| Background and ObjectiveVascular dementia (VD) is in the second place in dementias during the elderly man, and many studies have shown that VD is in the first place in the elderly patients with dementia, higher than Alzheimer’s disease (AD) in Asia. With the aging of society and life expectancy increasing, cerebrovascular disease is increasing more and more, which makes VD incidence increased year after year. As the disease has high morbidity and disability, poor treatment efficacy, and longer duration, it brings a heavy financial and emotional burden to society and the patients’s family. Therefore, the research of VD and its treatment is an important issue in the21st century, which causes widespread concern around the world by scholars and practitioners.Drugs for the treatment of VD in current were cholinesterase inhibitors, increasing the neurotrophic agents, agent metabolic activation of brain cells, nerve growth factor and nutritional factors, antioxidants and so on, but the effects are not clear. Dl-NBP was firstly used in the treatment of ischemic stroke, and has got satisfactory result. These years, it was used for VD’s treatment, but the effects and mechisms are poorly understood and studied.Ischemic is the based incidence of VD, and one of the mechanisms of VD is the nerve’s losing, or the synaptic’s decreasing, Therefore if we can repair and remodel the nerve after nerve injured,we may improve the symptoms associated with VD or cure VD. Nerve repairing and synaptic remodeling have become a hot topic of medical attention, and a large number of experiments have confirmed that nerve repair and synaptic remodeling are existed, and GAP-43and P38are the two important factors in the remodeling of nerve damage. In the study, we will observe that the learning and memory and GAP-43’s and P38’s changes in protein and gene expression level of VD rat mode and the efficacy after the treatment by Butylphthalide injection, and then learn the efficacy and mechanism of Butylphthalide on VD through the establishment of vascular dementia rat model.Materials and methods1Animals and groups80healthy male Wistar rats (From experimental Animal Center of Zhengzhou University), weighing350-450g, aged14-15weeks,were randomly divided into sham operation group (n=25), VD group (n=28), Butylphthalide (NBP) group (n=27);three of the VD group was killed and three of the Butylphthalide group was killed during the experiments.2Models and medication VD models were establistted using a permanent bilateral common carotid arteries occlusion(2-VO), Butylphthalide group was given Butylphthalide injection6mg/kg (made of emulsion before use) since operation by intraperitoneal injection once a day, a total of6weeks, and false surgery group and the VD group were given the same dose of saline intraperitoneally once a day, a total of6weeks.3Measurement of learning and memory The ability of learning and memory was measured by MG-Y-type maze after intraperitoneal injection. The rats which were subjected to electric shocks from the starting area after fleeing to safer areas directly are considered as the correct response, and nine correct reactions in continuously10tests are considered to reach the learning standards. Learning test: the numbers of electric shocks required before reaching the learing were counted standard. Memory capacity test:24hours after the learning test, numbers of the correct shock reaction in10electric shocks were recerded as a memory performance.4Specimens preparation After the learning and memory test, the rats were anesthetized, three rats of each group were took out to perfusion fixation through the heart, and then brain hippocampus was removed and fixed in formalin to observe the morphological changes by HE staining; the remaining rats were anesthetized and brains were removed quickly, isolated from the hippocampus (ice operation) was isolated quickly from their brains and immediately stored in liquid nitrogen, and performed into two detection systems:mRNA detection and protein detection. The specimens were almost the same in both systems.5Indices of observation The protein and mRNA quantitative of GAP-43and P38were determined by Western blotting and by RT-PCR technology; the morphological changes were detected by HE staining..6Statistical Methods All experimental data was expressed by mean±standard error (x±s), using AN OVA to analyze the comparison of data among multiple sets, and the S-N-K method between groups, the statistical software SPASS17.0for windows was used to analysis data, a=0.05Result1. Y maze learning and memory VD rats are the worst group (study grades71.60±3.97, the memory score4.24±1.20), Butylphthalide group center (study grades53.04±3.57, the memory score6.20±0.93), while the sham group are the most capable of learning and memory (study grades39.88±3.97, the memory score7.36±1.25), the results among the three groups are significantly different, P<0.05.2. Morphology of neurons in the hippocampus after HE staining In the light microscope after HE staining was observed, the specimens of in sham group: Neurons packed tightly, with more quantity, large clear nuclei, stained uniform, prominent nucleoli; in VD group:neurons rarely, arrangement, deeply stained nuclei, condensation, increased cytoplasm, the nucleolus disappears; in Butylphthalide group:normal cells are more than these in VD group, but less than these in the sham group, arranged in dense, oval or round nucleolus, rich in chromatin, condensation rare degeneration of neurons. 3. mRNA determination results The expression of GAP-43mRNA in butyl phthalite group and VD group was higher than the sham group (P<0.05), and Butylphthalide group was significantly higher than the VD group(P<0.05); in P38mRNA of the expression, Butylphthalide group were increased compared with the sham group,and VD group (all P<0.05), while no significant difference between the VD group and the sham group (P>0.05); Sham group, VD group, Butylphthalide group GAP-43mRNA expression were:0.49±0.05,1.11±0.18,1.31±0.06; P38mRNA were:0.58±0.06,0.60±0.06,1.28±0.04.4. Protein determination results In GAP-43protein expression, BNP group were increased compared with the sham group and the VD group (all P<0.05), while the sham group and the VD group, no significant difference (P>0.05); in P38protein expression, VD group, Butylphthalide group decreased compared with the sham group (P<0.05), but the VD group decreased more significantly (P<0.05).The Sham group, VD group, Butylphthalide GAP-43protein group were:1.17±0.05,1.19±0.05,1.61±0.04; P38protein were:1.71±0.04,1.32±0.06,1.57±0.05.Conclusion1The reduced expression of P38protein within the hippocampus is closely related with the learning and memory harm in rats which was the molecular mechanisms of VD leading to cognitive dysfunction;2The mRNA and protein of GAP-43±、P38mRNA are not be the molecular mechanisms involved in cognitive impairment of VD;3the mRNA and protein expression of GAP-43and P38are the molecular mechanisms of cognitive impairment improved by Butylphthalide injection on VD,4Butylphthalide can improve learning and memory in rats with VD, through increasing GAP-43protein expression which can increase P38protein expression directly or indirectly, thereby contributing to synaptic remodeling and new synapse formation,... |
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