Long‑term exposure to ethanol downregulates tight junction proteins through the protein kinase Cα signaling pathway in human cerebral microvascular endothelial cells

作者：Hao Yu, Changliang Wang, Xiaolong Wang, Hongbo Wang, Chunan Zhang, Jiabin You, Pengfei Wang, Chun-Mei Feng, Guohui Xu, Rui Zhao, Xu Wu, Guohua Zhang · 发表于：Experimental and Therapeutic Medicine · 年份：2017 · DOI：10.3892/etm.2017.5180 · 被引用次数：24 · 研究领域：Barrier Structure and Function Studies、Neurological Disease Mechanisms and Treatments、Ginger and Zingiberaceae research

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 ...