| Ischemic cerebrovascular disease(ICVD)is a common and frequently occurring disease among middle aged and elderly people,accounting for 80% of all kinds of strokes.The brain is sensitive to hypoxia,which not only causes serious local brain tissue damage and functional damage,but it also makes the ischemic damage turn into irreversible damage when the blood flow is restored.After isch emic cerebrovascular disease,the cognitive function of patients is often damaged,resulting in sensory dysfunction,motor nerve dysfunction,mental consciousness abnormality,change of judgment ability,decrease of IQ score,decline of memory ability,change of personality type,depression,and so on.Hippocampus is closely related to learning,memory and cognitive function.Cerebral ischemia-reperfusion leads to serious damage of hippocampal neurons,especially oxidative stress injury and increase of neuron apoptosis,which maybe the main cause of cognitive dysfunction.Butylphthalide,a synthetic compound based on l-3-n-butylphthalide,is isolated from celery seeds and used as the treatment of ischemic stroke,which has been approved by China food and Drug Administration in 2002.Many clinical studies show that butylphthalide not only improves the symptoms of ischemic stroke,but also contributes to long-term rehabilitation.The potential mechanism of butyl phthalide in the treatment of ischemic stroke may be related to the protection of neurons from multiple targets.The effects of butylphthalide on the cognitive function of rats after cerebral ischemia-reperfusion injury,as well as the effects of oxidative stress injury and mitochondrial dysfunction in its mechanism,have not been reported.Objective:Inthis study,the rat model of cerebral ischemia-reperfusion was established by the method of thread thrombus,and the improvement of neurologic function and cognitive function of the rats was observed.The study has explored whether butylphthalide can protect neurons by reducing oxidative stress and im proving mitochondrial function.Methods: 80 adult healthy male SD rats were randomly divided into sham operation group,cerebral ischemia-reperfusion model group,low-dose butylphthalid e treatment group and high-dose butylphthalide treatment group.The model of cerebral ischemia-reperfusion was established by the method of reperfusion after 2hours of thread occlusion,while the sham operation group did not insert thread occlusion.All the surviving rats were included in the experiment.On the secon d day,the treatment group was given 40 mg/kg butylphthalide and 80 mg/kg but ylphthalide by gavage once a day for 7 days.The sham operation group and the model group were given corn oil of the same volume.The neurological deficit score(longa method)was performed 7 days after the treatment of rats,and the behavioral test was performed about 1 month after the operation.Morris water maze was used to carry out positioning navigation experiment and space exploration experiment to evaluate the spatial learning ability and spatial memory ability of rats.HE staining was used to detect the morphological changes of hippocampal neurons,meanwhile,TUNEL staining and caspase-3 /caspase-9 expression of cortical neurons was used to detect apoptosis.The experiment has isolated the hippocampal neurons according to the potential change of cell mitochondrial membr ane by JC-1 staining flow cytometry and detected the content of ROS in neurons by DCFH-DA fluorescence staining,and assessed the mechanism of apoptosis of cerebral ischemia rats' cognitive dysfunction induced by mitochondrial membrane potential.Results:(1)Cerebral ischemia-reperfusion model in rats was established successfully by the method of thread thrombus.The rats in the sham operation group did not have neurological deficit,and the rats in the model group had the highest neur ological deficit score,while the rats in the low-dose butylphthalide treatment group had a downward trend,while the rats in the high-dose butylphthalide treatme nt group had a decline of nearly 40% compared with the model group,with a statistical difference(P <0.05).(2)One month after the establishment of the model,the spatial learning ability of the rats were detected by the navigation experiment.After five days,with the days passed,the escape latency of the rats in each group was significantly shortened,and the escape latency of the rats in the cerebral ischemia-reperfusion model group was significantly longer than that in the sham operation group(P<0.05);the rats in the low-dose and high-dose butylphthalide treatment group were significantly lower than those in the cerebral ischemia-reperfusion model group(P<0.05)With the increase of the dose of butylphthalide,the escape latency time was shorter(P<0.05).(3)On the second day after the completion of the navigation experiment,the spatial memory ability of the rats were detected,the space exploration experim ent was carried out to observe and record the target quadrant dwell time of the four groups,and it was found that the brain ischemia-reperfusion model group was significantly lower than the sham operation group,with significant difference(P<0.05);the butylphthalide treatment group was significantly higher than the bra in ischemia-reperfusion group,with significant difference(P<0.05);the high-dose butylphthalide treatment group was higher than the low dose butylphthalide treat ment group.(4)HE staining was used to observe the morphological changes of the brain tissue cells after cerebral ischemia-reperfusion.It was found that the loss of cells in the model group was serious,and the large area of neurons was necrotic,especially in the CA1 area of the hippocampus.After the treatment of low and high doses of butylphthalide,the damage of neurons decreased to a certain extent,indicating that butylphthalide has a protective effect on hippocampal neurons after cerebral ischemia-reperfusion in rats.(5)TUNEL was used to detect the apoptotic state of brain cells.It was found that the expression of TUNEL was significantly increased in the model group of cerebral ischemia-reperfusion.The number of TUNEL positive cells in the hippocampus of the high-dose butylphthalide treatment group was significantly lower than that of the low-dose butylphthalide treatment group and the model group of cerebral ischemia-reperfusion,the difference was statistically significant(P<0.001).(6)From the extraction of protein from hippocampus,the expression of apoptosis related protein Caspase-3 and caspase-9 were detected by Western blot.The results showed that the expression of Caspase-3 / caspase-9 was low in sham operation group,but significantly increased in cerebral ischemia-reperfusion model group.After treatment with butylphthalide,the expression of Caspase-3 / Caspas e-9 decreased,especially with the increase of the group dose of butylphthalide,t he expression of Caspase-3 / Caspase-9 decreased more obviously(P<0.05).(7)After JC-1 staining of isolated hippocampal neurons,the potential change of mitochondrial membrane was detected by flow cytometry.The results showed that the mitochondrial membrane potential in the model group was 47.7% lower than that in the sham operation group,and that in the butylphthalide group,the mitochondrial membrane potential was significantly increased,especially in the h igh-dose butylphthalide group,the effect was best recovered to 80% of that in the sham operation group(P<0.01),It is suggested that butylphthalide can significa ntly improve the mitochondrial damage of hippocampal neurons caused by cerebr al ischemia-reperfusion injury.(8)After labeling ROS in neurons with DCFH-DA fluorescent probe,through observing and taking photos by fluorescence microscope,the results showed th at there was only a small amount of green fluorescence of DCFH in the cells of sham operation group,while the green fluorescence of DCFH in the cells of cerebral ischemia-reperfusion model group increased significantly,which indicated that ROS was increased.However,the green fluorescence intensity was significant ly decreased after treatment with butylphthalide compared with model group(P <0.01),indicating that butylphthalide can significantly inhibit ROS produced in th e process of cerebral ischemia-reperfusion injury and reduce oxidative stress injury of neurons.Conclusion: Butylphthalide can improve the neural function and cognitive function of rats with cerebral ischemia-reperfusion injury,especially the large dose of butylphthalide can effectively improve the neural function score,improve the spatial learning ability and spatial memory ability of rats in the treatment group.At the same time,butylphthalide can significantly improve the mitochondrial membrane potential of hippocampal neurons in rats with cerebral ischemia-reperfusio n injury,reduce the production of reactive oxygen species in brain cells,reduce the expression of Caspase-3 / Caspase-9,inhibit the apoptosis of neurons,and it is beneficial to the functional recovery of neurons in hippocampus of rats with cerebral ischemia. |
PDF Full Text Request