Development of A LC-MS/MS Method for Simultaneously Determining Nucleosides and Methy-Nucleosides in Mice Liver mRNA of Epimedin C-Induced Liver Injury Model​

Abstract Background Epimedium, the crude drug used in clinical, has been proved to have the potential to cause liver damage in patients. As one of the main active ingredient of Epimedium, Epimedin C is reported to have the potential hepatotoxicity. However, the mechanism of Epimedin C-induced mice liver injury has not been studied. mRNA methylation is implicated in the regulation of many biological processes and diseases. The study of mRNA methylation in Epimedin C-induced liver injury may contribute to clarify the liver toxicity mechanism of Epimedin C. Therefore, an analysis method needs to be established to determine liver mRNA nucleoside and methy-nucleoside levels in epigenetic studies. Methods The Epimedin C groups were intragastrically administered single doses of 10 and 40 mg/kg body weight of Epimedin C for four weeks, and the normal control group were given the same volume of saline. The results of hematoxylin and eosin staining of the liver and serum aminotransferase levels were used to evaluate liver injury. 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. Results In this study, a selective, rapid and sensitive method was established using high performance liquid chromatography-tandem mass spectrometry for the simultaneous determination of six nucleosides (adenosine, uridine, cytidine, guanosine, N6-methyladenosine and N5-methylcytidine) in mRNA. In this method, linear concentrations of adenosine, cytidine, N6-methyladenosine and N5-methylcytidine were 40-20000 pg/mL, guanosine was 0.2–100 ng/mL, and uridine was 2-1000 ng/mL. N6-methyladenosine and N5-methylcytidine modification levels of the mice liver mRNA increased after Epimedin C treatment. Epimedin C led to the elevation of serum aminotransferase levels, and increased the inflammatory cell infiltration and vacuolar degeneration in liver, indicated the liver injury in mice. Conclusion This method was successfully applied to the detection of six liver mRNA nucleosides levels in Epimedin C-induced liver injury with good precision and accuracy. The results indicated that mRNA methylation might be associated with Epimedin C-induced liver injury. This study offers a method for the research on the mechanism of Epimedin C-induced liver injury, and will facilitate the research on the hepatotoxicity of herbal medicine in epigenetic studies.