| BackgroundsThere are the elderly dementia in some chronic diseases has attracted more and more social attention, which has become an important global health problems and have great influence on the family and the national economy, due to the continuous population aging, this problem is expected to grow dramatically in the future. Therefore, understanding of the pathogenesis of dementia disease, the development of prevention and treatment is the priority. The damage to the central nervous system, no matter what the reason, can lead to cognitive dysfunction. The clinical phenotype caused by brain damage is the result of many factors, including the size and location of brain lesions, the loss of neurons, and in which the physiological and pathological changes of brain tissue occurred. Previously considered vascular dementia is the second common types of dementia after Alzheimer’s disease. But recently, vascular related cognitive decline is thought to be more common than previously thought of independent or associated with a neurodegenerative disease.The early view began in early twentieth century, dementia disease is caused by cerebral arteriosclerosis. In the 60’s, the researchers concluded that infarction volume in patients with greater development risks may exist for senile dementia. In the 70’s, Hachinski et al proposed a new term multi infarct dementia, implies a cumulative consequences after multiple cerebral infarction. With the rapid development of neuroimaging, some small vessel ischemic lesions, such as white matter lesions, lacunar infarction, eventually can cause cognitive impairment. VD the most common pathological mechanism of cerebral ischemia damage is caused. In our country vascular dementia prevalence rate is about the level of 1.1-1.3%, the annual incidence rate in the 5-9%, and with the Chinese people life improvement and the change of social environment on working pressure, showing a rising trend year by year. Vascular dementia (Vascular dementia, VD) will not only affect the patient’s self adjustment ability, but also greatly reduce the quality of life, will make the patient’s family appear larger economic difficulties, and even cause social heavy economic burden, so the research field of the prevention and treatment of vascular dementia is more important. So the research field of the prevention and treatment of vascular dementia is more important, is also a hot field, it need of attention and solution to deal with the society and medical problem at present in our country.Glycosides of cistanche (GC) is extracted from Xin Jiang Cistanche, which has been widely used as a Chinese herb with neuroprotective effects. Cistanches Herba extract enhance learning and memory of mice through promoting neuronal cell differentiation, neurite growth, and synapse formation. GC can prevent apoptosis in cerebellar granule neurons and exert anti-apoptosis effect by inhibiting the activition of caspase-3 and caspase-8. Echinacoside, a major active component of Cistanches Herba, is sufficient to protect neuronal cells from rotenone injury by activating Trk signaling. However, the mechanisms by which GC improves cognitive impairment in VD are still largely unknown.Part 1 The making of vascular demenia rat model and the effect of GC on the behavior test of VD ratsVD is built on the basis of cerebrovascular diseases, in field of memory, behavior, cognitive impairment, the most common pathological mechanism of VD is caused by cerebral ischemia damage, as in second types of dementia after Alzheimer’s disease. Cerebral ischemia can lead to brain hypoxia, glucose metabolism, reduce the degree of the situation. The hippocampus is a senior activity of learning and memory, very sensitive to the brain tissue ischemia and hypoxia, the model of 2-VO rats, Wistar rats by permanent ligation of bilateral common carotid artery, this model has the advantages of simple manufacture, and stable, is the classic animal model to study the cognitive activity. At present, treating vascular dementia lack in effective drug, GC is extracted from Xinjiang, the alcohol glycosides as the main component, has protective effect on cerebral ischemia and hypoxia.Oxiracetam as drug control group, to evaluate the protective effect of glycosides of Cistanche on learning and memory function of vascular dementia model rats using the Morris water maze test method.Objective:To prepare the rat model of vascular dementia, the glycosides of Cistanche (glycosides of Cistanche, GC) improve the function of learning and memory on vascular dementia (vascular dementia, VD) and explore the possible mechanism of protection from proteomics and immunohistochemistry.Methods:The bilateral common carotid artery occlusion (2-VO) rat model of VD was established, and randomly divided into sham operation group, VD model group, GCs treatment group, oxiracetam drug control group, and then received daily i. p. administration of saline, GC (10 mg/kg/d) or oxiracetam (450mg/kg/d) for 14 days. The sham animals were injected with the same volume of normal saline, and then underwent the morris water maze testing (MWM) to elucidate cognitive performance.Results:(1) The Morris water maze experiment proved VD model group rats place navigation test escape latency was prolonged, there was significant difference compared with the sham operation group (p<0.01), from the first day, VD model group and sham operation group, the escape latency difference have no statistics significance, At fifth the days the escape latency of VD model group was 60.2 ± 18.9s, sham operation group was 32.5 ± 9.9s,the escape latency time of VD group was significantly prolonged, and found that the VD model group with the extension of training days, escape latency gradually shortened, the first day of training the escape latency was 82.7 ± 22.3s, the fifth days escape latency gradually cut down to 60.2 ± 18.9s, may be related to physical training rats.(2)After GC treatment, in the fourth, fifth day VD rats escape latency differently were 41.2 ± 20.0s,38.6 ± 15.2s, there was significant difference compared with the VD model group (p<0.05); GC group, oxiracetam drug control group and sham operation group had no statistical difference.Conclusion:This experiment in rats through Morris water maze platform escape latency were compared, found from the first day, VD model group and sham operation group, the escape latency was not significantly different between the analysis.According to the route map recorded in rats, the rats in the initial search platform for random or edge based, time that find the platform needs spend a relatively long time. The fifth days VD model group escape latency difference increased significantly than those in the sham operation group. And found that the VD model group with the extension of training days, escape latency gradually shortened, because the VD rats look for platform from the beginning of the random or edge type, at last search platform type change straight type. In the use of glycosides of Cistanche after treatment, rat escape latent period compared with the VD model group was significantly shorter. Through Morris water maze training a few days later, VD rat model were gradually observed improvement of the ability of learning and memory.Conclusion:This experiment in rats through Morris water maze platform escape latency were compared, found from the first day, VD model group and sham operation group, the escape latency was not significantly different between the analysis.According to the route map recorded in rats, the rats in the initial search platform for random or edge based, time that find the platform needs spend a relatively long time. The fifth days VD model group escape latency difference increased significantly than those in the sham operation group. And found that the VD model group with the extension of training days, escape latency gradually shortened, because the VD rats look for platform from the beginning of the random or edge type, at last search platform type change straight type. In the use of glycosides of Cistanche after treatment, rat escape latent period compared with the VD model group was significantly shorter. Through Morris water maze training a few days later, VD rat model were gradually observed improvement of the ability of learning and memory.Part 2 The effect of glycosides of Cistanche on the expression of p-tau protein and proteomics in vascular dementia ratVD, the second leading senile dementia, is mainly caused by ischemic cerebrovascular disease. Although mechanisms of VD underlying cognitive impairment are heterogeneous, these mechanisms can affect hippocampus and other limbic neural cells, which result in memory, cognitive function impairment. GC is widely used as a Chinese herb with neuroprotective effects. Using modern pharmacology, numerous studies indicate that GC has become a critical focus of basic research that could yield potential novel therapeutic tools to overcome the disease.The tau protein is a microtubule associated protein, mainly to promote microtubule assembly, maintain the stability and integrity of the microtubule cytoskeleton, further stabilizing the axoplasmic transport of normal. Found the hyperphosphorylation of tau protein in AD of patients with brain tissue, suggesting that this protein leads to a decrease in microtubule assembly ability, microtubule stability is poor, affecting the synthesis, transport, uptake and release of synaptic neurotransmitter, may eventually lead to the decline of cognitive function in patients with AD. Proteomics as a new discipline, research protein components and activity orderliness in various physiological and pathological state. This article aims to use proteomic analysis of VD model of hippocampal proteome composition and change of proteome composition after using GC.The present study is to evaluate the effects of GC on leaning-memory function in a rat model of VD, using proteomics approach and p-tau immunohistochemical analysis, which analyzed the hippocampus to identify the possible molecular events.To provide the basis for further study of VD in possible pathogenic mechanisms and the experimental basis for the cognitive function from the pharmacological studies angle of GC.Purpose:GC extracted from Xin Jiang Cistanche, is widely used as a Chinese herb with neuroprotective effects. To get a better insight into this herb, the present work is to deduce changes in hippocampus of effects of GCs on hippocampus of VD rat model.Materials and Methods:VD model was established by ligature of the bilateral common carotid artery in adult Wistar rats, which received daily administration of GC (10mg/kg/d i. p.) during an experimental period of 14 days, and then underwent the morris water maze testing (MWM) to elucidate cognitive performance. (the same as the first part).After completing behavioral studies, using proteomics approach and immunohistochemical method identified molecular events of GC.Results:(1) In MWM test, GC group showed significantly lower escape latency than VD group at 4 and 5 days after operation, but showed no significant difference compared with sham-operated group and oxiracetam control group. (the same as the part 1)(2) In the hippocampus, using ProFound software to search proteins in NCBI database, 21 protein spots in the VD model group showed the different expression compared with sham-operated group. And ultimately levels of 4 proteins, as followed thioredoxin-like protein 1 (TXL 1), dual specificity mitogen-activated protein kinase kinase 1 (MAPKK 1) and Dihydropyrimidinase-related protein 2 (DPYSL 2), were distinctly upregulated, and 1 protein, glutathione synthetase (GCL) was significantly downregulated. Compared with VD group,15 protein spots in the GC group showed the different expression. And ultimately levels of 3 proteins(Tab 3 Fig 3), as followed:heat shock protein 75 (HSP 75) and actin-related protein 3 (ARP 3), were distinctly upregulated, and 1 protein keratin, type II cytoskeletal 6A (KRT 6A).(3) Immunohistochemistry analysis showed that P-tau protein level was significantly higher in VD model group than sham-operated group (P< 0.05). After GC treatment, P-tau protein level in VD model rats showed significant decrease compared with VD group treated with saline (P< 0.05).Conclusions:These findings indicate that GC alters the levels of some targeted proteins,21 protein spots in the VD model group showed the different expression compared with sham-operated group. And ultimately levels of 4 proteins, as followed thioredoxin-like protein 1 (TXL 1), dual specificity mitogen-activated protein kinase kinase 1 (MAPKK 1) and Dihydropyrimidinase-related protein 2 (DPYSL 2), were distinctly upregulated, and 1 protein, glutathione synthetase (GCL) was significantly downregulated. Compared with VD group,15 protein spots in the GC group showed the different expression. And ultimately levels of 3 proteins as followed:heat shock protein 75 (HSP 75) and actin-related protein 3 (ARP 3), were distinctly upregulated, and 1 protein keratin, type II cytoskeletal 6A (KRT 6A) was significantly downregulated. Function of these proteins involved in energy metabolism, anti oxygen free radical, signal transduction, neuronal polarity and axon formation and many pathophysiological processes, which would provide a basis for further research to explore the pathogenic mechanism of VD possible. The immunohistochemical results suggest that the VD model group, tau protein phosphorylation level was significantly higher than those in the sham operation group, and tubulin binding decreased, resulting in maintaining microtubule function stability decreases, cause nerve fiber degeneration or loss of function. Phosphorylation level of tau protein may be the main pathological changes of vascular dementia, after GCs intervened, phosphorylation of tau protein level decreased significantly, can improve the activity of microtubule assembly, further improve the synthesis, transport, uptake and release of neurotransmitters. |
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