Song Zhizhen, Li Zeyun, Wen Xueqian, Liu Ruijuan, Tian Xin
Department of Pharmacy, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, People's Republic of China.
Henan Key Laboratory of Precision Clinical Pharmacy, Zhengzhou, People's Republic of China.
Chin Med. 2021 Sep 21;16(1):91. doi: 10.1186/s13020-021-00501-7.
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.
淫羊藿苷C是淫羊藿的主要活性成分之一，据报道具有潜在的肝毒性。然而，淫羊藿苷C诱导肝损伤的机制尚未得到研究。mRNA甲基化主要包括N6-甲基腺苷和N5-甲基胞嘧啶，参与许多生物过程和疾病的调控。对淫羊藿苷C诱导肝损伤小鼠中mRNA甲基化变化进行定量研究，可能有助于阐明其肝毒性机制。因此，需要建立一种分析方法来测定肝脏mRNA中的核苷和甲基核苷水平。
建立并验证了一种超高效液相色谱-串联质谱（UPLC-MS/MS）方法，用于同时测定肝脏mRNA中的六种核苷（腺苷、尿苷、胞苷、鸟苷、N6-甲基腺苷和N5-甲基胞嘧啶）。此外，通过每日灌胃给予10或40mg/kg淫羊藿苷C持续4周，研究淫羊藿苷C诱导的肝损伤小鼠模型。小鼠肝脏mRNA的核苷样品经酶消化后，用0.1%甲酸水和甲醇在UPLC柱上进行制备和分离。然后用Qtrap 6500质谱仪进行检测。
该方法中，六种核苷的校准曲线在其浓度范围内均显示出良好的线性。腺苷、胞苷、N6-甲基腺苷和N5-甲基胞嘧啶的线性范围为40-20,000pg/mL，鸟苷为0.2-100ng/mL，尿苷为2-1000ng/mL。成功建立了淫羊藿苷C诱导的肝损伤小鼠模型，血清转氨酶水平升高、肝脏炎症细胞浸润增加以及空泡变性可证明这一点。淫羊藿苷C处理后，小鼠肝脏mRNA的N6-甲基腺苷和N5-甲基胞嘧啶水平，以及N6-甲基腺苷与腺苷、N5-甲基胞嘧啶与胞苷的比值均显著升高。
所建立的方法成功应用于淫羊藿苷C诱导的肝损伤小鼠模型和对照组肝脏mRNA中六种核苷水平的测定。结果表明，mRNA甲基化可能与淫羊藿苷C诱导的肝损伤有关。本研究将有助于淫羊藿苷C肝毒性机制的研究。
