The protective effect of eupatilin on indomethacin-induced cell damage in cultured feline ileal smooth muscle cells: Involvement of HO-1 and ERK

Chronic users of non-steroidal anti-inflammatory drugs frequently develop ulcerative lesions in their intestines. The purpose of the present study was to investigate whether eupatilin, an active ingredient derived from Artemisia plants, prevents this side effect in vitro. Extracts of the whole herb of Artemisia asiatica Nakai have been used in oriental medicine for the treatment of inflammation. As measured by the MTT assay, the treatment of cultured feline ileal smooth muscle cells (ISMCs) with 2.5 mM indomethacin for 2 h decreased the cell viability to 43%. Pretreatment with eupatilin resulted in dose-dependent inhibition on indomethacin-induced cell damage. This cytoprotective effect of eupatilin required concentrations of more than 150 μM and incubation periods of longer than 16 h. Pretreatment of ISMC with cycloheximide, an inhibitor of protein synthesis, attenuated the cytoprotective effect of eupatilin, suggesting that eupatilin induces proteins that are responsible for the cytoprotection. Heme oxygenase-1 (HO-1), which is known as a cytoprotective enzyme due to its anti-inflammatory actions, is a candidate protein since ZnPP, an HO-1 inhibitor, repressed the protective effect of eupatilin on indomethacin-induced cell damage in a concentration-dependent manner. Western blot analysis revealed that eupatilin-mediated HO-1 induction occurred in a concentration- and time-dependent manner. We also found that PD98059, a MEK (MAPK/ERK kinase) inhibitor, attenuated the eupatilin-induced HO-1 expression and nuclear translocation of transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2). Taken together, the data imply that eupatilin protects ISMC from cell damage caused by indomethacin, and that its cytoprotective action could be attributed to eupatilin-mediated HO-1 induction via ERK and Nrf2 signaling in ISMC.