P38MAPK And JNK Signaling Pathway With Matrix-Metalloproteinases Involved In Lipopolysaccharide-Induced Damage Of Blood-Brain Barrier

PART Ⅰ The effect of p38MAPK and JNK signal pathways on LPS induces dysregulation of tight junction proteins.Objective:To investigate the effect of p38MAPK and JNK signal pathways on LPS induced dysregulation of tight junction proteins.Methods:1. In this study, human cerebral micro vascular endothelial cells (hCMEC/D3) were exposed to different concentrations of LPS, p38 MAPK inhibitor (SB203580) or JNK inhibitor (SP600125) for 24h, the viability of hCMEC/D3 was tested by MTT assay.2. hCMEC/D3 were treated with different concentrations of LPS for 24h, the expression of TJ proteins (Occuludin and Zonula Occludens (ZO)-1) was determined by Western blot.3. hCMEC/D3 cells were treated with SB203580 or SP600125 for different time, phosphorylation of p38 MAPK and JNK were determined by Western blot.4. Then hCMEC/D3 were pretreated with SB203580 or SP600125 1h prior to LPS treatment, the expression of TJ proteins and mRNA (Occludin and ZO-1) were measured with Western blot and quantitative Real Time-PCR (qRT-PCR), respectively.Results:1.LPS, SB203580 or SP600125 under 10μg/ml,7.69 μg/ml or 0.22 μg/ml concentrations respectively had no side effects on cell viability as determined by the MTT assay.2. Treatment with LPS decreased protein and mRNA levels of Occludin and ZO-1, and also activated the phosphorylation of p38 MAPK and JNK.3. These effects were attenuated by pretreatment with the SB203580 or SP600125, but not on the LPS-induced decrease in ZO-1 protein and mRNA levels.Conclusions:These results show that LPS induces dysregulation of Occludin on protein and mRNA levels in hCMEC/D3 via upregulating phosphorylation of p38MAPK and JNK signal pathways, but not on the LPS-induced decrease in ZO-1 protein and mRNA levels. PART ⅡThe effect of matrix metalloprotelnases on LPS induces dysregulation of tight junction proteins and their mechamisms of signaling pathway.Objective:To investigate the effects of matrix metalloproteinase-2/-9(MMP-2 and MMP-9) on LPS induces dysregulation of tight junction proteins and their mechanisms of signaling pathway.Methods:1. In this study, hCMEC/D3 were pretreated with SB203580 or SP600125 1 h prior to exposing to LPS, the expression of MMP-2 and MMP-9 were measured with Enzyme linked immunosorbent assay(ELISA) and qRT-PCR, respectively.2. hCMEC/D3 were pretreated with MMPs inhibitor(Doxycycline hyclate), MMP-2 inhibitor(SB-3CT 13.9nmol/l) or MMP-9 inhibitor(SB-3CT 600nmol/L) before exposed to LPS, the expression of Occludin were measured with Western blot.Results:1. Treatment with LPS induced overexpression of MMP-2 and MMP-9, which were attenuated by p38 MAPK and JNK inhibitors.2. LPS decreased protein level of Occludin, which was attenuated by pretreatment with the inhibitors of MMPs, MMP-2 and MMP-9.Conclusions:These data suggest that overexpression of MMP-2 and MMP-9 in hCMEC/D3 are pathological events associated with the decrease of Occludin induced by LPS, and may explain the molecular basis of BBB disruption, in which p38MAPK and JNK signaling pathways involved.