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

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 brain^3,4. It is well recognized that the two core pathological events in AD are extracellula....

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. Pre....

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 (

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
7500 Real-Time PCR System	APExBIO	S0080	Used for detecting apoptotic cells
A450 enzyme labeling instrument	Proteintech	50599-2-Ig	WB, dilution 1:1000
Annexin V-FITC apoptosis detection kit	Proteintech	60178-1-Ig	WB, dilution 1:1000
Anti-Bax antibody	Proteintech	10494-1-AP	WB, dilution 1:1000
Anti-Bcl-2 antibody	Abcam	ab32057	WB, dilution 1:1000
Anti-GAPDH antibody	Proteintech	28866-1-AP	WB, dilution 1:1000
Anti-p-Tau (Ser396) antibody	Biofroxx	1172	Blocking buffer for WB
Anti-p-Tau (Thr181) antibody	Vazyme Biotech	A311-01/02	Used for cell viability assay
Biofroxx skim milk powder	Solarbio Science & Technology	D8371	Solvent for compounds
CCK-8 kit	Wisent Biotechnology	319-105-CL	For cell culture
Dimethyl sulfoxide (DMSO)	APExBIO (USA)	K1231	WB detection reagent
DMEM high-glucose medium	Aladdin Biochemical Technology	G111309	Active compound
ECL chemiluminescent reagent	Promega	A6101	For qPCR amplification
Flow cytometer	Solarbio Science & Technology	BC6410	Lipid peroxidation assay
Gastrodin (GAS) powder, purity ≥98%	MedChemExpress	HY-N6785	Inducer of tau hyperphosphorylation
Gel imaging system	Beyotime Biotechnology	C0221A	Wash the cells
GoTaq qPCR Master Mix	Proteintech	SA00001-1	WB, dilution 1:5000
MDA assay kit (malondialdehyde)	Proteintech	SA00001-2	WB, dilution 1:5000
Okadaic acid (OA)	ATCC	CRL-2266	Cell
PBS	Beyotime Biotechnology	S0101M	For oxidative stress assay
Secondary antibody (Goat anti-mouse IgG, HRP-conjugated)	Solarbio Science & Technology	BC1315	Antioxidant capacity detection
Secondary antibody (Goat anti-rabbit IgG, HRP-conjugated)	Beyotime Biotechnology	R0016	RNA extraction
SH-SY5Y	Sigma-Aldrich	9002-07-7	Cell digestion
SOD assay kit (total superoxide dismutase)	Thermo Fisher Scientific	A45072	Multifunctional microplate reader for absorbance detection
Total antioxidant capacity (T-AOC) kit	Becton, Dickinson and Company	661310	For multi-parameter single-cell analysis and cell sorting.
Trizol reagent	Thermo Fisher Scientific	4351104	gene expression analysis
Trypsin	Bio Rad	1708195	DNA/RNA/protein gel imaging