their exact roles and underlying mechanisms remain unclear. Our attempt here was to investigate the influence elicited by hydrogen peroxide (H₂O₂) on functional changes of MECs. Our study showed that cell viability of MECs was inhibited after cells were exposed to oxidative agents. Cells subjected to H₂O₂ at the concentration of 150 µM for 24 h and 48 h exhibited an elevation of reactive oxygen species (ROS) activity, decrease of total antioxidant capacity (T-AOC) level and reduced mitochondrial membrane potential (ΔΨm) compared with control cells. 95 protein spots with more than twofold difference were detected in two dimensional electrophoresis (2DE) gels through proteomics assay following H₂O₂ exposure for 48 h, 10 proteins were identified through TOF/MS analysis. Among the proteomic changes explored, 8 proteins related to energy metabolism, mitochondrial function, structural regulation, and cell cycle control were downregulated. Our study provides key insights that enhance our understanding of the role of MECs in the pathology of brain and optic nerve disorders.

Figure 1

H₂O₂ treatment decreases cell viability of MECs. Cell viability of MECs was reduced after exposure to H₂O₂ at different concentration for 12 h (A), 24 h (B), 36 h (C), 48 h (D) respectively compared with controls. Data are represented as mean ± SD (n = 6),* p < 0.05, ^▲p < 0.01; ^#p < 0.001, control versus H₂O₂ treatment groups. MECs, meningothelial cells.

Figure 2

Morphology of MECs under the microscopy. MECs culture images of controls after the incubation for 24 h (A) and 48 h (C); MECs treated with H₂O₂ (150 µM) for 24 h (B) and 48 h (D). Scale bar: 100 μm. MECs, meningothelial cells.

Figure 3

H₂O₂ treatment inhibits T-AOC content of MECs. T-AOC content of MECs was decreased after cells were exposed to H₂O₂ (150 µM) for 24 h and 48 h respectively compared with untreated cells. Data are represented as mean ± SD (n = 6) *p < 0.05, **p < 0.01, ***p < 0.001. MECs, meningothelial cells; T-AOC, total antioxidant capacity.

Figure 4

H₂O₂ exposure activates intracellular ROS activity of MECs. Quantitative analysis of intracellular ROS in MECs exposure to H₂O₂ (150 µM) for 24 h and 48 h respectively. Values are shown as mean ± SD (n = 6). *p < 0.05, ***p < 0.001. MECs, meningothelial cells; ROS, reactive oxygen species.

Figure 5

Impact of H₂O₂ on mitochondrial membrane potential (ΔΨm) of MECs. Changes in the ΔΨm were analyzed using JC-1 after MECs exposure to H₂O₂ (150 µM) for 48 h. H₂O₂-exposed cells (B) with decreased J-aggregates representing low ΔΨm compared to the controls (A); A significant increase in monomer in cells following H₂O₂ exposure (C). Values are shown as mean ± SD (n = 4). ***p < 0.001. MECs, meningothelial cells; JC-1:5,5′,6,6′ tetrachloro-1,1′3,3′ tetraethylbenzimidazolcarbocyanine iodide.

Figure 6

Representative two-dimensional difference gel electrophoresis (2D-DIGE) gel images. Isoelectric focusing (IEF) was performed using 24 cm immobilized pH gradient (IPG) strips covering a linear pH 3–10. (A) Shows a 2D gel of total protein from controls; (B) shows a 2D gel of total protein from H₂O₂-treated cells.