Gastrodin Ameliorates Cognitive Dysfunction in Vascular Dementia Rats by Suppressing Ferroptosis via the Regulation of the Nrf2/Keap1-GPx4 Signaling Pathway

Abstract

Gastrodia elata Bl. has a long edible history and is considered an important functional food raw material. Gastrodin (GAS) is one of the main functional substances in G. elata BI. and can be used as a health care product for the elderly to enhance resistance and delay aging. This study investigated the ameliorative effect and mechanism of GAS on cognitive dysfunction in vascular dementia (VaD) rats, which provides a theoretical basis for development and utilization of functional food. The water maze test shows that GAS improves learning and memory impairment in VaD rats. Meanwhile; GAS significantly decreased the levels of Fe²⁺ and malondialdehyde (MDA); increased the content of glutathione (GSH); and significantly up-regulated the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and glutathione peroxidase 4 (GPx4), the key regulatory factors of ferroptosis; while it down-regulated the expression of kelch-like ECH-associated protein (Keap1) and cyclooxygenase 2 (COX2). However, GAS does not directly regulate GPx4 and COX2 to inhibit ferroptosis. Furthermore, compared with GAS alone, GAS combined with Bardoxolone (an agonist of Nrf2) did not further affect the increase in GPx4 levels and decrease in COX2 levels, nor did it further affect the regulation of GAS on the biochemical parameters of ferroptosis in HT22 hypoxia injury. These findings revealed that GAS inhibited ferroptosis in hippocampal neurons by activating the Nrf2/Keap1-GPx4 signaling pathway, suggesting its possible application as a functional food for improving vascular dementia by inhibiting ferroptosis.

Keywords: Nrf2/Keap1-GPx4; ferroptosis; functional food; gastrodin; vascular dementia.

Figure 1

Neuroprotective effect of GAS on learning and memory impairment in VaD rats. (A), Escape latency (B), swimming distance in less than 2 min (C), swimming track diagram, red circle: the location of the platform; yellow line: the track of the first landing on the platform. (D), The number of times crossing the platform (E), Percentage of time spent on target project. Results are shown as mean ± SD (n = 6). ** p < 0.01 specifies the differences between sham rat and VaD rat. ^## p < 0.01 compares between VaD rat and GAS-treated rat or ferrostatin1-treated rat.

Figure 2

Effects of GAS on ferroptosis indexes in the hippocampus of VD rats. (A–C), detection of biochemical indicators of ferroptosis, (D–G), Western blot analyses of ferroptosis-related protein expression. Results are shown as mean ± SD (n = 6).* p < 0.05, ** p < 0.01 specifies the differences between sham rat and VaD rat. ^# p < 0.05, ^## p < 0.01 compares between VaD rat and GAS-treated rat or ferrostatin1-treated rat.

Figure 3

GAS inhibits hypoxia-induced ferroptosis in HT22 Cells. (A), Western blot showing the levels of GPx4 and COX2 at different time points of hypoxia in HT22 cells (B), HT22 cells were pretreated with GAS for 1 h and cultured in hypoxia for 36 h, and then, the expressions of GPx4 and COX2 were detected by Western blotting (C), ROS levels were measured by flow cytometry (D), detection of GSH content by ELISA (E), When HT22 cells were cultured under hypoxia for 36 h in the presence or absence of GAS, the intracellular Fe²⁺ levels were measured by immunofluorescence microscopy using FerroOrange. * p < 0.05, ** p < 0.01 versus control, ^# p < 0.05, ^## p < 0.01 versus hypoxia.

Figure 4

Experimental study on the improvement of hypoxic injury of HT22 cells by GAS based on the regulation of ferroptosis by the GPx4 pathway. (A,B), GPx4 and COX2 were analyzed using Western blot. Pharmacological inhibitors; erastin (ferroptosis inducers), ferrostatin-1 (ferroptosis inhibitor) (C), intracellular ROS levels were labeled with 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA, 10 μM) and detected by flow cytometry (n = 3) (D), levels of GSH in HT22 cells treated with ferroptosis inducers (erastin) were detected with or without GAS treatment (100 μM) for 24 h (n = 3) (E), detection of intracellular Fe²⁺ changes using FerroOrange. * p < 0.05, ** p < 0.01 compared to the control group; ^# p < 0.05, ^## p < 0.01 compared to the hypoxia group; ^& p < 0.05, ^&& p < 0.01, compared to the hypoxia + ferrostatin-1 or hypoxia + erastin group.

Figure 5

GAS activates the Nrf2 signaling pathway to ameliorate hypoxic injury in HT22 cells (A), HT22 cells were incubated in a normal incubator (5% CO₂, 37 °C) with GAS for 1 h and then placed in a hypoxia incubator (5% O₂, 5% CO₂, 90% N₂, 37 °C) for 36 h, and then the protein levels of GPx4 and COX2 were detected by Western blot (B), Immunofluorescence microscopy images of Nrf2 in HT22 after hypoxia treatment in the presence, or absence, of GAS for 36 h, and cell nuclei were stained with 4,6-diamino-2-phenyl indole (DAPI, blue fluorescence). Mean fluorescence intensity values were calculated by Image J software (n = 3). Magnification, ×400; scale bars, 25 µm. Results are presented as mean ± SD. ** p < 0.01, control versus hypoxia; ^# p < 0.05, ^## p < 0.01, hypoxia versus hypoxia plus GAS.

Figure 6

GAS inhibits ferroptosis via the Nrf2/GPx4 Pathway to improve hypoxic injury in HT22 cells. (A,B), levels of GPx4 and COX2 in HT22 cells treated with Nrf2 inhibitors (ML385) or Nrf2 agonists (Bardoxolone) were assessed with or without GAS treatment (100 μM) for 36 h (n = 3) (C), intracellular ROS levels were labeled with DCFH-DA (10 μM) and detected by flow cytometry (n = 3) (D), levels of GSH in HT22 cells treated with an agonist of Nrf2 (Bardoxolone) were detected with or without GAS treatment (100 μM) for 36 h (n = 3) (E), detection of intracellular Fe²⁺ changes using FerroOrange. * p < 0.05, ** p < 0.01, compared to the control group; ^# p < 0.05, ^## p < 0.01 compared to the hypoxia group; NS indicates no significance.

Figure 7

GAS ameliorates cognitive dysfunction in VaD rats by suppressing ferroptosis via the regulation of the Nrf2/Keap1-GPx4 signaling pathway. The illustration represents how the GAS up-regulates the Nrf2 gene, promotes Nrf2 release from the complex of Keap1 and Nrf2. Then, Nrf2 enters the nucleus and binds with antioxidant response elements (ARE), thereby. increasing the expression of GPx4 to decrease iron deposition, strengthen antioxidant capacity and decrease lipid peroxidation.