Identification of Proteomic Markers for Monitoring Direct Toxic Liver Injury (DTLI): Profiling Hepatoprotective...

The aim of this study was to examine the hepatoprotective activity of multicomponent mixtures of natural origin in the BALB/C mouse model (n = 59), with acute direct toxic liver injury (DTLI) induced by the administration of streptozotocin (STZ) (100 mg/kg) in combination with a high-fat and high-fructose diet (HFFD). The hepatoprotective activity of activated hydrolytic lignin (Bp-Cx-1), methanolic fraction of Bp-Cx-1 (Bp-Cx-M) and isoflavones from kudzu Pueraria lobata roots (IFL) were evaluated on molecular level using mass spectrometry (MS)-based omics technologies. Untargeted label-free DIA quantitation resulted in 7214 protein groups identification (FDR 1%) after filtering across 40 liver tissue extracts. All treatment groups were closer to the control samples on the liver proteomic landscape compared to the untreated DTLI group, with the best results shown for the Bp-Cx-M and IFL groups. In order to identify differences between specific groups, we applied the post hoc Dunn's test and used Hedges' g as the effect size metric, revealing 64 proteins that tended to return to their normal level after treatment. In-depth proteomic liver tissue analysis enabled us not only to reveal the main pathways such as inflammation and oxidative stress, which are in a good agreement with DTLI and non-alcoholic liver disease pathophysiology, but also to evaluate hepatoprotective activity of multicomponent mixtures of natural origin containing polyphenols and mostly associated with protein metabolism (e.g., PSMD7, HCFC1) and deubiquitination pathways (e.g., UCHL3). It is worth noting that the Bp-Cx-1 isolated methanol fraction (Bp-Cx-M) demonstrated a pronounced increased hepatoprotective activity compared to the parent material due to the enrichment with active components such as polyphenols. Consistent with the proteomic findings of restored ubiquitin-proteasome function, assessment by comet assay revealed that treatments with Bp-Cx-M and IFL significantly reduced DNA damage by 50% compared to the untreated DTLI group. The developed MS-based multi-omics approach may be implemented for the robust and high-throughput screening method during assessment of new hepatoprotective agents of synthetic or natural origin.