Inhibitory effect of Wogonin on hepatocellular carcinoma through modulation of lactate metabolism and suppression of regulatory T cell differentiation[]
Description
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.
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Naïve CD4+ T cells were isolated from mouse spleens and cultured in 1640 medium (Corning, NY, USA) with 10% fetal bovine serum (BC-SE-FBS01) (BioChannel Biological Technology Co., Ltd., Nanjing, China), 1% penicillin/streptomycin (211092) (NEST, NY, USA), anti-mouse 3 μg/mL anti-CD3, 3 ng/mL TGF-β, 3 μg/mL anti-CD28 (BioLegend, China). For in vitro experiments, triplicate replicates were performed for each condition to account for biological variability and ensure reproducibility. Some culture bottles and dishes required for cell culture (CCB06-025, CCB06-075, CCD06-100A), were purchased from Bioland Biotechnolog 2.9 Enzyme-linked immunosorbent assay (ELISA) The level of α-KG, 2HG was analyzed by ELISA kit (Meilian, China). The activation of LDHA was analyzed by ELISA kit (Yifeixue, China). All metabolites and enzyme activities were evaluated after 3 days stimulation. 2.13 H&E staining The tumor tissues were fixed with 4% paraformaldehyde. After dehydration, the samples were embedded in paraffin, sectioned, and mounted. Six sections in each group were stained with H&E and pathological changes were evaluated under a microscope. Data were analyzed using Image J and Image-Pro Plus.
Institutions
- Nanjing University of Chinese Medicine