姜黄素通过抑制有氧糖酵解效应促进甲状腺癌细胞凋亡

Curcumin Induces Apoptosis of Thyroid Cancer Cells Through the Inhibition of Aerobic Glycolysis

摘要:

目的：探讨姜黄素是否通过调控有氧糖酵解（Warburg） 效应诱导人甲状腺癌细胞凋亡及其潜在机制。方法：以人甲状腺癌细胞SW579和CAL-62为模型，进行以下实验：采用细胞计数试剂盒-8（CCK-8） 法检测不同浓度姜黄素处理24 h、48 h、72 h后的细胞增殖活力；TUNEL染色观察细胞凋亡；高通量转录组测序筛选差异表达基因。并结合网络药理学预测姜黄素的作用靶点，进行基因本体（GO） 功能注释和京都基因与基因组百科全书（KEGG） 通路富集分析及分子对接验证；蛋白质印迹法检测凋亡相关蛋白B 细胞淋巴瘤-2（Bcl-2）、Bcl-2相关X蛋白（Bax）、活化的半胱天冬酶3（C-Caspase3） 的表达；试剂盒法检测乳酸生成量（LAC）、葡萄糖摄取量（Glu） 及超氧化物歧化酶（SOD） 含量以评估Warburg效应。结果：CCK-8结果显示，与0 μmol/L姜黄素比较，经20、40、60、80、100 μmol/L姜黄素处理24、48、72 h后SW579和CAL-62细胞的IC50逐渐降低，呈浓度和时间依赖性。TUNEL染色显示，与对照组比较，姜黄素组细胞红色荧光增多，细胞凋亡增加；与姜黄素组比较，姜黄素+核因子红细胞2相关因子2（NRF2） 抑制剂组的红色荧光染色减少，细胞凋亡减少。测序分析显示NRF2介导的氧化应激反应通路等被富集。网络药理学获得姜黄素靶点906个，疾病靶点569个，66个交集靶点。筛选出核心靶点为超氧化物歧化酶2（SOD2）、MYC原癌基因（MYC）、过氧化物酶体增殖物激活受体γ （PPARG）。KEGG分析结果显示，交集靶点富集于凋亡、丝裂原活化蛋白激酶（MAPK）、磷脂酰肌醇3-激酶-蛋白激酶B（PI3K-Akt） 等信号通路。分子对接提示姜黄素与SOD2、MYC、PPARG 等靶点具有较好的结合活性。蛋白质印迹法显示， 姜黄素组Bcl-2 蛋白表达低于对照组， Bax、C-Caspase3 蛋白表达高于对照组（P＜0.05）；姜黄素+NRF2 抑制剂组Bcl-2 蛋白表达高于姜黄素组，Bax、C-Caspase3蛋白表达低于姜黄素组（P＜0.05）。Glu、LAC及SOD含量检测显示，姜黄素组Glu、LAC含量均低于对照组（P＜0.05），SOD含量高于对照组（P＜0.05）；姜黄素+NRF2抑制剂组Glu、LAC含量均高于姜黄素组（P＜0.05），SOD含量低于姜黄素组（P＜0.05）。结论：姜黄素可能通过激活NRF2通路，调控SOD水平及凋亡相关蛋白表达，抑制甲状腺癌细胞的Warburg效应，从而促进细胞凋亡、抑制其生长。

Abstract:

Abstract： Objective： To investigate whether curcumin induces apoptosis in human thyroid cancer cells by regulating the aerobic glycolysis （Warburg effect） and to explore its underlying mechanisms. Methods：Human thyroid cancer cell lines SW579 and CAL-62 were used as models. The following experiments were performed： cell proliferation viability after treatment with different concentrations of curcumin for 24，48，and 72 hours was detected using the CCK-8 assay；cell apoptosis was observed by TUNEL staining；differentially expressed genes were screened by high-throughput transcriptome sequencing. Network pharmacology was combined to predict the targets of curcumin， followed by Gene Ontology （GO） functional annotation，Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis，and molecular docking validation. The expressions of apoptosis-related proteins B-cell lymphoma- 2 （Bcl-2）， Bcl-2-associated X protein （Bax）， and cleaved caspase-3 （C-Caspase3） were detected by Western blotting. The Warburg effect was evaluated by measuring lactate production （LAC）， glucose uptake （Glu）， and superoxide dismutase （SOD） content using commercial kits. Results ： CCK-8 results showed that compared with 0 μmol/L curcumin，the IC50 gradually decreased after 24，48，and 72 hours of curcumin treatment at 20，40，60， 80，and 100 μmol/L，showing a concentration and time dependence. TUNEL staining showed that compared with the control group， the curcumin group exhibited increased red fluorescence and increased apoptosis； compared with the curcumin group， the curcumin+NRF2 inhibitor group showed decreased red fluorescence and reduced apoptosis. Sequencing analysis revealed enrichment of pathways such as the NRF2-mediated oxidative stress response pathway. Network pharmacology identified 906 curcumin targets， 569 disease targets， and 66 intersecting targets. The core targets were identified as SOD2， MYC proto-oncogene （MYC）， and peroxisome proliferator-activated receptor gamma （PPARG）. KEGG analysis showed that the intersecting targets were enriched in signaling pathways such as apoptosis， mitogen-activated protein kinase （MAPK）， and phosphatidylinositol 3-kinase-protein kinase B （PI3KAkt）. Molecular docking suggested that curcumin had good binding activity with targets such as SOD2， MYC and PPARG. Western bloting showed that Bcl-2 protein expression was lower in the curcumin group than in the control group， while Bax and C-Caspase3 protein expressions were higher than those in the control group （P＜0.05）. Compared with the curcumin group，the curcumin+NRF2 inhibitor group showed higher Bcl-2 expression and lower Bax and C-Caspase3 expressions （P＜0.05）. Glu，LAC，and SOD content assays showed that Glu and LAC levels in the curcumin group were lower than those in the control group （P＜0.05），while SOD content was higher than that in the control group（ P＜0.05）. Compared with the curcumin group，the curcumin+NRF2 inhibitor group exhibited higher Glu and LAC levels （P＜0.05） and lower SOD content （P＜0.05）. Conclusion： Curcumin may promote apoptosis and inhibit the growth of thyroid cancer cells by activating the NRF2 pathway，regulating SOD levels and apoptosis-related protein expression，thereby suppressing the Warburg effect.