Licorice Total Flavonoids Alleviate Liver Fibrosis by Inhibiting TGF-β1 Signaling in Human Stellate Cells

Liver fibrosis is a critical, reversible pathological condition arising from chronic liver injury, serving as a precursor to severe complications like cirrhosis and hepatocellular carcinoma. Despite its prevalence and serious prognosis, effective, specifically licensed therapeutic drugs for liver fibrosis remain elusive, representing a significant unmet medical need. Licorice (Glycyrrhiza uralensis Fisch.), a widely recognized traditional Chinese medicine, contains licorice total flavonoids (LTF) as its primary active components. However, the precise anti-hepatic fibrotic mechanisms and therapeutic potential of LTF have historically been poorly understood, creating a gap in evidence-based applications.

Researchers investigated the anti-fibrotic potential of licorice total flavonoids (LTF) using a multi-pronged approach. Initially, network pharmacology was employed to predict core targets and signaling pathways. Molecular docking and high-throughput bio-layer interferometry (BLI) fishing, followed by liquid chromatography-mass spectrometry (LC-MS) analysis, were utilized to identify active components binding to key targets. For in vitro validation, human hepatocyte-derived stellate cells (LX-2) were treated with 16, 32, and 64 µg/mL LTF after induction with transforming growth factor-β1 (TGF-β1). The primary endpoints included assessing the inhibition of LX-2 cell activation and measuring the mRNA and protein expression levels of fibrotic markers like α-smooth muscle actin (α-SMA), Collagen I, and TGF-β1 via qPCR and Western blot.

Network pharmacology analysis revealed that LTF exerts its anti-fibrotic effects by modulating multiple targets and pathways, with the TGF-β signaling pathway identified as a central regulatory hub. In human hepatocyte-derived stellate cells (LX-2), LTF at concentrations of 16, 32, and 64 µg/mL significantly inhibited the transforming growth factor-β1 (TGF-β1)-induced activation of these cells. This inhibition was accompanied by a marked reduction in both mRNA and protein expression of key fibrotic markers, including α-smooth muscle actin (α-SMA), Collagen I, and TGF-β1 itself. These dose-dependent effects underscore LTF's direct impact on cellular processes driving fibrosis. Furthermore, LC-MS results, following high-throughput BLI analysis, provided compelling evidence:

Compared with the control group, 95 flavonoid active components within the total licorice flavonoids group were identified as strong candidate compounds capable of binding directly to TGF-β1, a core target in liver fibrosis. These findings collectively indicate that LTF likely exerts its anti-fibrotic actions primarily by regulating TGF-β-related signaling pathways through the direct inhibition of TGF-β1 expression and activity.