UPLC-Q-TOF-MS profiling of the hippocampus reveals metabolite biomarkers for the impact of Dl-3-n-butylphthalide on the lipopolysaccharide-induced rat model of depression

Figures & data

Figure 1 (A) Typical UPLC-Q-TOF-MS TIC of hippocampus tissue from the QC sample of ESI+. (B) Typical UPLC-Q-TOF-MS TIC chromatogram of hippocampus tissue from the QC sample of ESI−.

Abbreviations: UPLC-Q-TOF-MS, ultra-high-performance liquid chromatography-mass spectroscopy; TIC, total ion chromatogram; QC, quality control, ESI, electrospray ionization.

Figure 2 Multivariate statistical analysis between the LPS-induced depression and healthy Control groups at ESI+: (A) principal component analysis (PCA) scores plot; (B) partial least squares-discriminate analysis (PLS-DA) scores plot; (C) statistical validation of the PLS-DA model through 200× permutation testing; and (D) pair-wise orthogonal projections to latent structures discriminant (OPLS-DA) scores plot. Multivariate statistical analysis between the LPS-induced depression and healthy Control groups at ESI−: (E) PCA scores plot; (F) PLS-DA scores plot; (G) statistical validation of the PLS-DA model through 200×permutation testing; and (H) OPLS-DA scores plot.

Abbreviations: LPS, lipopolysaccharide; ESI, electrospray ionization.

Figure 3 Multivariate statistical analysis between the LPS-induced depression and NBP treatment groups (LPS+Ding) at ESI+: (A) PCA scores plot; (B) PLS-DA scores plot; (C) Statistical validation of the PLS-DA model through 200× permutation testing; and (D) OPLS-DA scores plot. Multivariate statistical analysis between the LPS-induced depression and NBP treatment groups at ESI−: (E) PCA scores plot; (F) PLS-DA scores plot; (G) statistical validation of the PLS-DA model by 200× permutation testing, and (H)OPLS-DA scores plot.

Abbreviations: LPS, lipopolysaccharide; ESI, electrospray ionization; NBP, Dl-3-n-butylphthalide; PCA, principal component analysis; PLS-DA, partial least squares-discriminate analysis; OPLS-DA, orthogonal partial least-squares discriminant analysis.

Table 1 Summary of the significantly different metabolites in the hippocampus between the LPS and Control groups at ESI+ and ESI−

NO	ESI	Metabolite	RT	Mass	VIP-value OPLS-DA^a	Fold change^b	p-value (Student's t-test)^a
1	na	γ-Aminobutyric acid	18.10	126.05	1.58	1.39	5.85E-03
2	H	Xanthine	18.06	153.02	1.33	1.21	3.08E-02
3	na	Purine	18.17	143.04	1.79	1.41	5.93E-04
4	H	Proline betaine	18.08	144.09	1.64	1.54	3.46E-03
5	H	Octadecanedioic acid	0.97	315.26	1.36	0.49	2.58E-02
6	na	Lysine	18.11	169.09	1.39	1.26	2.18E-02
7	na	L-Tryptophan	18.04	227.06	1.56	1.42	6.81E-03
8	na	L-Threonine	18.15	142.03	1.48	1.32	1.27E-02
9	na	Indole	18.19	140.04	1.32	0.83	3.22E-02
10	na	Histidine	18.4	178.06	1.86	1.47	2.33E-04
11	H	D-Ribose 5-phosphate	18.13	231.00	1.44	1.45	1.55E-02
12	na	D-Glucose 6-phosphate	18.05	283.03	1.33	1.30	3.04E-02
13	H	Cytidine diphosphate	18.08	403.99	1.68	1.49	2.43E-03
14	na	Citric acid	18.07	215.02	1.63	1.65	3.80E-03
15	H	cAMP	18.17	330.05	1.55	1.22	7.55E-03
16	H	Betaine aldehyde	18.15	102.09	1.67	1.24	2.51E-03
17	H	2E,6E,8E-decatrienoic acid	18.12	167.09	1.58	1.30	5.77E-03
18	H	Oxoglutaric acid	16.51	145.02	1.39	0.89	7.49E-03
19	cl	L-Thyronine	18.17	308.07	1.28	0.91	1.68E-02
20	cl	L-Glutamine	18.18	181.04	1.33	0.95	1.20E-02
21	H	Cinnamic acid	18.20	147.03	1.75	0.85	3.14E-05
22	cl	2E,4E,8Z,10E-dodecatetraenoic acid	18.24	227.07	1.48	0.93	3.01E-03
23	cl	13,16-docosadienoic acid	1.31	371.29	1.15	0.91	3.80E-02

Notes: ^aOnly metabolites with VIP>1.0 and p<0.05 were considered to exhibit statistically significant differences. ^bFold change was calculated as the average mass response (area) ratio between the two classes (ie, fold change=LPS/Control). Therefore, fold change values >1 show significantly higher levels in LPS group versus Control group, and fold change values <1 suggest significantly lower levels in LPS group versus Control group.

Abbreviations: LPS, lipopolysaccharide; NO, Number; ESI, electrospray ionization; RT, retention time; VIP, variable influence on projection; OPLS-DA,orthogonal partial least-squares discriminant analysis.

Table 2 Summary of the significantly different metabolites in the hippocampus between the LPS and LPS+NBP at ESI+ and ESI−

NO	ESI	Metabolite	RT	Mass	VIP-value OPLS-DA^a	Fold change^b	p-value (Student's t-test)^a
1	na	Hexatriacontanoic acid	13.95	559.58	1.78	1.80	1.18E-02
2	H	Glycerophosphocholine	0.80	258.09	1.49	1.31	4.61E-02
3	na	Cytosine	18.11	134.03	1.43	1.31	5.82E-02
4	h	Quinic acid	18.24	191.05	1.77	0.80	3.61E-04
5	cl	PC(2:0/0:0)	18.13	334.10	1.31	0.93	2.56E-02
6	cl	L-Thyronine	18.17	308.07	1.66	0.89	1.60E-03
7	h	L-Methionine	18.20	148.04	1.28	0.87	3.09E-02
8	cl	L-Glutamine	18.18	181.04	1.66	0.92	1.46E-03
9	h	L-Arginine	0.94	173.11	1.51	1.09	6.66E-03
10	cl	Ketoleucine	18.21	165.04	1.36	0.94	1.99E-02
11	cl	D-Mannitol	18.39	217.04	1.19	0.93	4.78E-02
12	h	Cinnamic acid	18.20	147.04	1.80	0.86	2.08E-04
13	cl	2E,4E,8Z,10E-dodecatetraenoic acid	18.24	227.07	1.50	0.93	7.34E-03

Notes: ^aOnly metabolites with VIP>1.0 and p<0.05 were considered to exhibit statistically significant differences. ^bFold change was calculated as the average mass response (area) ratio between the two classes (ie, fold change=LPS+NBP/LPS). Therefore, fold change values >1 show significantly higher levels in LPS+NBP group versus LPS group, and fold change values <1 suggest significantly lower levels in LPS+NBP group versus LPS group.

Abbreviations: LPS, lipopolysaccharide; NBP, Dl-3-n-butylphthalide; NO, Number; ESI, electrospray ionization; RT, retention time; VIP, variable influence on projection; OPLS-DA,orthogonal partial least-squares discriminant analysis.

Table 3 Result from pathway analysis from MetaboAnalyst

Group	Pathway name	Total	Expected	Hits	Raw p	-log(p)	Holm adjust	FDR	Impact
LPS versus control	Pentose phosphate pathway	19	0.23	2	2.10E-02	3.86E+00	1.00E+00	3.12E-01	3.20E-01
	Alanine, aspartate, and glutamate metabolism	24	0.29	2	3.27E-02	3.42E+00	1.00E+00	3.78E-01	2.10E-01
	Citrate cycle (TCA cycle)	20	0.24	2	2.31E-02	3.77E+00	1.00E+00	3.12E-01	1.20E-01
	Purine metabolism	68	0.82	4	7.47E-03	4.90E+00	5.82E-01	1.51E-01	4.00E-02
LPS+NBP versus LPS	Phenylalanine, tyrosine, and tryptophan biosynthesis	4	0.03	1	3.38E-02	3.39E+00	1.00E+00	6.82E-01	5.00E-01
	Arginine and proline metabolism	44	0.38	2	5.00E-02	2.96E+00	1.00E+00	7.03E-01	8.00E-02

Note: The table above shows the detailed results from the pathway analysis. Since we are testing many pathways at the same time, the statistical p-values from enrichment analysis are further adjusted for multiple testings. In particular, the Total is the total number of compounds in the pathway; the Hits is the actually matched number from the user uploaded data; the Raw p is the original p-value calculated from the enrichment analysis; the Holm p is the p-value adjusted by Holm–Bonferroni method; the FDR p is the p-value adjusted using False Discovery Rate; the Impact is the pathway impact value calculated from pathway topology analysis.

Abbreviations: LPS, lipopolysaccharide; NBP, Dl-3-n-butylphthalide.

Figure 4 Summary of the pathway analysis using MetaboAnalyst. (A) LPS group versus Control group (B) LPS+NBP group versus LPS group.

Abbreviations: LPS, lipopolysaccharide; NBP, Dl-3-n-butylphthalide.

Figure 5 Merged network combining major signaling networks related to the metabolites differentially expressed in the Ingenuity Pathways Analysis.

Note: For clarity, proteins and metabolites were merged with different geometric shapes in the relevant network in the hippocampus. Red indicates upregulation of metabolites, whereas green indicates downregulation of metabolites. Metabolites not correlated to proteins and treatment are shown as clear. Dotted lines show indirect interactions or regulations between the two parties, whereas solid lines show direct physical interactions (eg, binding) of the two parties.

Abbreviation: CP, correlation of pathway.