基于NMR的绿原酸抗乳腺癌代谢标记物的研究

Nuclear magnetic resonance spectroscopy-based metabonomics study on metabolic markers of chlorogenic acid against mouse breast cancer

目的基于核磁共振法对小鼠乳腺癌血清代谢组学的研究，揭示绿原酸抗肿瘤相关生物标记物和抗癌作用靶点。方法以小鼠（Balb—c／EMT-6）乳腺癌为疾病模型，设立绿原酸治疗组（CHA，20mg／kg，qd）、多西他赛（DX，5mg／kg，qod）治疗阳性对照组、肿瘤模型组、空白组；小鼠血清去蛋白后，测定并采集各样品的核磁共振（NMR）谱，对比不同周期各组血清波谱特征的差异；采用PLS—DA模式识别分析，计算出各组波谱参数的统计学关系。结果各个特征峰的归属分别为乳酸（δ1．32—1．36ppm）、丙氨酸（δ1．48ppm）、乙酸（δ1．92ppm）、缬氨酸（δ2．26ppm）、丙酮酸（δ2．38ppm）、柠檬酸（δ2．5～2．75ppm）、肌酸（δ3．05ppm）、胆碱（δ3．20～3．22ppm）、葡萄糖（δ3．75ppml；PLS—DA模式识别分析使各组参数得到有效区分；CHA组和DX组出现部分重叠，接近于空白组并区别于模型组，随着治疗周期的延长，这些现象呈现加强的趋势。经绿原酸治疗后，血清中胆碱、肌酸和柠檬酸等代谢组分与模型组相关代谢组分比较呈减小趋势（P〉0．1），乳酸、丙氨酸、乙酸、和丙酮酸呈现较明显下降趋势（P≤0．1）；随着治疗的深入，上述关联代谢组分的变化趋势随着抑瘤率的逐渐增加而得到加强。结论小鼠乳腺癌血清中乳酸、丙氨酸、乙酸、肌酸、柠檬酸、胆碱、丙酮酸等代谢物的变化水平与肿瘤的生长密切相关，意味着经过深入验证后可成为表达肿瘤生长状态的生物标记物。根据乳酸、丙氨酸、乙酸、肌酸、柠檬酸、胆碱、丙酮酸等无氧糖酵解代谢物的变化，推测绿原酸可能通过抑制肿瘤能量代谢途径发挥抗癌作用。

Objective To identify the serum metabolic biomarkers of chlorogenic acid on the breast cancer mouse model using the method of nuclear magnetic resonance spectrum （NMR）. Methods Balb-c/EMT-6 mice model of breast cancer were established. All mice were classified into four groups： chlorogenic acid treatment group （CHA, 20mg/kg, qd）, docetaxel treatment group （DX, 5mg/kg, qod） as the positive control, the tumor model group and the health control group. Serum samples were collected after 3 weeks of treatment and NMR of deproteinized serum was detected and collected. Differences in spectral parameters of different groups were calculated by a PLS-DA pattern recognition analysis. Results The attribution of each peak was as followings： δ1.32-1.36ppm for lactic acid, δ1.48 ppm for alanine, δ1.92 ppm for acetic acid, δ2.26 ppm for valine, δ2.38ppm for pyruvate, δ2.50-2.75ppm forcitric acid, 63.05 ppm for creatine, 63.20-3.22 ppm for choline, 63.75 ppm for glucose. Each set of parameters was effectively distinguished by PLS-DA pattern recognition analysis. The CHA group and the DX group overlapped in some region, which were closing to the blank group, different from the model group. With the extension of the treatment time, These phenomena showed a strengthening trend. After treatmented with chlorogenic acid, serum metabolic components of choline, creatine and citric acid showed a decreasing trend （P〉0.1）, compared to the metabolic components of the model group. Lactic acid, alanine, acetic acid and pyruvic acid decreased obviously （P〈_O. 1）. Along with the further treatment, the trend of the metabolic components get strengthened with increasing rate of tumor depression, Conclusion The metabolites change level of lactic acid, alanine, acetic acid, creatine, citric acid, choline and acetone acid in breast cancer mice serum were closely related with the growth of tumors. After in- depth verification, these metabolites can be as biomarkers representing tumor growth state. According to changes of anaerobic glycolysis metabolites of lactic acid, alanine, acetic acid, creatine, citric acid, choline and acetone acid, speculated that chlorogenic acid may play cancer-fighting properties by inhibiting tumor energy metabolic pathways.