UHPLC-QTOF-MS-based metabolomics joint high-throughput RNA sequencing transcriptomics approach for the analysis of fecal and liver biological samples and application in a case study for the mechanism of Qing-Kai-Ling oral liquid in treating MASLD.

Qing-Kai-Ling (QKL) oral liquid has been increasingly used in metabolic dysfunction-associated steatotic liver disease (MASLD). However, the specific metabolic differentials and metabolic pathway mechanisms that affect the MASLD regulated by QKL remained unclear. In this study, serum biochemical analyses and hematoxylin-eosin staining of the liver revealed QKL reduced liver injury and enhanced lipid metabolism ability, respectively. To clarify the therapeutic mechanism of the QKL in the treatment of MASLD, UHPLC-QTOF-MS non-target metabolomics and RNA-Seq high-throughput sequencing analysis were used to explore the mechanism of the QKL in the treatment of MASLD from the perspective of metabolic-gene interactions. UHPLC-QTOF-MS-based untargeted metabolomics further revealed that there were 196 common differentially expressed metabolites identified among 3 groups; QKL significantly up-regulated 44 metabolites, while 11 metabolites (including N-phenylacetylglutamic acid and glycocholic acid) were downregulated significantly. Moreover, the main metabolic pathways regulated by QKL included amino acids, peptides, bile acids, carbohydrates, linoleic acids, etc. Additionally, the result of the RNA sequencing-based transcriptomics showed that a total of 984 differential genes (DEGs) were identified and 9 important DEGs were obtained. The result of the Kyoto Encyclopedia of Genes and Genomes (KEGG) demonstrated that the 984 DEGs were linked to bile acid metabolism, glycerophospholipid metabolism, insulin resistance, AMPK signaling pathway, etc. Overall, this work was the first to show that QKL regulated metabolites and genes to alleviate MASLD by the UHPLC-QTOF-MS-based untargeted metabolomics joint high-throughput RNA sequencing-based transcriptomics analysis, providing the basis and research method for the treatment of metabolic diseases by QKL and other drugs.