通过 AKT/GSK-3β/Nrf2 通路，半合成异荭草素 GSK-3β 抑制剂对大鼠嗜铬细胞瘤 PC12 细胞和东莨菪碱诱导的 AD 模型小鼠的抗氧化应激和认知改善作用。

Anti-oxidative stress and cognitive improvement of a semi-synthetic isoorientin-based GSK-3β inhibitor in rat pheochromocytoma cell PC12 and scopolamine-induced AD model mice via AKT/GSK-3β/Nrf2 pathway.

机构信息

Science and Technology Innovation Center, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong Province, China.

Science and Technology Innovation Center, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong Province, China; College of Plant Protection, Henan Agricultural University, Wenhua Road No. 95, Zhengzhou, 450002, China.

出版信息

Exp Neurol. 2024 Oct;380:114881. doi: 10.1016/j.expneurol.2024.114881. Epub 2024 Jul 10.

DOI:10.1016/j.expneurol.2024.114881

PMID:38996864

Abstract

BACKGROUND

Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive cognitive deficits. Although the pathogenesis of AD is unclear, oxidative stress has been implicated to play a dominant role in its development. The flavonoid isoorientin (ISO) and its synthetic derivatives TFGF-18 selectively inhibit glycogen synthase kinase-3β (GSK-3β), a potential target of AD treatment.

PURPOSE

To investigate the neuroprotective effect of TFGF-18 against oxidative stress via the GSK-3β pathway in hydrogen peroxide (HO)-induced rat pheochromocytoma PC12 cells in vitro and scopolamine (SCOP)-induced AD mice in vivo.

METHOD

The oxidative stress of PC12 cells was induced by HO (600 μM) and the effects of TFGF-18 (2 and 8 μM) or ISO (12.5 and 50 μM) were observed. The AD mouse model was induced by SCOP (3 mg/kg), and the effects of TFGF-18 (2 and 8 mg/kg), ISO (50 mg/kg), and donepezil (DNP) (3 mg/kg) were observed. DNP, a currently accepted drug for AD was used as a positive control. The neuronal cell damages were analyzed by flow cytometry, LDH assay, JC-1 assay and Nissl staining. The oxidative stress was evaluated by the detection of MDA, SOD, GPx and ROS. The level of ACh, and the activity of AChE, ChAT were detected by the assay kit. The expressions of Bax, Bcl-2, caspase3, cleaved-caspase3, p-AKT (Thr308), AKT, p-GSK-3β (Ser9), GSK-3β, Nrf2, and HO-1, as well as p-CREB (Ser133), CREB, and BDNF were analyzed by western blotting. Morris water maze test was performed to analyze learning and memory ability.

RESULTS

TFGF-18 inhibited neuronal damage and the expressions of Bax, caspase3 and cleaved-caspase3, and increased the expression of Bcl-2 in vitro and in vivo. The level of MDA and ROS were decreased while the activities of SOD and GPx were increased by TFGF-18. Moreover, TFGF-18 increased the p-AKT, p-GSK-3β (Ser9), Nrf2, HO-1, p-CREB, and BDNF expression reduced by HO and SCOP. Meanwhile, MK2206, an AKT inhibitor, reversed the effect of TFGF-18 on the AKT/GSK-3β pathway. In addition, the cholinergic system (ACh, ChAT, and AChE) disorders were retrained and the learning and memory impairments were prevented by TFGF-18 in SCOP-induced AD mice.

CONCLUSIONS

TFGF-18 protects against neuronal cell damage and cognitive impairment by inhibiting oxidative stress via AKT/GSK-3β/Nrf2 pathway.

摘要

背景

阿尔茨海默病（AD）是一种以进行性认知功能障碍为特征的神经退行性疾病。虽然 AD 的发病机制尚不清楚，但氧化应激已被认为在其发展中起主导作用。类黄酮异牡荆素（ISO）及其合成衍生物 TFGF-18 选择性抑制糖原合酶激酶-3β（GSK-3β），这是 AD 治疗的一个潜在靶点。

目的

研究 TFGF-18 通过 GSK-3β 通路对过氧化氢（HO）诱导的体外大鼠嗜铬细胞瘤 PC12 细胞和东莨菪碱（SCOP）诱导的 AD 小鼠体内氧化应激的神经保护作用。

方法

用 HO（600 μM）诱导 PC12 细胞氧化应激，观察 TFGF-18（2 和 8 μM）或 ISO（12.5 和 50 μM）的作用。用 SCOP（3 mg/kg）诱导 AD 小鼠模型，观察 TFGF-18（2 和 8 mg/kg）、ISO（50 mg/kg）和多奈哌齐（DNP）（3 mg/kg）的作用。DNP 是一种公认的 AD 药物，用作阳性对照。通过流式细胞术、LDH 测定、JC-1 测定和尼氏染色分析神经元细胞损伤。通过检测 MDA、SOD、GPx 和 ROS 来评估氧化应激。通过试剂盒检测 ACh 水平和 AChE、ChAT 的活性。通过 Western blot 分析 Bax、Bcl-2、caspase3、cleaved-caspase3、p-AKT（Thr308）、AKT、p-GSK-3β（Ser9）、GSK-3β、Nrf2 和 HO-1 以及 p-CREB（Ser133）、CREB 和 BDNF 的表达。通过 Morris 水迷宫试验分析学习和记忆能力。

结果

TFGF-18 抑制体外和体内神经元损伤以及 Bax、caspase3 和 cleaved-caspase3 的表达，增加 Bcl-2 的表达。TFGF-18 降低 MDA 和 ROS 水平，增加 SOD 和 GPx 活性。此外，TFGF-18 增加了 HO 和 SCOP 降低的 p-AKT、p-GSK-3β（Ser9）、Nrf2、HO-1、p-CREB 和 BDNF 表达。同时，AKT 抑制剂 MK2206 逆转了 TFGF-18 对 AKT/GSK-3β 通路的作用。此外，TFGF-18 改善了 SCOP 诱导的 AD 小鼠的胆碱能系统（ACh、ChAT 和 AChE）紊乱和学习记忆障碍。