Research About The Effects And Mechanisms Of Natural Borneol On Blood-tumor Barrier In Vivo

1ObjectiveTo study the dose-and time-dependent manner of natural borneol improving DDP through BTB, and then try to research the mechanism of natural borneol opening BTB, so making preliminary clarifications on its effects and mechanism. And exspect to expiain its effect mordel by modern scientific language and offer some condtions for further research on efficient path of improving anti-cancer medicine tissue distribution.2Methods2.1Establishment of SD rat C6glioma tumor model in vivoTumor cells were injected into the right striatum on the SD rat, The needle was left in place for a further10min.After removing the needle, trepanation was occluded using wax for bone, and skin was sutured. Then the rat was placed back into the cage with free access to water and food. Tumor bearing rats (7,14,21days after implantation) were selected randomly and positioned into a clinical3.07T MR scanner, to acquire TwoTl-weighted and T2-weighted series before and after subcutaneous contrast injections. At the same time, tumor brain tissues were gotten for HE staining pathology, respectively. At last, we made sure that the inoculation quantity of tumor cells was2.5X106cells/25μL each rat and the rats were prepared for treatment fourteen days after tumor implantation.2.2Effects of borneol on the pharmacokinetics and brain tissue distribution of cisplatin on C6tumor bearing ratFourteen days after, the rats with tumor implants had been fed without forage but water for more than12h. The rats were then randomized into three groups (n=4per point, including7time points per group):control group, intragastric administration with0.5%CMCNa; low-dose group, intragastric administration with35mg/kg natural borneol; high-dose group, intragastric administration with140mg/kg natural borneol. Then, all the tumor-bearing rats were injected with10mg/kg cisplatin (dissolved in normal saline) into coccygeal vein1h after i. g. After injection all the tumor-bearing rats were collected brain tissues at corresponding time, successively.The concentration of DDP was determined with UV-HPLC at254nm on an Hydro-RP80A column C18(250mm x4.6mm,4um)with an Phenomenex C18(4mmX4mm) guard column, and we used a mixture of water/methanol (20:80, v/v) as the mobile phase at a fixed flow rate of1mL/min, column elution was carried out at28℃.2.3Effect of natural borneol on TJ associate proteins gene and expression of BTBDetermine the genes variation of Claudin-5、Occludin、Z0-1、cytoskeletal protein F-actin by quantitative real-time RT-PCR, and further determine protein variation of them through ELISA.3ResultsAn animal model of rat cerebral C6glioma was established successfully, and it is reliable and stable. The tumor growth property and pathological characteristics are similar to human cerebral glioma. Hence, the model is applicable satisfactorily for the clinical basic research of glioma. A sensitive, accuracy and precision pre derivatization HPLC-UV method was established, there are no interference between endogenous substance and DDP. And, the detection results showed that the AUC(0-8h)of borneol high-dose is about80%to borneol low-dose in plasma, while the AUC(0-8h)of borneol high-dose is about twice over borneol low-dose and their size order was AUC(o-8)high-dose> AUC(0-8)low-dose> AUC(0-8)control in brain tissue after statistics calculation, the variation is statistical significance(P<0.05).Quantitative real-time RT-PCR and ELISA to detect the effect of natural borneol on TJ proteins (Claudin-5, Occludin, Z0-1、F-actin) of BTB. PCR showed that level of Z0-1beginning to decrease from2min and the lowest of it in borneol high-dose and low-dose group was appeared respectively at30min、45min following initiation of borneol treatment (P<0.05), thereafter restored to the level of borneol0h. The manner of F-actin was the same as Z0-1, but amplitude of variation was lower. There were no significant differences in the levels of claudin-5or occludin before or after treatment in either group(P>0.05). The ELISA results were nearly consistent with PCR.4ConelusionNatural borneol can improve the permeability of BTB to a certain extent, hence increase distribution of DDP to brain tumor area. It showed that down-regulating Z0-1and F-actin gene and protein expression may play an important role in permeability of the blood tumor barrier following natural borneol treatment, while reversible translocation of claudin-5and occludin, still need to be further investigated.