Antistress Effects of San-Huang-Xie-Xin Decoction on Restraint-Stressed Mice Revealed by 1H NMR-Based Metabolomics and Biochemistry Analysis

Abstract

San-Huang-Xie-Xin decoction (SHXXD), composed of Rhei Radix et Rhizoma, Coptidis Rhizoma, and Scutellariae Radix, is a representative antipyretic and detoxifying prescription in traditional Chinese medicine. In this study, we investigated the antistress effects and underlying mechanisms of San-Huang-Xie-Xin decoction (SHXXD) on restraint-stressed mice by ¹H NMR-based metabolomics combined with biochemistry assay. A total of 48 male mice (5 weeks old, 18-22 g) were divided randomly into 6 groups (n = 8), including the normal group, restraint-stressed group, vitamin C group (positive drug, 17 mg/kg), and 3-dosage groups of SHXXD (200, 400, and 800 mg/kg). The stress model was induced by restraining mice in a polypropylene centrifuge tube for 6 h every day. The rotarod test was performed, and several biochemical indicators were measured. Moreover, other 24 animals were divided into 3 groups (n = 8) including the normal group, restraint-stressed group, and SHXXD group (800 mg/kg) for ¹H NMR-based metabolomics analysis. Our results showed that SHXXD significantly increased the rotarod time, thymus index, spleen index, and the levels of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and interleukin- (IL-) 2, but decreased the levels of malondialdehyde (MDA), IL-1β, tumor necrosis factor- (TNF-) α, corticosterone (CORT), and adrenocorticotropic hormone (ACTH) in restraint-stressed mice. Moreover, the contents of eight endogenous metabolites that were changed by restraint stress were significantly reversed by SHXXD. The results of both metabolomics and biochemical analysis indicated that SHXXD (800 mg/kg, p.o.) could improve the biochemical changes and metabolic disorders in restraint-stressed mice by antioxidation and anti-inflammation, enhancing the body's immune function and restoring several disturbed metabolic pathways (i.e., lipid metabolism, glycolysis and gluconeogenesis, inflammatory injury, and energy metabolism). Taken together, these results indicated that SHXXD has a potential antistress effect in restraint-stressed mice and could be considered as a candidate drug for stress-related disorders.

Figure 1

Experimental procedure of the antistress activity evaluation of SHXXD.

Figure 2

HPLC chromatograms of mixed standard compounds (a) and SHXXD extract (b). Nine chromatographic peaks were identified: (1) jatrorrhizine, (2) baicalin, (3) palmatine, (4) berberine, (5) wogonoside, (6) baicalein, (7) aloe-emodin, (8) rhein, and (9) wogonin.

Figure 3

Effects of SHXXD on rotarod time (a), thymus index (b), and spleen index (c). The data are presented as the mean ± S.D. (n = 8); ^∗p < 0.05 and ^∗∗p < 0.01 vs. the control group.

Figure 4

Effects of SHXXD on serum CORT (a) and ACTH (b) levels. The data are presented as the mean ± S.D. (n = 8); ^∗p < 0.05 and ^∗∗p < 0.01 vs. the control group.

Figure 5

Effects of SHXXD on serum cytokines (IL-1β (a), TNF-α (b), and IL-2 (c)) and GSH-Px (d), MDA (e), and SOD (f) in the brain of restraint-stressed mice. Each point represents an individual sample. Error bars indicate the mean ± S.D. (n = 8). ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 vs. the control group.

Figure 6

Representative ¹H NMR CPMG spectra of the serum samples of mice ((a) normal group, (b) SHXXD group, and (c) control group) in the range of 0.8-8.5 ppm. The low-field region (6.8-8.5 ppm) was magnified 12 times from the remaining region for the purpose of clarity. Signal assignments: (L1) LDL and VLDL, CH₃–(CH₂)_n–; (L2) LDL and VLDL, CH₃–(CH₂)_n–; L3 (lipids), –CH₂–CH₂–C=O; (L4) lipids, –CH₂–CH=CH–; (L5) lipids, =CH–CH₂–CH=; and (L6) lipids, –CH=CH–.

Figure 7

Score plots and corresponding loading plots generated from pairwise PLS-DA analysis of ¹H NMR data: (a) score plot of the normal and restraint-stressed groups; (b) loading plot of the normal and restraint-stressed (control) groups; (c) score plot of the restraint-stressed and SHXXD groups; (d) loading plot of the restraint-stressed and SHXXD groups.

Figure 8

Boxplot of relative levels of the 10 differential metabolites that were obtained from PLS-DA in the serum of the normal, restraint-stressed (control), and SHXXD groups of mice. Data were normalized to the total spectral area, and so the bar plots show the normalized values, and Y axes are represented as relative units.