[Changes and regulatory mechanism of tight junction proteins in in vitro model of lead-induced blood-brain barrier injury].

OBJECTIVE

To clarify the relationship between the expressions of tight junction (TJ) proteins and the changes of MAPK, AKT signaling pathways in the process of lead-induced blood-brain barrier (BBB) injury in vitro and observe the regulatory mechanisms of related signaling pathway inhibitors.

METHODS

The in vitro model of lead-induce BBB injury was established using Transwell™ chamber co-culture system of human umbilical vein endothelium-derived ECV304 cells and rat glioma C6 cells. The system was supplemented with different concentrations of PbC4H6O4. The transendothelial electrical resistance (TEER) and permeability of FITC-labeled dextran were tested to evaluate the changes of permeability of in vitro BBB cell model. The expressions and locations of TJ proteins (ZO-1, occludin and claudin-5) were determined by Western blotting and immunocytochemical staining, respectively. Signaling pathway inhibitors were used to clarify the causal relation between TJ protein expressions and signaling pathways.

RESULTS

Lead inhibited ECV304 growth and this inhibition was time- and dose-dependent (P<0.05). TEER value decreased and the permeability of FITC-labeled dextran increased significantly as the time of lead exposure went by (P<0.05). Western blotting showed that the expressions of ZO-1, occludin and claudin-5 were significantly reduced after 2.5 μmol/L lead treatment (P<0.05); the phosphorylation levels of ERK1/2, JNK, p38, AKT473 and AKT308 were elevated significantly (P<0.05); ERK1/2 inhibitor PD98059 and PI3K inhibitor LY294002 reversed the lead effects. These results suggested that lead could inhibit the TJ protein expressions through the ERK1/2 and PI3K/AKT pathways.

CONCLUSION

The TJ proteins are important target molecules of lead-induced structural and functional damage of BBB. The MAPK and PI3K/AKT signal transduction pathways are involved in the regulation on the expressions of TJ proteins.