Department of Anatomy and Embryology, Peking University Health Science Center, 38 Xueyuan Road, Haidian Qu, Beijing 100083, China.
Exp Neurol. 2008 Nov;214(1):37-46. doi: 10.1016/j.expneurol.2008.07.006. Epub 2008 Jul 15.
Our previous study demonstrated that p53 plays an orchestrating role in the vasospasm and apoptotic cell death after subarachnoid hemorrhage (SAH). We now hypothesize that p53 also plays an important role in brain edema by up-regulating the expression of MMP-9 via the NF-kappaB molecular signaling pathway. Adult male rats (300-350 g) were divided into five groups (n=20 each): Sham, SAH treatment with DMSO or PFT-alpha (0.2 mg/kg and 2.0 mg/kg), intraperitoneally. The monofilament puncture model was used to induce SAH and animals were subsequently sacrificed at 24 h. The blood-brain barrier (BBB) disruption, brain water content, MMP-9 activity, immunohistochemistry, treble fluorescence labeling, Western blot, and ultra-structural observations were performed. Evans blue extravagation, BBB diffuse leakage of IgG protein and brain water content were significantly reduced by PFT-alpha treatment; and the expression of p53, NF-kappaB and MMP-9 were significantly increased. The tight junction protein (Occludin) in endothelia cells and Collage IV in basal lamina were decreased in the brain of SAH rats, and were also modified by PFT-alpha treatment. Ultra-structural changes included disruption of endothelial tight junction and widening of the inter-endothelial spaces. Treble labeling showed p53 colocalized with NF-kappaB and MMP-9 in cerebral endothelia cells. We thus conclude that the level of p53 in cerebral microvasculature significantly affects the BBB permeability and brain edema after 24 h of SAH in rats. This can be at least partially ascribed to p53 inducing a significant up-regulation of MMP-9 via NF-kappaB in the endothelium, which in turn opened the tight junction by degrading Occludin and disrupting the basal lamina by degrading collagen IV.
我们之前的研究表明,p53 在蛛网膜下腔出血(SAH)后血管痉挛和凋亡性细胞死亡中发挥着协调作用。我们现在假设,p53 通过 NF-κB 分子信号通路上调 MMP-9 的表达,在脑水肿中也发挥着重要作用。成年雄性大鼠(300-350g)分为五组(每组 20 只):假手术组、SAH 治疗组(DMSO 或 PFT-α,0.2mg/kg 和 2.0mg/kg,腹腔内注射)。使用单丝穿刺模型诱导 SAH,动物在 24 小时后处死。进行血脑屏障(BBB)破坏、脑水含量、MMP-9 活性、免疫组化、三重荧光标记、Western blot 和超微结构观察。PFT-α 治疗显著减少 Evans 蓝外渗、BBB 中 IgG 蛋白弥散渗漏和脑水含量;并显著增加 p53、NF-κB 和 MMP-9 的表达。SAH 大鼠脑内皮细胞中的紧密连接蛋白(Occludin)和基底层中的 Collage IV 减少,PFT-α 治疗也改变了这一情况。超微结构变化包括内皮紧密连接的破坏和内皮细胞间空间的扩大。三重标记显示 p53 与 NF-κB 和 MMP-9 在脑内皮细胞中存在共定位。因此,我们得出结论,大鼠 SAH 后 24 小时,脑微血管中 p53 的水平显著影响 BBB 通透性和脑水肿。这至少可以部分归因于 p53 通过 NF-κB 在内皮细胞中显著上调 MMP-9,从而通过降解 Occludin 打开紧密连接,通过降解 Collage IV 破坏基底层。