UPLC-MS/MS method for simultaneously determining nucleosides and methyl-nucleosides in liver mRNA of Epimedin C-induced liver injury mouse model.
UPLC-MS/MS method for simultaneously determining nucleosides and methyl-nucleosides in liver mRNA of Epimedin C-induced liver injury mouse model.
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UPLC-MS/MS同时测定Epimedin C诱导肝损伤小鼠模型肝脏mRNA中核苷和甲基核苷的方法
DOI：
10.1186/s13020-021-00501-7
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发表时间：
2021-09-21
期刊：
影响因子：
4.9
通讯作者：
Tian X
中科院分区：
文献类型：
--
作者：
Song Z;Li Z;Wen X;Liu R;Tian X
淫羊藿苷C是淫羊藿的主要活性成分之一，有报道称其具有潜在的肝毒性。然而，淫羊藿苷C诱导肝损伤的机制尚未研究。mRNA甲基化主要包括N6-甲基腺苷和N5-甲基胞苷，涉及许多生物过程和疾病的调节。通过对朝藿定C诱导的肝损伤小鼠肝组织mRNA甲基化水平的定量研究，可能有助于阐明其肝毒性的机制。因此，需要建立一种分析方法来确定肝脏mRNA中的核苷和甲基核苷水平。建立了一种同时测定肝脏mRNA中腺苷、尿苷、胞苷、鸟苷、N6-甲基腺苷和N5-甲基胞苷6种核苷的超高效液相色谱-串联质谱（UPLC-MS/MS）方法。此外，通过以10或40 mg/kg的每日剂量灌胃给予朝藿定C持续4周来研究朝藿定C诱导的肝损伤小鼠模型。制备小鼠肝脏mRNA的核苷样品，并在酶消化后使用0.1%甲酸水和甲醇在UPLC柱上分离。然后通过Qtrap 6500质谱仪检测样品。在该方法中，6个核苷的校准曲线在其浓度范围内显示出良好的线性。腺苷、胞苷、N6-甲基腺苷和N5-甲基胞苷的线性范围为40- 20，000 pg/mL，鸟苷的线性范围为0.2-100 ng/mL，尿苷的线性范围为2-1000 ng/mL。成功建立了淫羊藿苷C诱导的小鼠肝损伤模型，血清转氨酶水平升高，肝脏炎性细胞浸润增多，空泡变性。结果表明，淫羊藿苷C处理后，小鼠肝脏mRNA的N6-甲基腺苷和N5-甲基胞苷水平以及N6-甲基腺苷与腺苷和N5-甲基胞苷与胞苷的比值均显著升高。该方法成功地应用于淫羊藿苷C诱导的肝损伤小鼠模型和对照组肝脏mRNA中6种核苷的含量测定。结果表明，mRNA甲基化可能与朝藿定C诱导的肝损伤有关。本研究为进一步研究朝藿定C的肝毒性机制奠定了基础。
Epimedin C, one of the main active ingredients of Epimedium, has been reported to have potential hepatotoxicity. However, the mechanism of Epimedin C-induced liver injury has not been studied. mRNA methylation, mainly including N6-methyladenosine and N5-methylcytidine, is implicated in the regulation of many biological processes and diseases. The study of quantifying mRNA methylation alterations in Epimedin C-induced liver injury mice may contribute to clarify the mechanism of its hepatotoxicity. Therefore, an analysis method needs to be established to determine nucleoside and methyl-nucleoside levels in liver mRNA. An ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to simultaneously determine six nucleosides (adenosine, uridine, cytidine, guanosine, N6-methyladenosine and N5-methylcytidine) in liver mRNA. Besides, the Epimedin C-induced liver injury mouse model was studied by intragastrical administration Epimedin C at a daily dose of 10 or 40 mg/kg for 4 weeks. The nucleoside samples of the mice liver mRNA were prepared and separated on an UPLC column using 0.1% formic acid water and methanol after enzymatic digestion. Then the sample was detected by a Qtrap 6500 mass spectrometer. In this method, calibration curves of the six nucleosides showed good linearity over their concentration ranges. The linear ranges were 40–20,000 pg/mL for adenosine, cytidine, N6-methyladenosine and N5-methylcytidine, 0.2–100 ng/mL for guanosine, and 2–1000 ng/mL for uridine. Epimedin C-induced liver injury mouse model was successfully established,which could be proved by the elevation of serum aminotransferase levels, and the increased inflammatory cell infiltration as well as vacuolar degeneration in liver. The N6-methyladenosine and N5-methylcytidine levels, and the ratios of N6-methyladenosine to adenosine and N5-methylcytidine to cytidine of the mice liver mRNA were all significantly increased after Epimedin C treatment. The established method was successfully applied to the determination of six nucleosides levels in liver mRNA of the Epimedin C-induced liver injury mice model and the control group. The results indicated that mRNA methylation might be associated with Epimedin C-induced liver injury. This study will facilitate the mechanism research on the hepatotoxicity of Epimedin C.
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