Study On The Effect And Mechanism Of Bilobalide On Modulating Blood-Brain Barrier Permeability

Objective:The blood-brain barrier(BBB)offers protection to the central nervous system(CNS)by maintaining its homeostasis.Besides restricting the entry of harmful xenobiotics and endogenous molecules to CNS,BBB also limits the entry of therapeutic agents.The extremely low permeability of BBB is the bottle neck for brain drug delivery and CNS therapeutic efficiency.Our team firstly proposed the idea that "Activating blood circulation and opening the barrier to benefit the body,overcoming the blood-brain barrier to promote brain uptake of therapeutics".Our pervious study found that the active components of ginseng in the brain tissues of rats were significantly increased when co-treated with ginkgo biloba extract,the underlying mechanism was related with ginkgo biloba extract regulating BBB permeability.Previous in vitro studies have demonstrated that bilobalide is the main active component of ginkgo biloba extract that regulated the BBB permeability.The present study aims to investigate the modulatory effect of bilobalide on BBB permeability and the mechanisms involved.Methods:1.Co-culture of the immortalized human cerebral microvascular endothelial cell line(hCMEC/D3)and immortalized human astrocyte cell line(HEB)was established as an in vitro BBB model.Transendothelial electrical resistance(TEER)and fluorescein sodium(Na-F)penetration rate were analyzed to valuate bilobalide’s effect on the in vitro BBB permeability.2.Mice were treated with different doses(2.5,5,10 mg/kg)of bilobalide and then intravenous injected with the tracer 10 kDa FITCdextran.Brain parenchymal FITC-dextran leakage was measured by fluorescence quantitative analysis and immunofluorescence methods to valuate BBB permeability.3.Explored the effect of bilobalide on hCMEC/D3 cells tight junctions.Expression of adhesion junction protein VE-cadherin,tight junction proteins Occludin and Claudin-3 were measured by Western Blot,and the ultrastructure of intercellular tight junctions of hCMEC/D3 was performed by transmission electronic microscopy.4.Explored the effect of bilobalide on actin-binding proteins ERM and MLC.The phosphorylation of ERM and MLC in hCMEC/D3 were detected by Western Blot,and the phosphorylation expressions of ERM and MLC in mice brain microvascular were detected by immunofluorescence.5.Explored the role of adenosine A1 receptor(A1R)in bilobalideinduced BBB permeability.We designed the siRNA to silence the A1 R gene in hCMEC/D3.The changes of TEER,protein expression of pERM/p-MLC and tight junction ultrastructure were detected after bilobalide treatment.Results:1.The results showed that bilobalide led to remarkable reductions of the TEER value and the minimal values were observed at 2 h posttreatment(P < 0.01 vs.control).Notably,TEER almost return to control level at 4 h.Similarly,Na-F penetration rate reached a maximum at 2 h(P < 0.05 vs.control)and back to the baseline at 4 h.These results implied bilobalide could reversibly increase the paracellular permeability of in vitro BBB while maintained BBB integrated.2.In mice,BBB permeability was significantly enhanced after bilobalide(5 mg/kg)administration,evidenced by the markedly increased concentration of FITC-dextran leakage in the brain parenchyma(P < 0.05 vs.control),and there was no significant difference in serum(P > 0.05).3.The expression levels of the tight-junction and adherent junction protein(Occludin,Claudin3 and VE-Cadherin)between bilobalide group and control group were not significant different(P > 0.05).But the tightjunction ultrastructure of the hCMEC/D3 was changed after bilobalide treatment.4.The phosphorylation of actin-binding proteins ERM and MLC in hCMEC/D3 cells were significantly up-regulated after bilobalide treatment(P < 0.01 vs.control).The phosphorylation level of ERM and MLC reached peak at 90 and 120 min respectively and all dephosphorylated at 4 h.Similarly,the phosphorylation levels of ERM and MLC in brain microvessels of mice were also significantly up-regulated.5.When A1 R gene was silenced by the siRNA,the TEER reduction,the up-regulation of p-ERM and p-MLC and the changes of tight-junction ultrastructure induced by bilobalide were largely neutralized.It was suggested that A1 R signaling pathway might be involved in bilobalideincreased BBB permeability.Conclusion:Our study demonstrated that bilobalide might reversibly modulate BBB permeability through A1R-mediated phosphorylation of ERM and MLC,which could lead to cytoskeletal re-organization and increase the cell-to-cell junctional spaces to cause an increased BBB permeability.This study provides a promising strategy to enhance BBB permeability and may be helpful for brain drug delivery.