The regulation of tight junction protein via PKCα/β for abnormal permeability of brain microvascular endothelial cells exposed to paraquat

Abstract

Objective: To explore if conventional protein kinase C (cPKC: PKCα and PKCβ) contributes to paraquat (PQ) -induced abnormal permeability of mouse brain microvascular endothelial cells (BMECs) via the regulation of tight junction (TJ) proteins. Methods: The immortalized mouse brain endothelial cell line (bEnd.3) was used to establish a monolayer blood-brain barrier (BBB) model. In order to evaluate the function of the in vitro BBB model, the transendothelial electrical resistance (TEER) and permeability were measured by a Millicell-ERS volt-ohmmeter and sodium fluorescent (Na-FLU) , respectively. MTT assay was used to determine the relative survival rate of cells. The dose-response relationship was determined by treating cells with 0, 50, 100, 200, and 300 μmol/L PQ for 24 hours. The time-response relationship was determined by treating cells with 200 μmol/L PQ for 6, 12, 24, 48, and 72 hours. After the treatment of cells with 0, 100, 200, and 300 μmol/L PQ for 24 hours, the protein and mRNA expression levels of ZO-1, Occludin, and Claudin-5 were measured by immunofluorescence (IF) and quantitative real-time RT-PCR (qRT-PCR) , respectively; the expression of PKCα, PKCβ, phosphorylated (p) -PKCα, and p-PKCβ was determined by Western blot. After the treatment of cells with 200? mol/L PQ for 24 hours following the pretreatment with a classical PKC inhibitor (Go 6983, 1 μmol/L) for 1 hour, the protein expression of ZO-1, Occludin, Claudin-5, p-PKCα, and p-PKCβ was measured by Western blot. Results: The TEER of the bEnd. 3 cells increased gradually with the cell culture time, and reached a peak value of 114.3±6.9 Ω·cm(2) on day 6. According to the permeability analysis by Na-FLU, cell permeability gradually decreased with the cell culture time, and reached 1.7±0.2 cm/min on day 6, suggesting a well-behaved barrier function of cells. Compared with the control group, the survival rates of the bEnd.3 cells were significantly reduced after exposure to 100, 200, or 300 μmol/L PQ for 24 hours (P<0.05) , or after exposure to 200 μmol/L PQ for 6, 12, 24, 48, or 72 hours (P <0.05) , indicating a dose-and time-dependent relationship. The IF and qRT-PCR results showed that the protein and mRNA expression levels of ZO-1, Occludin, and Claudin-5 were significantly reduced with the increase in the concentration of PQ (P<0.05) . The Western blot analysis showed that compared with the control group, cells exposed to PQ had significantly higher protein expression of p-PKCα and p-PKCβ and significantly lower protein expression of ZO-1, Occludin, and Claudin-5 (P<0.05) . Compared with the PQ treatment group, the Go 6983 intervention group had significantly higher protein expression of ZO-1, Occludin, and Claudin-5 and significantly lower protein expression of p-PKCα and p-PKCβ (P<0.05) . Conclusion: By activation of cPKC (PKCα and PKCβ) , PQ reduces the protein and mRNA expression of TJ proteins and enhances the permeability of murine BMECs.