Effect of luteolin on ceftiofur intestinal absorption: the rational use of Caco-2 monolayer cell model and everted gut sac model

Background: The clinical utility of ceftiofur primarily stems from its broad-spectrum antibacterial properties. However, its limited oral bioavailability significantly constrains therapeutic effectiveness. Although natural flavonoids have been well-documented to facilitate intestinal drug transport, the potential of luteolin to enhance ceftiofur intestinal absorption and the mechanistic basis for this effect remain to be elucidated. Purpose: This study aims to investigate the effects of luteolin on duck intestinal absorption of ceftiofur and elucidate its molecular mechanisms. Methods: This study employed duck everted gut sacs and Caco-2 cell monolayers to examine ceftiofur intestinal absorption in the absence and presence of luteolin. The specific inhibitors verapamil (P-glycoprotein, P-gp), Ko143 (breast cancer resistance protein, BCRP), and probenecid (multidrug resistance-associated protein 2, MRP2) were used to identify absorption-limiting transporters. The real-time quantitative PCR (qRT-PCR) and Western blot were performed to assess the transcription and translation of efflux transporters and tight junction proteins. Molecular docking evaluated the binding affinity of luteolin toward key transporters. Results: Ceftiofur was poorly absorbed in the duck intestine via an active transport mechanism. Critically, luteolin enhanced the intestinal absorption of ceftiofur. Furthermore, probenecid, the MRP2 inhibitor, promoted ceftiofur intestinal absorption. The qRT-PCR and Western blot detection results revealed that luteolin downregulated ceftiofur-related efflux transporter MRP2 and tight junction proteins (Occludin, Claudin-1, and ZO-1). Molecular docking results indicated that luteolin exhibited strong binding affinity to these key transporter proteins. Conclusion: Luteolin increased ceftiofur intestinal absorption by downregulating the transcription and translation level of MRP2 or tight junction proteins (Occludin, Claudin-1, and ZO-1), supporting its role as an oral bioavailability enhancer.