| Ischemic stroke is a major problem in modern medical research,which is caused by various reasons including vascular occlusion.Compound angelica injection(CAI)is made from three traditional Chinese medicines of angelica,Chuanxiong,and safflower,having a good effect of promoting blood circulation.Previous studies found that CAI has a certain therapeutic effect on cerebral ischemic injury model.The concept of neurovascular unit(NVU)broadens the research field of cerebral ischemic injury.Blood-brain barrier(BBB)and neurons are important components of NVU,and are also important targets in the study of cerebral ischemic injury.This study will be based on the previous experimental results of the research group,starting from the neurovascular unit,to study the effect of CAI on cerebral ischemic injury.Objective:By establishing a model of permanent middle cerebral artery occlusion(MCAO)in rats,to study the effect of CAI on neurobehavioral function,brain tissue morphology and the expression of matrix metal protein(MMP)-2 and MMP-9,both of which are closely related to the integrity of BBB.The conditioned culture medium after CAI action on Brain micro vascular endothelial cells(BMECs)was used to neurons.To explore the effect of CAI on neuronal model of ischemic injury and its possible molecular mechanismMethods:(1)Behavior and morphology observation of MCAO rats:the sham operation group(normal saline 3 mL/kg)was randomly divided from SD rats.The rest of the rats were established on the right MCAO cerebral ischemia model.Z.Longa grade 5 scoring method was used to divide the MCAO rats into model group(3 mL/kg normal saline),positive control group(3 mL/kg edaravone injection)and CAI group(10 mL/kg CAI).After 1 day,3 days,7 days,and 14 days of intervention to evaluate the effect of CAI on neurological behavior in rats with cerebral ischemic injury.Hematoxylin-Eosn(HE)staining was used to observe the pathological and morphological changes of the brain tissue of rats in each group.(2)Immunohistochemical method was used to analyze the expression of MMP-2 and MMP-9 in the cortical tissues of cerebral ischemic area of rats in each group.(3)BMECs were primary cultured and passed to the third generation.and CD31 related antigens were used for immunofluorescence identification.BMECs were divided into normal group,model group and CAI low(L=1.25 ?L/mL),middle(M=2.5 ?L/mL),high(H=5 ?L/mL)dose groups.The model group and the CAI group used the Oxygen and glucose deprivation(OGD)method to establish the BMECs quasi-ischemic injury model.Collect BMECs' normal conditioned medium(N-CM),Ischemic-Conditioned medium(I-CM)and conditioned medium of different dose CAI groups after ischemic injury(IC-CM),in which 1.25?L/mL CAI acts on BMECs' IC-CM as IC-CML,2.5 ?L/mL CAI acts on IC-CM as IC-CMM,5?L/mL CAI acts on BMECs IC-CM as IC-CMH.Then,Neurons were primary cultured and immunofluorescence identified by NSE-associated antigens.The neurons were divided into 5 groups(N-CM,I-CM,IC-CML,IC-CMM,IC-CMH group),the corresponding endothelial cell conditioned medium was applied to neurons.Except for the N-CM group,the remaining groups were modeled with OGD for 6 h,and then the activity of each group of neurons was detected by CCK-8.(4)PI3K/Akt and MAPK/Erk pathway related index detection:PCR method was used to detect the expression level of Akt mRNA in each group of neurons,and Western blot method was used to detect the phosphorylation level(p-Akt/Akt)and Erk Phosphorylation level(p-Erk/Erk).Results:(1)The neurological damage in the model group gradually increased,the most severe on the 7th day,and the CAI can reduce the neurological damage in the rats.On the 7th and 14th days,the difference between the CAI group and the model group was statistically significant.HE staining results showed that the brain tissue of the model group at each time point showed obvious traumatic changes with obvious necrosis.The positive control group and the CAI group improved significantly after 7 and 14 days of intervention compared with the model group.(2)When cerebral ischemia,the expression of MMP-2 in the ischemic cortical area gradually increased with the increase of ischemic time.The expression peaked at 7 d and decreased at 14 d.Compared with the model group,CAI drug can reduce the expression of MMP-2,and the difference was statistically significant at 3 and 7 days.The high expression of MMP-9 appeared in the early stage,showing the highest at 1d,decreased at 3 and 7 d,and increased at 14 d.Compared with the model group,the expression level of CAI group decreased at 1 and 3 days,and statistical significance.(3)After OGD modeling,the activity of neurons in the I-CM treatment group was significantly lower than that in the N-CM treatment group.The neuron activity in the IC-CM treatment group after CAI intervention gradually increased with the increase of drug concentration.(4)Compared with the N-CM treatment group,the Akt mRNA expression of neurons in the I-CM treatment group was significantly reduced.The expression of Akt mRNA in neurons of IC-CM treatment groups increased gradually with increasing concentration.The difference between Akt mRNA in IC-CMH group and I-CM group was statistically significant The expression of p-Akt/Akt in neurons has similar trend as Akt mRNA expression results.Compared with the I-CM group,the IC-CML group was statistically significant,and the IC-CMM and IC-CMH groups were significantly different Compared with the N-CM group,the p-Erk/Erk of the neurons in the I-CM group increased significantly,and all IC-CM groups showed a decrease trend,but there was no difference in statistics compared to the I-CM group.Conclusion:CAI can improve the neurological function and pathological injury of model rats.Its effect may be related to the regulation of BBB-related mmp-2 and mmp-9 expression in the cortex of rats with cerebral ischemia injury.CAI-mediated BMECs conditioned fluid increased the survival rate of ischemic neurons,which may be related to the activation of PI3K/Akt signaling pathway. |
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