| BackgroundAcute cerebral infarction is caused by acute cerebral ischemia due to various factors.The ischemic area may involve cerebral cortex,subcortical gray matter nucleus and subcortical white matter.Most previous studies focused on the gray matter region,and acute subcortical white matter infarction can lead to serious clinical consequences,which deserves attention.Its clinical symptoms can be manifested as light hemiplegia or partial body sensory disorder,aphasia,executive ability decline caused by vascular dementia.The clinical pathology was lacunar infarction in subcortical white matter,brainstem,thalamus and basal ganglia.Early therapeutic intervention can not only delay the development of cerebral infarction,but also promote the recovery of neurological function and alleviate clinical symptoms.In traditional Chinese medicine,acute cerebral infarction is classified into the category of ischemic stroke,and its etiology and pathogenesis can be summarized into six aspects:deficiency,fire,wind,phlegm,qi and blood,among which blood stasis is both the pathogenic factor and pathological product.In recent years,academician Wang Yongyan proposed the etiology theory of "poison damage brain collaterals",which has enriched the understanding of the etiology of ischemic stroke in traditional Chinese medicine.The etiological theory of toxic damage to the brain collaterals reflects the abnormal transformation between relevant material bases and signal pathways in vivo,including vascular endothelial cells,smooth muscle,vasoactive substances,cytokines and signal transduction pathways.In clinical practice,for such patients with acute subcortical white matter infarction with blood stasis and toxin damage to brain collaterals,the application of the representative drug of activating blood circulation,detoxifying toxins and dredging collaterals-Kudiezi injection,can effectively relieve the damage of cerebral infarction.However,the mechanism of how Kudiezi injection regulates ephrin/Eph signal transduction pathway to repair damaged blood-brain barrier and remodeling neurons is still unclear.Therefore,this study aims to explore the correlation between the syndrome of blood stasis and toxin damage and the activation of ephrin-A5 signaling pathway in acute subcortical white matter infarction,and the mechanism of neural remodeling effect of Kudiezi injection on acute subcortical white matter infarction by regulating ephrin-A5 signaling pathway.ObjectiveTo observe the changes of ephrin-A5,Eph B2 gene and protein expression of white matter and hippocampus on the ischemic side of acute subcortical white matter infarction in rats with blood stasis and toxic damage syndrome,and to explore the relationship between these changes and acute cerebral ischemia he dynamic changes of Ephrin-A5,Eph B2 gene and protein expression in white matter and hippocampus of rats with acute subcortical white matter infarction with blood stasis and toxic damage,and to explore the relationship between these changes and ischemic damage,investigating the neural remodeling effect of Kudiezi injection on the syndrome of blood stasis and poison damage in acute subcortical white matter infarction and its mechanism from the level of gene and protein.Methods100 Wistar rats were randomly divided into three groups:model group,treatment group,and sham operation group.A total of 6 rats were included in the sham operation group,36 rats were included in each of the other two groups,and the remaining 22 rats were reserved.The model group and the treatment group were firstly injected intraperitoneally with carrageenan,and then the left middle cerebral artery was occluded by thread embolism to establish the rat model of acute subcortical white matter infarction with blood stasis and toxic damage syndrome.The treatment group was intraperitoneally injected with Kudizi injection(8.4 g/(kg·d))at different time points after operation,and the sham operation group and the model group were intraperitoneally injected with normal saline.The injection dose,frequency and time were the same as that of the treatment group.The samples were taken at 1,3,6,24,72 h and 7 d after modeling,then Western Blotting and RT-PCR were used to detect the expression of ephrin-A5 and Eph B2 in the ischemic white matter and hippocampus of rats in each group.Results1.Neurological function score of rats in each group:The behavioral performance of the sham operation group without neurological function deficit was rated as 0.The treatment group and the model group showed different degree of neurological impairment in behavioral manifestations.Compared with the sham operation group,the scores of the model group and the treatment group increased at 1,3,6,24,72 h and 7 d after ischemia.The scores of the model group were higher than those of the treatment group at 24,72 h and 7 d after ischemia(P<0.05),and there was no significant difference between the model group and the treatment group at 1,3 and 6h after ischemia(P>0.05).Intra-group comparison:There was no statistical difference in the scores of model group at each time point after ischemia(P>0.05),and the scores of the 7 d group were significantly lower than that of the 24 h group(P<0.05).2.Expression of ephrin-A5 and Eph B2 genes in ischemic white matter and hippocampus:The white matter:Compared with sham operation group,ephrin-A5 mRNA(P<0.01)and Eph B2 mRNA(P<0.05)were highly expressed in model group on day 7.The hippocampus:Compared with sham operation group,ephrin-A5 mRNA expression was higher at 1 h and 7 d after ischemia(P<0.01),and Eph B2 mRNA expression was higher in model group at 1,3,6 h and 7 d after ischemia(P<0.01).3.Effects of Kudiezi injection on the expression of ephrin-A5 and Eph B2 Genes in ischemic white matter and hippocampus:The white matter:The expression level of ephrin-A5 mRNA in the treatment group was lower than that in the model group and the sham group at 72 h and 7 d after ischemia(P<0.01).The expression level of ephrin-A5 mRNA in the treatment group was lower than that in the model group and the sham group at 1,24,72 h and 7d after ischemia(P<0.01 or P<0.05).The hippocampus:At 1,6,24 h and 7 d after ischemia,the expression level of ephrin-A5 mRNA in the treatment group was lower than that in the model group and sham group(P<0.01 or P<0.05).At 1,3,6 h and 7 d after ischemia,the expression of Eph B2 mRNA in the treatment group was lower than that in the model group(P<0.01).The expression of Eph B2 mRNA in the treatment group was lower than that in the sham group at 1,72 h and 7 d after ischemia(P<0.01 or P<0.05).4.Expression of ephrin-A5 and Eph B2 proteins in ischemic white matter and hippocampus:The white matter:Compared with the sham operation group,the expression of ephrin-A5 protein was significantly higher in the model group at 1,3,6,24,72 h and 7 d after ischemia(P<0.01 or P<0.05),and the expression of Eph B2 protein was significantly higher in the model group at 1,3,24 h and 7 d after ischemia(P<0.01 or P<0.05).The hippocampus:Compared with the sham operation group,the expression of ephrin-A5 protein was significantly higher in the model group at 1,3,6,24,72 h and 7 d after ischemia(P<0.01 or P<0.05),and the expression of Eph B2 protein was significantly higher in the model group at 1,3,6,24,72 h and 7 d after ischemia(P<0.01 orP<0.05).5.Effects of Kudiezi injection on the expression of ephrin-A5 and Eph B2 Genes in ischemic white matter and hippocampus:The white matter:At 1,3,6,24,72 h and 7 d after ischemia,the expression of ephrin-A5 protein in the treatment group was lower than that in the model group(P<0.01 or P<0.05),and the expression of ephrin-A5 protein in the treatment group was lower than that in the sham group on day 7(P<0.01).At 1,3,24 h and 7 d after ischemia,the expression of Eph B2 protein in the treatment group was lower than that in the model group(P<0.01 or P<0.05).The expression of Eph B2 protein in the treatment group was lower than that in the sham group on day 7(P<0.01).The hippocampus:The expression level of ephrin-A5 protein in the treatment group was lower than that in the model group at 1,24,72 h and 7 d after ischemia(P<0.01 or P<0.05).The expression level of ephrin-A5 protein in the treatment group at 7d was lower than that in the sham group(P<0.01).At 1,6,24 h and 7 d after ischemia,the expression of Eph B2 protein in the treatment group was lower than that in the model group(P<0.01).The expression of Eph B2 protein in the treatment group was lower than that in the sham group on day 7(P<0.05).Conclusion1.After acute cerebral ischemia,the neural function of rats with acute subcortical white matter infarction was changed,and the expression of ephrin-A5 and Eph B2 gene and protein in the ischemic white matter and hippocampus were significantly changed.These results indicate that the ephrin-A5 signaling pathway is closely related to the occurrence and degree of nerve cell damage after acute subcortical white matter infarction2.Kudiezi injection can improve the neurological function deficit of rats with acute subcortical white matter infarction with blood stasis and toxic damage,interfere with ephrin-A5 signal transduction system,regulate the overexpression of ephrin-A5 and Eph B2 genes and proteins,so as to reduce the acute cerebral ischemia damage,repair the damage of blood-brain barrier and promote axon germination.It plays a role in neural remodeling.3.Traditional Chinese medicine of activating blood,detoxifying toxin and dredging collaterals,kudiezi injection may play a role in neural remodeling by interfering with ephrin-A5 signal transduction system after acute cerebral ischemia,providing a new idea and entry point for the treatment of acute subcortical white matter infarction with traditional Chinese medicine. |
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