GW0742对凝血酶诱导的体外血脑屏障破坏的保护作用

Protective effect of GW0742 on thrombine-induced destruction of the blood-brain barrier in vitro

摘要:

目的 探讨GW0742对凝血酶诱导的体外血脑屏障破坏的保护作用。方法 选取新生3～7日龄的SD大鼠若干，提取原代脑微血管内皮细胞（BMEC）及星形胶质细胞（AC）。于体外构建BMEC单层培养模型和BMEC+AC共培养模型，分为BMEC单层培养模型组、BMEC+AC共培养组及Blank组（只加培养基）；取体外血脑屏障（BBB）共培养模型，分为Control组和脑出血组（ICH组），Control组加入完全培养基处理，ICH组加入40 u/mL凝血酶处理，分别处理12 h模拟体外ICH模型，并通过4 h渗漏试验、荧光素钠通透性试验评价BBB及ICH模型的通透性。另取BBB共培养模型大鼠分成5个组：Control组、ICH组，以及GW0742低、中、高剂量组（1.25、2.50、5.00 μmol/L），给药24 h后采用Western blotting检测MMP-9、ZO-1、Occludin蛋白表达。结果 4 h渗漏试验结果显示：与Blank组比较，BMEC单层培养组能维持一定的液面差；与BMEC单层培养组比较，BMEC+AC共培养组能维持较好的液面差。荧光素钠通透性试验结果显示各组通透性为：Blank组> BMEC单层培养组> BMEC+AC共培养组。荧光素钠通透性试验结果显示：与Blank组比较，BMEC单层培养组在0.5、1.0、2.0 h时通透性降低（P <0.05）；与BMEC单层培养组比较，BMEC+AC共培养组在0.5、1.0、2.0 h时通透性降低（P <0.05）。4 h渗漏试验结果显示：与Control组相比，ICH组液面差降低，通透性增加。荧光素钠通透性试验结果显示：与Control组比较，ICH组在0.5、1.0、2.0 h时通透性增加（P <0.05）。细胞形态学观察结果显示：与Control组比较，ICH组内皮细胞形态明显皱缩、拉长；与ICH组比较，给药组内皮细胞形态未见明显皱缩、拉长，呈剂量依赖。与Control组比较，ICH组MMP-9蛋白相对表达量升高（P <0.05），ZO-1、Occludin蛋白相对表达量降低（P <0.05）；与ICH组比较，GW0742高剂量组MMP-9蛋白相对表达量降低（P <0.05），ZO-1、Occludin蛋白相对表达量升高（P <0.05）。结论 GW0742对凝血酶诱导的血脑屏障破坏具有保护作用，其保护作用可能与抑制MMP-9的过度表达及促进紧密连接蛋白ZO-1、Occludin的表达有关。

Abstract:

Objective To investigate the protective effects of GW0742 against thrombin-induced in vitro blood-brain barrier (BBB) disruption.Methods Neonatal SD rats aged 3 to 7 days were utilized to extract primary brain microvascular endothelial cells (BMEC) and astrocytes (AC). In vitro, BMEC monolayer culture and BMEC + AC co-culture models were established, including the BMEC monolayer culture model group, BMEC + AC co-culture group, and Blank group (culture medium only). For the in vitro BBB co-culture model, rats were assigned to the Control group and intracerebral hemorrhage (ICH) group. The Control group received complete culture medium, while the ICH group was treated with 40 u/mL thrombin for 12 hours to simulate the in vitro ICH model. BBB and ICH model permeability were evaluated through a 4-hour leakage test and sodium fluorescein permeability test. Rats in the BBB co-culture model were divided into five groups: Control group, ICH group, and GW0742 low, medium, and high dose groups (1.25, 2.50 and 5.00 μmol/L). After 24 hours of administration, Western blotting was used to detect MMP-9, ZO-1, and Occludin protein expressions.Results The 4-hour leakage test results showed that compared to the Blank group, the BMEC monolayer culture group maintained a certain fluid level difference, and the BMEC+AC co-culture group maintained a better fluid level difference than the BMEC monolayer culture group. The sodium fluorescein permeability test results demonstrated the following permeability order: Blank group > BMEC monolayer culture group > BMEC + AC co-culture group. In the sodium fluorescein permeability test, the BMEC monolayer culture group showed reduced permeability at 0.5, 1.0, and 2.0 hours compared to the Blank group (P < 0.05), and the BMEC + AC co-culture group exhibited decreased permeability at 0.5, 1.0, and 2.0 hours compared to the BMEC monolayer culture group (P < 0.05). The 4-hour leakage test results revealed that the ICH group had a decreased fluid level difference and increased permeability compared to the Control group. In the sodium fluorescein permeability test, the ICH group demonstrated increased permeability at 0.5, 1.0, and 2.0 hours compared to the Control group (P < 0.05). Morphological observations indicated that compared to the Control group, endothelial cells in the ICH group exhibited evident shrinkage and elongation. In comparison to the ICH group, the treatment groups showed no significant shrinkage or elongation of endothelial cells, exhibiting a dose-dependent response. Western blotting results demonstrated that compared to the Control group, the ICH group exhibited increased MMP-9 protein expression (P < 0.05) and decreased ZO-1 and Occludin protein expressions (P < 0.05). In comparison to the ICH group, the GW0742 high-dose group showed decreased MMP-9 protein expression (P < 0.05) and increased ZO-1 and Occludin protein expressions (P < 0.05).Conclusion GW0742 exerts protective effects against thrombin-induced blood-brain barrier disruption, likely mediated by inhibiting excessive MMP-9 expression and promoting the expression of tight junction proteins ZO-1 and Occludin.