*Purpose: Tacrolimus (FK506) is an immunosuppressant agent that is frequently used to prevent rejection of solid organs upon transplant. However, nephrotoxicity due to apoptosis and inflammatory response mediated by FK506 limit its usefulness. Eupatilin, a pharmacologically active flavone derived from Artemisia species, is known to have antioxidant and antiinflammatory activities. In the present study, the protective effect of eupatilin against FK506-induced damage in LLC-PK1 pig kidney epithelial cells was investigated.

*Methods: LLC-PK1 cells were exposed to FK506 with eupatilin, and cell viability and glutathione (GSH) were measured. Western blotting and RT-PCR analyses evaluated protein expression of mitogen-activated protein kinases (MAPKs), caspase-3, kidney injury molecule-1 (KIM-1), and Toll-like receptor-4 (TLR-4) expression was assessed. The number of apoptotic cells was measured using an annexin V/PI staining with flow cytometry.

*Results: Reduction in cell viability by 50mM FK506 was ameliorated significantly by cotreatment with eupatilin. Eupatilin protected LLC-PK1 cells by preventing FK506-induced GSH decrease. The phosphorylation of p38, extracellular signal-regulated kinases, and KIM-1, TLR-4 and cleaved caspase-3, increased markedly in LLC-PK1 cells treated with FK506 and significantly decreased after cotreatment with eupatilin. Moreover, flow cytometry assay showed that apoptotic cell death was increased by FK506 treatment, whereas it was decreased after cotreatment with eupatilin.

*Conclusions: These results collectively provide therapeutic evidence that eupatilin ameliorates the FK506-induced renal damage via antioxidant effect and inhibiting apoptosis.

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