Fig 1.
Venn diagram of overlapped targets between AD-related genes and targets from PkO.
The green circle represents AD-related genes from DrugBank, GeneCards, and OMIM databases. The blue circle represents the targets from ingredients in PkO from TCMSP database.
Fig 2.
The network construction of PkO-ingredients-targets.
The green node represents 13 active ingredients of PkO, and the blue node represents the predicted targets from these 13 active ingredients.
Fig 3.
The GO and KEGG enrichment analysis of the common gene targets.
A, Gene ontology terms of candidate targets of PkO against AD. The top 10 terms in each GO category with P Adjust Value < 0.05 were selected. BP, biological process; CC, cellular component; MF, molecular function. The axis Y designates the GO entry, and the square measure of the axis X and bar chart designate the figure of genes pertains to GO in candidate targets. B, KEGG pathway enrichment of candidate targets of PkO against AD. 20 terms with P Adjust Value < 0.05 and sorted by gene counts. The axis Y designates the name of pathways, axis X, and orbicular area designate the number of genes pertains to this signal pathway in candidate targets.
Fig 4.
Three-dimensional view of the docking mode of three representative ingredients and Syk identified and conducted by Autodock and Pymol based on the model prediction.
A, the docking mode of hancinone and Syk. The pink solid line represents hydrophobic interaction. B, the docking mode of kadsurenin k and Syk. The pink solid line represents hydrophobic interaction and the blue solid line represents hydrogen bonding interaction. C, the docking mode of kadsurenin m and Syk. The pink solid line represents hydrophobic interaction.
Fig 5.
The GO and KEGG enrichment analysis of hancinone targets.
A, Gene ontology terms of candidate targets of hancinone against AD. The top 10 terms in each GO category with P Adjust Value < 0.05 were chosen. BP, biological process; CC, cellular component; MF, molecular function. The axis Y designates the GO entry, and the square measure of the axis X and circular chart represent the number of genes pertaining to GO in candidate targets. B, KEGG pathway enrichment of candidate targets of hancinone against AD. 20 terms with P Adjust Value < 0.05 and sorted by gene counts. The axis Y designates the name of pathways, axis X, and bar area designate the number of genes pertains to this signal pathway in candidate targets.
Fig 6.
A, The primary PPI network of all overlapped targets of hancinone for treatment of AD by STRING database.
Nodes represent proteins, and edges represent protein-protein associations. The top 30 relational targets arrange the circle layout on the periphery of the network. B, The network pharmacology approach to excavate the key 15 targets and 20 pathways in hancinone for further screening of 20 pathways and 15 genes related to hacinone and demonstration of correlations between them. The azure diamond node is the main pathway and the violet circular node is the key protein.
Fig 7.
Effect of different concentrations of Syk inhibitor on the expressions of TREM2, Syk, and p-Syk HMC3 cells for 24 h.
A, Western blot analysis showed the levels of TREM2, Syk, and p-Syk proteins in different concentrations of Syk inhibitor-treated HMC3 cells for 24 h. B, the bar graph shows the TREM2 protein expression level. C, the bar graph shows the Syk protein expression level. D, the bar graph shows the p-Syk protein expression level. Data represent the mean ± S.D. of three independent experiments, whereas * P < 0.05, ** P < 0.01, *** P < 0.001 versus control. # P < 0.05 versus the second group.
Fig 8.
Effect of different concentrations of β-amyloid (1-42) oligomer on the expressions of TREM2, Syk, and p-Syk HMC3 cells for 24 h.
A, Western blot analysis showed the levels of TREM2, Syk and p-Syk proteins in different concentrations of β-amyloid (1-42) oligomer-treated HMC3 cells for 24 h. B, the bar graph shows the TREM2 protein expression level. C, the bar graph shows the Syk protein expression level. D, the bar graph shows the p-Syk protein expression level. Data represent the mean ± S.D. of three independent experiments, whereas * P < 0.05, ** P < 0.01, *** P < 0.001 versus control.
Fig 9.
A, Effects of hancinone on phagocytic ability in 100 nM Syk inhibitor-induced HMC3 cells using 5-FITC-(Acp)-
β-amyloid (1-42) staining assay. HMC3 cells were respectively treated with culture medium (control), 100 nM Syk inhibitor, 100 nM Syk inhibitor + 0.5 μM hancinone, 100 nM Syk inhibitor + 2.5 μM hancinone and 100 nM Syk inhibitor + 10 μM hancinone for 24 h. B, Histogram showed the percentage of cell phagocytosis rates after different treatments. The percentage of cell phagocytosis rates means the ratio of cell containing Aβ fluorescence to the total cells. ## P < 0.01 versus control. ** P < 0.01 versus the model group induced by 100 nM Syk inhibitor. Data represent the mean ± S.D. of three independent experiments.
Fig 10.
A, Effects of hancinone on phagocytic ability in 2.5 μmol/L Aβ1-42 oligomer-induced HMC3 cells using 5-FITC-(Acp)-
β-amyloid (1-42) staining assay. HMC3 cells were respectively treated with culture medium (control), 2.5 μmol/L Aβ1-42 oligomer, 2.5 μmol/L Aβ1-42 oligomer + 0.5 μmol/L hancinone, 2.5 μmol/L Aβ1-42 oligomer + 2.5 μmol/L hancinone and 2.5 μmol/L Aβ1-42 oligomer + 10 μmol/L hancinone for 24 h. B, Histogram showed the percentage of cell phagocytosis rates after different treatments. ## P < 0.01 versus control. ** P < 0.01 versus the model group induced by 2.5 μmol/L Aβ1-42 oligomer. Data represent the mean ± S.D. of three independent experiments.
Fig 11.
Effect of hancinone on the expressions of TREM2, Syk, p-Syk, p-PI3K, p-AKT, and mTOR in Syk inhibitor-induced HMC3 cells for 24 h.
A, Western blot analysis showed the levels of TREM2, Syk, p-Syk, p-PI3K, p-AKT, PI3K, AKT, and mTOR proteins in Syk inhibitor-treated HMC3 cells for 24 h. B, the bar graph shows the TREM2 protein expression level. C, the bar graph shows the p-Syk protein expression level. D, the bar graph shows the Syk protein expression level. E, the bar graph shows the p-PI3K protein expression level. F, the bar graph shows the p-AKT protein expression level. G, the bar graph shows the mTOR protein expression level. Data represent the mean ± S.D. of three independent experiments, whereas # P < 0.05, ## P < 0.01 versus control. * P < 0.05, ** P < 0.01 versus the group treated with Syk inhibitor.
Fig 12.
Effect of hancinone on the expressions of TREM2, Syk, p-Syk, p-PI3K, p-AKT, and mTOR in A
β1-42 oligomer-induced HMC3 cells for 24 h. A, Western blot analysis showed the levels of TREM2, Syk, p-Syk, p-PI3K, p-AKT, PI3K, AKT, and mTOR proteins in Aβ1-42 oligomer-treated HMC3 cells for 24 h. B, the bar graph shows the TREM2 protein expression level. C, the bar graph shows the p-Syk protein expression level. D, the bar graph shows the Syk protein expression level. E, the bar graph shows the p-PI3K protein expression level. F, the bar graph shows the p-AKT protein expression level. G, the bar graph shows the mTOR protein expression level. Data represent the mean ± S.D. of three independent experiments, whereas # P < 0.05, ## P < 0.01 versus control. * P < 0.05, ** P < 0.01 versus the group treated with β-amyloid (1-42) oligomer.
Fig 13.
A, the transformation of phenotype in 100 nmol/L Syk inhibitor-induced HMC3 cells and effects of hancinone on phenotypic change using CD68 antibody and CD206 (MMR) antibody staining assay.
HMC3 cells were respectively treated with culture medium (control), 100 nmol/L Syk inhibitor, 100 nmol/L Syk inhibitor + 0.5 μmol/L hancinone, 100 nmol/L Syk inhibitor + 2.5 μmol/L hancinone and 100 nmol/L Syk inhibitor + 10 μmol/L hancinone for 24 h. B, Histogram showed the percentage of CD206/CD68 after different treatments. ## P < 0.01 versus control. * P < 0.05 versus the model group induced by 100 nmol/L Syk inhibitor. Data represent the mean ± S.D. of three independent experiments.
Fig 14.
A, the transformation of phenotype in 2.5 μmol/L Aβ1-42 oligomer-induced HMC3 cells and effects of hancinone on phenotypic change using CD68 antibody and CD206 (MMR) antibody staining assay.
HMC3 cells were respectively treated with culture medium (control), 2.5 μmol/L Aβ1-42 oligomer, 2.5 μmol/L Aβ1-42 oligomer + 0.5 μmol/L hancinone, 2.5 μmol/L Aβ1-42 oligomer + 2.5 μmol/L hancinone and 2.5 μmol/L Aβ1-42 oligomer + 10 μmol/L hancinone for 24 h. B, Histogram showed the percentage of CD206/CD68 after different treatments. ## P < 0.01 versus control. ** P < 0.01 versus the model group induced by 2.5 μmol/L Aβ1-42 oligomer. Data represent the mean ± S.D. of three independent experiments.