The Role Of Chloride Channels In The Enhanced Transport Of Adiamycin And Rhodamine Across The Blood Brain Barrier Induced By Borneol

Aim:The borneol can promote drug transport across the blood brain barrier(BBB), but the action mechanism is unclear. Our previous work has demonstrated that the Cl C-3 chloride channel is involved in the transport of substances across the cell membrane. However, there is no report whether chloride channels are associated with drug transport across BBB. The aim of this study was to investigate the roles of chloride channels in the borneol-induced increase of adriamycin and rhodamine 123 transport across BBB.Method: Wide type and Cl C-3 over-expressed FVB mice(equal number in female and male) of the weight of 20-30 g were used for experiments. The standard curve for measuring the concentration of adriamycin and rhodamine was established using an automatic microplate reader. The adriamycin and rhodamine concentration in the cerebrospinal fluid(CSF) and serum was determined by using the standard concentration curve and the fluorescence value detected by the automatic microplate reader. Immunohistochemistry was used to detect the expression and distribution of Cl C-3 proteins in the brain of wide type and Cl C-3 over-expressed mice. Transmission electron microscope was used to detect the changes of brain small blood vessels. The borneol-activated chloride currents were recorded by the patch clamp technique in human umbilical vein endothelial cells(HUVEC). Time-lapse imaging was used to analyze the effect of borneol and the chloride channel blockers tamoxifen and DIDS on the cell volume.Results: The results revealed that the adriamycin(ADM) concentration in the serum was much higher than that in the CSF after intraperitoneal injection of adriamycin in FVB mice(P <0.01), suggesting that adriamycinis not easy to pass through BBB. However, the adriamycin concentration in CSF was increased by 2.48 times and reached 37.8% of that in serum in the adriamycin group pretreated with borneol by intragastric administration. There was no significant change in serum adriamycin concentration induced by borneol. The results reveal that borneol can enhance adiamycin transport across the blood brain barrier. The chloride channel blockers tamoxifen and DIDS attenuated the effect of borneol and decreased the adriamycin concentration in CSF(P<0.01).Compared with the group of rhodamine alone, intragastric administration of borneol increased rhodamine concentration in CSF by 1.90 times in the group of rhodamine plus borneol(P <0.01). The chloride channel blockers tamoxifen and DIDS attenuated the effect of borneol and decreased the rhodamine concentration in CSF(P<0.01). There was no significant change of rhodamine concentration in serum induced by borneol(P >0.05).Based on the PCR results of mouse tail gene detection, the mice with the positive 440 bp stripes in agarose gels were classed as Cl C-3 over-expressed mice. Compared with the control group, the adriamycin and rhodamine concentration in CSF was increased in the Cl C-3 over-expressed group and this increment of drug concentration in CSF could be attenuated by the chloride channel blocker tamoxifen and DIDS(P<0.01), suggesting that Cl C-3 chloride channels can enhance the permeability of BBB. Compared with the groups of Cl C-3 overexpression alone and borneol alone, intragastric administration of borneol further increased radiamycin and hodamine concentration in CSF in the group of Cl C-3 overexpression plus borneol(P<0.05). Compared with that in wide type mice, the promoting effect of borneol on drug transport across BBB was much greater in Cl C-3 over-expressed mice, suggesting that the role of borneol in opening BBB is much more obvious in the Cl C-3 over-expressed mice.The results of immunohistochemistry revealed that Cl C-3 was highly expressed in the blood vessels of the meninges in the Cl C-3 over-expressed mice, with an increment of 29% in Cl C-3 protein expression compared with the wide type group(P<0.01). It was showed by the transmission electron microscopy that the number of pinosomes was increased and the tight junctions were loosen in the endothelia of the meninges blood vessels in Cl C-3 over-expressed mice.The results of patch clamp experiments revealed that borneol could activate chloride channels in HUVEC. Under the isotonic condition, the basal current was small. Perfusion of 20 ?M borneol could activate a chloride current(P<0.01). The outward current at +80 m V(32.65±4.97 p A·p F-1) was bigger than the inward current at-80 m V(-23.90±3.21 p A·p F-1). The current was outwardly rectified, did not show time or voltage-dependent inactivation and was volume-sensitive(it could be inhibited by hypertonic bath solution). The chloride channel blocker DIDS decreased the borneol-activated current at +80m V from 28.93±6.32 p A·p F-1 to 6.63±1.50 p A·p F-1(P<0.01) with an inhibition rate of 95.4±3.9%. The difference in inhibition rates was not significant between the effects on outward and inward currents. The chloride channel blocker tamoxifen decreased the borneol-activated current at +80m V from 32.10±5.67 p A·p F-1 to 6.49±1.38 p A·p F-1(P<0.01) with an inhibition rate of 94.2±3.8%. The inhibition of outward current was not significantly different from that of inward current. These results further prove that the current activated by borneol is a chloride current mediated by chloride channels.The results obtained by the time-lapse imaging demonstrated that extracellular perfusion of 20 ?M borneol solution decreased the cell volume by 12.6 ± 2.1% in HUVEC(P<0.01). The borneol-induced decrease of cell volume was inhibited by the chloride channel blockers,tamoxifen and DIDS(P<0.01). There was no significant difference in the inhibitory efficiency between tamoxifen and DIDS(P>0.05).Conclusion: 1. Borneol can increase the permeability of BBB in mice, and promote adiamycin and rhodamine transport into brain tissue. 2. Cl C-3 over expression can promote adiamycin and rhodamine across BBB. 3. Borneol can activate Cl C-3 chloride channels in HUVEC, resulting in the outflow of Cl- and water and shrinkage of endothelial cells, which may in turn widen the intercellar gap and loosen the tight junction and open BBB. Chloride channels play an important role in borneol-induced opening of BBB.