Protection of cultured brain endothelial cells from cytokine-induced damage by α-melanocyte stimulating hormone

Abstract

The blood–brain barrier (BBB), an interface between the systemic circulation and the nervous system, can be a target of cytokines in inflammatory conditions. Pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) induce damage in brain endothelial cells and BBB dysfunction which contribute to neuronal injury. The neuroprotective effects of α-melanocyte stimulating hormone (α-MSH) were investigated in experimental models, but there are no data related to the BBB. Based on our recent study, in which α-MSH reduced barrier dysfunction in human intestinal epithelial cells induced by TNF-α and IL-1β, we hypothesized a protective effect of α-MSH on brain endothelial cells. We examined the effect of these two pro-inflammatory cytokines, and the neuropeptide α-MSH on a culture model of the BBB, primary rat brain endothelial cells co-cultured with rat brain pericytes and glial cells. We demonstrated the expression of melanocortin-1 receptor in isolated rat brain microvessels and cultured brain endothelial cells by RT-PCR and immunohistochemistry. TNF-α and IL-1β induced cell damage, measured by impedance and MTT assay, which was attenuated by α-MSH (1 and 10 pM). The peptide inhibited the cytokine-induced increase in brain endothelial permeability, and restored the morphological changes in cellular junctions visualized by immunostaining for claudin-5 and β-catenin. Elevated production of reactive oxygen species and the nuclear translocation of NF-κB were also reduced by α-MSH in brain endothelial cells stimulated by cytokines. We demonstrated for the first time the direct beneficial effect of α-MSH on cultured brain endothelial cells, indicating that this neurohormone may be protective at the BBB.

Simvastatin, edaravone and dexamethasone protect against kainate-induced brain endothelial cell damage

“…The tightness of the BBB model was tested by the measurement of P app for marker molecules fluorescein (4.72 × 10 −6 cm/s) and albumin (0.31 × 10 −6 cm/s) which were in the range we measured in previous studies [29,41]. Kainate treatment (100 μM) significantly increased the permeability of the BBB model for both markers at the 1 and 24 h time points (Fig.…”

“…This method has been established to follow not only cell attachment and growth, but cell viability as well. We have verified by endpoint colorimetric tests and morphological methods that impedance changes can sensitively detect cell damage [23,29,33] and protection [23,29]. The RTCA-SP system (ACEA Biosciences, CA, USA) registers the impedance of cells automatically every 10 min.…”

“…The isolation of primary rat brain endothelial cells, glial cells and pericytes and the co-culture BBB model were done as described in our previous studies [28,29] and in Additional file 1: Detailed protocol. Primary rat brain endothelial cells were isolated from 3-week-old outbred male and female Wistar rats (Harlan Laboratories, USA).…”

“…The concentrations of the marker molecules were measured by a fluorescence multiwell plate reader (Fluostar Optima, BMG Labtechnologies, Germany) at 584 nm excitation and 680 nm emission wavelengths for EBA, and 485 nm excitation and 520 nm emission wavelengths for SF. The apparent permeability coefficients (P app ) were calculated as described in our earlier studies [29,38].…”

“…M: 1 Kb Plus DNA ladder used as a reference inducer of ROS in the assay (Fig. 4), as in our previous study [29], while sodium nitroprusside was given to increase NO production [40].…”