| ObjectiveThe subject is based on the theory of "blood-brain" of traditional Chinese medicine.It takes SCF/c-kit signal regulation pathway as the research entry point and establishes MCAO/R rat model.Using the method of “Nao Luo Xin Tong” which is the representative party of Xin'an Wang's internal medicine characteristic method to intervene it to observe the angiogenesis in the ischemic lateral parietal cortex of rats and the change of STAT3,Raf-1,MMP-9,CD105 and other proteins.On the other hand,it explores the effect of and mechanism of Yiqi Huoxue Tongluo on angiogenesis after cerebral ischemia,which providing experimental basis for the rational application and development of Yiqi Huoxue Tongluo Method and its representative party(Nao Luo Xin Tong).MethodsThere were 84 healthy male Sprague-Dawley rats,weighing 300±20g,and those weree randomly divided into sham operation group,model group,Tongxinluo group and Naoluoxintong group.Each group had 21 rats and three subgroups.Reperfusion was performed for 3 days,7 days,and 14 days,with 7 in each subgroup.The MCAO/R rat model was prepared by modified suture method,and reperfused after 2 hours of obstruction.According to the Zea Longa scoring criteria,they were selected which the neurological deficit score was scored 2-3 points.Equivalent measurement was carried out according to the conversion coefficient of human and rat body surface area.Naoluoxintong and Tongxinluo were intragastrically administered twice a day at a dose of 1.143g/kg and 0.033g/kg.Respectively,after 3 days,7 days and 14 days,the brain was opened.The HE staining was used to observe the pathological changes of the ischemic cortex in different groups of rats at different time points.Immunohistochemical Envision two-step method was used to detect the positive area of MMP-9 and CD105 protein expression in the ischemic cortex of rats in different groups at each time point.Western-Blot was used to detect the relative expression of STAT3 and Raf-1 proteins in the ischemic cortex of rats in different groups at each time point.The obtained measurement data were expressed as mean±standard deviation(x±s),and statistical processing was performed using IBM SPSS Statistics 23.0.Meanwhile,One-way ANOVA was used between different groups at the same time point and LSD was used for pairwise comparison,which P<0.01 or P<0.05 was statistically significant.The charts were created using GraphPad Prism 5 software and the statistics were created using word 2010 software.ResultsThe results of HE staining showed that the neurons in the sham operation group were arranged neatly and the tissue morphology was normal.Compared with the sham operation group,the phenomenon of the edema and neuronal degeneration and necrosis were obvious in the model group at various time points,accompanied by inflammatory cell infiltration,and the reaction was most obvious in 3 days.After the intervention of Naoluoxintong and Tongxinluo,the damage of each group at each time point was reduced to a different extent than that of the model group.Neuronal degeneration,necrosis decreased,nuclear pyknosis decreased,and inflammatory cell infiltration improved.There was no significant difference between the Naoluoxintong group and the Tongxinluo group.The results of immunohistochemistry showed that MMP-9 was mainly expressed in the cytoplasm of neuronal cells,vascular endothelial cells and glial cells in the frontal cortex of the ischemic side.The yellow-brown sediment of CD105 is mainly in the cytoplasm of vascular endothelial cells in the cortex of the frontal parietal lobe of the ischemic side.The expressions of MMP-9 and CD105 in the different groups were significantly different at 3,7 and 14 days after reperfusion(P<0.01).In the sham operation group,MMP-9 and CD105 proteins in the frontal cortex of the brain were expressed in small amounts at different time points.The positive area of MMP-9 and CD105 protein expression in the model group peaked in the 3-day group,and decreased in 7 days and 14 days.Compared with the sham operation group,the positive areas of MMP-9 and CD105 protein expression in the simultaneous model group were significantly increased,and the difference was statistically significant(P<0.01).The positive areas of MMP-9 and CD105 protein expression in the Naoluoxintong group and Tongxinluo group increased significantly at each time point,and were the highest in the 3-day group,and gradually decreased in 7 days and 14 days.They were significantly higher than the model group.Compared with Tongxinluo group,there was no significant difference in the positive area of MMP-9 protein expression at different time points in the Naoluoxintong group(P>0.05).Western-Blot detection showed that the expression of STAT3 and Raf-1 in the different groups were significantly different at 3d,7d and 14d(P<0.01).There were a small amount of STAT3 and Raf-1 expression in the brain tissue of the sham operation group.Compared with the sham operation group,the expression levels of STAT3 and Raf-1 protein in the model group increased significantly(P<0.01).The expression levels of STAT3 and Raf-1 protein in the Naoluoxintong group and Tongxinluo group were significantly higher than those in the model group(P<0.01).There was no significant difference in the expression of STAT3 and Raf-1 protein between the Naoluoxintong group and the Tongxinluo group(P>0.05).ConclusionOn the basis of previous studies on SCF,c-kit protein and regional cerebral blood flow,this experiment found that the Yiqi Huoxue Tongluo method and its representative Naoluoxintong could up-regulate the expression of CD105 protein and increase the number of vascular regeneration after cerebral ischemia,as well as up-regulating the expression of STAT3,Raf-1,MMP-9 protein,regulating SCF/C-kit signaling pathway and promoting vascular regeneration after cerebral ischemia.Vascular regeneration after cerebral ischemia-reperfusion plays a therapeutic role in preventing focal cerebral ischemia-reperfusion injury. |
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