Xu Yajie, Lu Yu, Xu Rukun, Zhang Yong, Zhang Chen, Yin Jialin, Bao Hongguang, Wang Xiaoliang
Department of Anesthesiology, Nanjing First Hospital, Nanjing Medical University, Nanjing, China.
Department of Anesthesiology, Nanjing First Hospital, Nanjing Medical University, Nanjing, China.
Brain Res. 2024 Jan 1;1822:148607. doi: 10.1016/j.brainres.2023.148607. Epub 2023 Oct 6.
Perioperative neurocognitive disorder (PND) remains a prevalent complication following anesthesia and surgery. Recent studies have revealed the therapeutic potential of gastrodin (GAS) in treating cognitive disturbances. This study delves deeper into the mechanisms through which GAS impacts PND.
Male C57BL/6 mice (18 months old) underwent laparotomies and were administered GAS orally daily for three weeks preceding surgery and one week post-surgery. Thirty minutes before GAS administration, an intraperitoneal injection of Compound C was given. In vitro, HO-incubated SH-SY-5Y cells, with or without Nrf2-siRNA transfection, were set up and subjected to GAS or Compound C treatments. Cell viability was assessed via MTT assays, and apoptosis levels were assessed through flow cytometry. Cognitive function was evaluated using the Morris water maze, novel object recognition, and Y-maze tests. Oxidative stress markers, including MDA, SOD, GSH, GSH-px, and intracellular ROS (determined through immunofluorescence), were quantified. The expression of the genes Caspase3, Bax, Bcl-2, GST, and NQO1 was gauged using real-time RT-PCR. Brain, cortex and hippocampal pathologies were examined with hematoxylin-eosin (HE) and NeuN/TUNEL costaining. Finally, Nrf2 and p-AMPK were analyzed using Western blotting (WB) and immunofluorescence assays.
GAS improved cognitive dysfunction in PND mice and reduced oxidative stress, neuro-apoptosis, and ROS levels both in vivo and in vitro experiment. In vivo, Immunofluorescence and Western blot outcomes indicated that postoperative p-AMPK and Nrf2 levels in the hippocampus were mitigated but were augmented by GAS. In vitro studies revealed GAS's protective effect against HO-induced oxidative stress and apoptosis and its upregulation of p-AMPK and Nrf2 in SH-SY-5Y cells. Notably, this protective effect was negated when Nrf2 siRNA was introduced. ELISA and PCR results highlighted the role of GAS in enhancing GST and NQO1 activity in both the mice hippocampus and SH-SY-5Y cells. Compound C, an AMPK inhibitor, both in vitro and in vivo, reversed the beneficial effects of GAS on Nuc-Nrf2/Cyt-Nrf2 expression and counteracted the positive influence of GAS on cognitive functions in PND mice.
GAS facilitates the nuclear translocation of Nrf2 via AMPK activation, offering a therapeutic avenue for alleviating postoperative cognitive impairments in mice, with a significant reduction in oxidative stress.
围手术期神经认知障碍（PND）仍是麻醉和手术后常见的并发症。最近的研究揭示了天麻素（GAS）在治疗认知障碍方面的治疗潜力。本研究更深入地探讨了GAS影响PND的机制。
雄性C57BL/6小鼠（18个月大）接受剖腹手术，并在手术前3周和手术后1周每天口服GAS。在给予GAS前30分钟，腹腔注射化合物C。在体外，建立用或不用Nrf2-siRNA转染的HO孵育的SH-SY-5Y细胞，并进行GAS或化合物C处理。通过MTT试验评估细胞活力，通过流式细胞术评估凋亡水平。使用莫里斯水迷宫、新物体识别和Y迷宫试验评估认知功能。对氧化应激标志物，包括丙二醛（MDA）、超氧化物歧化酶（SOD）、谷胱甘肽（GSH）、谷胱甘肽过氧化物酶（GSH-px）和细胞内活性氧（通过免疫荧光测定）进行定量。使用实时RT-PCR测定Caspase3、Bax、Bcl-2、谷胱甘肽S-转移酶（GST）和醌氧化还原酶1（NQO1）基因的表达。用苏木精-伊红（HE）和NeuN/TUNEL共染色检查脑、皮质和海马的病理情况。最后，使用蛋白质免疫印迹法（WB）和免疫荧光试验分析Nrf2和磷酸化腺苷酸活化蛋白激酶（p-AMPK）。
在体内和体外实验中，GAS改善了PND小鼠的认知功能障碍，降低了氧化应激、神经细胞凋亡和活性氧水平。在体内，免疫荧光和蛋白质免疫印迹结果表明，海马中术后p-AMPK和Nrf2水平降低，但GAS使其升高。体外研究揭示了GAS对HO诱导的氧化应激和细胞凋亡的保护作用，以及其在SH-SY-5Y细胞中上调p-AMPK和Nrf2。值得注意的是，当引入Nrf2 siRNA时，这种保护作用被消除。酶联免疫吸附测定（ELISA）和PCR结果突出了GAS在增强小鼠海马和SH-SY-5Y细胞中GST和NQO1活性方面的作用。化合物C，一种AMPK抑制剂，在体外和体内均逆转了GAS对核Nrf2/胞质Nrf2表达的有益作用，并抵消了GAS对PND小鼠认知功能的积极影响。
GAS通过激活AMPK促进Nrf2的核转位，为减轻小鼠术后认知障碍提供了一条治疗途径，同时显著降低了氧化应激。
