The Influence Of Gnsenoside Rb1 On Expression Of Caspase-3, Tau Protein And PP-2A In Vascular Dementia Rats Induced By Chronic Cerebral Ischemia

Vascular dementia(VD) is an acquired intelligence impairment, such as decrease cognitive and remembrance function, caused by brain disorder. VD has close relationship with stroke, the same risk factors include systolic or diastolic hypertension, diabetes, hypercholesterolemia, cigarette smoking, heavy alcohol consumption, artherosclerosis, heart disease, and so on. Transient Ischemic Attack is high risk factor. VD not only damage healthy and living quality of patient, but also bring heavy burden to society and family. Meanwhile, VD is one of the preventable and treatable dementia, so is a significant topic in geratology domain, the research on VD cause generally attention.Recently, many experiments authenticate that neuron death is not simple cytonecrosis after transient cerebral ischemia. Apoptosis always accompany with ischemic neuron death, especially delayed neuronal death. Neurocyte apoptosis is considered play an important role in VD. Apoptosis is regulated by gene. Caspase famliy play an important role in apoptotic signal transduction pathway. After cerebral ischemia, the change of apoptosis related gene and their expression production can induce cell apoptosis. Tau belongs to the family of microtubule associated proteins(MAPs) with the functions of modulating microtubule assembly, dynamic behavior and spatial organization. Alzheimer's disease(AD) is pathologically characterized by the occurrence of neurofibrillary tangles(NETs) composed primarily of arrays of paired helicalfilaments(PHF). PHF are anomalous structures generated by self aggregation of hyperphosphorylated forms of tau protein. Recent research had found increased expression of tau protein ater cerebral ischemia. The imbalance of protein kinase and phosphatase system may be the reason of hyperphosphorylated of tau protein. Gnsenoside Rb1 , which belong to panoxadiol saponin, have the function of cleaning free radicle, resisting tissue damage, antiinflammation, immune regulation, metabolism of central nervous system neurotransmitter. The research found that Gnsenoside Rb1 can resist apoptosis, relieve hyperphosphorylation of tau protein in the brain of AD model rat.The present study was aimed to investigate the protection mechanism of Gnsenoside Rb1 on central nervous system through apoptosis,tau protein and PP-2A , the role of Tau protein on the mechanism of VD, to afford the basic theory and clinical treatment for vascular dementia.1. The establishment of vascular dementia model rats induced by chronic cerebral ischemia and the effect of Gnsenoside Rb1 on ethology and histology of model rats1.1 permanent Occluding of bilateral common carotid arteries to set up vascular dementia model ratsThis study choose healthy Wistar rats of 3-5 months weighted 250-350g.We select swift, activated rats by Morris Water Maze and randomly divided them into five groups: normal control group, sham group, dementia group,low dose Gnsenoside Rb1 group,large dose Gnsenoside Rb1 group.Every group divided into four experimental time points, which is 4w,8w,12w,16w groups. Every experimental time point have 12 rats. Six for pathologic study, and the other for westernblotting detect. The normal control group accept no treatment. The other groups were anesthetized by peritoneal injection 10% chloral hydrate(0.33ml/100g), backlying and fixing. after sterilizing, The rat was cut in the middle of neck, and disconnected bilateral common carotid arteries. Sham group rats only disconnected bilateral common carotid arteries, not ligating and shearing them. Dementia group and Gnsenoside Rb1 treatment group ligated distal and proximal end of the bilateral common carotid arteries, and sheared the middle of it. Since the last month, every rat of treatment group had injected Gnsenoside Rb1 solution one time per two days. Each rat of low dose treatment group injected 2.5mg/ml Gnsenoside Rb1 solution 0.5ml ,and each rat of large dose treatment group injected 5mg/ml Gnsenoside Rb1 solution 0.5ml. Meanwhile,Every rat of dementia group and sham group injected saline 0.5ml.1.2 Detection of learning and memory ability of model ratsWe detected the learning and memory ability of model rats by Morris Water Maze which include hidden-platform acquisition training and spatial probe test. The escape latencies and times of going through the platform were recorded. Rats of better learning and memory ability have the shorter escape latencies and more times of going through the platform.Experimental results show that the escape latencies in dementia group were significant longer than that in normal control group and sham group, and the times of going through the platform were significant decreased.But there were no significant differences between the latter two groups. The results demonstrate that memory ability decreases when the rats of permanent bilateral common carotid arteries are occluded since the first month. Compared with dementia group, rats of Gnsenoside Rb1 treatment group in 1m and 2m experimental time points have the shorter escape latencies and more times of going through the platform, but have no significant differences in 3m and 4m experimental time points. The results reveal Gnsenoside Rb1 can protect the learning and memory ability of 2-vo model rats and early treatment is important. Gnsenoside Rb1 treatmentgroup compared with normal control group and sham group demonstrate that the escape latencies prolonged and the the times of going through the platform decreased, which showed that memory ability of Gnsenoside Rb1 group can't reach the normal level.The low dose Gnsenoside Rb1 group compared with the large dose treatment group showed the escape latencies prolonged and the the times of going through the platform decreased, which indicates that the therapeutic efficacy of large dose Gnsenoside Rb1 is better.1.3 Results of HE staining in frontal lobe and hippocampus of ratsHE staining of normal control group and sham group shows pyramidal cell of frontal lobe and hippocampus line up in order,uniformity,cellular structure is integrity. HE staining of dementia group shows that at the beginning, the diffused cerebral edema and exudation were apparent, then attenuated gradually. Then the degeneration and detachment of neuron could be seen, so did the necrosis of the neuron and hypertrophic neuroglial cells. We thought these pathological changes are due to chronic and steady reduction of regional cerebral blood flow, while not induced by acute cerebral ischemia. But the ischemic changes of Gnsenoside Rb1 treatment group are relieved apparently. Experimental results confirm that permanent bilateral common carotid arteries occlusion result in neuronal chronic persistent ischemic impairment of frontal lobe and hippocampus,and cause the degression of learning and memory ability of rats. Gnsenoside Rb1 can relieve the ischemic impairment.2. Effection of Gnsenoside Rb1 on the expression of Caspase-3 in frontal lobe and hippocampus of vascular dementia model ratsOur research used permanent occluding of bilateral common carotid arteries to set up vascular dementia model rats, then observed the expression of Caspase-3 in frontal lobe and hippocampus of vascular dementia model rats and the effection of Gnsenoside Rb1 by immunohistochemistry staining. The results showed that Caspase-3 seldom expressed in normal control group and sham group. Since the first month after permanent occluding of bilateral common carotid arteries, the expression of Caspase-3 increased significantly, and were gradually decreased from the second month. So chronic cerebral ischemia can increase the expression of Caspase-3. Compared with dementia group, rats of Gnsenoside Rb1 treatment group in 1m and 2m experimental time points showed Caspase-3 positive cell obviously decreased in frontal lobe and hippocampus, but have no significant differences in 3m and 4m experimental time points. The results reveal Gnsenoside Rb1 can reduce the expression of Caspase-3 protein to inhibit apoptosis and encourage neuronal survival,especially at early stage of chronic cerebral ischemia. Gnsenoside Rb1 treatment group compared with normal control group and sham group demonstrate that the expression of Caspase-3 were increased, which showed that Gnsenoside Rb1 can't reduce the expression of Caspase-3 protein to the normal level.The low dose Gnsenoside Rb1 group compared with the large dose treatment group showed the expression of Caspase-3 protein were increased, which indicates that the therapeutic efficacy of large dose Gnsenoside Rb1 is better.The experimental result indicates that permanent occluding of bilateral common carotid arteries can induce the expression of Caspase-3. Gnsenoside Rb1 can significantly reduce the expression of Caspase-3 protein so as to inhibit apoptosis and improve the learning and memory ability of vascular dementia rats.5. Effection of Gnsenoside Rb1 on the expression of Tau-pThr231 and PP-2A in frontal lobe and hippocampus of vascular dementia model ratsOur research used permanent occluding of bilateral common carotid arteries to set up vascular dementia model rats, then observed the expression of Tau-pThr231 and PP-2A in frontal lobe and hippocampus of vascular dementia model rats and the effection of Gnsenoside Rb1 by immunohistochemistry staining and westernblotting.The results showed that compared with normal control group and sham group, Expression of Tau-pThr231 in dementia group were increased. Since the first month after permanent occluding of bilateral common carotid arteries, the expression of Tau-pThr231 increased significantly, and were gradually decreased from the second month. So chronic cerebral ischemia can induce the phosphorylation of tau protein. Compared with dementia group, rats of Gnsenoside Rb1 treatment group in 1m and 2m experimental time points showed Tau-pThr231 obviously decreased in frontal lobe and hippocampus, but have no significant differences in 3m and 4m experimental time points. The results reveal Gnsenoside Rb1 can reduce the phosphorylation of tau protein,especially at early stage of chronic cerebral ischemia. Gnsenoside Rb1 treatment group show higher expression of Tau-pThr231 compared with normal control group, which indicated that Gnsenoside Rb1 can't reduce the phosphorylation of tau protein to the normal level.The low dose Gnsenoside Rb1 group compared with the large dose treatment group showed the expression of Tau-pThr231 were increased, which indicates that the therapeutic efficacy of large dose Gnsenoside Rb1 is better.PP-2A positive cell in dementia group were more than that in normal control group and sham group. Since the first month after permanent occluding of bilateral common carotid arteries, the expression of PP-2A decreased significantly, and were gradually increased from the second month. So chronic cerebral ischemia can reduce the expression of PP-2A. Compared with dementia group, rats of Gnsenoside Rb1 treatment group in 1m and 2m experimental time points showed PP-2A positive cell obviously increased in frontal lobe and hippocampus, but have no significant differences in 3m and 4m experimental time points. The results reveal Gnsenoside Rb1 can increase the expression of PP-2A,especially at early stage of chronic cerebral ischemia, Gnsenoside Rb1 treatment group show lower expression of PP-2A compared with normal control group, which indicated that Gnsenoside Rb1 can't increase the expression of PP-2A to the normal level.The low dose Gnsenoside Rb1 group compared with the large dose treatment group showed the expression of PP-2A were decreased, which indicates that the therapeutic efficacy of large dose Gnsenoside Rb1 is better.The experimental result indicates that permanent occluding of bilateral common carotid arteries can increase the expression of tau protein and inhibit the secretion of PP-2A. we suppose that chronic cerebral ischemia decrease the secretion of PP-2A so to enhance the expression of hyperphosphorylated tau protein. Gnsenoside Rb1 can inhibit this effection of chronic cerebral ischemia, so has neural protection on central nervous system, can improve the learning and memory ability of vascular dementia model rats.In conclusion, Gnsenoside Rb1 can regulate the expression of Caspase-3 to inhibit apoptosis, and it can regulate PP-2A, decrease the expression of hyperphosphorylated form of tau protein to reduce the impairment of hyperphosphorylated tau protein against nerve cell.Therefore, we consider Gnsenoside Rb1 can protect nerve cell and improve the learning and memory ability of vascular dementia model rats though inhibiting apoptosis and reducing the impairment of hyperphosphorylated tau protein against nerve cell. This research afford the basic theory and clinical treatment for vascular dementia.