| ObjectiveAs we all know, the reason of bradykinin-induced blood-brain barrier (BBB) opening is that B2 receptor, which presents on the vessel endothelial cell, combines with bradykinin, then changes the structure and function of BBB. Recently studies indicate that there is no expression of B2 receptor on capillary in both brain and tumor tissue, it be found on the tumor cells, and is highly correlated with the opening of BBB. According to the above results, bradykinin may directly combines with B2 receptor on tumor cells to cause it releasing cytokine and/or other mediators, then changes the structure as well as the function of BBB, and induces opening of BBB. But there is no relation with the B2 receptor on vessel endothelial cell. However, the actual mechanism is still unclear.Inamura reported that when bradykinin (small dose sustained) had been infused via carotid to glioma-bearing rats for 15min, BTB opened to the maximum, declined thereafter and remained to close. Even though the infusion continued during the experiment, BTB was out of function. This process called tachyphylaxis. So far there was no any convincing evidences to confirm the mechanism of bradykinin opening the BTB or the tachyphylaxis.This study aimed to make clear that kind of cytokine and mediators would be released by tumor cells; how to affect the BBB by them; as well as the mechanisms of tachyphylaxis on BBB, after bradykinin combined with B2 receptor on gliomas.Methods1. Establishment of rat brain C6 tumor model 1×10~5/5μL C6 cells were injected into the right caudate nucleus using the stereotaxic apparatus in rats, according to Nicolau. Tumor bearing models of rat were successfully prepared after 15 days.2. Detection of blood-brain barrier permeability as well as the changes of tight junction, using evans blue and electron microscope on tumor-bearing rat before and after bradykinin treatment.3. Detection of blood-brain barrier permeability as well as the changes of tight junction, using evans blue and electron microscope on tumor-bearing rat before and after tumor necrosis factor-α(TNF-α) treatment.4. Detection of TNF-αconcentration in the C6 culture medium, using radioimmunoassay before and after bradykinin treatment.5. Investigation of heat shock factor 1(HSF1) and heat shock protein(HSP70) protein expression in C6 cells, using Western blotting analysis before and after bradykinin treatment.6. Investigation of the TNF-αmRNA in C6 cells, using Reverse Transcription Polymerase Chain Reaction (PT-PCR) before and after bradykinin treatment.7. Investigation of occludin protein on microvessels in tumor-bearing rat, using immunohistochemistry before and after TNF-αtreatment.Results1. Model of C6 glioma-rat was successfully established.2. The permeability of blood-brain barrier and opening of tight junction were increased to the peak at 15min, and then approached to normal control levels at 60min after C6 glioma-rat were treated with bradykinin.3. The permeability of blood-brain barrier and opening of tight junction were increased to the peak at 15min, and then approached to normal control levels at 60min after C6 glioma-rat were treated with TNF-α.4. The concentration of TNF-αwas increased to the peak at 15min in the culture medium, and higher than the control group at 60min after C6 cells were treated with bradykinin.5. Expressions of HSF1 and HSP70 protein were gradually increased after C6 cells were treated with bradykinin, reaching to the peak at 5min and 30min respectively, and both higher than the control group at 60min after bradykinin treatment.6. Level of TNF-αmRNA was increased after C6 cells were treated with bradykinin, and the peak occurred at 10min, declined thereafter, and reached to the minimum value at 60min.7. The expression of occludin on brain tumor microvessels decreased after the C6 glioma-rat was treated with TNF-α.Conclusions1. Permeability of BTB and opening of tight junction were increased to the peak at 15min, and then approached to normal control levels at 60min after C6 glioma-rat were treated with bradykinin or TNF-αalone.2. Bradykinin promoted the expression of TNF-αand TNF-αmRNA in glioma-cells, and then increased release of TNF-αfrom glioma-cells. Their differences were achieving to the peak at 5min and 10min and 15min, after C6 glioma-rat were treated with bradykinin, respectively. The results showed that the expression of HSF1 may promote the expression of TNF-αmRNA and release of TNF-α, this is one of mechanisms on bradykinin selectivity opening the blood-tumor barrier.3. The concentration of TNF-αto the peak and expression of occludin protein to the minimum at the same time in glioma-tissue, after C6 glioma-rat were treated with bradykinin. In the meantime, opening of BTB to the maximum by bradykinin. The results suggested that the effect of TNF-αon bradykinin-induced BTB opening might due to changing the expression of occludin protein on brain microvessels.
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