Mechanistic insights into the Ganfule capsule in alleviating alcoholic liver disease.

OBJECTIVE

Using a combination of network pharmacology and experimantal validation, the study aims to elucidate the specific therapeutic effects of Ganfule (GFL), a traditional Chinese medicinal formula approved for the treatment of advanced liver cancer in China, on alcoholic liver disease (ALD) and its interactions with the biological pathways associated with ALD.

METHODS

GFL was administrated orally to ethanol (EtOH)-induced mice daily for 50 days. The therapeutic efficacy of the GFL in mice was assessed using biochemical assays, H&E staining, and TUNEL staining. These findings were further validated in an ALD cell model pretreated with GFL through cell viability assay, oil red O staining, and flow cytometry. Network pharmacology was employed to predict key therapeutic targets of GFL against ALD, followed by transcriptome sequencing analysis. Molecular docking analysis and molecular dynamics simulation were conducted to evaluate the binding interactions between active chemical constitutes and their potential targets. The predicted targets and signaling pathways were further verified using molecular biology techniques, including qRT-PCR and Western blot.

RESULTS

Network pharmacology analysis revealed that the common targets between GFL and ALD are predominantly associated with lipid metabolism, oxidative stress, inflammatory reaction, and apoptotic pathways. Transcriptome sequencing further indicated that the therapeutic effect of GFL on ALD may primarily be mediated through the modulation of these signaling pathways. GFL alleviates lipid deposition in hepatocytes under ALD conditions by upregulating PGC-1α and downregulating genes involved in lipid metabolism. Pretreatment with GFL suppresses the elevation of malondialdehyde (MDA) and reactive oxygen species (ROS) levels while restoring reduced glutathione (GSH) levels by downregulating CYP2E1 within hepatocytes. Additionally, GFL inhibits the inflammatory response under ALD conditions by suppressing the IL-6/STAT3 pathway and reducing the transcription levels of IL-17RA and certain matrix metalloproteinases (MMPs)-related genes. Furthermore, by downregulating pro-apoptotic genes and upregulating anti-apoptotic genes, GFL prevents EtOH-induced hepatocyte apoptosis and improves liver function in mice. Subsequent molecular docking and molecular dynamics simulation suggested that GFL may exert its effects through the binding of its abundant quercetin (QCT) and phytolaccagenin to IL-6.

CONCLUSION

GFL capsule exerts a protective effect against ALD in mice and hepatocytes, potentially through the inhibition of inflammation, oxidative stress and apoptosis. This study provides a solid theoretical foundation for the potential application of GFL capsule in the prevention and clinical management of ALD.