Transforming growth-beta 1 contributes to isoflurane postconditioning against cerebral ischemia-reperfusion injury by regulating the c-Jun N-terminal kinase signaling pathway
Transforming growth-beta 1 contributes to isoflurane postconditioning against cerebral ischemia-reperfusion injury by regulating the c-Jun N-terminal kinase signaling pathway
复制标题
转化生长-β 1 通过调节 c-Jun N 末端激酶信号通路有助于异氟醚后处理对抗脑缺血再灌注损伤
DOI：
10.1016/j.biopha.2016.01.030
复制
发表时间：
2016-03-01
影响因子：
7.5
通讯作者：
Yao, Shanglong
中科院分区：
文献类型：
--
作者：
Wang, Sheng;Yin, Jiangwen;Yao, Shanglong
目的：脑缺血再灌注（I/R）损伤是围手术期的严重并发症。转化生长因子β（Transforming growth factor beta，TGF-β）是参与I/R损伤后修复和调控过程反应的关键蛋白。异氟烷广泛用于神经外科。已有研究表明异氟醚预处理在神经保护中起重要作用。然而，异氟醚后处理对脑I/R损伤的影响尚未阐明。本研究旨在探讨异氟醚后处理对脑I/R损伤的保护作用，并探讨TGF-β信号通路及其下游的c-Jun N-末端激酶（JNK）信号通路在神经保护机制中的作用。方法：采用SD大鼠大脑中动脉闭塞90 min，再灌注24 h的脑缺血/再灌注模型，制备大鼠脑缺血再灌注损伤模型。在缺血再灌注开始时间点，通过吸入不同浓度（1.5%、3.0%和4.5%）的异氟烷进行后处理1 h。采用2，3，5-三苯基氯化四氮唑和碘化丙啶（PI）染色法进行神经功能缺损评分。TUNEL法检测海马CA 1区细胞凋亡。通过免疫组化和Western blot检测TGF-β 1、Smad 2/3、p-Smad 2/3、JNK和p-JNK的表达水平。与I/R组相比，1.5%和3.0%异氟醚后处理显著降低神经行为缺陷评分和梗死体积，但I/R组和4.5%异氟醚后处理组的神经行为缺陷评分无显著差异。此外，与I/R组相比，1.5%异氟烷后处理减少了再灌注后24 h PI阳性细胞的数量。TGF-β 1和p-Smad 2/3蛋白在I/R损伤后逐渐增加，在1.5%和3%异氟烷后处理组中观察到最高值。Smad 2/3蛋白表达在各组间无差异。在诱导TGF-β/SMAD 3信号通路特异性阻断剂（LY 2157299）后，神经功能缺损评分增加，梗死体积扩大，凋亡增加，海马中PI阳性CA 1细胞也增加。TGF-β 1和p-Smad 2/3蛋白表达下调。预注射LY 2157299期间，TGF-β 1和pSmad 2/3的表达水平明显下降，但与假手术组相比，p-JNK的表达水平明显升高。当LY 2157299注射取消时，p-JNK的表达显著降低。当同时注射LY 2157299和SP 600125时，p-JNK和TGF-β 1的表达水平显著降低。然而，SP 600125介导的保护作用完全消失，LY 2157299的作用占据主导地位。结论：1.5%异氟醚可上调TGF-β 1的表达，下调p-JNK的表达，从而减轻I/R损伤，减轻脑损伤。然而，当TGF-β 1信号通路被LY 2157299阻断时，这种保护作用被废除。总之，本研究结果提供了有效的证据，证明TGF-β 1通过抑制JNK信号通路有助于异氟烷后处理对抗脑I/R损伤。(C)2016 Elsevier Masson SAS。All rights reserved.
Aim: Cerebral ischemia-reperfusion (I/R) injury is a devastating complication in the perioperative period. Transforming growth factor beta (TGF-beta) is a key protein that can participate in the repair and control process responses after I/R injury. Isoflurane is widely used in neurosurgery. Previous studies have shown that isoflurane preconditioning plays an important role in neuroprotection. However, the effects of isoflurane postconditioning on cerebral I/R injury have not yet been elucidated. In the present study, we evaluated the protective effect of isoflurane postconditioning against cerebral I/R injury and investigated the role of the TGF-b signaling pathway and the downstream c-Jun N-terminal kinase (JNK) signaling pathway in neuroprotective mechanism. In particular, the JNK signaling pathway emerges as a possible target for brain repair after stroke.Methods: Cerebral I/R injury was produced in SD rat by using the middle cerebral artery occlusion model for 90 min, followed by 24 h reperfusion. Postconditioning by inhalation of isoflurane was performed at different concentrations (1.5%, 3.0%, and 4.5%) for 1 h after ischemia at the starting time point of reperfusion. The protective effect was tested by neurological deficit scoring with 2,3,5-triphenyl tetrazolium chloride and propidium iodide (PI) staining. Apoptosis of CA1 cells in the hippocampus was detected by TUNEL method. Expression levels of TGF-beta 1, Smad 2/3, p-Smad2/3, JNK, and p-JNK were determined by immunostaining and Western blot.Results: Postconditioning by isoflurane at 1.5% and 3.0% concentrations significantly decreased the neurobehavioral deficit scores and infarct volume compared with the I/R group, but no significant difference in neurobehavioral deficit score was detected between the I/R and 4.5% isoflurane postconditioning groups. Additionally, 1.5% isoflurane postconditioning decreased the numbers of PI-positive cells at 24 h after reperfusion compared with the I/R group. TGF-beta 1 and p-Smad2/3 protein gradually increased after I/R injury, with the highest values observed in the 1.5% and 3% isoflurane postconditioning groups. For Smad2/3 protein expression, no differences existed among all groups. After inducing the TGF-beta/SMAD3 signaling pathway specific blocker (LY2157299), the neurological deficit scores increased, infarct volumes enlarged, apoptosis increased, and PI-positive CA1 cells in the hippocampus also increased. The expression levels of TGF-beta 1 and p-Smad2/3 proteins were downregulated. During the pre-injection of LY2157299, the expression levels of TGF-beta 1 and pSmad2/3 decreased significantly, but compared with the sham group, the expression level of p-JNK significantly increased. When the injection of LY2157299 was abolished, the expression of p-JNK significantly decreased. The expression levels of p-JNK and TGF-beta 1 significantly decreased when LY2157299 and SP600125 were injected simultaneously. However, the protective effect mediated by SP600125 completely disappeared, and the role of LY2157299 became dominant. Compared with the sham group, the expression of TGF-beta 1 was almost unchanged by the injection of SP600125 alone, but the expression of p-JNK significantly decreased.Conclusions: Up to 1.5% isoflurane can upregulate the expression of TGF-beta 1 and downregulate that of p- JNK, which significantly mitigated I/R injury, leading to cerebral injury. However, this protective effect was abrogated when the TGF-beta 1 signaling pathway was blocked by LY2157299. Overall, the present results provided valid evidence to demonstrate that TGF-beta 1 contributes to isoflurane postconditioning against cerebral I/R injury by inhibiting the JNK signaling pathway. (C) 2016 Elsevier Masson SAS. All rights reserved.