Hepatoprotective action of Sonchus oleraceus against paracetamol-induced toxicity via Nrf2/KEAP-1/HO-1 pathway in relation to its metabolite fingerprint and in silico studies

Abstract

Background: Paracetamol overdose causes severe hepatotoxicity. Sonchus oleraceus is traditionally used to treat liver disorders, but its potential against paracetamol-induced liver injury is unexplored. This work aimed to investigate the protective mechanisms of an S. oleraceus extract (SOEtOH) using in vivo, histological and biochemical assessments along with metabolomics profiling and in silico studies, including molecular docking and dynamic simulations (MD).

Methods and findings: SOEtOH was administered to rats with paracetamol-induced hepatotoxicity at 50, 100, and 200 mg/kg doses. Serum enzymes, hepatic antioxidants, and histopathology were evaluated. UPLC-MS characterized bioactive metabolites and molecular docking and assessed their anti-inflammatory potential. SOEtOH significantly restored serum ALT and AST toward normal levels in a dose-dependent manner. It also replenished depleted hepatic glutathione (up to 3.9-fold) and superoxide dismutase (up to 4.7-fold). Immunohistochemistry revealed SOEtOH progressively attenuated caspase-3 expression related to apoptosis. It also ameliorated characteristic histopathological alterations like necrosis, inflammation, and sinusoidal congestion. Thirty-two bioactive metabolites, including flavonoids, phenolic acids, and terpenes, were identified. Molecular docking revealed potent anti-inflammatory effects via JNK inhibition, with luteolin-O-dihexoside, isorhamnetin-O-hexoside, di-O-caffeoylquinic, and kaempferol-O-hexoside having the strongest binding affinities. MD simulations demonstrated that these compounds' complexes significantly contribute to JNK1 and JNK2's catalytic binding site.

Conclusion: This integrated study demonstrates that SOEtOH protects against paracetamol hepatotoxicity by mitigating oxidative stress and inhibiting pro-inflammatory/apoptotic signaling. Our results reveal therapeutic lead compounds that may be further explored for clinical applications.

Fig 1. The herb Sonchus oleraceus L.

A) Plant shoot system, B) close view of leaves, C and D) close view of closed and open flowers.

Fig 2. Effect of SOEtOH on serum ALT and AST levels of paracetamol intoxicated rats.

ALT (A), AST (B). Each bar represents the mean ± SEM (n = 5). Statistical analysis was carried out by one-way analysis of variance (ANOVA) and followed by Tukey’s multiple comparison test.* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, ns: non-significant, (SOEtOH) Sonchus oleraceus extract.

Fig 3. Effect of S. oleraceus extract (SOEtOH) on hepatic content of paracetamol intoxicated rats.

(A) GSH, (B) SOD, (C) Nrf2, (D) Keap1 and (E) HO-1. Each bar represents the mean ± SEM (n = 5). Statistical analysis was carried out by one-way analysis of variance (ANOVA) and followed by Tukey’s multiple comparison test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, ns: non-significant, (SOEtOH) Sonchus oleraceus extract.

Fig 4. Histopathological examination of liver from different experimental groups (H&E).

Fig 5. Immunoreactivity of active caspase-3 protein in hepatocytes of experimental groups.

Fig 6. UPLC-qTOF-MS/MS base peak chromatograms of S. oleraceus ethanolic extract.

Negative (A) and positive (B) ionization modes.

Fig 7. Binding mode in the active site of JNK1 (PDB ID: 3V3V) and JNK2 (PDB ID: 3NPC).

(A, B) 3D and 2D binding mode of the luteolin-O-di-hexoside in the active site of JNK1, (C, D) 3D and 2D binding mode of the isorhamnetin-O-hexoside in the active site of JNK1, (E, F) 3D and 2D binding mode of the kaempferol-O-hexoside in the active site of JNK2, (G, H) 3D and 2D binding mode of the 3,4-di-O-caffeoylquinic acid in the active site of JNK2.

Fig 8. Results of Molecular Dynamic (MD) simulation and system stability on JNK1 and JNK2 protein.

[A] RMSD of Cα atoms of the protein backbone atoms on JNK1, [B] RMSF of each residue of the protein backbone Cα atoms of protein residues on JNK1, (C) ROG of Cα atoms of protein residues on JNK1, and (D) solvent accessible surface area (SASA) of the Cα of the backbone atoms relative (black) to the starting minimized over 40 ns for the catalytic binding site with isorhamnetin-O-hexoside – JNK1 (red), and luteolin-O-di-hexoside-JNK1(blue), [E] RMSD of Cα atoms of the protein backbone atoms on JNK2, [F] RMSF of each residue of the protein backbone Cα atoms of protein residues on JNK2, (G) ROG of Cα atoms of protein residues on JNK2, and (H) solvent accessible surface area (SASA) of the Cα of the backbone atoms relative (black) to the starting minimized over 40 ns for the catalytic binding site with kaempferol-O-hexoside–JNK2 (red) and 3,4-di-O-caffeoylquinic acid–JNK2 (blue).

Fig 9. Per-residue decomposition plots.

Showing the energy contributions to the binding and stabilization of isorhamnetin-O-hexoside [A], and luteolin-O-di-hexoside [B] into catalytic binding site of JNK-1, as well as kaempferol-O-hexoside [C], and 3,4-di-O-caffeoylquinic acid [D] into catalytic binding site of JNK-2. Corresponding inter-molecular interactions are shown [a], [b], [c], and [d].

Fig 10. The mechanistic pathway associated with JNK1 and JNK2 proteins.