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

Abstract

Purpose: An increasing body of evidence reveals that inflammation is involved in the pathological mechanisms of depression. Our previous basic research confirmed that Dl-3-n-butylphthalide (NBP) possess anti-inflammatory properties. However, studies investigating metabolite biomarkers for the involvement of NBP in hippocampus tissue in the lipopolysaccharide (LPS)-induced rat model of depression are currently limited. Thus, the aim of this study was to identify metabolite biomarkers in the hippocampus for the impact of NBP in this model of depression.

Material and methods: Male Sprague-Dawley rats were randomly allocated to one of the following three groups (n=6): Control, LPS-induced rat model of depression (LPS), and NBP involvement in the LPS-induced rat model of depression (LPS+NBP). Ultra-high-performance liquid chromatography-mass spectroscopy was used to determine the hippocampal metabolites. Multivariate statistical analysis was performed to identify differentially expressed hippocampal metabolites in the three groups.

Results: Most of the identified differentially expressed metabolites were related to amino acid, lipid, energy, and oxidative stress metabolism. Additionally, metabolites were eventually connected to different pathways and metabolic networks, which may partly account for the pathophysiological process of depression.

Conclusion: The present findings provide insight into the anti-inflammatory effects of NBP, and further elucidate the pathophysiological mechanisms underlying inflammation-induced depression.

Keywords: Dl-3-n-butylphthalide; inflammation; lipopolysaccharide; metabolite biomarkers; ultra-high-performance liquid chromatography-mass spectroscopy.

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.

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.