Characterizing metabolites and potential metabolic pathways changes to understanding the mechanism of medicinal plant Phellodendri Amurensis cortex against doxorubicin-induced nephritis rats using UPLC-Q/TOF-MS metabolomics

Phellodendri Amurensis cortex (PAC), a famous traditional Chinese herb with good anti-inflammatory efficacy, is used to treat various liver and kidney sickness in clinical practice. However, the potential mechanisms protecting against nephritis of PAC have not been comprehensively elucidated. The aim of this research was to explore the mechanism of PAC against doxorubicin- induced nephritis in rats by characterizing metabolites and potential metabolic pathways changes. The rat models of nephritis were established using 6.5 mg/kg doxorubicin injection from caudal vein for five weeks. The rats in the treatment group were respectively received PAC extract at the dose of 216, 432, and 864 mg/kg once a day during the experiment. Then, urine metabolomics strategy based on ultra-performance liquid chromatography-quadrupole time of flight-tandem mass spectrometry (UPLC-Q/TOF-MS) has been employed to discover the possible significant metabolites and metabolic pathway of nephritis rats. At the end of the experiment, serum, urine and kidney tissue were collected for biochemical and pathological examination. The results showed that PAC treatment notably decreased urinary protein, serum Cr content and renal tissue lesions, and increased serum TP and ALB content. A total of potential twenty- eight metabolites such as 5'-methylthioadenosine, cGMP, dehydroepiandrosterone sulfate, salbuta, 2-phenylaminoadenosine contributing to nephritis rat model were selected and identified in the urine samples. Compared with the model group, the high-dose PAC group can recall 18 metabolites level, the medium-dose group can recall 13 metabolites level, and the low-dose PAC group can recall 8 metabolites level, which were involved in nine primary metabolic pathways such as steroid hormone biosynthesis, alanine,aspartate and glutamate metabolism, cysteine and methionine metabolism as well as glyoxylate and dicarboxylate metabolism. The protein expressions of key enzymes involving methylthioadenosine phosphorylase (Mtap), cytidine deaminase (Cda), thymidine kinase (Tk), argininosuccinate synthase (Ass) in metabolic pathways were further verified by Western blot. The results showed that Phellodendron chinense up-regulated the protein expressions of Cda and Tk and down-regulated the protein expressions of Mtap and Ass. In conclusion, PAC possesses renoprotective effect against doxorubicin-induced nephritis, which may be mediated via regulating differential metabolites, reducing oxidative stress response, improving renal function, enhancing the ability of the immune system, regulating the role of key enzymes.

Keywords: Mass spectrometry; Metabolomics; Nephritis; Phellodendri Amurensis cortex; Urine.