Cytochrome P450 Mediated Bioactivation of Rutaevin, a Bioactive and Potentially Hepatotoxic Component of Evodia Rutaecarpa.

Rutaevin is one of the major bioactive constituents isolated from Evodia rutaecarpa, a well-known herbal medicine that has been widely prescribed for the treatment of gastrointestinal disorders in China. However, oral administration of rutaevin has been shown to cause hepatotoxicity in mice. Bioactivation was suggested to be involved in rutaevin-induced hepatotoxicity. The aim of this study was to investigate the bioactivation of rutaevin in rat and human liver microsomes fortified with NADPH. Rutaevin was metabolized into the reactive intermediate cis-butene-1,4-dial (BDA) that was dependent on NADPH. The rutaevin-derived BDA intermediate was trapped by nucleophiles such as glutathione (GSH), N-acetyl-lysine (NAL), and methoxylamine (MOA) in the microsomal incubation system. A total of 10 conjugates resulting from the conjugation of the intermediate with GSH, NAL, or MOA were detected and structurally characterized by liquid chromatography combined with high-resolution tandem mass spectrometry. M1, structurally confirmed by NMR spectroscopic analysis, was identified as a cyclic mono(GSH) conjugate of the BDA intermediate, which was also found in the biliary samples of rutaevin-treated rats. Further inhibitory experiments suggested that ketoconazole showed strong inhibitory effect on the formation of the rutaevin-derived BDA intermediate. CYP3A4 was demonstrated to be the major enzyme responsible for rutaevin bioactivation by using cDNA-expressed human recombinant cytochrome P450 enzymes. Additionally, it was found that rutaevin was a mechanism-based inactivator of CYP3A4, with inactivation parameters of KI = 15.98 µM, kinact = 0.032 min-1, and t1/2 inact = 21.65 min. In summary, these findings are of great significance in understanding the bioactivation mechanism of rutaevin, the potential mechanism of rutaevin-caused hepatotoxicity, and the drug-drug interactions associated with rutaevin mainly via CYP3A4 inactivation.

Tetramethylpyrazine protects against high glucose-induced vascular smooth muscle cell injury through inhibiting the phosphorylation of JNK, p38MAPK, and ERK.

Objectives High glucose-induced alterations in vascular smooth muscle cell behavior have not been fully characterized. We explored the protective mechanism of tetramethylpyrazine (TMP) on rat smooth muscle cell injury induced by high glucose via the mitogen-activated protein kinase (MAPK) signaling pathway. Methods Vascular smooth muscle cells (VSMCs) isolated from rat thoracic aortas were divided into control, high glucose (HG), and pre-hatching TMP groups. The effect of different glucose concentrations on cell viability and on the migration activity of VSMC cells was examined using MTT analysis and the wound scratch assay, respectively. Superoxide dismutase (SOD) and malondialdehyde (MDA) levels were measured using enzyme-linked immunoassays. The levels of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38MAPK, and MAPK phosphorylation were assessed by western blotting. Results Cell proliferation was remarkably increased by increased glucose concentrations. Compared with the HG group, the migratory ability of VSMC cells was reduced in the presence of TMP. TMP also decreased the MDA content in the supernatant, but significantly increased the SOD activity. Western blotting showed that TMP inhibited the phosphorylation of JNK, p38MAPK, and ERK. Conclusions TMP appears to protect against HG-induced VSMC injury through inhibiting reactive oxygen species overproduction, and p38MAPK/JNK/ERK phosphorylation.