Study on the mechanism of acute liver injury protection in Rhubarb anthraquinone by metabolomics based on UPLC-Q-TOF-MS

Abstract

As a traditional Chinese medicine, rhubarb has been used in a variety of liver diseases and it is widely used in clinic to prevent and treat acute liver injury. Anthraquinone, as the main medicinal component of rhubarb, can reverse the further development of liver fibrosis caused by acute liver injury. In this study, metabonomics was used to explore the mechanism of different doses of rhubarb anthraquinone on acute liver injury in rats. Rhubarb anthraquinone was administered intragastric to rats at doses of 3.9, 7.8 and 15.6 mg/kg, respectively, for 7 days, and then 30% CCl₄ was injected intraperitoneally at the dose of 1 ml/kg to replicate the acute liver injury model. The biochemical indicators content of ALT, AST, ALP, γ-GT, TG, TC, LDL, HDL in serum and GSH, Hyp, SOD, TNF-α, IL-6 and IL-8 in liver tissue extract were tested respectively, and liver tissue was histopathologically analysis. At the same time, UPLC-Q-TOF-MS combined with non-targeted metabolomics were used to study the metabolites and metabolic pathways of rhubarb anthraquinone in treating acute liver injury. Compared with normal rats, the contents of ALT, AST, ALP, TG, TC, LDL, γ-GT in serum and Hyp, MDA, IL-6, IL-8, TNF-α in the liver tissue extract were significantly increased in model rats (p < 0.05, p < 0.01), and the content of HDL in the serum was significantly decreased (p < 0.05); the activities of GSH and SOD in liver tissue extract were also significantly decreased (p < 0.05). After administration of rhubarb anthraquinone, compared with the model group, with the increase of dosage, some biochemical indexes showed opposite changes, and gradually approached to normal rats. 12 different metabolites were identified by metabonomics, and the biosynthesis and metabolism of phenylalanine, tyrosine and tryptophan, the metabolism of amino sugars, nucleotide sugars and pyrimidines metabolism, and the biosynthesis of steroid hormone were identified based on the biomarker analysis. Based on the biochemical analysis and metabonomics analysis of rats with acute liver injury treated with different doses of rhubarb anthraquinone, combined with histopathological observation, the results show that the protective effect of rhubarb anthraquinone on acute liver injury is related to the dosage; Meanwhile, the metabolic pathway analysis suggested that rhubarb anthraquinone alleviate acute liver injury by regulating inflammation, oxidative stress and fibrosis disorders. This study explained the therapeutic effect of rhubarb anthraquinone on acute liver injury from both material basis and action pathway, and provided safe and effective research ideas for clinical application of rhubarb.

Keywords: Rhubarb anthraquinone; UPLC-Q-ToF-MS; acute liver injury; metabolomics; traditional Chinese medicine.

FIGURE 1

The effects of rhubarb anthraquinone on liver tissue of rats with acute liver injury refers to balloon degeneration and punctate necrosis of hepatocytes; refers to the infiltration of lymphocytes and neutrophils.

FIGURE 2

PCA score plots and PLS-DA score plots of different groups in ESI+ and ESI- mode respectively. The score plots of PCA of NC, ALI, RA-L, RA-M and RA-H groups from PCA analysis in ESI+ (A) and ESI- (B); The score plots of PLS-DA of NC, ALI, RA-L, RA-M and RA-H groups from PLS-DA analysis in ESI+ (C) and ESI- (D); 200-permutation test of PLS-DA model in ESI+ (E) and ESI- (F).

FIGURE 3

OPLS-DA score plots of different groups in ESI+ and ESI- mode respectively. The score plots of OPLS-DA of NC, ALI, RA-L, RA-M and RA-H from OPLS-DA analysis in ESI+ (A) and ESI- (B); Metabolites with VIP >1 of ALI vs. NC were marked with a red triangle in ESI+ (C) and ESI- (D); Metabolites with VIP >1 of ALI vs. RA-H were marked with a red triangle in ESI+ (E) and ESI- (F).

FIGURE 4

Variations in the trends of the difference metabolites. There were shown the variations in the trends of Phenylacetic acid, 4-Hydroxybenzoic acid, 5-Aminolevulinic acid, Pyrroline hydroxycarboxylic acid, Kynurenic acid, 9-OxoODE, S-Adenosylmethionine, 5-Hydroxyindoleacetic acid, Methylmalonic acid, Succinic acid semialdehyde, 4-Aminobutyraldehyde, 3, 4- Dihydroxyphenylglycol respectively. Compared with the NC group, *p < 0.05, **p < 0.01; Compared with the ALI group, ^# p < 0.05, ^## p < 0.01.

FIGURE 5

Schematic diagram of metabolic pathway related to rhubarb anthraquinone treated rat acute liver injury. The boxes bordered in red represent metabolites that are significantly higher in NC, RA-L, RA-M and RA-H than in ALI, respectively. The light blue boxes indicate metabolites significantly higher in ALI than in NC, RA-L, RA-M and RA-H respectively.