| ObjectiveThrough establishment blood tumor barrier model in vitro, to research the dose-and time-dependent relationship and the regulation of the tight junction proteins; Combined animal models in vivo to research the pharmacodynamics of natural borneol increased antitumor drug transport blood tumor barrier, to clarify effects and mechanism of borneol regulating blood tumor barrier opening a new way forclinical treatment of intracranial diseases such as glioma.Methods1.The establishment of BTB model in vitroThrough non-contact co-culturing of C6 rat glioma cells and human umbilical vein endothelial cells(HUVECs)to establish cell BTB model in vitro. Using endothelial cell membrane impedance(TEER) and HRP flow model to evaluate its performance.2.Determinate the content of d-borneol in natural borneolThrough gas chromatography to Determinate the content of d-borneol in natural borneol. GC conditions:chromatographic column:ZB-WAX capillary column(30M×0.25mm×0.25μm); graded temperature,90℃ retain 2min, with 10℃ /minup to 110℃, retain 2min.with 30℃/min up to 160℃, retain 2min; the inlet temperature is 210℃; FID detector temperature is 250℃; the pressure of high purity nitrogen is 5psi; split ratio 10:1;air flow rate is 450mL/min; hydrogen flow rate is 45mL/min; injection amount is 1μL.3.The effect of permeability and expression of proteins tight junction related about nature borneol on BTB model.The experiment was divided into 4 groups:control group, borneol low dose group(25μg/mL), borneol middle dose group(50μg/mL), borneol high dose group(100 μg/mL), each group has three wells. HRP flow to determine the permeability of different doses of natural borneol in vitro BTB model; Tmmunofluorescence and ELISA methods to determinate expression of Claudin-5,Occludin, ZO-1, F-actin proteins.4.The preparation of Rat C6 glioma model in vivoSD rats were fasted water for 12h,10% chloral hydrate(3.5mg/kg)anaest hetized by intraperitoneal injection.C6 glioma cells(2.5×106) (25μL)were injected in right caudate nucleus, sterile bone wax closed bone hole, sutu re skin, incision disinfection, conventional breeding.5. Study the pharmacokinetics and tissue distribution of natural borneol on methotrexate in C6 rat glioma model in vivo.The successful model in rats were randomly divided into control group (0.5% sodium carbonxymethyl cellulose), borneol low-dose group (35mg/kg) and borneol high-dose group(140mg/kg)fed by equal volume(10mL/kg)solvent or borneol solution. And 1 h later, MTX(20mg/kg) were given to three groups by intravenous injection. Plasma and brain tumor tissue samples were collected at corresponding time after intravenous injection, samples of plasma and brain at each time point are four rats.Chromatographic conditions:Column:Synergi Hydro-RP C18(250mm×4.6mm,4 μm); mobile phase:methanol-0.3% acetic acid/0.25% triethylamine(26:74); flow rate:imL/min; autosampler temperature:4℃; column temperature:35℃; detection wavelength:302nm.Results1. The successfully established BTB cell model in vitro.be better for reflectting the situation in vivo, can be used to simulate in vivo environment, and for the study of drugs across the blood tumor barrier.2. D-borneol of natural borneol in this experiment reached 97.025% by gas chromatography, conforms to the Chinese Pharmacopoeia 2010 edition, may be used for subsequent dosing experiment.3. HRP flow to determine the permeability of different doses of natural borneol in vitro BTB model. Results show that compared with the blank control group,natural borneol low dose group(25μg/mL), middle dose group(50μg/mL), high dose group(100μg/mL), HRP permeability gradually increased with the extension of time, are statistical significance (P<0.01); where the low-dose group compared with the control group at the corresponding time points 10,30,60,120,240min, the permeability improved significantly, is statistical significance(P<0.01);the permeability of middle-and high dose group are higherthan control group in all time points. It can be seen there is a dose-trend from the permeability at the same time:high dose group>middle dose group> low dose group. From the transmittance of adjacent time point, low、middle、 high dose groups are in 10-30min,30-60min, enhancement of HRP is most obvious,upgrade rate gradually decreased at 60-120min,returned to normal levels at 120-240min.4. ELISA and immunofluorescence to determinate the expression of tight junction associated proteins Claudin-5、Occludin、Z0-1、F-actin on blood tumor barrier.The ELISA results showed that:expression of Claudin-5 protein firstly decrease and then increase gradually, restored to the level before the medicine, and in the 30min expression level was the lowest, and there are dose-dependent at 30、60min, high dose group<middle dose group<low dose group. The determination of Occludin protein result that there are no significant changes in the amount of protein expression on different groups, suggests that natural borneol low dose group, middle dose group, high dose group are no significant(P>0.05)within 4h. Natural borneol low dose group, expression of ZO-1 protein at different time points compared with the previous administration has no statistical difference (P>0.05); low dose group and high dose group, the expression of Z0-1 protein decreased firstly and then increased gradually, and respectively in 60,30min was the lowest (P<0.01). The expression of F-actin in three groups is firstly decreased and then increased gradually, in 30、60min expression is significantly lower than control group, and there was a dose dependent trend. The results of four protein expression by Immunofluorescence method are basically identical with ELISA experimental results.5.Rat C6 brain glioma model was successfully prepared; established the HPLC method for determinating concention of methotrexate in plasma and brain homogenate.ConelusionBorneol by regulating tight junction-related proteins Claudin-5, Z0-1, F-actin expression and distribution, thereby regulating the permeability of BTB cell in vitro model;in vivo experiment further confirmed the natural borneol can regulate the permeability of the blood tumor barrier, enhance bioavailability of antitumor drug methotrexate in brain tissue. |
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