Inhibitory effect of Wogonin on hepatocellular carcinoma through modulation of lactate metabolism and suppression of regulatory T cell differentiation[]

Abstract
Background: Wogonin, an active flavonoid derived from the traditional Chinese medicinal (TCM) herb Scutellaria baicalensis, has been reported to regulate cellular metabolism. Increasing evidence indicates that wogonin may inhibit regulatory T cell (Treg) differentiation and modulate immune responses both in vivo and in vitro. However, the specific molecular mechanisms remain unclear. Our previous findings demonstrated that lactic acid, a metabolic byproduct of glycolysis in hepatocellular carcinoma (HCC) cells, promotes differentiation of naïve CD4+ T cells into Tregs.
Objective: To investigate the molecular mechanisms underlying the anti-HCC effects of wogonin, particularly its impact on lactate metabolism and Treg differentiation.
Methods: The effects of wogonin (1.25, 5, and 20 μg/mL) on naïve CD4+ T-cell differentiation were assessed in the presence of lactic acid. Treg proportions were measured using flow cytometry, while protein and mRNA expression levels of TGF-β, p-Smad2, p-Smad3, LDHA, p-mTOR, HIF-1α, ATP5B, and FOXP3 were analyzed by Western blotting and RT-PCR. Levels of IL-10, α-KG, and 2HG were quantified using ELISA. To further assess in vivo efficacy, a mouse hepatoma transplantation model was established via subcutaneous injection of H22 cells. After 7 days, mice received wogonin treatments (12.5 or 25 mg/kg). Three weeks later, mice were euthanized, tumors were histologically analyzed using HE is staining, CD4+ and CD8+ T-cell populations were assessed by immunohistochemistry and flow cytometry, LDHA enzyme activity and intra- and extracellular lactate and glucose concentrations were determined by ELISA, and Foxp3 and IL-10 mRNA levels were measured by RT-PCR.
Results: Upon cytokine stimulation (anti-CD3, anti-CD28, and TGF-β) with supplemental lactic acid, the proportion of Treg cells was significantly elevated. Conversely, treatment with wogonin significantly and dose-dependently decreased Treg differentiation and simultaneously inhibited LDHA expression. Through inhibitor screening, c-Myc was identified as a crucial molecular target of wogonin. Mechanistically, wogonin suppressed c-Myc activity, thereby downregulating LDHA expression and activity, reducing abnormal accumulation of 2HG, normalizing the 2HG/α-KG ratio, and ultimately attenuating the expression of the Treg-specific transcription factor Foxp3 and cytokine IL-10.
Conclusion: Wogonin suppresses Treg cell differentiation and exhibits anti-HCC effects by inhibiting c-Myc-mediated LDHA expression and disrupting the 2HG/ATP5B/mTOR/HIF-1α signaling pathway. Given its potential to reshape the immunosuppressive tumor microenvironment, we propose further investigation into the synergistic therapeutic effects of wogonin in combination with PD-1/PD-L1 inhibitors.