Serum Metabolomics Based on GC-MS Reveals the Antipyretic Mechanism of Ellagic Acid in a Rat Model

Abstract

Ellagic acid (EA) is a polyphenol dilactone that has been reported to have antipyretic, anti-inflammatory, anti-tumor, and antioxidant activities, but the mechanism of action has not been reported. In this study, serum metabolomics was used to explore the mechanism of EA on rat fever induced by beer yeast, and to screen out marker metabolites to provide a reference for the antipyretic effect of EA. The acute fever model of male Sprague Dawley rats involved subcutaneous injection with 20% aqueous suspension of yeast (15 mL/kg) in their back. At the same time of modeling, EA was given orally by 10 mL/kg intragastric administration for treatment. During the experiment, the temperature and its change values of rats were recorded, and Interleukin-6 (IL-6), Tumor Necrosis Factor-α (TNF-α), Prostaglandin E2 (PGE2), Cyclic Adenosine Monophosphate (cAMP), Superoxide Dismutase (SOD) and Malondialdehyde (MDA)—six physiological and biochemical indexes of rats—were detected after the experiment. In addition, the hypothalamus of each rat was analyzed by Western blot (WB), and the levels of Phospho Nuclear Factor kappa-B (P-NF-κB P65) and IkappaB-alpha (IKB-α) were detected. Then, the serum metabolites of rats in each group were detected and analyzed by gas chromatograph mass spectrometry and the multivariate statistical analysis method. Finally, when screening for differential metabolites, the potential target metabolic pathway of drug intervention was screened for through the enrichment analysis of differential metabolites. Pearson correlation analysis was used to systematically characterize the relationship between biomarkers and pharmacodynamic indicators. EA could reduce the temperature and its change value in yeast induced fever rats after 18 h (p < 0.05). The level of IL-6, TNF-α, PGE2, cAMP, SOD and MDA of the Model group (MG) increased significantly compared to the Normal group (NG) (p < 0.001) after EA treatment, while the levels of the six indexes in the serum and cerebrospinal fluid of yeast-induced rats decreased. The administration of yeast led to a significant increase in Hypothalamus P-NF-κB P65 and IKB-α levels. Treatment with EA led to a significant decrease in P-NF-κB P65 levels. Moreover, combined with VIP > 1 and p < 0.05 as screening criteria, the corresponding retention time and characteristic mass to charge ratio were compared with the NIST library, Match score > 80%, and a total of 15 differential metabolites were screened. EA administration significantly regulated 9 of 15 metabolites in rat serum. The 15 differential metabolites involved linoleic acid metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, galactose metabolism, biosynthesis of unsaturated fatty acids and glycerolipid metabolism. Pharmacodynamic correlation analysis was conducted between 15 different metabolites and six detection indexes. There was a significant correlation between 13 metabolites and six detection indexes. D-(−)-lactic acid, glycerin, phosphoric acid, 5-oxo-L-proline were negatively correlated with TNF-α, and p values were statistically significant except for L-tyrosine. In addition, glycerin was negatively correlated with IL-6, PGE2 and MDA, while phosphoric acid was negatively correlated with IL-6. In conclusion, EA may play an antipyretic anti-inflammatory role through the inhibition of the IKB-α/NF-κB signaling pathway and five metabolic pathways, which may contribute to a further understanding of the therapeutic mechanisms of the fever of EA.

Keywords: IKB-α; Western blot; ellagic acid; fever; gas chromatograph-mass spectrometer; metabolomics.

Figure 1

The structural formula of ellagic acid.

Figure 2

Antipyretic effects of Aspirin and EA. (A): The temperatures were measured at 0, 2, 4, 6, 8 and 18 h after yeast administration (n = 8). (B): Temperatures change value at 2, 4, 6, 8 and 18 h after yeast administration (n = 8). p value is for individual time point. (### p < 0.001, vs. NG, indicates significantly different results compared with NG; * p < 0.05, ** p < 0.01, *** p < 0.001, vs. MG, indicate significantly different results compared with the model that was incubated with 15 mL/kg yeast).

Figure 3

Anti-inflammation effects of aspirin and EA. (A): Effects of aspirin and EA in serum TNF-α secretion (n = 8). (B): Effects of aspirin and EA in serum IL-6 secretion (n = 8). (C): Effects of aspirin and EA in serum MDA secretion (n = 8). (D): Effects of aspirin and EA in serum SOD activity (n = 8). (E): Effects of aspirin and EA in cerebrospinal fluid PEG2 secretion (n = 8). (F): Effects of aspirin and EA in cerebrospinal fluid cAMP activity (n = 8). (# p < 0.05, ## p < 0.01, ### p < 0.001, vs. NG, indicates significantly different results compared with NG; * p < 0.05, ** p < 0.01, vs. MG, indicate significantly different results compared with the model that was incubated with 15 mL/kg yeast).

Figure 4

Effects of ellagic acid on P-NF-κB p65 and IKB-α protein expression in hypothalamus tissue of pyretic rats induced by brewer’s yeast as measured by Western blot. A~F:NG, MG, APG, EALG, EAMG, EAHG.

Figure 5

PCA of serum metabolites. (A): scores scatter plot. (B): loading.

Figure 6

OPLS-DA of serum metabolites. (A): scores scatter plot. (B): loading of six groups. (C): 3D plot. (D): random displacement test (n = 200) and the explanatory rate of the model.

Figure 7

Box diagram of 15 differential metabolites in serum # p < 0.05, ## p < 0.01, ### p < 0.001 vs. NG; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. MG.

Figure 8

Metabolic pathway analysis. 1: Linoleic acid metabolism. 2: Phenylalanine, tyrosine and tryptophan biosynthesis. 3: Galactose metabolism. 4: Biosynthesis of unsaturated fatty acids. 5: Glycerolipid metabolism.

Figure 9

EA plays an antipyretic and anti-inflammatory role by regulating IKB-α/NF-κB pathway and inhibiting inflammatory factors.

Figure 10

Potential target metabolic pathway of EA in the treatment of fever in rats. The green box shows the differential metabolites, ↑ level up, ↓ level down; The red box shows the regulated inflammatory factors. # p < 0.05, ## p < 0.01 vs. NG; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. MG.