| ObjectivesAltered cerebral microcirculation,and alterations of blood-brain barrier(BBB) permeability play a key role in the development of brain edema after traumatic brain injury.ET-1 is one of the most potent vasoconstrictor peptides in humans.ET plays an important role in the pathophysiology of brain edema.ET itself may cause brain edema by enhanceing the permeability of BBB.The rhizome of Salvia Miltiorrhiza Bunge,also known as "Tanshen",is an important herb for promoting the circulation of blood and eliminating stasis in chinese traditional medicine.Therefore Tan can affect many aspects of the mediator cascade that can cause a permeability defect in the BBB. However,the relationship between cerebral edema,especially the BBB permeability and TanshinoneIIA(Tan IIA)has not been well established,we investigated the effect of combination of mannitol with TanIIA on brain edema in rats in response to cortical freezing in rats.Furhermore,we examine and compare the biochemical and molecular responses of the ET system:(ET-1,ECE-1,ETA and ETB mRNA)in cultured BMVECs to TNF-αand Tan IIA in an attempt to elucidate possible cerebrovascular effects of Tan IIA.MethodsPART ONE:(1)80 wister rats were were randomly divided into 4 groups after craniectomy and cold injury.They were treated with deionized water,mannitol (10ml·kg-1·d-1,ⅳ).Tan plus mannitiol treatment:mannitiol(10ml·kg-1,ⅳ)and tanshinoneIIA(25 mg·kg-1·d-1,ⅳ),starting from 30 min after surgery(2)Cold injury. A cold lesion was produced by the application of a cooper rod and cooled via the application of liquid nitrogen to the dura for 30 s.(3)Neurological deficit score were evaluated by the Zea-Longa scale(4)The pathologic changes of the brain were oberserved by light microscope and electron microscopy examination.(5)Water Content: Freshly dissected tissue samples from the peripheral part of the lesion in the injured hemisphere,and the area symmetrical to the injury site in the undamaged hemisphere were weighed.Water content was calculated as([wet weight-dry weight]/wet weight)×100%.(6)BBB permeability:The integrity of the BBB permeability was investigated with Evans blue dye(EB)extravasation.After decapitation,the brain was removed quickly,and fresh tissue samples taken from the above-mentioned areas were obtained and weighed on preweighed aluminum foil,then placed in 2 ml of formamide for dye extraction.The samples were incubated in formamide overnight,and the concentration of Evans blue extracted into formamide was measured at 620 nm.The tissue content of Evans blue was calculated as micrograms of dye per gram of wet tissue.PART TWO:(1)The whole blood viscosity were measured.(2)Plasma ET-1 levels:Blood samples were collected in chilled vials containing 30ml EDTA2NA and 20 ml Aprotinin,ET-1 in serum and were measured using radioimmunoassay or the kits supplied by Radioimmunological Institute of PLA General Hospita,following manufacturer's instructions of the kitsPART THREE:(1)Evaluation of cell viability was conducted by MTT.(2) Extraction and assay of ET-1,big ET-1 Media samples were collected.ET-1 and big ET-1 levels were determined using radio immunoassay kits.Aliquots of the reconstituted samples were loaded in duplicate onto the wells and the assay was carried out according to the manufacturer's protocol.(3)Quantitation of mRNA expression by RT-PCR Whole-cell RNA was isolated.The cDNA representing 50 ng input RNA was amplified by PCR using Taq polymerase.Specific primer pairs,constructed from the reported rat gene sequence for ET-1,ECE-1,ETA,ETB were applied as described previously.Both primer pairs were added simultaneously to the PCR reaction vials. Samples were analyzed by agarose gel electrophoresis.(4)Radioligand binding studies: Radiolabeled ET-1 binding was studied in living cells.Cells were washed three times with 1.0 mL binding buffer.1×10-11mol/L 125I-ET-1 and increasing concentrations of unlabeled ET-1 in the presence or absence of TNF-αand Tan IIA at indicated concentrations were added to each well.Cell-bound radioactivity was counted with a Wallac 1470 gamma counter.ResultsPART ONE(1)Neurological deficit score of C,D group were significantly lower than the B group(p＜0.01 or P＜0.01).(2)In the Tan and Tan+mannitol groups,The disruption of BBB was less severe than the mannitol group,morphological abnormalities of endothelial cells,basal laminas,and neurons were not evident.BBB almost remained ultrastructural integrity.(3)Water content and EB of brain tissues levels of C,D group were significantly lower than the B group(p＜0.01),D group was markedly lower than the C groups(p＜0.01or p＜0.05).PART TWO(1)The whole blood viscosity of D group was lower than that of B group(P＜0.01 or P＜0.05),but there was no signigificant diffence between B and C groups(P＞0.05)(2)In the control group,the ET-1 levels of plasms were significantly higher than the sham-operated group.The infusion of TanIIA and TanIIA +mannitol showed significant attenuation of the increase of ET-1 cold injury.The infusion of manntiol alone did not modify the ET-1 level significantly.PART THREE(1)Effects of Tan IIA on cell viability:The concentrations (0-20μg/ml)of Tan IIA used here had no effect on the viability of BMVECs.The TNF-αat 5μg/ml reduced the viability of BMVECs with cell viability of 82%±3%, but Tan IIA reversed significantly TNF-α-induced reduction of cell viability at concentrations of 10μg/ml and 20μg/ml.(2)TNF-αincrease significantly ET-1 concentration in the media when BMVECs were treated with Tan IIA.TNF-α-induced ET-1 elevation was suppressed in a dose dependent manner.Big ET-1 levels decreased in response to TNF-α.Conversely,big ET-1 levels progressively increased in response to Tan IIA in a dose dependent manner.(3)Effects of Tan IIA on the regulation of endothelin system(expression of ET-1,ECE-1,ETA and ETB mRNA) TNF-α-induced ECE-1 activation in BMVECs):ECE-1 mRNA expression was significantly upregulated by TNF-α.TNF-α-induced ECE-1 activation was suppressed significantly by Tan IIA at concentrations of 10μg/ml and 20μg/ml.The expression of ET-1 significantly increased with TNF-αexposure.However,no alteration in ET-1 mRNA could be detected after incubation of the cell with Tan IIA at indicated concentrations for 24 h prior to TNF-αcompared with BMVECs stimulated with TNF-αalone.TNF-αexposure result in a significant increased mRNA expression of ETA.However,there was no appreciative effect on ETB receptor mRNA expression.Compared with TNF-αalone,Tan IIA exposure resulted in a significant decrease in ETA mRNA expression at concentrations of 10μg/ml and 20μg/ml Tan IIA casused a significant increase of ETB mRNA(4)Endothelin receptor binding was not altered in BMVECs stimulated with TNF-αalone or in combined TNF-αand Tan IIA.Conclusion(1)The treatment with mannitol plus TanIIA is better for attenuating blood-brain barrier permeability and the extent of brain edema formation in response to cortical freezing in rats,partly by TanIIA' decreasing the level of plasma ET-1.(2)Tan IIA may inhibit ET-1 production in TNF-α-induced BMVECs through the suppression of ECE-1 synthesis. |
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