Metabolite-related antidepressant action of diterpene ginkgolides in the prefrontal cortex

Figures & data

Figure 1 (A) Sucrose preference (%). (B–F) Open field test and EPM test results. (B) Distance in the open arm. (C) Time spent in the open arm. (D) Distance traveled in the center. (E) Immobility time in the center. (F) Rearing. *p<0.05.

Abbreviations: CON, control; DG, diterpene ginkgolide; EPM, elevated plus maze.

Figure 2 PCA is an unsupervised multidimensional statistical analysis method, which can explain the overall metabolic differences between groups and the variability within each group.

Notes: The main parameters of PCA are the two principal components, including R²X and Q². Generally, the main parameter for judging the quality of the model is R²X, which indicates how well the model explains the data. A value of >0.4 indicates that the model is reliable. (A–C) show the PCA scores comparing the metabolites among the three groups.
Abbreviations: CON, control; DG, diterpene ginkgolide; PCA, principal component analysis.

Figure 3 PLS-DA is a supervised analysis method. In the supervised PLS-DA, R²Y and Q² are the two important parameters. Q² >0.5 indicates that the model performs well in discriminant analysis.

Notes: PLS-DA score plots for pairwise comparisons between the (A) VLX and DG groups, (B) the VLX and CON groups and (C) the DG and CON groups. Model validation for each group: (D) VLX vs DG; (E) VLX vs CON; (F) DG vs CON.
Abbreviations: CON, control; DG, diterpene ginkgolide; PLS-DA, partial least squares-discriminant analysis; VLX, venlafaxine.

Table 1 Key differential metabolites in the DG and CON groups in the mouse prefrontal cortex

DG vs CON
Metabolites	Metabolic pathway	r.t (min)	Mass	Fold change	VIP score	t-test (p-value)
l-proline	Amino acid metabolism	9.61	142	0.54	2.76	0.001
Thioctamide	Amino acid metabolism	23.14	131	0.67	1.34	0.002
Urocanic acid	Amino acid metabolism	7.1	79	0.68	1.25	0.04
N-Acetyl-l-aspartic acid	Amino acid metabolism	13.52	73	0.79	2.42	0.009
Alanine	Amino acid metabolism	6.79	116	0.82	3.92	0.023
l-Valine	Amino acid metabolism	8.17	144	0.9	1.19	0.042
3-Aminoisobutyric acid	Amino acid metabolism	7.29	102	1.12	1.97	0.027
l-Asparagine	Amino acid metabolism	7.72	72	1.32	1.57	0.01
Methionine	Amino acid metabolism	12.17	128	3.98	1.44	0.001
Citric acid	Energy metabolism	15.04	73	1.24	1.87	0.031
Methylmalonic acid	Lipid metabolism	7.51	147	0.56	1.46	0.038
Ethanolamine	Lipid metabolism	8.06	130	0.65	4.98	0.041
Cholesterol	Lipid metabolism	27.95	119	1.14	3.83	0.047
d-Alanyl-d-alanine	Others	6.62	116	0.71	1.54	0.015
Octanal	Others	6.96	101	0.72	1.3	0.101
(2R)-2-Amino-3-phosphonopropanoic acid	Others	10.19	340	0.73	1.52	0.021
Malonic acid	Others	6.6	221	1.65	2.67	0.013
Phosphomycin	Others	9.03	211	2.3	3.58	0

Notes: Values of >1 indicate higher levels in the DG group relative to the CON group, while values of <1 indicate higher levels in the CON group relative to the DG group. VIP was obtained from PLS-DA (threshold of 1.0).

Abbreviations: CON, control; DG, diterpene ginkgolide; PLS-DA, partial least squares-discriminant analysis; VIP, variable importance in projection.

Figure 4 Heat map of the differential metabolites in the CON and DG groups.

Abbreviations: CON, control; DG, diterpene ginkgolide.

Figure 5 VLX and control groups.

Abbreviations: CON, control; VLX, venlafaxine.

Figure 6 Red shows upregulation, green shows downregulation and yellow represents the opposite changes of the metabolites in the DG and VLX groups.

Abbreviations: DG, diterpene ginkgolide; VLX, venlafaxine.

Figure 7 (A) The network involved 12 metabolites, including citric acid, cholesterol, 3-aminoisobutyric acid, methionine, asparagine, malonic acid, alanine, octanal, ethanolamine, N-acetyl-l-aspartic acid, methylmalonic acid and proline. Red indicates an increase in the metabolite, while blue indicates a decrease. Other metabolites are uncolored. (B) The relationship of metabolites in the network of venlafaxine.

Abbreviation: GABA, γ-aminobutyric acid.

Figure 8 (A) Metabolite pathways in the DG group. (B) Metabolite pathways in the VLX group.

Notes: (a) Alanine, aspartate and glutamate metabolism. (b) Aminoacyl-tRNA biosynthesis. (c) Citrate cycle (TCA cycle). (d) Arginine and proline metabolism. (e) d-Glutamine and d-glutamate metabolism.
Abbreviations: DG, diterpene ginkgolide; TCA, tricarboxylic acid; VLX, venlafaxine.

Figure 9 Differential metabolites were correlated with behavior.

Notes: The color and size of the circles in the matrix represent the level of correlation: red represents a negative correlation and blue represents a positive correlation. Correlation analysis revealed a significant positive association of N-methylhydantoin concentration in the sucrose preference test.
Abbreviations: EPM, elevated plus maze; OFT, open field test.

Figure 10 Correlation analysis of the DG group revealed a positive association of citric acid and phosphomycin in the sucrose preference test, a negative correlation of l-proline and thioctamide in the sucrose preference test and a negative association of phosphomycin in the EPM.

Abbreviations: DG, diterpene ginkgolide; EPM, elevated plus maze.