Yu Hao, Wang Changliang, Wang Xiaolong, Wang Hongbo, Zhang Chunan, You Jiabin, Wang Pengfei, Feng Chunmei, Xu Guohui, Zhao Rui, Wu Xu, Zhang Guohua
Department of Forensic Pathology, School of Forensic Medicine, China Medical University, Shenyang, Liaoning 110122, P.R. China.
Exp Ther Med. 2017 Nov;14(5):4789-4796. doi: 10.3892/etm.2017.5180. Epub 2017 Sep 21.
Brain microvascular endothelial cells (BMECs) are the primary component of the blood-brain barrier (BBB). Tight junction (TJ) proteins, including claudin, occludin and zonula occludens (ZO)-1, ZO-2 and ZO-3, maintain the structural integrity of BMECs. Ethanol activates the assembly and disassembly of TJs, which is a process that is regulated by protein kinase C (PKC). In addition, ethanol treatment leads to the loss of structural integrity, which damages the permeability of the BBB and subsequently affects central nervous system homeostasis, thus allowing additional substances to enter the brain. However, the mechanisms underlying ethanol-induced loss of BBB structure remain unknown. It has been hypothesized that long-term exposure to ethanol reduces the expression of claudin-5, occludin and ZO-1 via the PKC signaling pathway, thereby affecting BBB structural integrity. In the current study, the human cerebral microvascular endothelial cell line, HCMEC/D3, was treated with 50, 100, 200 and 400 mM ethanol for 24, 48 and 72 h. Cell viability was determined using an MTS assay. The expression of claudin-5, occludin and ZO-1 protein and mRNA was measured using western blot analysis and reverse transcription-quantitative polymerase chain reaction, respectively. Following the pretreatment of HCMEC/D3 cells with the PKCα-specific inhibitor, safingol (10 µmol/l), the expression of claudin-5, occludin, ZO-1 and phosphorylated (p)-PKCα was measured using western blot analysis, and PKCα localization was determined by immunofluorescence. With increasing concentrations of ethanol, the expression of claudin-5, occludin and ZO-1 protein decreased, while the expression of claudin-5, occludin and ZO-1 mRNA increased. Exposure to ethanol significantly increased the expression of p-PKCα, whereas no significant effect on the expression of PKCα was observed. Following 48 h treatment with 200 mM ethanol, the expression of claudin-5, occludin and ZO-1 protein was significantly decreased when compared with the control. By contrast, the expression of p-PKCα was increased, and increased translocation of PKCα from the cytoplasm to the nuclear membrane and nucleus was observed. In addition, the results demonstrated that safingol significantly reversed these effects of ethanol. In conclusion, long-term exposure to ethanol downregulates the expression of claudin-5, occludin and ZO-1 protein in HCMEC/D3 s, and this effect may be mediated via activation of PKCα.
脑微血管内皮细胞（BMECs）是血脑屏障（BBB）的主要组成部分。紧密连接（TJ）蛋白，包括闭合蛋白、闭锁蛋白和闭合小环蛋白（ZO）-1、ZO-2和ZO-3，维持着BMECs的结构完整性。乙醇激活TJ的组装和拆卸，这一过程由蛋白激酶C（PKC）调控。此外，乙醇处理会导致结构完整性丧失，损害血脑屏障的通透性，进而影响中枢神经系统的稳态，使其他物质得以进入大脑。然而，乙醇诱导血脑屏障结构丧失的潜在机制仍不清楚。据推测，长期暴露于乙醇会通过PKC信号通路降低闭合蛋白-5、闭锁蛋白和ZO-1的表达，从而影响血脑屏障的结构完整性。在本研究中，人脑血管内皮细胞系HCMEC/D3分别用50、100、200和400 mM乙醇处理24、48和72小时。使用MTS法测定细胞活力。分别使用蛋白质印迹分析和逆转录-定量聚合酶链反应测量闭合蛋白-5、闭锁蛋白和ZO-1蛋白及mRNA的表达。在用PKCα特异性抑制剂沙芬戈（10 µmol/l）预处理HCMEC/D3细胞后，使用蛋白质印迹分析测量闭合蛋白-5、闭锁蛋白、ZO-1和磷酸化（p）-PKCα的表达，并通过免疫荧光确定PKCα的定位。随着乙醇浓度的增加，闭合蛋白-5、闭锁蛋白和ZO-1蛋白的表达降低，而闭合蛋白-5、闭锁蛋白和ZO-1 mRNA的表达增加。暴露于乙醇显著增加了p-PKCα的表达，而对PKCα的表达未观察到显著影响。用200 mM乙醇处理48小时后，与对照组相比，闭合蛋白-5、闭锁蛋白和ZO-1蛋白的表达显著降低。相比之下，p-PKCα的表达增加，并且观察到PKCα从细胞质向核膜和细胞核的转位增加。此外，结果表明沙芬戈显著逆转了乙醇的这些作用。总之，长期暴露于乙醇会下调HCMEC/D3中闭合蛋白-5、闭锁蛋白和ZO-1蛋白的表达，这种作用可能是通过激活PKCα介导的。
