Anoectochilus roxburghii polysaccharide prevents carbon tetrachloride-induced liver injury in mice by metabolomic analysis

Anoectochilus roxburghii (Wall.) Lindl, a traditional Chinese medicine, is used for the effective treatment of liver disease in China. Anoectochilus roxburghii polysaccharide (ARPT) is an important constituent of Anoectochilus roxburghii. ARPT exerts a hepatoprotective effect and contributes directly to the therapeutic benefit of Anoectochilus roxburghii. However, the hepatoprotective mechanism of ARPT requires further elucidation. The present study was designed to assess the effects and underlying mechanism of ARPT when used to pretreat carbon tetrachloride (CCl₄)-induced liver injury in mice. Mice were randomly divided into three groups: control group (no ARPT treatment or liver injury), model group (liver injury induced with CCl₄), and the ARPT group (ARPT pretreatment followed by liver injury). A metabolomic method, based on liquid chromatography combined with mass spectrometry (LC-MS) and pattern recognition analysis, was applied. The data were analyzed with principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA), to determine differentiating metabolites in the serum and liver tissue between the experimental groups. The PCA and OPLS-DA scores plots of the serum and liver tissue samples based on the LC-MS data showed a clear separation between the control and liver injury model group, while the ARPT-treated group showed a trend of close with the control. There were eleven metabolites [PS(O-18:0/0:0), phosphocholine, phenylalanine, hippuric acid, α-ketoisovaleric acid, metyrosine, leucinic acid, ketoleucine, Cer(d18:1/19:0), α-kamlolenic acid, and 4-formyl indole] were identified as candidate biomarkers in the serum samples, eight such metabolites (valine, phosphohydroxypyruvic acid, phosphocholine, ornithine, indole, xanthine, uridine, and glucose 6-phosphate) were found in the liver tissue samples, and one metabolite (phosphocholine) was observed in both the serum and liver tissue samples. These endogenous metabolites are considered to be in response to the hepatoprotective effects of ARPT and are involved in amino acid metabolism, lipid metabolism, gut bacteria metabolism, energy metabolism, and the methylation pathway. These findings suggest that ARPT mitigates the metabolic effect of CCl₄-induced hepatotoxicity in mice by affecting at least five different pathways. LC-MS-based metabolomics provides a powerful approach for identifying potential biomarkers and for elucidating the protective mechanisms of traditional Chinese medicines against disease.

Keywords: Anoectochilus roxburghii; Carbon tetrachloride; Liver injury; Metabolomics; Polysaccharide.