Study On The Auxo-action Of Borneol Accelerating The Penetration Of BBB And P-glycoprotein-mediated Mechanism

Background:Blood-brain barrier is the interface of central nervous system and blood , which protects brain tissue from the blood changes, but it also limits the drug to achieve therapeutic concentration. In recent years,the incidence of intracranial tumors increase year by year. Conventional chemotherapeutics, such as fat-soluble alkylating agent and mixed type of nitrosamines are difficult to achieve the desired therapeutic concentration, even the latest generation of alkylating agent– Temozolomide,its concentration in central nervous system is only 40% of the blood, the water-soluble chemotherapeutics is even worse.To find effective method to enhance the permeability of the blood-brain barrier is becoming a hot research, chinese medicine is noticed as its high-performance and low toxicity. Borneol as a small molecule,can not only through the blood-brain barrier quickly, but also promote the permeability of other drugs. However, most of them is qualitative research.Therefore, to detect accuratly the dose- effect and time-effect and explore the possible mechanism of borneol have great significance,it can provide experimental basis for clinical application in future.Objective:To investigate the auxo-action of Borneol, we evaluate the dose-effect and time- effect relationship,exploring on the possible mechanisms through P-glycoprotein.Methods:⑴Making rapid frozen sections of rat brain, we qualitative detect the content of Evans Blue in rat brain by fluorescence microscope, and evaluate the role of borneol; we also detect it by fluorescence spectrophotometer,to evaluate the dose-effect and time-effect relationship of borneol.⑵The expression of P-gp on the rat brain is detected by immunohistochemistry, compared with blank group and the control group, evaluating the change of P-gp, to explore the mechanism of borneol.⑶T he concentration of vincristine in brain is detected by high-performance liquid chromatography (HPLC), compared with the classic P-gp blocker- verapamil, exploring on the mechanism of competitive inhibition through P-glycoprotein.Result:⑴General observation: when the rat is injected with EB, the animal's muscles,skin and sclera will change into blue after 30s. The uneven distribution of blue dye can be seen on all the cerebral cortex and cerebellum in experimental group, which is not in the control group.⑵Fluorescence microscopy: A large number of oval erythema can be seen in experimental group, compared with control group having only occasionally erythema(P<0.01).⑶Fluorescence spectrophotometer: Dose-effect test results show: the different doses of borneol can affect the content of EB in brain(P﹤0.01).As the dose of 0.25 g / kg.d, the permeability-promoting effect of borneol is the best..It have statistical difference between the control group and each dose group of 0.125 g/kg.d,0.25 g/kg.d,0.5 g/kg.d,1 g/kg.d(P﹤0.05), but it have no statistical significance between the blank group and the control group(P﹥0.05). Time-effect test results show: the different time of borneol can affect the content of EB in brain(P﹤0.01),The concentration of EB in rat brain has maintained higher from 0.5h to 48h(P﹥0.05),but have statistical significance between with other two points(0h and 72h)(P﹤0.05).⑷Immunohistochemistry: Positive expression in experimental group is 55%,the blank group and control group are 50%.There have no statistically significant between experimental group,control group and blank group(P>0.05).⑸High Performance Liquid Chromatography: Borneol of 0.25 g/kg.d can obviously increase the content of VCR in brain.There have statistically significant between experimental group and blank group(P﹤0.05), which is similar with the classic Pgp blocker- Ver.Conclusion:⑴Borneol can improve vincristine and Evans Blue's penetration of the blood-brain barrier;⑵As the dose of 0.25 g / kg.d, the permeability-promoting effect of borneol is the best, the concentration of EB in rat brain has maintained higher from 0.5h to 48h after injecting;⑶borneol can not affect the expression of Pgp on rat brain, but can obviously increase the content of VCR in brain through competitive inhibition,which is similar with the classic Pgp blocker- Ver. I believe this is one mechanism of borneol in opening blood-brain barrier.