A Metabonomics Investigation into the Therapeutic Effects of BuChang NaoXinTong Capsules on Reversing the Amino Acid-Protein Interaction Network of Cerebral Ischemia

Abstract

Background: Amino acids (AAs) in cerebrospinal fluid (CSF) play a pivotal role in cerebral ischemia (CI). BuChang NaoXinTong Capsules (BNC) are widely prescribed in Chinese medicine for the treatment of cerebrovascular and cardiovascular diseases.

Methods: In order to investigate the therapeutic effects and pharmacological mechanisms of BNC on reversing CI from a system level, an amino acid-protein interaction imbalanced network of CI containing metabolites of AAs, key regulatory enzymes, and proteins was constructed for the first time. Furthermore, a novel method for detecting the ten AAs in CSF was developed by UPLC-QQQ-MS in an effort to validate the imbalanced networks and the therapeutic effects of BNC via analysis of metabolites.

Results: Based on a middle cerebral artery occlusion (MCAO) rat model, the dynamic levels of amino acids in CSF 3, 6, 12, and 24 h after MCAO were analyzed. Up to 24 h, the accumulated nine AA biomarkers were found to significantly change in the MCAO group compared to the sham group and exhibited an obvious tendency for returning to baseline values after BNC treatment. In addition, based on the imbalanced network of CI, four key enzymes that regulate the generation of BNC-mediated AA biomarkers were selected and validated using an enzyme-linked immunosorbent assay and western blotting. Finally, aromatic-L-amino-acid decarboxylase (AADC) was found to be one of the putative targets for BNC-mediated protection against CI.

Conclusion: This study provides new strategies to explore the mechanism of cerebral ischemia and help discover the potential mechanism of BNC.

Figure 1

The CI imbalanced network of AAs-enzymes-CI-related proteins. Light purple square: 23 AAs that were collected after CI from articles published from 1992 to 2016; peach square: the ten AAs in CSF detected in this study; blue diamond: key regulatory enzymes that were involved in the regulation of two or more metabolisms of AAs; orange diamond: four key enzymes regulating the generation of BNC-mediated AA-biomarkers measured by ELISAs and western blotting in CSF; pink circle: the proteins associated with CI.

Figure 2

Neurological score by BNC pretreatment: neurobehavioral score (a), infarct area (b), and TTC staining of the brain (c). ^∗ p < 0.05, ^∗∗ p < 0.01, and ^∗∗∗ p < 0.001 in the MCAO group versus the sham group; ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001 in the BNC group versus the MCAO group.

Figure 3

Typical overlap chromatograms of 10 AAs and internal standard. Alanine (Ala), valine (Val), taurine (Tau), leucine (Leu), isoleucine (Ile), glutamine (Gln), glutamate (Glu), phenylalanine (Phe), (S)-tyrosine (Tyr), tryptophan (Trp), and phenylalanine-d5 (internal standard (IS)).

Figure 4

The score plot using the first two principal components for the PCA model of sham, MCAO, and BNC-treated rats. Green circle: sham; blue circle: MCAO; red circle: BNC.

Figure 5

The line chart of 10 amino acid levels in CSF. Blue: sham group; red: MCAO model group; green: BNC-treated group. ^∗ p < 0.05, ^∗∗ p < 0.01, and ^∗∗∗ p < 0.001 in the MCAO group versus the sham group; ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001 in the BNC group versus the MCAO group.

Figure 6

Verification of the enzymes regulated by BNC against CI in CSF based on ELISA and western blotting. ELISA test for GOT2 (a) and AADC (b); western blotting test for AADC (c); three individual samples were analyzed in each group. Results were analyzed by one-way ANOVA. ^∗ p < 0.05 and ^∗∗ p < 0.01 in the MCAO group versus the sham group; ^# p < 0.05 in the BNC group versus the MCAO group.