Abstract
PURPOSE: Aerobic glycolysis is crucial in the proliferation, metastasis, immunosuppression of hepatocellular carcinoma (HCC). We established mice model with HCC to explore whether electroacupuncture and moxibustion can alleviate HCC mice by inhibiting NSUN2-mediated aerobic glycolysis. METHODS: HCC mice model was established by intraperitoneal injection of diethylnitrosamine combined with carbon tetrachloride. In the moxibustion and electroacupuncture groups, the acupoints "Ganshu" and "Zusanli" (bilateral) were selected for intervention. Enzyme-linked immunosorbent assay quantified the serum levels. Immunohistochemistry/Western blotting, quantitative real-time PCR assessed protein and mRNA expressions of aerobic glycolysis. MRM-based targeted metabolomics quantified hepatic energy metabolism alterations. RESULTS: After HCC model completion on week 28, macroscopic examination revealed pronounced hepatomegaly with evident morphological distortion. The hepatic surface exhibited a coarse and irregular texture, accompanied by varying degrees of adhesions between adjacent liver lobes. Notably, large, well-defined tumors presenting in either whitish or dark-red appearances were observed scattered across the liver parenchyma. Following a total intervention period of 26 weeks, electroacupuncture and moxibustion significantly decreased the number of liver tumor, tumor load, mean tumor diameter and tumor volume compared to the model group (P < 0.05). Furthermore, the spleen and liver index were also significantly lowered (P < 0.05). A significant decrease was observed in the protein levels of serum AFP and AFP-L3 after treatment (P < 0.05). The protein and mRNA expression levels of NSUN2 and PKM2 were significantly downregulated (P < 0.01). Consistent with the inhibition of glycolytic flux, the protein expression of enzyme hexokinase 2 (HK2) was profoundly reduced (P < 0.001). Meanwhile, moxibustion decreased in the hepatic content of early glycolytic intermediates, glucose-6-phosphate (G6P) and fructose 6 phosphate (F6P) (P < 0.01), and electroacupuncture just decreased G6P (P < 0.001). Conversely, the level of phosphoenolpyruvate, a high-energy intermediate preceding the final step of glycolysis, was significantly elevated and increased (P < 0.001). CONCLUSION: The study reveals that both electroacupuncture and moxibustion alleviate tumor proliferation in HCC mice by suppressing the NSUN2-PKM2 glycolytic axis, thereby highlighting this pathway as a novel metabolic target for non-pharmacological intervention; moreover, moxibustion uniquely exhibits the potential to induce a more extensive metabolic stress response. The observed reductions in tumor burden and key biomarkers corroborate the therapeutic potential of this approach. Collectively, these preclinical findings suggest that electroacupuncture and moxibustion, as non-pharmacological adjunctive therapies, hold translational potential for modulating tumor metabolism and slowing HCC progression, warranting further clinical investigation.