| ObjectiveBuilding upon the rats model of middle cerebral artery embolism (MCAO) and rat brain microvascular endothelial cells (BMECs) hypoxia model, this article explains the effect of Buyang Huanwu decoction (BHD) on NO, vWFv 6-keto-PGF1?, PAT-1?tPA?ICAM-1 and VCAM-1 in the model rats of MCAO (vivo experiments) and the effect of DCP and drug-containing serum (DCS) on hypoxic rat BMECs (ex vivo experiments), as well as compare approaching degree of the expression of true value between vivo experiments and ex vivo experiments,provide specification for Chinese medicine Plasma pharmacology method (CPPM).MethodsThe model of MCAO was made by using the method of thread embolizing of the internal carotid artery in rats and was intervented with BHD, Nerve symptom scores were observed for 1?3?5? days after the treatment. HE staining, TUNEL method were used to observe the brain tissue pathological morphology for 1?3?5?7 days after the treatment. After taking the plasma after intragastric administration for 1? 3?5?7 days, The method of Enzyme-linked immuneosorbent (ELISA) was used to measure the expression of vWF?6-keto-PGF1??PAT-1 and TPA levels, Nitrate reductive enzymatic to the NO levels, immune protein imprinting method (Western blot) to the TCAM-1?VCAM-1 protein content in brain tissue, real-time fluorescent quantitative reverse transcription polymerase chain reaction (real time PCR) to ICAM-1 and VCAM-1 mRNA expression.The model of rat BMECs hypoxia model was made and intervented with BHD DCP and DCS of the 1s?3rd?5th?7th day, testing index and method were the same as vivo experiments.Results:Compared with the model group, the scope of cerebral infarction?cell apoptosis rate and score of neural symptoms in the BHD group decreased markedly at the same time points (P< 0.01),the cell apoptosis rate reduced the most obviously on the 3rd day (P< 0.01), the scope of cerebral infarction and the score of neural symptoms in administration was significantly lower on the 7th day (P<0.01). Compared with the model group on the 7th day after treatment, the expression of NO in the edaravone group and the BHD group increased markedly at the same time points (P< 0.01),and the expression of NO in the BHD group increased the most obviously on the 7th day (P< 0.01); Compared with the model group on the 3rd and 7th day after treatment, the expression of vWF in the edaravone group and the BHD group decreased markedly at the same time points (P< 0.01),and the expression of vWF in the BHD group decreased the most obviously on the 3rd day (P< 0.01); On the 7th day 6-keto-PGF1? in the BHD group significantly increased than the model group at the same time point(P<0.01).The expression of PAI-1 in the BHD group of after administration for 3 days decreased significantly compared with the edaravone group (P< 0.01); the expression of tPA in the edaravone group and BHD group after administration for 3,5,7 days was significantly lower than the sham operation group (P< 0.01), ICAM-1 and VCAM-1 mRNA expression in the edaravone group and the BHD group after administration for 1,3,5S 7 days significantly reduced than the model group (P< 0.01), and the BHD group decreased significantly after administration for 7 days (P< 0.01); and ICAM-1 protein expression in the BHD group after administration for 7 days significantly decreased than the edaravone group (P< 0.01). Compared with the model group, VCAM-1 protein expression in the BHD group after administration for 1?3?5?7 days significantly decreased (P< 0.01), to minimum values on the 7th day(P< 0.01).Compared with the model group, the expression of NO in the 3rd,5th,7th DCP group?the 5th?7th DCS group and the edaravone group significantly increased (P< 0.05), to maximum values on the 7th day; compared with the model group, the expression of vWF in the edaravone group, the technical group, the 3rd,5th,7th DCP group?the 1st?rd?5th?7th DCS group significantly decreased (P< 0.05), to minimum values on 3rd day; compared with the model group, the expression of 6-keto-PGF1? in the edaravone group?the technical group, the 1st,3rd,5th,7th DCP group?the 1st?3rd?5th?7th DCS group significantly increased (P< 0.05), to maximum values on 7th day; compared with the model group and the blank plasma group, the expression of PAI-1 in the edaravone group?the technical group?the 3rd?5th?7th DCP group significantly decreased (P< 0.05), to minimum values on 3rd day; compared with the model group, blank plasma group and blank serum group, the expression of tPA in the edaravone group, the 1st,5th,7th DCP group and the 1st,5th,7th DCS group significantly increased, the technical group was significantly lower (P< 0.05), to maximum values on 1st day; compared with the technical group, ICAM-1 expression in the 7th DCP group and the 7th DCS group decreased most significantly (P< 0.01); compared with the model group, the blank plasma group, the blank serum group, edaravone group and the technical group VCAM-1 expression in the 5th,7th DCP group and the 5th,7th DCS group was significantly reduced (P< 0.05), to the lowest value on 7th day; compared with edaravone group, ICAM-1mRNA and VCAM-1 mRNA expression in the 5th,7th DCP group and the 5th,7th DCS group decreased significantly (P< 0.05), to the lowest value on 7th day.BHD DCP and DCS can significantly reduce the content of vWF in BMECs with hypoxia culture, increased NO and 6-keto-PGFla levels, there was significant difference compared with the blank plasma and the blank serum (P< 0.05); DCP is more effective to reduce the content of PAI-1 and enhanced the level of tPA than DCS, DCP and DCS can significantly reduce the content of ICAM-1, VCAM-1, ICAM-1mRNA and VCAM-1mRNA, there was significant difference compared with the blank plasma and the blank serum (P< 0.05); The correlation coefficient of vWF in the DCP group and vivo experiment was higher than the serum group, there was significant difference; The'correlation coefficient of NO and 6-keto-PGF1? in the DCP group and vivo experiment was close to the DCS group, there was no difference; The correlation coefficient of PAI-1, tPA, ICAM-1, VCAM-1, ICAM-1mRNA and VCAM-1mRNA in the DCP group and vivo experiment was higher than the serum group, there was significant difference.ConclusionsBHD can obviously lower the cell apoptosis rate, there is the most obvious after treatment for 3 days; as well as decrease the scope of cerebral infarction and improve neurological symptoms integral, there is the most obvious after treatment for 7 days. BHD can inhibit platelet aggregation, inflammatory cell adhesion, aggregation and activation, inhibit antagonize ischemic brain injury, protect brain tissue by significantly elevating tPA expression, the most obviously on 1st day after treatment; decreasing vWF?PA I-1 level,the most obviously on 3rd day after treatment; increasing NO?6-keto-PGF1? level and reducing ICAM-1, VCAM-1, ICAM-1 and VCAM-1 mRNA,, the most obviously on 7th day after treatment.BHDDCP and DCS can significantly decrease the levels of vWF and PAT-1, increase no,6-keto-PGF1? and tPA, decrease the expression of adhesion molecules. The 3rd day DCP reduce vWF and PAI-1 levels most obviously, the 1st day DCP elevate of tPA level most significantly, The 7th day DCP reduce adhesion molecules level to the lowest value, increase NO,6-keto-PGF1? to the highest value. When BHD DCP or DCS is used in ex vivo experiment, it is suggested that blood is taken after administration for 3 days when detect the indexes of vWF and PAI-1; after administration for 1 day when detect the index of tPA; after administration for 7 days when detect the index of NO,6-keto-PGF1??VCAM-1 and ICAM-1.When Chinese herbal medicine compound is used to study the expression of vWF,6-keto-PGF1??PAI-1, tPA. VCAM-1?ICAM-1 and mRNA in the ex vivo experiment, it is suggested that the plasma pharmacology method should be priority preferred. and when study the expression of NO, both of plasma pharmacology and serum pharmacology method can be used. |
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