PKCα/β通过调控紧密连接蛋白参与百草枯致脑微血管内皮细胞的通透性异常

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

目的 探讨经典型蛋白激酶C(cPKC:PKC α、PKC β)是否通过调控紧密连接蛋白(tight Junction,TJ)的表达,引起百草枯(paraquat,PQ)致小鼠脑微血管内皮细胞通透性的异常。 方法 体外培养小鼠脑组织来源的血管内皮细胞株(bEnd.3)作为单层血脑屏障模型,通过Millicell-ERS细胞电阻仪测定跨内皮细胞电阻(TEER)、荧光素钠(Na-FLU)通透性测定以评价体外血脑屏障模型的功能。四甲基偶氮唑蓝(MTT)法测定细胞相对存活率:终浓度0、50、100、200、300 μmol/L PQ处理细胞24 h,确定剂量-效应关系;终浓度200 μmol/L PQ分别处理细胞6、12、24、48、72 h,确定时间-效应关系。终浓度0、100、200、300 μmol/L PQ处理细胞24 h,免疫荧光(IF)和实时荧光定量PCR(qRT-PCR)分别测定紧密连接蛋白(ZO-1、Occludin、Claudin-5)和基因的表达水平,免疫印迹法(Western blot)测定PKC α、PKC β、p-PKC α、p-PKC β蛋白表达水平;经典PKC抑制剂(Go 6983)1 μmol/L预处理细胞1 h后,200 μmol/L PQ染毒细胞24 h,Western blot法测定ZO-1、Occludin、Claudin-5及p-PKC α、p-PKC β蛋白表达水平。 结果 bEnd. 3细胞的TEER随培养时间延长逐渐升高,于第6 d达到峰值[(114.3±6.9)Ω· cm^2],荧光素钠通透性检测显示,细胞通透性随培养时间延长逐渐降低,于第6天降至(1.7±0.2)cm/min,说明细胞屏障功能良好。与对照组比较,100、200、300 μmol/L PQ染毒组细胞存活率明显降低,200 μmol/L PQ染毒细胞12、24、48、72 h,细胞存活率明显降低,差异有统计学意义(P<0.05),呈剂量依赖和时间依赖关系。IF和qRT-PCR显示,随着PQ浓度的升高,ZO-1、Occludin、Claudin-5蛋白和基因表达量明显减少,差异均有统计学意义(P<0.05)。Western blot法检测显示,与对照组比较,PQ染毒组的p-PKC α、p-PKC β蛋白表达水平明显升高,ZO-1、Occludin、Claudin-5蛋白表达量明显降低,差异均有统计学意义(P<0.05);Go 6983干预组的ZO-1、Occludin、Claudin-5蛋白表达水平较染毒组明显升高,p-PKC α、p-PKC β蛋白表达水平明显下降,差异均有统计学意义(P<0.05)。 结论 PQ通过激活cPKC(PKC α、PKC β)导致紧密连接蛋白/基因表达降低,引起小鼠脑微血管内皮细胞通透性升高。

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.