Mesquita-Britto Maria Helena Rodrigues, Mendonça Monique Culturato Padilha, Soares Edilene Siqueira, de Oliveira Giovanna, Solon Carina Silva, Velloso Lício Augusto, da Cruz-Höfling Maria Alice
Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas (UNICAMP), Campinas, São Paulo, Brazil; Department of Biochemistry and Tissue Biology, Institute of Biology, State University of Campinas (UNICAMP), Campinas, São Paulo, Brazil.
Department of Biochemistry and Tissue Biology, Institute of Biology, State University of Campinas (UNICAMP), Campinas, São Paulo, Brazil.
Toxicon. 2020 Oct 15;185:76-90. doi: 10.1016/j.toxicon.2020.06.019. Epub 2020 Jul 7.
This study was undertaken to elucidate why VEGF/VEGFR-2 is elevated in the hippocampus of rats injected with Phoneutria nigriventer spider venom (PNV). PNV delays Na channels inactivation; blocks Ca and K channels, increases glutamate release, causes blood-brain breakdown (BBBb), brain edema and severe excitotoxicity. Analytical FT-IR spectroscopy showed profound alteration in molecular biochemical state, with evidences for VEGFR-2 (KDR/Flk-1) signaling mediation. By blocking VEGF/VEGFR-2 binding via pre-treatment with itraconazole we demonstrated that animals' condition was deteriorated soon at 1-2 h post-PNV exposure concurrently with decreased expression of VEGF, BBB-associated proteins, ZO-1, β-catenin, laminin, P-gp (P-glycoprotein), Neu-N (neuron's viability marker) and MAPKphosphorylated-p38, while phosphorylated-ERK and Src pathways were increased. At 5 h and coinciding with incipient signs of animals' recuperation, the proteins associated with protection (HIF-1α, VEGF, VEGFR-1, VEGFR-2, Neu-N, occludin, β-catenin, laminin, P-gp efflux protein, phosphorylated-p38) increased thus indicating p38 pathway activation together with paracellular route strengthening. However, the BBB transcellular trafficking and caspase-3 increased (pro-apoptotic pathway activation). At 24 h, the transcellular route reestablished physiological state but the pro-survival pathway PI3K/(p-Akt) dropped in animals underwent VEGF/VEGFR-2 binding inhibition, whereas it was significantly activated at matched interval in PNV group without prior itraconazole; these results demonstrate impaired VEGF' survival effects at 24 h. The inhibition of VEGF/VEGFR-2 binding identified 5 h as turning point at which multi-level dynamic interplay was elicited to reverse hippocampal damage. Collectively, the data confirmed VEGFR-2 signaling via serine-threonine kinase Akt as neuroprotective pathway against PNV-induced damage. Further studies are needed to elucidate mechanisms underlying PNV effects.
本研究旨在阐明注射黑腹栉足蛛毒液（PNV）的大鼠海马中血管内皮生长因子（VEGF）/血管内皮生长因子受体-2（VEGFR-2）升高的原因。PNV会延迟钠通道失活；阻断钙通道和钾通道，增加谷氨酸释放，导致血脑屏障破坏（BBBb）、脑水肿和严重的兴奋性毒性。傅里叶变换红外光谱分析显示分子生化状态发生了深刻变化，有证据表明VEGFR-2（KDR/Flk-1）信号介导。通过用伊曲康唑预处理阻断VEGF/VEGFR-2结合，我们发现动物在PNV暴露后1-2小时状况迅速恶化，同时VEGF、血脑屏障相关蛋白、紧密连接蛋白1（ZO-1）、β-连环蛋白、层粘连蛋白、P-糖蛋白（P-gp）、神经元核抗原（Neu-N，神经元活力标志物）和丝裂原活化蛋白激酶磷酸化p38的表达降低，而磷酸化细胞外信号调节激酶（ERK）和Src途径增加。在5小时，与动物开始恢复的迹象一致，与保护相关的蛋白（缺氧诱导因子-1α、VEGF、VEGFR-1、VEGFR-2、Neu-N、闭合蛋白、β-连环蛋白、层粘连蛋白、P-gp外排蛋白、磷酸化p38）增加，从而表明p38途径激活以及细胞旁途径增强。然而，血脑屏障的跨细胞运输和半胱天冬酶-3增加（促凋亡途径激活）。在24小时，跨细胞途径恢复生理状态，但在接受VEGF/VEGFR-2结合抑制的动物中，促生存途径磷脂酰肌醇-3激酶/（磷酸化Akt）下降，而在未预先使用伊曲康唑的PNV组中，在相同时间间隔该途径被显著激活；这些结果表明在24小时时VEGF的生存效应受损。VEGF/VEGFR-2结合的抑制确定5小时为转折点，此时引发了多层次的动态相互作用以逆转海马损伤。总体而言，数据证实通过丝氨酸-苏氨酸激酶Akt的VEGFR-2信号传导是对抗PNV诱导损伤的神经保护途径。需要进一步研究以阐明PNV作用的潜在机制。
