Blocking the EGFR/p38/NF-κB Signal Pathway Alleviates Disruption of BSCB and Subsequent Inflammation After Spinal Cord Injury

Abstract Background:Epidermal growth factor receptor (EGFR) activation may play an important role in blood spinal cord barrier (BSCB) disruption and secondary injury after SCI as it is significantly upregulated in the astrocytes (AS) and microvascular endothelial cells (MEC), which are the main component of cells in BSCB. EGFR inhibition alleviates the disruption of BSCB and improves the functional recovery in rats following spinal cord injury (SCI). However, the biological mechanisms underlying EGFR activation mediating secondary damage after SCI remain unclear. Methods:An in vitro model of Oxygen and glucose deprivation/reoxygenation (OGD/R) induced BSCB damage and in vivo rat SCI model was employed to define the role of EGFR activation and its induced inflammatory injury during this pathological process in AS and MEC. AS and MEC were exposed to EGFR or p38 MAPK up-regulation in the presence and absence of EGFR inhibitor, p38 MAPK inhibitor, NF-κB inhibitor, and/or appropriate shRNA. RT-PCR, ELISA and western blotting were used for mRNA and protein expression analyses of TNF-α, iNOS, COX-2 and IL-1β. Immunohistochemical staining and confocal microscopy were used to demonstrate cellular EGFR activation and to investigate the expression of tight junction (TJ) protein (ZO-1 and occludin). Measurement of transendothelial electrical resistance (TEER) and transendothelial FITC-dextran permeability were used to measure permeability of BSCB in vitro, while Evans blue dye extravasation test and evaluation spinal cord edema were used to detect permeability of BSCB in vivo.Results:The expression of pEGFR was significantly increased in BSCB component cells (AS and MEC) after SCI and BSCB damage model. EGFR activation induced inflammation injury by upregulating the expression of TNF-α, iNOS, COX-2, and IL-1β and BSCB disruption with loss of TJ protein by downregulating the expression of ZO-1 and occludin in BSCB damage model and SCI rats. Moreover, EGFR or p38 activation leads to NF-κB nuclear translocation in primary AS and MEC after OGD/R. The EGFR inhibitor as well as shRNA against EGFR markedly attenuated pro-inflammatory factor excessive producing and loss of TJ protein, and activation of the EGFR/p38/NF-κB pathway. While, EGFR overexpression significantly increased the expression of TNF-α, iNOS, COX-2, and IL-1β and decrease the expression of ZO-1 and occludin, inducing activation of the EGFR/p38/NF-κB pathway in both AS and MEC. Conclusion:This study strongly suggests that EGFR activation in BSCB component cells after SCI and BSCB damage model mediates both upregulation of pro-inflammation expression and downregulation of TJ protein downregulation via the EGFR/p38/NF-κB pathway. These findings contribute to a better understanding of the biological mechanisms underlying BSCB disruption and secondary injury following SCI mediating by EGFR activation.