| Objective:To observe and compare the protective effect of Tongqiao Huoxue Decoction on cerebral ischemia-reperfusion injury by three different methods,and to explore-glutamine circulating metabolism pathway in astrocytes.Methods:The adult male Sprague-Dawley rats of grade SPF were randomly divided into sham operation group and to preparation model,s group.The sham operation group only separated blood vessels and did not enter the line.The other rats were modified to prepare the rat model of middle cerebral artery occlusion reperfusion injury.Then the rats were randomly divided into model group,water preparation of Tongqiao Huoxue Decoction group,wine preparation of Tongqiao Huoxue Decoction group and alcohol extraction preparation of Tongqiao Huoxue Decoction group.The sham operation group and model group were treated with equal volume of saline,water preparation of Tongqiao Huoxue Decoction group,wine preparation of Tongqiao Huoxue Decoction group and alcohol extraction preparation of Tongqiao Huoxue Decoction group were treated with 6.3 g·kg-1·d-1solution.One day after the operation began to gavage medication,continuous treatment for 7 days.Observe the general situation of rats and score the m NSS neurofunction defect to detect the recovery of neurofunction in rats;HE staining observed the morphological changes of brain tissue in rats,Western Blot detection of GFAP、GLT-1 and GS protein expression in rat brain tissue.HPLC was used to detect Glu changes in rat brain tissue.Results:1.Compared with the sham operation group,the weight of the model group was significantly decreased,and the difference was statistically significant（P<0.01）.Compared with the model group,the weight of rats in the three groups treated with Tongqiao Huoxue Decoction increased significantly（P<0.05）.There was no significant difference between the three groups treated with Tongqiao Huoxue Decoction.2.The m NSS scores in sham operation group and model group compared to prior treatment in the same group are no significant difference.The m NSS scores in three groups treated with Tongqiao Huoxue Decoction were significantly reduced（P<0.05）.After 7 days of continuous treatment,the m NSS score in model group was significantly higher compared with the sham operation group,and the difference was statistically significant（P<0.01）.Compared with the model group,the m NSS scores of water preparation of Tongqiao Huoxue Decoction group,wine preparation of Tongqiao Huoxue Decoction group and alcohol extraction preparation of Tongqiao Huoxue Decoction group were significantly decreased,and the difference was statistically significant（P<0.01）.There was no significant difference between the three groups treated with Tongqiao Huoxue Decoction（P>0.05）.3.He staining showed that neurons in the cerebral cortex and hippocampus of the sham operation group were arranged neatly,with complete morphological structure,normal cell size and clear nucleoli.In the model group,the cerebral cortex presented large necrosis,disordered arrangement,cell edema and inflammatory infiltration.Compared with the model group,the morphology and structure of cerebral cortex and hippocampal nerve cells in the three groups treated with Tongqiao Huoxue Decoction were improved,and the degree of injury was reduced to some extent.4.Immunofluorescence results showed that compared with the sham group,the colocalization of GLT-1 and GFAP and the colocalization of GS and GFAP in the model group brain tissues were significantly decreased（P<0.01）.Compared with the model group,the colocalization of GLT-1 and GFAP in water preparation of Tongqiao Huoxue Decoction group and the colocalization of GS and GFAP in wine preparation of Tongqiao Huoxue Decoction group were significantly increased（P<0.05）,and the colocalization of GS and GFAP in water preparation of Tongqiao Huoxue Decoction group was increased,but the differences are not statistically（P>0.05）,the colocalization of GLT-1 and GFAP in wine preparation of Tongqiao Huoxue Decoction group were significantly increased（P<0.01）,the colocalization of GLT-1 and GFAP and the colocalization of GS and GFAP in alcohol extraction preparation of Tongqiao Huoxue Decoction group were significantly increased（P<0.01）.Compared with water preparation of Tongqiao Huoxue Decoction group,the colocalization of GLT-1 and GFAP and the colocalization of GS and GFAP in the wine preparation of Tongqiao Huoxue Decoction group were no significant difference,the colocalization of GLT-1and GFAP and the colocalization of GS and GFAP in the alcohol extraction preparation of Tongqiao Huoxue Decoction group were significantly increased（P<0.05）.Compared with wine preparation of Tongqiao Huoxue Decoction group,the colocalization of GLT-1 and GFAP and the colocalization of GS and GFAP in alcohol extraction preparation of Tongqiao Huoxue Decoction group were increased,but the differences are not statistically（P<0.05）.5.After the treatment,Western Blot analysis showed that the expression of GFAP in the model group was significantly increased,the expression of GLT-1 was significantly decreased than that in the sham group（P<0.01）,the expression of GS was significantly decreased than that in the sham group（P<0.05）.Compared with the model group,the expressions of GFAP and GLT-1 in water preparation of Tongqiao Huoxue Decoction group were significantly increased（P<0.05）,GS expression was increased in the water preparation of Tongqiao Huoxue Decoction group,but the differences are not statistically（P>0.05）,the expression of GFAP,GS and GLT-1 were significantly increased in alcohol extraction preparation of Tongqiao Huoxue Decoction group（P<0.01）.Compared with water preparation of Tongqiao Huoxue Decoction group,the expression of GFAP and GLT-1were not significantly increased in the wine preparation of Tongqiao Huoxue Decoction group（P>0.05）,the expression of GS in wine preparation of Tongqiao Huoxue Decoction group was significantly increased（P>0.05）,the expression of GFAP in alcohol extraction preparation of Tongqiao Huoxue Decoction group was significantly increased（P<0.01）,the expression of GLT-1 and GS in alcohol extraction preparation of Tongqiao Huoxue Decoction group were significantly increased（P<0.05）.Compared with wine preparation of Tongqiao Huoxue Decoction group,the expression of GFAP in alcohol extraction preparation of Tongqiao Huoxue Decoction group was significantly increased（P<0.01）,and the expression of GLT-1 in alcohol extraction preparation of Tongqiao Huoxue Decoction group was significantly increased（P<0.05）,the expression of GS in alcohol extraction preparation of Tongqiao Huoxue decoction group was not statistically significant（P>0.05）.6.HPLC results showed that compared with the sham operation group,the expression of Glu was significantly increased in model group and the difference was statistically significant（P<0.01）.Compared with the model group,the expression of Glu decreased significantly in the three groups treated with Tongqiao Huoxue Decoction,and the difference was statistically significant（P<0.05）.Compared with water preparation of Tongqiao Huoxue Decoction group,the expression of Glu were significantly decreased in alcohol extraction preparation of Tongqiao Huoxue Decoction group,and the difference was statistically significant（P<0.05）,there was no statistical difference in wine preparation of Tongqiao Huoxue Decoction group（P>0.05）;Compared with wine preparation of Tongqiao Huoxue Decoction group,the expression of Glu were significantly decreased in alcohol extraction preparation of Tongqiao Huoxue Decoction group,and the difference was statistically significant（P<0.05）.Conclusion:The three kinds of Tongqiao Huoxue Decoction can improve the nerve function of model rats with CIRI.The alcohol extract group of Tongqiao Huoxue Decoction had the most obvious effect,followed by wine preparation of Tongqiao Huoxue Decoction group and water preparation of Tongqiao Huoxue Decoction group.The mechanism may be achieved by promoting the further activation of astrocytes,increasing the expression of GLT-1 and GS,promoting the clearance of Glu accumulated in the synaptic cleft by astrocytes through the glutamate-glutamine cycle,and reducing the excitatory toxicity of Glu. |
