Determining the protective effects of Yin-Chen-Hao Tang against acute liver injury induced by carbon tetrachloride using 16S rRNA gene sequencing and LC/MS-based metabolomics.

Yin-Chen-Hao Tang (YCHT), consisting of Artemisia annua L., Gardenia jasminoides Ellis, and Rheum Palmatum L., has been used to relieve liver diseases in China for thousands of years. Several protective mechanisms of YCHT on liver injury have been investigated based on metabolomics, but the effects of YCHT on the alterations in the gut microbiota are still unclear. In this study, an integrated approach based on 16S rRNA gene sequencing combined with high-performance liquid chromatography-mass spectrometry (HPLC-MS) metabolic profiling was performed to assess the effects of YCHT on liver injury induced by carbon tetrachloride (CCl₄) through the regulation of the relative abundances of gut microbiota and their relationships with biomarker candidates. A total of twelve significantly altered bacterial genera and nine metabolites were identified, which returned to normal levels after YCHT treatment. The relative abundances of the identified microbiota, including significantly elevated amounts of p_Firmicutes, c_Clostridia, o_Clostridiales, f_Ruminococcaceae, g_[Eubacterium]_coprostanoligenes_group, s_uncultured_bacterium_f_Lachnospiraceae and remarkedly increased amounts of p_Bacteroidetes, c_Bacteroidia, o_Bacteroidales, f_Bacteroidaceae, g_Bacteroides and s_uncultured_bacterium_g_Bacteroides, were found in model rats compared with controls. Potential biomarkers, including lower levels of LysoPC (16:1(9Z)/0:0), LysoPC (20:3(5Z,8Z,11Z)), LysoPC (17:0), LysoPC (20:1(11Z)) and 3-hydroxybutyric acid and higher amounts of ornithine, L-kynurenine, hippuric acid and taurocholic acid are involved in several custom metabolic pathways, such as arginine and proline metabolism, tryptophan metabolism, glycerophospholipid metabolism and primary bile acid biosynthesis. Interestingly, there was a strong correlation between the perturbed gut microbiota in genera c_Clostridia and o_Clostridiales and the altered plasma metabolite 3-hydroxybutyric acid. This finding means that the hepatoprotective effects of YCHT may be due to the regulation of the production of the functional metabolite 3-hydroxybutyric acid through changes in the proportions of c_Clostridia and o_Clostridiales. These results showed that the hepatoprotective effects of YCHT not only focused on custom metabolic pathways but also depended on the changes in the gut microbiota in liver injury. These findings suggest that the 16S rRNA gene sequencing and LC-MS based metabolomics approach can be applied to comprehensively evaluate the effects of traditional Chinese medicines (TCMs).