Transport Of Neferine In The Central Nervous System And Regulatory Effect Of P-Glycoprotein On Neferine Transport

OBJECTIVE: To observe the permeability of neferine (Nef) through the blood -brain barrier (BBB), to explore the relationship between doses and the concentrations of Nef in brain, heart and kidney of mice, and to study the regulatory effect of P-glycoprotein (P-gp) on transport of Nef in the central nervous system. METHODS: ① After intravenous injection of Nef, the concentrations of Nef in mice brains obtained at different time were determined by HPLC.With administration of different doses of Nef, the concentrations of Nef in brains were compared with those in hearts and kidneys.② Cytotoxicity of Nef, dichlorvos (DDVP) and verapamil (Ver) were tested by MTT method in RBE4 cells.③ Time and concentration courses for uptake of Nef and DDVP by RBE4 cells were gotten. Based on these courses and ascertained inhibitory rates of Nef and DDVP on cells, the optimal culture time and concentration for Nef and DDVP uptake were obtained, respectively.④ Ver, a P-gp inhibitor, was used to observe its effect on Nef and DDVP uptake by RBE4 cells. Effect of DDVP on Nef uptake was also studied in RBE4 cells.RESULTS: ① Nef was not detected in brain tissue of mice when Nef was injected at the dose of 5μg·kg-1. After administration of Nef at the dose of 10-30μg·kg-1, Nef was detected in brain tissue and the concentration of Nef increased with the enhanced dose. Peak concentration of Nef was attained at 20 min after medication. Under the same dosage, there was a significant difference (P<0.01) between the concentration of Nef in brain and that in heart and kidney.② The inhibitory rates were lower than 10% when Nef, DDVP and Ver were in the range of 1-5μg·ml-1, 1-12μg·ml-1and 1-15μg·ml-1, respectively.③ The uptake of Nef and DDVP by RBE4 cells were time-dependent and the steady-state of uptake appeared at 60 min and 90 min, respectively. The uptake amount of Nef and DDVP increased in concentration-dependent manner. Peak concentrations of them were attained when the concentration of Nef was 5μg·ml-1 and the concentration of DDVP was 12μg·ml-1. So the optimal culture time and concentration for Nef and DDVP uptake were 60 min/5μg·ml-1and 90 min/12μg·ml-1, respectively.④ Ver (5-15μg·ml-1) significantly increased the uptake of Nef (5μg·ml-1) by RBE4 cells (P<0.01), but Ver (2.5-15μg·ml-1) had no significant effect (P＞0.05) on that of DDVP (12μg·ml-1). There was no significant change in the uptake of Nef (5μg·ml-1) by RBE4 cells (P＞0.05) in the present of DDVP (2-12μg·ml-1). CONCLUSIONS: Nef can be transported into the brain through the BBB; the permeability of Nef into the brain is restricted by P-gp, but Nef is both an inhibitor and a substrate for P-gp and the reversal effect of Nef on itself makes it keeping an effective therapeutic concentration in the brain; DDVP is neither a substrate nor an inhibitor of P-gp and has no effect on the transport of Nef in the central nervous system.