基于RP-UPLC-MS和HILIC-UPLC-MS的骨疏丹对糖皮质激素性骨质疏松模型大鼠干预作用的尿液代谢组学研究

A Urinary Metabonomic Research on the Intervention Effects of Gushudan on Prednisolone-Induced Oesteoprosis Rats by RP-UPLC-MS and HILIC-UPLC-MS

目的建立反相超高效液相色谱-质谱(RP-UPLC-MS)及亲水相互作用超高效色谱-质谱(HILIC-UPLC-MS)代谢组学方法,研究骨疏丹干预糖皮质性骨质疏松模型大鼠尿液代谢物的变化,探索与该病症密切相关的代谢模式及骨疏丹抗骨质疏松作用的代谢途径。方法基于代谢组学研究策略,建立正常大鼠、糖皮质性骨质疏松模型大鼠和给予骨疏丹干预大鼠尿液的RP-UPLC-MS与HILIC-UPLC-MS代谢指纹谱,采用主成分分析法(PCA)研究给药前后内源性代谢物变化,寻找潜在生物标志物,探讨骨疏丹的作用机制。结果在RP-UPLC-MS模式下发现了如苯乙酰甘氨酸、N2-琥珀酰-L-鸟氨酸等极性较小的代谢物,而在HILIC-UPLC-MS模式下发现了如甜菜碱、次黄嘌呤等极性较大的代谢物,2种检测模式下共发现了22个潜在生物标志物,这些标志物主要和氨基酸代谢、脂代谢、能量代谢、肠道菌群代谢及肾损伤和体内的氧化应激状态相关。而骨疏丹可以调节骨质疏松模型大鼠体内上述代谢通路并使其向正常状态恢复。结论该研究结果表明,整合RP-UPLC-MS和HILIC-UPLC-MS 2种检测手段可以较全面的表征大鼠体内内源性代谢物轮廓,同时也表明代谢组学在中药整体药效评价、作用机制的研究等方面具有很好的应用前景。

OBJECTIVE To develop an intergrated urinary metabonomic strategy based on RP-UPLC-MS and HILIC-UPLC-MS method and to investigated the mechanism of osteoporosis and the prevention effect of glucocorticoid osteoporosis of Gushudan. METHODS The RP-UPLC-MS and HILIC-UPLC-MS method were developed for rat urine metabolite profiling study among control group, model group and Gushudan treatment group. Principal component analysis （PCA） were used to find the biomarkers, further more, to explore the mechanism of the prevention effect on glucocorticoid osteoporosis of Gushudan. RESULTS In the mode of RP- UPLC-MS, low polarity metabolites like phenylacetylglyeine, N2-succinic acid-L-ornithine were found while relatively high polarity me- tabolites like betaine, hypoxanthine were found in the mode of HILIC-UPLC-MS. And twenty-two potential biomarkers have been totaly found in the urine of glucocorticoid osteoporosis, primiarily related to amino acid metabolism, energy metabolism, lipid metabolism, in- testinal flora metabolism and kidney damage. Gushudan has intervention effects on rats with osteoprosis via the regulation of muhi- plemetabolic pathways. CONCLUSION The combination of RP-UPLC-MS and HILIC-UPLC-MS method can give a more comprehen- sive profiling of endogenous metabolites and it also indicates metabonomic strategy has become a powerful tool to evaluate the overall ef- ficacy of traditional Chinese medicine （TCM） and to calrify the mechanism of TCM.