Transmembrane Transport of Anemoside B4 in HEK293 Cells
Objective:To observe the transport behavior of anemoside B4(B4)in HEK293 cells and explore the transport mechanism.Methods:Cell viability was detected by the cell counting kit-8(CCK-8)assay to obtain the safe concentration of B4 for cell treatment.An ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)method was established for determining B4 content.UPLC-MS/MS was adopted to determine the content of B4 in the cells treated with B4 at different concentrations and for different time periods,which was expected to reveal the transport of B4.The inhibitors of organic anion transporters(OATs)and organic cation transporters(OCTs)were used to co-treat the cells with B4,and the intracellular content of B4 was measured to evaluate the effects of the inhibitors on the transmembrane transport of B4.The inhibitor and small interfering RNA(siRNA)of P-glycoprotein(P-gp)were employed to investigate whether P-gp was involved in the efflux of B4.Results:B4 at a concentration below 156 μg·mL-1 showed no cytotoxicity within 48 h.The content of B4 reached the peak after treatment for 1 h and increased in a concentration-dependent manner.Cimetidine,the inhibitor of OCTs,significantly inhibited B4 transport into cells,while inhibition of P-gp had no significant effect on the content of B4 in cells.Conclusion:B4 enters HEK293 cells through passive transport.OCTs were involved in B4 transport into the cells,and the efflux of B4 was independent of P-gp.
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