Fig. 3

NRG-1 attenuates heme-induced damage of BBB in an in vitro BBB model consisting of human brain microvascular endothelial cells (hCMEC/D3) and neuroglial cells/astrocytes of human A172 glioma cells. a–d When hCMEC/D3 was co-cultured with human neuroglial cells/ astrocytes (human A172 glioma), Claudin 5 was expressed on cell membranes (arrows) and disappeared following treatment with 30 μM heme for 24 h. Additionally, heme reduced Claudin5 expression while NRG-1 attenuated heme-induced reduction in claudin5 expression. e Quantitation of claudin5 staining of hCMEC/D3 co-cultured with A172 astrocytes by different treatment as indicated. Total immunofluorescence intensity with claudin5 antibody (red) was determined with ImageJ software program, and the average value for the indicated number of cells examined (n = 500) is shown. Quantitative analysis of immunoreactive signals of claudin5 antibody in hCMEC/D3 cells indicated decreased staining from pronounced (401.80 ± 39.00) in control to moderate (303.21 ± 32.67) after treatment with heme (n = 500, p < 0.05), while staining for claudin5 proteins in cells treated with NRG-1 increased significantly (385.00 ± 39.97, p < 0.05). The asterisks indicate significant difference with a p value of 0.05 as determined with Student’s paired t test. f–i When hCMEC/D3 was co-cultured with human neuroglial cells/ astrocytes (A172 glioma), tight junction protein marker ZO-1 was expressed on cell membranes (arrows). The membranous staining was disrupted and cytoplasmic staining was more dense after cells were treated with 30 μM heme for 24 h. In addition, heme reduced ZO-1 expression while NRG-1 increased ZO-1 expression reduced by heme. j Quantitation of ZO-1 staining of hCMEC/D3 co-cultured with A172 astrocytes by different treatment as indicated. Total immunofluorescence intensity with ZO-1antibody (red) was determined with ImageJ software program, the average value for the indicated number of cells examined (n = 500) is shown. Briefly, the average immunofluorescence intensity for ZO-1decreased to moderate 114.69 ± 11.60 in heme-treated cells as compared to control (192.17 ± 20.01, p < 0.05), while the intensity of this staining returned to pronounced levels (187.46 ± 16.78) when the cells were treated with NRG-1 (p < 0.05). The asterisks indicate significant difference with a p value of 0.05 as determined with Student’s paired t test. k–n When hCMEC/D3 was co-cultured with human neuroglial cells/astrocytes from human A172 glioma, tight junction protein marker occludin was expressed on cell membranes (arrows). The membrane-bound expression disappeared after cells were treated with 30 μM heme for 24 h. In addition, heme reduced occludin expression, and NRG-1 increased occludin expression reduced by heme. o Quantitation of occludin staining in hCMEC/D3 cells treated with heme, NRG-1, combination of heme and NRG-1, and corresponding control. Total immunofluorescence intensity with occludin antibody (red) was determined with ImageJ software program, and the average value for the indicated number of cells examined (n = 500) is shown. The average relative intensity of occludin in cells treated with heme decreased to 170.11 ± 15.89 as compared to control 303.34 ± 28.98 (n = 500, p < 0.05). The average immunofluorescence intensity with occludin antibody increased to 350.00 ± 47.92 when NRG-1 was added to the cells treated with heme (p < 0.05). Together, these results suggest that NRG-1 protects against heme-induced damage of brain vascular endothelial cells in in vitro BBB model