溶血磷脂酸对体外血脑屏障模型通透性的影响

目的 探讨溶血磷脂酸(LPA)对血脑屏障通透性影响及其可能机制.方法 将体外建立的血脑屏障模型随机分为空白对照组、LPA组和蛋白激酶C抑制剂(Ro31-8220)+LPA组.γ计数仪检测模型对125I-BSA的通过率;流式细胞仪检测PKC-α表达阳性的微血管内皮细胞(BMEC)比例;类鬼笔环肽染色观察BMEC的F-肌动蛋白变化;电子显微镜观察血脑屏障-紧密连接(BBB-TJ)改变;Western印迹法检测BBB-TJ内闭合蛋白5表达.结果 LPA组125I-BSA通过率(364 cmp±15cmp)和PKC-α阳性细胞率(77%±7%)均高于空白对照组和Ro31-8220+LPA组(184 cmp±10 cmp、223 cmp±9 cmp,42%±4%、52%±3%,均P＜0.01);闭合蛋白5表达则低于空白对照组和Ro31-8220+LPA组(0.353±0.04、1.00±0.03、0.574±0.07,均P＜0.01),同时LPA使BBB-TJ开放、F-肌动蛋白重组.结论 LPA可促使BBB-TJ开放,增加血脑屏障通透性,其机制可能与PKC-α信号途径激活进而促使闭合蛋白5表达下调以及F-肌动蛋白重组有关.

Effect of lysophosphatidic acid increase the permeability of blood-brain barrier model

OBJECTIVE: To explore if lysophosphatidic acid (LPA) can regulate the permeability of blood-brain barrier (BBB) and the possible mechanism. METHODS: Astrocytes and blood microvascular endothelial cells (BMECs) of neonatal SD rat were co-cultured to establish BBB model in vitro. The BBB models were divided randomly into 3 groups: blank control group (Group C), LPA group (Group L, treated with LPA 10 micromol/L for 2 h), and Ro31-8220 plus LPA group. (Group Ro + L, pretreated with Ro31-8220, a selective protein kinase inhibitor 5 micromol/L for 2 h and then treated with LPA 10 micromol/L for 2 h). 12I-bovine serum albumin (BSA) was added into the culture fluid of the BBB models and then the permeability of the BBB models was detected by gamma-events-per-unit-time meter. The cells expressing PKC-alpha were detected by flow cytometry (FCM). Phalloidin staining and fluorescence microscopy were used to detect the expression of F-actin. The BBB-tight-junction (TJ) was observed by electron microscopy. The expression of claudin-5 was detected by Western blotting. RESULTS: The throughput of 125I-BSA of Group L was 364 cmp +/- 15 cmp, significantly higher than those of the Group C and Group RO + L (184 cmp +/- 10 cmp and 223 cmp 9 cmp respectively, both P < 0.01). FCM showed that the rate of PKC-alpha expressing-BMECs of Group L was 77% +/- 7%, significantly higher than those of Groups C and Ro + L (42% +/- 4% and 52% +/- 3% respectively, both P < 0.01). The claudin-5 expression of Group L was 0.035 +/- 0.004, significantly lower than those of Groups C and Ro + L (1.00 +/- 0.03 and 0. 574 +/- 0.07 respectively, both P < 0.01). Phalloidin staining showed that a zone of F-actin filaments in Group C, intercellular spaces were seen in Group L, and F-actin filaments were recombined in Group Ro + L. Transmission electron microscopy showed that there was continuous high-density BBB-TJ in Group C, the BBB-TJ of Group L was obviously opened, and the BBB-TJ of Group Ro + L was partially opened at a lower degree. CONCLUSION: LPA accommodates the BBB-TJ and the permeability of BBB via the activation of PKC-alpha channel which down-regulates the caudin-5 expression and F-actin recombination. Lysophosphatidic acid;