Use of cellular metabolomics and lipidomics to decipher the mechanism of Huachansu injection-based intervention against human hepatocellular carcinoma cells

Abstract

Huachansu (HCS) injection has been used for the clinical treatment of hepatocellular carcinoma (HCC) patients in China for more than thirty years. However, the underlying mechanisms of HCS injection in inhibiting HCC remains unclear. In this study, we aimed to evaluate the effects of HCS injection on HCC both in vitro and in vivo and to systematically explore the underlying mechanisms of HCS's actions against HCC using an ultrahigh-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS)-based cellular metabolomics and lipidomics approach. MTT and DAPI staining were performed to evaluate the effects of HCS injection on cell proliferation and apoptosis, respectively, in human HepG2 HCC cells with and without HCS treatment. An H22 hepatoma-bearing mouse model was employed to investigate the anti-HCC action of HCS injection in vivo. A UPLC-HRMS-based cellular metabolomics and lipidomics method was employed to characterize cellular metabolite fingerprinting, to identify potential biomarkers associated with the effect of HCS injection on the inhibition of HCC and to explore the anti-HCC mechanisms of HCS injection from the perspective of cellular metabolism. As a result, HCS injection inhibited the proliferation of HepG2 cells and induced their apoptosis. Body weight loss, sarcoma weights and volumes of H22 hepatoma-bearing mice in the HCS treatment group were significantly attenuated compared to those in the model group. Twenty-five biomarkers responsible for the intervention effect of HCS were screened and annotated using UPLC-HRMS-based cellular metabolomics and lipidomics. Among these biomarkers, two cellular metabolites, uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) and glutathione, were confirmed unambiguously using the reference substances. Metabolic pathway analysis revealed that the anti-HCC effects of HCS injection primarily involved the metabolism of glutathione, cysteine and methionine, sphingolipid, fatty acid biosynthesis, amino sugar and nucleotide sugars. As evidenced by western blotting experiments, the changes in UDP-GlcNAc content in response to HCS injection were linked to the inhibition of UDP-GlcNAc pyrophosphorylase 1 (UAP1) expression in HepG2 cells. In conclusion, HCS injection has a curative anti-HCC effect, and a UPLC-HRMS-based cellular metabolomics and lipidomics approach combined with further confirmatory experiments is a promising means of discovering its underlying mechanisms.

Keywords: Hepatocellular carcinoma; Huachansu injection; Lipidomics; Metabolomics; Ultrahigh-performance liquid chromatography-high-resolution mass spectrometry.