Analysis of Serum Metabolome in Mice with Gastric Cancer Treated with JPYZXZ Prescription Based on Liquid Chromatography-Mass spectrometry
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摘要: 目的 通过代谢组学方法，观察给予健脾养正消癥方的前后胃癌小鼠血清代谢产物变化规律，寻找其与治疗胃癌疗效相关特征分子，探讨健脾养正消癥方在调控胃癌内源性机制方面作用。方法 建立人胃癌裸鼠皮下移植瘤模型，并随机分为模型组，健脾养正消癥方低剂量组和健脾养正消癥方高剂量组。采用超高效液相色谱串联四级杆飞行时间质谱(UPLC-Q-TOF/MS)技术，对各组小鼠血清样本进行分析，结合主成分分析法(PCA)和正交偏最小二乘法判别分析(OPLS-DA)来筛选差异性代谢物，利用MetaboAnalyst网站分析相关代谢通路。结果 健脾养正消癥方高、低剂量组小鼠瘤体平均瘤质量明显小于模型组(P < 0.05)。代谢组学结果经分析，模型组、低剂量组和高剂量组血清样本能够得到很好的区分，健脾养正消癥方给药后胃癌小鼠的内源性代谢物水平发生不同程度的回调，并初步鉴定出5个差异代谢物和4条相关代谢通路。结论 健脾养正消癥方能够明显抑制小鼠肿瘤的生长，其作用机制可能与花生四烯酸水平的升高以及其激活的α-亚麻酸和亚油酸代谢和花生四烯酸代谢通路有关。
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关键词:
- 胃癌 /
- 健脾养正消癥方 /
- 代谢组学 /
- 超高效液相色谱-质谱 /
- 差异代谢物
Abstract: OBJECTIVE To observe the changes of serum metabolites in mice with gastric cancer before and after giving JPYZXZ prescription by metabolomics, and to find the characteristic molecules related to the efficacy of the prescription in the treatment of gastric cancer, and to explore the role of JPYZXZ prescription in regulating the local microenvironment and endogenous mechanism of gastric cancer.METHODS A subcutaneous transplantation tumor model of human gastric cancer in nude mice was established and randomly divided into a model group, a low-dose group of JPYZXZ prescription, and a high-dose group of JPYZXZ prescription. The serum samples of each group were detected by ultra performance liquid chromatography-mass spectrometry (UPLC-Q-TOF/MS), the differential metabolites were screened by principal components analysis (PCA) and orthogonal partial least squares- discriminant analysis (OPLS-DA), and MetaboAnalyst was used to analyze the relevant metabolic pathways.RESULTS The mean tumor weight of mice in the high and low dose groups was significantly smaller than that of the model group (P < 0.05). The results of metabolomics showed that the serum samples of the model group, the low-dose group, and the high-dose group could be well distinguished, and the levels of endogenous metabolites in the mice with gastric cancer were varying degrees of callbacks after the administration of JPYZXZ prescription, and 5 different metabolites and 4 related metabolic pathways were identified preliminarily.CONCLUSION JPYZXZ prescription can significantly inhibit the growth of tumors in mice, and its mechanism of action may be related to the increase of arachidonic acid level and the activation of α-linolenic acid and linoleic acid metabolism and arachidonic acid metabolic pathway. -
图 1 不同组别血清样本在正负离子模式下的UPLC-Q-TOF/MS总离子流图
注：正离子模式下, A.模型组; B.健脾养正消癥方低剂量组; C.健脾养正消癥方高剂量组；负离子模式下, D.模型组; E.健脾养正消癥方低剂量组; F.健脾养正消癥方高剂量组
图 3 正离子模式下OPLS-DA得分图与置换检验
注：A.健脾养正消癥方低剂量组与模型组对比的OPLS-DA得分图；B.健脾养正消癥方高剂量组与模型组对比的OPLS-DA得分图；C.健脾养正消癥方低剂量组与模型组对比的置换检验图；D.健脾养正消癥方高剂量组与模型组对比的置换检验图
图 4 负离子模式下OPLS-DA得分图与置换检验
注：A.健脾养正消癥方低剂量组与模型组对比的OPLS-DA得分图；B.健脾养正消癥方高剂量组与模型组对比的OPLS-DA得分图；C.健脾养正消癥方低剂量组与模型组对比的置换检验图；D.健脾养正消癥方高剂量组与模型组对比的置换检验图
表 1 不同组别裸鼠各阶段体质量比较(x±s)
组别 0 d 5 d 10 d 15 d 20 d 25 d 30 d 模型组 21.75±1.28 23.29±1.30 23.63±1.28 23.53±0.69 24.54±0.79 24.48±0.92 25.39±0.83 低剂量组 21.79±1.28 23.02±1.25 23.73±0.90 23.54±1.20 24.32±1.13 24.34±1.17 25.11±1.13 高剂量组 21.73±1.65 23.32±1.01 23.70±0.69 23.23±0.86 24.49±1.03 24.35±1.29 25.29±0.95 
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表 2 各组裸鼠皮下移植瘤瘤质量及抑瘤率比较
组别 肿瘤质量/g 抑瘤率/% 模型组 2.68±0.26 0 低剂量组 2.04±0.35* 23.88 高剂量组 1.78±0.33* 33.58 注：与模型组比较，*P < 0.05。
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表 3 低剂量组和模型组比较、高剂量组和模型组比较的差异代谢物
序号 差异代谢物 tR/min m/z 采集模式 LDMEL vs Model LDMEH vs Model VIP P FC VIP P FC 1 D-甘露糖 0.92 215.032 84 ESI- 2.13 < 0.001 1.38 1.37 0.004 1.37 2 1, 3-二甲基尿酸 0.92 217.029 83 ESI- 2.03 0.003 1.37 1.31 0.007 1.41 3 十六碳二酸 8.36 309.204 45 ESI+ 1.73 0.006 1.19 1.39 < 0.001 1.32 4 花生四烯酸 9.62 303.232 7 ESI- 1.51 0.043 1.31 1.74 < 0.001 1.86 5 美睾酮 9.63 305.248 26 ESI+ 2.40 < 0.001 0.48 1.49 < 0.001 0.42
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表 4 代谢通路分析
代谢通路 匹配 P -logP HolmP FDR 影响力 α-亚麻酸和亚油酸代谢 1/17 0.08 1.09 1 1 0.10 果糖和甘露糖的降解 1/28 0.13 0.88 1 1 0 半乳糖代谢 1/31 0.15 0.84 1 1 0 花生四烯酸代谢 1/65 0.28 0.55 1 1 0.30 注：Holm P为富集分析采集Holm-Bonferroni统计方法的P，FDR为多重检验中伪发生率错误控制的P。
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