Department of Anesthesiology, First Affiliated Hospital, School of Medicine, Shihezi University, Shihezi, 832002, China.
Department of Physiology, School of Medicine, Shihezi University and the Key Laboratory of Xinjiang Endemic and Ethnic Diseases, Shihezi, 832002, China.
Biomed Pharmacother. 2019 Feb;110:420-430. doi: 10.1016/j.biopha.2018.11.143. Epub 2018 Dec 5.
The Wnt/β-catenin signaling pathway plays an important role in ischemia-reperfusion(I/R) injury, and the transforming growth factor(TGF)-β/Smad signaling pathway participates in the neuroprotection effect induced by isoflurane(ISO) postconditioning. In this study, we aimed to explore the role of the Wnt/|[beta]|-catenin β-catenin signaling pathway in the neuroprotection effect induced by ISO postconditioning, and investigate the interaction of Wnt/β-catenin and TGF-β/Smad signaling pathway in this neuroprotection effect.
Cerebral I/R injury was established in Sprague-Dawley rats by using the middle cerebral artery occlusion (MCAO) model for 90 min followed by 24 h reperfusion. Postconditioning by inhalation of ISO was performed for 60 min after ischemia at the onset of reperfusion. Neurological deficit scoring, 2,3,5-triphenyl tetrazolium chloride staining and Nissl staining were adopted to evaluate brain injury. Apoptosis of the hippocampus and cortex neurons was detected by TUNEL staining. The expression levels of Wnt3a, GSK-3β, β-catenin, Cyclin D1, VEGF, Caspase 3, TGF-β1, Smad3 and p-Smad3 were determined by immunofluorescence (IF) staining, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot. Various targeted inhibitors were administered via intraperitoneal injection or lateral ventricle injection.
In the cortex region, the neurological deficit score, infarct volumes and neuron apoptosis increased, and the expression level of the Wnt3a, GSK-3β, β-catenin, VEGF and Cyclin D1 decreased in the MCAO group compared with the Sham group. In the MCAO + ISO group, the neurological deficit score, infarct volumes and neuron apoptosis reduced significantly, the expression levels of Wnt3a, β-catenin, VEGF and Cyclin D1 increased, while the expression level of GSK-3β and Caspase 3 decreased relative to MCAO group. When Wnt inhibitor(DKK-1) was given in advance followed by ISO postconditioning, the neurological deficit score, infarct volumes, neuron apoptosis and the expression level of GSK-3β and Caspase 3 increased. qRT-PCR and IF showed similar changes in the protein levels of all groups. However, the expression level of β-catenin in nuclear and cytoplasm both decreased significantly after pre-injection with the TGF-β1 inhibitor(LY2157299) and Smad3 inhibitor(SIS3), whereas the expression levels of TGF-β1, Smad3 and p-Smad3 were almost unchanged. The expression levels of all the related proteins and morphological changes in the hippocampus region were consistent with that of the cortex.
ISO postconditioning can reduce cerebral I/R injury by activating the Wnt/β-catenin signaling pathway and may be related to the TGF-β/Smad3 signaling pathway.
Wnt/β-连环蛋白信号通路在缺血再灌注（I/R）损伤中发挥重要作用，转化生长因子（TGF）-β/Smad 信号通路参与异氟醚（ISO）后处理诱导的神经保护作用。本研究旨在探讨 Wnt/β-连环蛋白信号通路在 ISO 后处理诱导的神经保护作用中的作用，并研究 Wnt/β-连环蛋白与 TGF-β/Smad 信号通路在该神经保护作用中的相互作用。
采用大脑中动脉闭塞（MCAO）模型 90 min 后再灌注 24 h 建立 Sprague-Dawley 大鼠脑 I/R 损伤模型。在再灌注开始时 ISO 吸入 60 min 进行后处理。采用神经功能缺损评分、2,3,5-三苯基四氮唑氯化物染色和尼氏染色评估脑损伤。TUNEL 染色检测海马和皮质神经元凋亡。免疫荧光（IF）染色、实时定量聚合酶链反应（qRT-PCR）和 Western blot 检测 Wnt3a、GSK-3β、β-连环蛋白、Cyclin D1、VEGF、Caspase 3、TGF-β1、Smad3 和 p-Smad3 的表达水平。通过腹腔注射或侧脑室注射给予各种靶向抑制剂。
在皮质区，与假手术组相比，MCAO 组的神经功能缺损评分、梗死体积和神经元凋亡增加，Wnt3a、GSK-3β、β-连环蛋白、VEGF 和 Cyclin D1 的表达水平降低。在 MCAO+ISO 组，神经功能缺损评分、梗死体积和神经元凋亡明显减少，Wnt3a、β-连环蛋白、VEGF 和 Cyclin D1 的表达水平增加，而 GSK-3β 和 Caspase 3 的表达水平降低。预先给予 Wnt 抑制剂（DKK-1）后再给予 ISO 后处理，神经功能缺损评分、梗死体积、神经元凋亡以及 GSK-3β 和 Caspase 3 的表达水平均增加。qRT-PCR 和 IF 显示各组蛋白水平均有类似变化。然而，在预先注射 TGF-β1 抑制剂（LY2157299）和 Smad3 抑制剂（SIS3）后，核和细胞质中β-连环蛋白的表达均显著降低，而 TGF-β1、Smad3 和 p-Smad3 的表达水平几乎不变。海马区的所有相关蛋白表达水平和形态变化与皮质区一致。
ISO 后处理通过激活 Wnt/β-连环蛋白信号通路减轻脑 I/R 损伤，可能与 TGF-β/Smad3 信号通路有关。
