Ginkgo biloba extract and its diterpene ginkgolide constituents ameliorate the metabolic disturbances caused by recombinant tissue plasminogen activator in rat prefrontal cortex

Figures & data

Figure 1 Metabolomic analysis of PFC samples from CON, rtPA, rtPA+DG, and rtPA+GBE rats. (A) PLS-DA score plot for the comparison of CON, rtPA, rtPA+DG, and rtPA+GBE rats, (B) PLS-DA score plots for pairwise comparisons between CON and rtPA, (C) rtPA and rtPA+DG, or (D) rtPA and rtPA+GBE. (E) Statistical validation of the PLS-DA model by permutation testing of CON, rtPA, rtPA+DG, and rtPA+GBE rats, statistical validation of the PLS-DA model by permutation testing between (F) CON and rtPA, (G) rtPA and rtPA+DG, or (H) rtPA and rtPA+GBE. The criterion used for determination of validity was that all blue Q² values to the left were lower than the original points to the right.

Abbreviations: PFC, prefrontal cortex; rtPA, recombinant tissue plasminogen activator; DG, diterpene ginkgolide; GBE, Ginkgo biloba extract; PLS-DA, partial least squares discriminant analysis; CON, control.

Figure 2 Heat map of the differential metabolites in the rat PFC for comparisons of rtPA vs CON, rtPA+DG vs rtPA, and rtPA+GBE vs rtPA.

Abbreviations: PFC, prefrontal cortex; rtPA, recombinant tissue plasminogen activator; DG, diterpene ginkgolide; GBE, Ginkgo biloba extract; CON, control.

Figure 3 (A) Venn diagram of the differential metabolites in rat PFC for the comparisons of rtPA versus CON, rtPA+DG versus rtPA, and rtPA+GBE versus rtPA. (B) Correlation network between these differential metabolites and the three comparisons. The line colors represent the direction of the fold change in the differential metabolites. Red lines indicate that levels are upregulated in the comparison, whereas green boxes indicate the levels are downregulated. The line widths represent the VIP scores for the differential metabolites.

Abbreviations: PFC, prefrontal cortex; rtPA, recombinant tissue plasminogen activator; DG, diterpene ginkgolide; GBE, Ginkgo biloba extract; VIP, variable importance in the projection; CON, control.

Table 1 Differential metabolites among the comparisons of rtPA/CON, rtPA+DG/rtPA, and rtPA+GBE/rtPA

Metabolite	Metabolic pathways	Mass	r.t. (min)	RtPA/CON			RtPA+DG/RtPA			RtPA+GBE/RtPA
				VIP score	t-test (P-value)	Fold change	VIP score	t-test (P-value)	Fold change	VIP score	t-test (P-value)	Fold change
Glycine	Neurotransmitter metabolism	102	7.30	1.51	0.029	0.74	2.73	0.001	0.48	3.68	0.000	0.39
Glutamic acid	Neurotransmitter metabolism	84	12.77	1.19	0.001	1.26	1.37	<0.001	0.69	–	–	–
Aspartic acid	Neurotransmitter metabolism	100	11.70	2.60	<0.001	1.31	1.68	0.021	0.82	3.12	0.016	0.83
N-Acetyl-l-aspartic acid	Neurotransmitter metabolism	116	14.94	2.52	0.001	1.36	1.80	0.008	0.80	–	–	–
Gamma-aminobutyric acid	Neurotransmitter metabolism	100	11.53	2.38	<0.001	1.64	–	–	–	–	–	–
Glutamine	Neurotransmitter metabolism	211	12.48	1.77	<0.001	2.77	1.07	0.002	0.65	1.10	0.001	0.33
Asparagine	Neurotransmitter metabolism	69	8.43	–	–	–	1.35	<0.001	12.07	1.59	<0.001	9.32
Aminooxyacetic acid	Amino acid metabolism	100	8.07	3.10	<0.001	0.30	1.37	<0.001	2.04	3.25	0.003	2.90
N-Carbamylglutamate	Amino acid metabolism	73	12.68	1.93	0.009	0.72	–	–	–	2.89	0.007	1.36
N-Acetyl-beta-alanine	Amino acid metabolism	73	22.35	1.04	<0.001	2.04	–	–	–	–	–	–
l-Allothreonine	Amino acid metabolism	101	8.27	–	–	–	1.02	0.030	0.59	1.16	0.039	0.52
Phenylalanine	Amino acid metabolism	91	13.56	–	–	–	–	–	–	1.05	0.001	0.55
Pyroglutamic acid	Amino acid metabolism	59	13.15	1.09	0.014	1.16	1.12	<0.001	0.82	–	–	–
Proline	Amino acid metabolism	142	9.61	–	–	–	2.52	0.035	2.88	–	–	–
Myo-inositol	Energy metabolism	103	16.73	1.95	0.022	1.21	2.33	<0.001	0.71	–	–	–
Lactic acid	Energy metabolism	117	6.40	7.60	<0.001	1.43	7.51	<0.001	0.60	–	–	–
Glucose 6-phosphate	Energy metabolism	299	21.23	1.19	0.017	2.92	–	–	–	1.08	0.002	0.33
Creatine	Energy metabolism	73	15.84	–	–	–	–	–	–	2.97	0.002	0.50
Palmitic acid	Lipid metabolism	117	18.55	1.09	0.017	0.71	1.29	0.003	1.50	–	–	–
O-Phosphoethanolamine	Lipid metabolism	299	14.95	2.58	0.002	2.85	2.45	0.013	1.71	–	–	–
Cholesterol	Lipid metabolism	129	27.95	–	–	–	–	–	–	5.20	0.024	0.38
d-Glycerol 1-phosphate	Lipid metabolism	299	14.52	–	–	–	2.03	0.001	0.61	–	–	–
Phosphate	Nucleotide metabolism	299	9.57	9.46	<0.001	0.94	9.39	<0.001	1.08	2.69	<0.001	0.68
Inosine	Nucleotide metabolism	103	24.20	1.45	<0.001	1.47	1.18	<0.001	0.67	2.53	<0.001	0.62
Adenosine	Nucleotide metabolism	103	24.50	–	–	–	–	–	–	2.45	<0.001	2.27
Hypoxanthine	Nucleotide metabolism	84	18.07	–	–	–	–	–	–	1.16	<0.001	0.72
2,3-Dihydroxypyridine	Others	240	10.07	1.25	0.001	0.28	–	–	–	1.35	0.038	2.65
Hexachlorobenzene	Others	79	6.32	1.38	<0.001	0.39	–	–	–	1.32	0.009	2.18
Thioctamide	Others	131	21.62	–	–	–	1.03	0.003	1.98	–	–	–
2-Amino-3- phosphonopropionic acid	Others	340	10.19	–	–	–	1.54	<0.001	1.89	1.36	0.008	0.46
N-Methylhydantoin	Others	116	6.62	–	–	–	2.95	0.005	4.55	–	–	–
N-Ethylglycine	Others	130	8.06	2.94	0.001	0.33	1.23	0.002	1.60	3.48	0.011	2.58

Abbreviations: rtPA, recombinant tissue plasminogen activator; DG, diterpene ginkgolide; GBE, Ginkgo biloba extract; VIP, variable importance in the projection; CON, control.

Figure 4 PLS-DA score plots for pairwise comparisons between (A) CON and rtPA+DG or (B) CON and rtPA+GBE. Statistical validation of the PLS-DA model by permutation testing between (C) CON and rtPA+DG or (D) CON and rtPA+GBE. The criterion used for determination of validity was that all blue Q² values to the left were lower than the original points to the right.

Abbreviations: rtPA, recombinant tissue plasminogen activator; DG, diterpene ginkgolide; GBE, Ginkgo biloba extract; PLS-DA, partial least squares discriminant analysis; CON, control.

Figure 5 (A) Correlation network between these differential metabolites and the three comparisons of rtPA versus CON, rtPA+DG versus rtPA, and rtPA+DG versus CON. (B) Correlation network between these differential metabolites and the three comparisons of rtPA versus CON, rtPA+GBE versus rtPA, and rtPA+GBE versus CON.

Abbreviations: rtPA, recombinant tissue plasminogen activator; DG, diterpene ginkgolide; GBE, Ginkgo biloba extract; CON, control.

Figure 6 (A) Significant pathways of rtPA treatment-related differential metabolites. (B) Significant pathways of differential metabolites in the comparison set of rtPA+DG versus rtPA. (C) The relative intensity of the differential metabolites in the four groups. ^*P<0.05, ^**P<0.01, ^***P<0.001.

Abbreviations: rtPA, recombinant tissue plasminogen activator; DG, diterpene ginkgolide; GBE, Ginkgo biloba extract.

Figure 7 Simplified schematic diagram of the metabolic changes induced by administration of rtPA, DG, and GBE. Red boxes indicate upregulation in the comparison, yellow boxes indicate no change, and green boxes indicate downregulation.

Abbreviations: TCA, tricarboxylic acid; rtPA, recombinant tissue plasminogen activator; DG, diterpene ginkgolide; GBE, Ginkgo biloba extract; CON, control; ADP, adenosine diphosphate; ATP, adenosine triphosphate.

Figure 8 Network of “amino acid metabolism, cell-to-cell signaling and interaction, molecular transport” associated with key differential metabolites in the comparison of rtPA and CON groups in rats PFC with a score of 38 and 14 differential metabolites involved.

Abbreviations: rtPA, recombinant tissue plasminogen activator; PFC, prefrontal cortex; CON, control.

Figure 9 Network of “amino acid metabolism, cell-to-cell signaling and interaction, molecular transport” associated with key differential metabolites in the comparison of rtPA+GBE and CON groups in rats PFC with a score of 40 and 15 differential metabolites involved.

Abbreviations: rtPA, recombinant tissue plasminogen activator; GBE, Ginkgo biloba extract; PFC, prefrontal cortex; CON, control.

Figure 10 Network of “cellular compromise, lipid metabolism, small molecule biochemistry” associated with key differential metabolites in the comparison of rtPA+DG and CON groups in rats PFC with a score of 38 and 14 differential metabolites involved.

Abbreviations: rtPA, recombinant tissue plasminogen activator; DG, diterpene ginkgolide; PFC, prefrontal cortex; CON, control.

Figure S1 Principal component analysis (PCA) score plot of all samples. R²X =0.987, Q² =0.859. 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.

Abbreviations: rtPA, recombinant tissue plasminogen activator; DG, diterpene ginkgolide; GBE, Ginkgo biloba extract; QC, quality control; CON, control.

Table S1 Key differential metabolites in the comparison of rtPA+DG and CON groups in rat PFC

Metabolites	Mass	R.t (min)	VIP score	t-test (P-value)	Fold change
2,3-Dihydroxypyridine	240	10.07	1.66	0.001	0.22
Glycine	102	7.30	3.89	<0.001	0.36
Phosphate	299	12.84	3.15	<0.001	0.51
N-Ethylglycine	130	8.06	2.59	0.016	0.52
N-Carbamylglutamate	73	12.68	3.04	0.001	0.62
Aminooxyacetic acid	100	8.07	1.91	0.019	0.62
d-Glycerol 1-phosphate	299	14.52	2.66	0.001	0.67
3-Hydroxy-l-proline	73	13.52	1.88	0.005	0.76
Alanine	116	6.80	1.96	0.008	0.85
Gamma-aminobutyric acid	100	11.53	2.97	<0.001	1.49
Ethanolamine	100	8.31	1.22	<0.001	1.67
Glutamine	211	12.48	1.40	<0.001	1.80
Malonic acid	147	14.25	1.10	<0.001	2.64
Proline	142	9.61	2.43	0.005	2.89
1-Methylhydantoin	116	6.62	2.95	0.012	2.91
2-Amino-3-phosphonopropionic acid	340	10.19	2.24	<0.001	3.05
l-Cysteine	211	15.21	1.02	0.031	4.11
O-Phosphorylethanolamine	299	14.95	5.39	<0.001	4.86
Glucose-1-phosphate	103	14.51	1.01	<0.001	7.24
Asparagine	69	8.43	1.68	<0.001	9.01

Abbreviations: rtPA, recombinant tissue plasminogen activator; DG, diterpene ginkgolide; PFC, prefrontal cortex; VIP, variable importance in the projection; CON, control.

Table S2 Key differential metabolites in the comparison of rtPA+GBE and CON groups in rat PFC

Metabolites	Mass	R.t (min)	VIP score	t-test (P-value)	Fold change
Glycine	102	7.30	4.57	<0.001	0.29
Methyl phosphate	133	9.06	1.14	<0.001	0.41
3-Hydroxy-l-proline	73	13.52	1.27	0.019	0.69
Phosphate	299	9.57	7.30	0.032	0.96
Pyroglutamic acid	59	13.15	1.65	<0.001	1.28
N-Acetyl-l-aspartic acid	116	14.94	2.25	0.001	1.30
d-Fructose 1,6-bisphosphate	299	21.49	1.05	0.006	1.30
Glutamic acid	84	12.77	1.31	0.001	1.34
Gamma-aminobutyric acid	100	11.53	2.47	<0.001	1.48
Lactic acid	117	6.40	7.69	0.015	1.53
Ethanolamine	100	8.31	1.07	0.001	1.58
Glutamine	211	12.48	1.09	0.002	2.30
Malonic acid	147	14.25	1.38	<0.001	2.75
Adenosine	103	24.50	2.43	<0.001	3.28
O-Phosphorylethanolamine	299	14.95	3.94	<0.001	4.32
Asparagine	69	8.43	2.06	<0.001	6.95
Glucose-1-phosphate	103	14.51	1.08	<0.001	7.21

Abbreviations: rtPA, recombinant tissue plasminogen activator; GBE, Ginkgo biloba extract; PFC, prefrontal cortex; VIP, variable importance in the projection; CON, control.