Screening of Active Substances Regulating Alzheimer's Disease in Ginger and Visualization of the Effectiveness on 6-Gingerol Pathway Targets

Abstract

Ginger has been reported to potentially treat Alzheimer's disease (AD), but the specific compounds responsible for this biological function and their mechanisms are still unknown. In this study, a combination of network pharmacology, molecular docking, and dynamic simulation technology was used to screen active substances that regulate AD and explore their mechanisms. The TCMSP, GeneCards, OMIM, and DisGeNET databases were utilized to obtain 95 cross-targets related to ginger's active ingredients and AD as key targets. A functional enrichment analysis revealed that the pathways in which ginger's active substances may be involved in regulating AD include response to exogenous stimuli, response to oxidative stress, response to toxic substances, and lipid metabolism, among others. Furthermore, a drug-active ingredient-key target interaction network diagram was constructed, highlighting that 6-Gingerol is associated with 16 key targets. Additionally, a protein-protein interaction (PPI) network was mapped for the key targets, and HUB genes (ALB, ACTB, GAPDH, CASP3, and CAT) were identified. Based on the results of network pharmacology and cell experiments, 6-Gingerol was selected as the active ingredient for further investigation. Molecular docking was performed between 6-Gingerol and its 16 key targets, and the top three proteins with the strongest binding affinities (ACHE, MMP2, and PTGS2) were chosen for molecular dynamics analysis together with the CASP3 protein as the HUB gene. The findings indicate that 6-Gingerol exhibits strong binding ability to these disease targets, suggesting its potential role in regulating AD at the molecular level, as well as in abnormal cholinesterase metabolism and cell apoptosis, among other related regulatory pathways. These results provide a solid theoretical foundation for future in vitro experiments using actual cells and animal experiments to further investigate the application of 6-Gingerol.

Keywords: 6-Gingerol; Alzheimer’s disease (AD); ginger; molecular dynamics (MD); network pharmacology.

Figure 1

Flow chart showing the experimental design.

Figure 2

The involvement of active compounds in related pathways in AD. (A) Venn diagram showing the relevant targets in AD. (B) Venn diagram showing the disease targets (1088) alongside the targets (298) associated with ginger active constituents. (C) GO enrichment results for disease targets (top 10 for BP, CC, and MF, respectively; 30 entries in total). (D) KEGG enrichment notes for disease targets (top 10). The size of different circles in the bubble chart represents different gene numbers, and different colors represent different p.adjust. The color from blue to red indicates a strong degree of significance in (C,D).

Figure 3

Construction of networks. (A) Pathway–key gene interaction network for GO. The orange circle represents the key gene, and the purple, red, and green circles represent Top 10 in BP, Top 10 in CC, and Top 10 in MF, respectively. (B) Pathway–key gene interaction network for KEGG. The orange circle represents the key genes, and the red represents the Top 10 in KEGG signaling pathways. (C) The regulatory network of drug–active ingredient–key target regulatory network (191 nodes, 444 edges). The golden hexagon represents one kind of traditional Chinese medicine, the purple triangles represent the active ingredients, and the red ellipses represent the key targets.

Figure 4

Protein interaction network analysis results of key targets (95 nodes, 958 edges).

Figure 5

Cell viability test using the CCK-8 kit. (A) PC12 cells exposed to different concentrations of Aβ1-42 culture medium. (B) Different concentrations of 6-Gingerol are used to regulate a PC12 cell injury model induced by Aβ1-42.

Figure 6

Molecular docking results of 6-Gingerol and the targets. (A) Docking model of ACHE–6-Gingerol with the lowest binding affinity (−8.3 kcal/mol). (B) Docking model of CASP3–6-Gingerol with the lowest binding affinity (−6.0 kcal/mol). (C) Docking model of MMP2–6-Gingerol with the lowest binding affinity (−8.7 kcal/mol). (D) Docking model of PTGS2–6-Gingerol with the lowest binding affinity (−7.9 kcal/mol).

Figure 7

Molecular dynamics simulation analysis of the ACHE-6-Gingerol complex. (A) RMSD curve of 6-Gingerol (red line), ACHE (blue line), and the ACHE–6-Gingerol complex (green line). (B) RMSF curve of ACHE. (C) Rg curve of ACHE. (D) Hydrogen bonds of the ACHE–6-Gingerol complex. (E,F) Three-dimensional and two-dimensional Gibbs free energy landscape of the ACHE–6-Gingerol complex.

Figure 8

Molecular dynamics simulation analysis of the CASP3-6-Gingerol complex. (A) RMSD curve of 6-Gingerol (blue line), CASP3 (purple line), and the CASP3–6-Gingerol complex (orange line). (B) RMSF curve of CASP3. (C) Rg curve of CASP3. (D) Hydrogen bonds of the CASP3–6-Gingerol complex. (E,F) Three-dimensional and two-dimensional Gibbs free energy landscape of the CASP3–6-Gingerol complex.

Figure 9

Molecular dynamics simulation analysis of the MMP2-6-Gingerol complex. (A) RMSD curve of 6-Gingerol (green line), MMP2 (red line), and the MMP2–6-Gingerol complex (blue line). (B) RMSF curve of MMP2. (C) Rg curve of MMP2. (D) Hydrogen bonds of the MMP2–6-Gingerol complex. (E,F) Three-dimensional and two-dimensional Gibbs free energy landscape of the MMP2–6-Gingerol complex.

Figure 10

Molecular dynamics simulation analysis of the PTGS2-6-Gingerol complex. (A) RMSD curve of 6-Gingerol (orange line), PTGS2 (red line), and the PTGS2–6-Gingerol complex (purple line). (B) RMSF curve of PTGS2. (C) Rg curve of PTGS2. (D) Hydrogen bonds of the PTGS2–6-Gingerol complex. (E,F) Three-dimensional and two-dimensional Gibbs free energy landscape of the PTGS2–6-Gingerol complex.