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Research Article

Gastrodin Alleviates Tau Hyperphosphorylation Associated with AKT/GSK-3β Signaling Changes in an Alzheimer's Disease Cell Model

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DOI:

10.3791/70432

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March 24th, 2026

 ,  ,  ,  ,  , 
1College of Life Sciences and Medicine, Zhejiang Sci-Tech University, 2Zhejiang Sci-Tech University Shaoxing Academy of Biomedicine

* These authors contributed equally

In This Article

Summary

Here, we present a protocol for establishing an okadaic acid-induced Alzheimer's disease cellular model in SH-SY5Y cells and evaluating the neuroprotective effects of gastrodin through oxidative stress and AKT/GSK-3β pathway analysis.

Abstract

Alzheimer's disease (AD) is a common neurodegenerative disease for which there are currently limited effective drugs. As the principal bioactive component derived from Gastrodia elata, Gastrodin (GAS) has shown clear therapeutic potential for treating AD; however, the molecular mechanism of its action remains to be elucidated. In this study, we aim to investigate the mechanism of the effect of GAS on AD model cells. Network pharmacology is employed to analyze the targets and signaling pathways affected by GAS in AD. An AD cell model is constructed by inducing human neuroblastoma SH-SY5Y cells with Okadaic acid (OA). Cell viability was assessed using the CCK-8 assay, while the levels of SOD, MDA, and T-AOC were measured. Apoptosis rate was determined through Annexin V-FITC/PI double staining, and expression of apoptosis-related factors, as well as AKT, GSK-3β, p-Tau (Ser396), and p-Tau (Thr181), was analyzed using RT-qPCR and Western blotting techniques. Network pharmacology analysis suggests that GAS has the potential to regulate cellular apoptosis and associated signaling pathways, including PI3K/AKT. Our experiments demonstrate that GAS can inhibit MDA levels, increase T-AOC and SOD in the AD model cells, and reduce cell apoptosis. Western blotting results indicate that GAS mitigates OA's inhibitory effects on p-AKT (Ser473) and p-GSK-3β (Ser9) expression. Additionally, it attenuates the overexpression of p-Tau (Ser396) and p-Tau (Thr181), suppresses Bax expression, and enhances Bcl-2 expression. GAS demonstrates the ability to ameliorate oxidative stress injury induced by OA and mitigate apoptosis. GAS may suppress Tau hyperphosphorylation, which is associated with changes in the AKT/GSK-3β signaling pathway, thereby exerting potential neuroprotective effects.

Introduction

Alzheimer's disease (AD) is a progressive neurodegenerative disorder primarily characterized by memory loss and cognitive impairment, representing the most prevalent form of dementia worldwide, accounting for approximately 60% to 80% of cases1. The underlying pathogenesis of AD remains complex and not yet fully elucidated2. Key pathological hallmarks observed in clinical AD cases include the accumulation of Senile Plaques (SP) and Neurofibrillary Tangles (NFTs) within the brain3,4. It is well recognized that the two core pathological events in AD are extracellular deposition of β-amyloid protein (Aβ) to form senile plaques and intracellular aggregation of hyperphosphorylated Tau protein to form neurofibrillary tangles5,6,7. Additionally, oxidative stress and neuroinflammation are supplementary factors that can contribute to the exacerbation of AD8,9,10,11.

As the global population aging trend intensifies, the number of dementia patients has significantly increased12,13. However, the treatment options for AD remain limited, predominantly comprising single-target drugs such as memantine, donepezil, and galantamine14. However, their therapeutic effects are primarily palliative rather than disease-modifying, and certain adverse effects such as gastrointestinal disturbances, dizziness, and bradycardia have been reported. Therefore, the development of safer and more effective therapeutic strategies remains an important research priority15,16. Recently launched monoclonal drugs, such as Lecanemab, have brought new hope for patients with mild AD. Yet their high costs and limitations fail to meet the substantial therapeutic demand. The prevention and treatment of AD continue to pose a significant challenge17,18. The findings from various studies have demonstrated that medicinal plants, particularly traditional Chinese medicines, exhibit multi-component effects that target multiple pathways involved in neurodegenerative disorders such as AD. Studies have shown that active compounds from Ganoderma lucidum can alleviate oxidative damage in the brain tissue of AD mice, inhibit apoptosis, reduce hippocampal neuron necrosis, and improve cognitive impairment19. In AD research, Bao et al. utilized network pharmacology to identify key targets of Astragaloside IV in AD-related inflammation. Their findings revealed that epidermal growth factor receptor (EGFR) and interleukin-1β (IL-1β) are crucial targets, providing potential therapeutic candidates and disease targets for the clinical treatment of neurodegenerative diseases20. Dartigues et al.21 analyzed Ginkgo biloba extract and demonstrated that it has beneficial effects on reducing the risk of dementia and mortality in elderly individuals. Patients treated with Ginkgo biloba extract exhibited a lower mortality rate22.

Gastrodia elata, belonging to the Orchidaceae family, is a traditional Chinese herb, edible and medicinal. The rhizome of Gastrodia elata is abundant in Gastrodin (GAS), gastrodia polysaccharides, and alkaloids. Recent pharmacological research has demonstrated that these active compounds possess memory-enhancing and cognitive-improving effects, as well as neuroprotective properties, and efficacy in treating insomnia and dizziness23. The main bioactive compound of Gastrodia elata is GAS, which is extracted from the tuber and possesses medicinal properties. GAS, also known as 4-hydroxybenzyl alcohol 4-O-β-d-glucoside, belongs to the class of phenolic glycosides24. The chemical structure of GAS is illustrated in Figure 1, exhibiting a molecular weight of 286.278. The relatively low molecular mass of glycosides and monoglycosides facilitates their efficient penetration through the blood-brain barrier, thus serving as a fundamental basis for GAS's neuroprotective role25. Moreover, their high absorbability further enhances their efficacy26. In recent years, numerous studies have demonstrated the potential of GAS in the treatment of various brain disorders, including AD27,28,29,30, Parkinson's disease (PD)31, and cerebral ischemia32. GAS exhibits a wide range of pharmacological properties, including antioxidative, hypolipidemic, anti-inflammatory, and anti-aging effects33,34. Its mechanism of action involves multiple signaling pathways such as Nrf2, NF-κB, and AMPK35. GAS exerts neuroprotective effects in a mouse model of Pb-induced neurological injury by modulating the Wnt/Nrf2 pathway36, and also exhibits an anti-apoptotic effect in the treatment of neurodegenerative disorders through regulation of the Nrf2 signaling pathway37. GAS also exerted a beneficial effect on AD by suppressing hippocampal BACE1 expression through the protein kinase/eukaryotic initiation factor-2α (PKR/eIF2α) signaling pathway38. Moreover, GAS exhibited inhibitory effects on TCDD-induced NF-κB signaling activation, thereby attenuating apoptosis of PC12 neurons and astrocyte activation39.

Chemical structure diagram of arbutin, a glycoside, showing phenol and glucose components.
Figure 1: Chemical structural formula of GAS. Please click here to view a larger version of this figure.

OA, derived from the black sponge of the Halichondria genus, belongs to a class of long-chain fatty acids and serves as the principal component of Diarrhetic Shellfish Poison (DSP). It effectively inhibits the activities of protein phosphatases, particularly PP1A and PP2A, leading to cellular hyperphosphorylation and disruption of cellular homeostasis40,41. This characteristic renders it a valuable tool for investigating diseases associated with protein phosphorylation. Therefore, the utilization of OA enables the construction of AD cell models featuring alterations in Tau protein phosphorylation. This study aims to investigate the impact and mechanism of GAS, the primary active component of Gastrodia elata, on AD cell model induced by OA, and to clarify its neuroprotective effect on AD model cells by targeting AKT/GSK-3β signaling pathway.

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Protocol

All network pharmacology operations were conducted in accordance with the Guidelines for Network Pharmacology Evaluation Methods. All experimental procedures were performed in compliance with Zhejiang Sci-Tech University laboratory management regulations.

Network pharmacology
Prediction of potential targets of GAS: The canonical SMILES of GAS (PubChem CID: 1150) was retrieved from the PubChem database42. The SMILES structure was uploaded to the SwissTargetPrediction database (http://www.swisstargetprediction.ch/)43. The species parameter was set to Homo sapiens. Predicted targets with a probability value > 0.1 were retained to reduce false-positive predictions. All predicted protein targets were standardized to official gene symbols using the UniProt database43 (species limited to Homo sapiens), and duplicate entries were removed.

AD-related genes were collected from the GeneCards database (https://www.genecards.org/)44,45 and the OMIM database (https://www.omim.org/)46 using "Alzheimer's disease" as the search term. In GeneCards, genes with a Relevance Score > 10 were selected. Targets obtained from GeneCards and OMIM were merged, duplicates were removed, and gene names were standardized using UniProt.

The predicted GAS targets and AD-related genes were imported into the Venny 2.1 online platform to obtain overlapping targets. A total of 52 intersecting genes (See Supplementary Table 1) were identified as potential therapeutic targets of GAS in AD.

The 52 intersecting targets were uploaded to the STRING database (https://string-db.org/)47 for PPI analysis. The species was set to Homo sapiens, and the minimum required interaction score was set to 0.4 (medium confidence). Disconnected nodes were hidden. The resulting interaction data were exported and visualized using Cytoscape software (version 3.9.1).

GO and KEGG enrichment analysis: The 52 intersecting targets were uploaded to the Metascape database (https://metascape.org/)48 for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The species was set to Homo sapiens. The enrichment criteria were defined as p-value < 0.01, minimum overlap ≥ 3, and enrichment factor > 1.5. KEGG pathways were ranked according to p-value, and the top 20 significantly enriched pathways were selected for visualization. Enrichment results were visualized using an online bioinformatics platform.

The protein structure file of GSK-3β was obtained from the RCSB PDB database. The structure file of GAS in SDF format was downloaded from the PubChem database and subsequently converted to Mol2 format using the Open Babel GUI software. The files mentioned above were processed using AutoDock software to add hydrogen atoms, remove water molecules, and assign charges. The processed files were subsequently saved in the PDBQT format. After detecting and processing torsion bonds in the ligand molecules, the files were then stored in PDBQT format. The lowest binding energy between the two molecules was obtained by performing molecular docking using AutoDock Vina software. Subsequently, the docking results were visualized and analyzed utilizing PyMOL.

Stock solution preparation
The OA powder (25 µg) was completely dissolved in 1200 µL of 10% dimethyl sulfoxide (DMSO) prepared with sterile phosphate-buffered saline (PBS) to generate a 25 µM stock solution. The stock solution was aliquoted and stored at -80 °C. Before each experiment, OA was freshly diluted with complete Dulbecco's Modified Eagle Medium (DMEM) (see Table of Materials) to the desired working concentrations. GAS powder (20 mg) was dissolved in 1.4 mL of sterile PBS, filtered through a 0.22 µm membrane for sterilization, and prepared as a 50 mM stock solution, which was stored at -20 °C. Prior to use, the stock solution was diluted with complete DMEM to the indicated working concentrations.

Construction of the AD cell model
Human neuroblastoma SH-SY5Y cells were obtained and used in this study (see Table of Materials). SH-SY5Y cells were induced with gradient concentrations of OA (0, 10, 20, 40, 80, 160 nM) for durations of 12, 24, and 36 h, respectively, to establish the AD cell model.

Cell viability assay
To determine the optimal concentration and induction duration of OA for establishing the AD cell model, SH-SY5Y cells were treated with gradient concentrations of OA (0, 10, 20, 40, 80, and 160 nM) for different time periods (12, 24, and 36 h), and cell viability was evaluated using the CCK-8 assay49. SH-SY5Y cells were seeded into 96-well plates at a density of 7.5 × 103 cells per well in 100 µL of complete DMEM. Plates were incubated at 37 °C in a humidified incubator with 5% CO₂ for 24 h to allow cell attachment. After 24 h attachment, 100 µL of OA working solution was added to each well to achieve final concentrations of 0, 10, 20, 40, 80, and 160 nM in a total volume of 200 µL per well. The control group (CON) received an equal volume of complete DMEM without OA, whereas the OA group received OA at the indicated concentrations. Cells were incubated for 12 h, 24 h, or 36 h under standard culture conditions. Each concentration and time point was set with six replicate wells. Three blank wells (medium only) were included for background correction.

Determination of cellular oxidative stress index
The SH-SY5Y cells in the logarithmic growth phase were seeded into a 6-well plate. Following cell treatment, the previous medium was discarded from the wells. Subsequently, the wells were washed three times with pre-chilled PBS at 4 °C. After that, cell lysis buffer was added, and the cells were lysed on ice using the appropriate method as instructed by the kit. The protein content of the samples was quantified using the BCA protein assay kit (see Table of Materials). The activities of T-AOC, SOD, and MDA were determined according to the instructions provided with the respective assay kits (see Table of Materials).

Cell apoptosis detection
Cell apoptosis was evaluated using an Annexin V-FITC/PI double staining kit (see Table of Materials) according to the manufacturer's instructions. Briefly, both floating and adherent cells were collected. Adherent cells were digested using trypsin without EDTA to avoid artificial membrane damage and combined with floating cells. Cells were washed twice with cold PBS and resuspended in 500 µL of 1× Binding Buffer at a density of 0.5–1 × 106 cells.

Subsequently, 5 µL of Annexin V-FITC and 5 µL of Propidium Iodide (PI) were added to each sample, gently mixed, and incubated at room temperature in the dark for 10–20 min. Samples were placed on ice and analyzed within 1 h using a flow cytometer.

For flow cytometric analysis, unstained controls and single-stained controls (Annexin V-FITC only and PI only) were used to set compensation parameters and quadrant gates. Forward and side scatter (FSC/SSC) gating was first applied to exclude debris and cell aggregates. A minimum of 10,000 events per sample was acquired. Data were analyzed using FlowJo software.

Real-time quantitative PCR analysis (RT-qPCR)
According to the instructions provided by the AG reverse transcription kit, the extracted RNA was subjected to reverse transcription to generate complementary DNA (cDNA), which was subsequently diluted 10-fold with sterile water to serve as the template. The RT-qPCR reaction system was prepared following the protocol of the Green Master Mix (No Rox) kit, and analysis was performed using a fluorescent quantitative PCR instrument (see Table of Materials). The obtained Ct values were processed and analyzed, and the expression levels of target genes were calculated using the 2-ΔΔCt method, where ΔCt represents the difference between Ct values in the treatment group and GAPDH, and ΔΔCt represents the difference between ΔCt values in the treatment group and control group. The list of primers can be found in Supplementary Table 2.

Western blotting analysis
The cell lysis buffer, along with a protease inhibitor and a phosphatase inhibitor, was combined in a ratio of 100:1:1. The cells were lysed to extract cellular proteins. The protein concentration was determined using the BCA assay kit. Proteins (20–30 µg per lane) were separated on 10% SDS-PAGE gels at 80 V for the stacking gel and 120 V for the resolving gel. Proteins were transferred to PVDF membranes (0.45 µm) at 300 mA for 90 min using wet transfer. The membrane was blocked with 5% skim milk at room temperature for 2 h, followed by overnight incubation with primary antibody (see Table of Materials) at 4 °C. After incubating with the secondary antibody at room temperature for 1 h, the membrane was washed four times with TBST for 5 min each. The bands were visualized using enhanced chemiluminescence (ECL) detection reagents (see Table of Materials), and detection was performed with a gel imaging system. The grayscale values were analyzed using ImageJ software for data insights.

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Results

GAS-related target screening for AD treatment
The identification of GAS's targets for treating AD involved database searches and software analysis. The Swiss Target Prediction database yielded 100 potential targets, while OMIM and GeneCards (with a Relevance score > 10) were queried for AD-related targets, resulting in a total of 2376 potential targets. Subsequently, employing the Venny 2.1 online platform enabled the identification of 52 potential therapeutic targets for GAS in AD (

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Discussion

In recent years, there has been a global increase in the prevalence of AD. This condition is characterized by a chronic course, irreversible nature, disability, and elevated mortality rates, making it a significant challenge within the context of an aging global population. Despite its prominence, the etiology of AD is intricate, and its pathogenesis remains incompletely understood51.

Traditional Chinese medicine offers significant advantages in disease prevention and t...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

This research was supported by the Open Fund of Zhejiang Sci-Tech University, Shaoxing Academy of Biomedicine (SXAB202007).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
7500 Real-Time PCR SystemAPExBIO S0080Used for detecting apoptotic cells
A450 enzyme labeling instrumentProteintech 50599-2-IgWB, dilution 1:1000
Annexin V-FITC apoptosis detection kitProteintech 60178-1-IgWB, dilution 1:1000
Anti-Bax antibodyProteintech 10494-1-APWB, dilution 1:1000
Anti-Bcl-2 antibodyAbcam ab32057WB, dilution 1:1000
Anti-GAPDH antibodyProteintech 28866-1-APWB, dilution 1:1000
Anti-p-Tau (Ser396) antibodyBiofroxx 1172Blocking buffer for WB
Anti-p-Tau (Thr181) antibodyVazyme Biotech 
A311-01/02		Used for cell viability assay
Biofroxx skim milk powderSolarbio Science & Technology D8371Solvent for compounds
CCK-8 kitWisent Biotechnology319-105-CLFor cell culture
Dimethyl sulfoxide (DMSO)APExBIO (USA)K1231WB detection reagent
DMEM high-glucose mediumAladdin Biochemical Technology G111309Active compound
ECL chemiluminescent reagentPromega  A6101For qPCR amplification
Flow cytometerSolarbio Science & Technology BC6410Lipid peroxidation assay
Gastrodin (GAS) powder, purity ≥98%MedChemExpressHY-N6785Inducer of tau hyperphosphorylation
Gel imaging systemBeyotime BiotechnologyC0221AWash the cells
GoTaq qPCR Master MixProteintech SA00001-1WB, dilution 1:5000
MDA assay kit (malondialdehyde)Proteintech SA00001-2WB, dilution 1:5000
Okadaic acid (OA)ATCCCRL-2266Cell
PBSBeyotime BiotechnologyS0101MFor oxidative stress assay
Secondary antibody (Goat anti-mouse IgG, HRP-conjugated)Solarbio Science & Technology BC1315Antioxidant capacity detection
Secondary antibody (Goat anti-rabbit IgG, HRP-conjugated)Beyotime Biotechnology R0016RNA extraction
SH-SY5YSigma-Aldrich 
9002-07-7		Cell digestion
SOD assay kit (total superoxide dismutase)Thermo Fisher ScientificA45072Multifunctional microplate reader for absorbance detection
Total antioxidant capacity (T-AOC) kitBecton, Dickinson and Company661310For multi-parameter single-cell analysis and cell sorting.
Trizol reagentThermo Fisher Scientific4351104gene expression analysis
TrypsinBio Rad1708195DNA/RNA/protein gel imaging

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