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目的:研究追毒方对三阴性乳腺癌细胞MDA-MB-231增殖、侵袭迁移的影响，并阐述其分子机制。方法:首先采用MTT细胞毒性试验，Transwell小室侵袭试验和伤口愈合试验观察不同浓度追毒方对三阴性乳腺癌细胞增殖及侵袭迁移能力的影响；通过流式细胞术和乳酸试剂盒评估不同浓度追毒方处理后三阴性乳腺癌细胞糖摄取及乳酸生成、外排水平，以观察其有氧糖酵解水平；Western blot法检测追毒方对三阴性乳腺癌细胞的细胞外基质金属蛋白酶诱导因子(CD147)糖基化及葡萄糖转运蛋白(GLUT1)、单羧酸转运蛋白(MCT4)表达的影响；为了进一步证明追毒方对TNBC细胞有氧糖酵解的调控与CD147糖基化有关，使用糖基转移酶GnT-V激动剂处理细胞，观察其对追毒方抑制效应的逆转作用。结果:与空白对照组相比，追毒方0.1、0.2、0.4、0.8、1.6、3.2μg/mL组的细胞增殖活性显著降低(P<0.01),24 h IC50为5.00μg/mL,48 h IC50为0.25μg/mL,72 h IC50为0.02μg/mL,追毒方0.4、0.8、1.6μg/mL组和2-DG 2 101μg/mL组的侵袭能力和迁移能力显著降低，CD147糖基化水平显著下降(P<0.01),糖摄取能力显著降低(P<0.01),细胞内和培养上清的乳酸含量显著降低(P<0.01),有氧糖酵解关键跨膜蛋白GLUT1/MCT4的表达显著下调(P<0.01)。视黄酸处理模型中，与空白对照组相比，视黄酸30μg/mL组的糖摄取能力、细胞内及培养上清乳酸含量，CD147糖基化水平降低，GLUT1/MCT4的表达明显上调(P<0.05或P<0.01);与追毒方0.8μg/mL组相比，视黄酸30μg/mL组+追毒方0.8μg/mL组的上述检测指标明显上调(P<0.05或P<0.01)。结论:追毒方通过调控CD147糖基化抑制GLUT1、MCT4表达，从而下调三阴性乳腺癌有氧糖酵解葡萄糖通量及乳酸生成和外排，最终抑制三阴性乳腺癌增殖及侵袭迁移。
Abstract：Objective:To study the effects of Zhuidu(追毒) Formula(ZDF) on the proliferation, invasion, and migration of triple-negative breast cancer(TNBC) MDA-MB-231 cells and explore the underlying molecular mechanisms. Methods:MTT assay, Transwell assay, and wound healing assay were employed to observe the effects of different concentrations of ZDF on the proliferation, invasion, and migration of TNBC cells, respectively. Then, flow cytometry and the lactate assay kit were employed to evaluate glucose uptake, intracellular lactate production, and extracellular lactate efflux of TNBC cells treated with different concentrations of ZDF,thus assessing the aerobic glycolysis levels. Western blotting was performed to study the effects of ZDF on the glycosylation of cluster of differentiation 147(CD147,also known as the extracellular matrix metalloproteinase inducer) and the protein levels of glucose transporter 1(GLUT1) and monocarboxylate transporter 4(MCT4) in TNBC cells. To further confirm that ZDF regulation of aerobic glycolysis in TNBC cells was related to CD147 glycosylation, this study treated the cells with the glycosyltransferase GnT-V activator retinoic acid and observed its reversal effect on the inhibitory effect of ZDF. Results:Compared with the blank control group, the ZDF groups(0.1,0.2,0.4,0.8,1.6,and 3.2 μg/mL) showed reduced cell proliferation(P<0.01),with IC50 values of 5.00,0.25,and 0.02 μg/mL at the time points of 24 h, 48 h, and 72 h, respectively. The ZDF groups(0.4,0.8,and 1.6 μg/mL) and the 2-DG(2 101 μg/mL) group showed weakened cell invasion and migration(P<0.01),reductions in CD147 glycosylation level(P<0.01),glucose uptake(P<0.01),and lactate content in cells and the supernatant(P<0.01),and down-regulated expression of aerobic glycolysis-related transmembrane proteins GLUT1/MCT4(P<0.01). Compared with the blank control group, the retinoic acid(30 μg/mL) group showed increases in glucose uptake, intracellular and supernatant lactate content, CD147 glycosylation level, and GLUT1/MCT4 expression(P<0.01 or P<0.05). Compared with the ZDF(0.8 μg/mL) group, the retinoic acid(30 μg/mL) + ZDF(0.8 μg/mL) group demonstrated up-regulation of the aforementioned indicators(P<0.01 or P<0.05). Conclusion:ZDF regulates CD147 glycosylation to suppress GLUT1 and MCT4 expression and down-regulate aerobic glycolysis-mediated glucose flux and lactate production and efflux, thus inhibiting the proliferation, invasion, and migration of TNBC.
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基本信息:
DOI：10.13412/j.cnki.zyyl.20250603.002
中图分类号:R285
引用信息:
[1]刘绍俊,王宗傲,张明慧,等.追毒方调控CD147糖基化介导的有氧糖酵解抑制三阴性乳腺癌增殖及侵袭迁移[J].中药药理与临床,2026,42(02):10-16.DOI:10.13412/j.cnki.zyyl.20250603.002.
基金信息:
国家自然科学基金(编号：82274423); 苏州市科学技术局苏州市姑苏卫生人才科研项目(编号：GSWS2022078)
2025-06-03
2025-06-03
2025-06-03