Inhibitory effects of wogonin on hepatocellular carcinoma through modulation of lactate metabolism and suppression of regulatory T-cell differentiation

Introduction

Wogonin, an active flavonoid derived from the traditional Chinese medicinal herb Scutellaria baicalensis, has been reported to regulate cellular metabolism. Increasing evidence suggests that wogonin inhibits regulatory T-cell (Treg) differentiation and modulates immune responses both in vitro and in vivo. However, the underlying molecular mechanisms remain poorly understood. Our previous findings demonstrated that lactic acid, a metabolic byproduct of glycolysis in hepatocellular carcinoma (HCC) cells, promotes the differentiation of naïve CD4⁺ T cells into Tregs.

Methods

The effects of wogonin (1.25, 5, and 20 μg/mL) on naïve CD4⁺ T-cell differentiation were examined in the presence of lactic acid. The proportion of Tregs was analyzed using flow cytometry. Protein and mRNA expression levels of TGF-β, p-Smad2, p-Smad3, LDHA, p-mTOR, HIF-1α, ATP5B, and FOXP3 were measured by western blotting (WB) and RT-PCR, respectively. Levels of IL-10, α-KG, and 2-HG were quantified using ELISA. To further assess in vivo efficacy, a mouse hepatoma transplantation model was established by subcutaneous injection of H22 cells. Seven days after inoculation, mice were treated with wogonin (12.5 or 25 mg/kg). After three weeks, mice were euthanized. Tumors underwent histological analysis by HE staining, while CD4⁺and CD8⁺ T-cell populations were assessed via immunohistochemistry and flow cytometry. LDHA enzyme activity, intracellular and extracellular lactate and glucose concentrations were measured by ELISA. FOXP3 and IL-10 mRNA levels were evaluated by RT-PCR.

Results

Supplemental lactic acid significantly increased Treg differentiation following cytokine stimulation (anti-CD3, anti-CD28, and TGF-β). Conversely, wogonin treatment significantly and dose-dependently reduced Treg differentiation and LDHA expression. Through inhibitor screening, c-Myc was identified as a critical molecular target of wogonin. Mechanistically, wogonin suppressed c-Myc activity, subsequently downregulating LDHA expression and enzyme activity, reducing abnormal accumulation of 2-hydroxyglutarate (2-HG), normalizing the 2-HG/α-KG ratio, and ultimately decreasing FOXP3 and IL-10 expression.

Conclusions

Wogonin suppresses Treg differentiation and exerts anti-HCC effects by inhibiting c-Myc-mediated LDHA expression and disrupting the 2-HG/ATP5B/mTOR/HIF-1α signaling pathway. Given its potential to reshape the immunosuppressive tumor microenvironment, further investigation into its combination with PD-1/PD-L1 inhibitors is warranted.