Dissertation
Dissertation > Medicine, health > Chinese Medicine > Of Pharmacy > Pharmacology > Chinese medicine Experimental Pharmacology

The Gastrodia effective ingredients group 1210 effects and mechanisms of prevention and treatment of vascular dementia

Author ZhaoZuo
Tutor SunJianNing
School Beijing University of Traditional Chinese Medicine
Course Pharmacology
Keywords Vascular dementia VAD Choline acetytransferaseChAT Glutamate Glu gamma aminobutyric acid GABA N-Methyl-D-Aspartate Receptor NMDAR Excitatory amino acid carrier GABA transporter GAT
CLC R285.5
Type PhD thesis
Year 2008
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Vascular dementia is an acquired lasting disturbance of intelligence syndromes due to cerebral vascular disease. Vascular dementia is the common type of senile dementia only secondly to Alzheimer disease. There is not an affirmative method to treat vascular dementia because of the complex etiology. The therapy of vascular dementia is to ameliorate the circulation of brain and oxygen supply of cerebral cell in order to prevent the new thrombus and infarct occurrence. At present, the common medicines to treat vascular dementia are the medicines to ameliorate the circulation of brain and the activator of cerebral energy and the accelerator of cholinergic system and so on. These medicines only have functions on the vascular dementia pathological partly course and have not satisfactory prevention and cure effects so that it is a significant subject in the field of the basic and clinical medicine.The paper deeply discussed the pharmacological function of the effective fraction of Rhizoma Gastrodiae using behavior science and biochemistry and molecular biotechnology. The contents of the paper include two parts:The first part is to appraise the pharmacological function of the effective fraction 1210 of Rhizoma Gastrodiae using two rat models—chronic lower perfusion vascular dementia rats model and middle cerebral artery occlusion rats model.The second part is to probe mechanism of the effective fraction 1210 of Rhizoma Gastrodiae on rat models from oxygen free radical metabolism and expression of synaptophysin (Syn) construction protein as well as rebuild the homeostasis between the Glu and GABA.1 Protection of nerve injury and spatial learning and memory of the effective fraction 1210 of Rhizoma Gastrodiae on vascular dementia rat model.1.1 Effects of the effective fraction 1210 of Rhizoma Gastrodiae on spatial learning and memory deficits of vascular dementia rat modelObjective: to observe the effective fraction of Rhizoma Gastrodiae on MACO and chronic lower reperfusion vascular dementia rat two models estimate its pharmacodynamics.Method: to observe the ratsspatial learning and memory deficits using Morris water maze. MACO vascular dementia rats were given medicine 30d continuously after replication and then examined. Chronic lower reperfusion vascular dementia rats were given medicine 60d continuously after replication and then examined.Result: the escape latencies and route of swim have been prolonged in MACO and chronic lower reperfusion vascular dementia rat two models. The rats in model group have orientation disorder of exploration and the times of passing through the platform have been decreased. Meanwhile memory ability of these rats was obviously damaged. After the platform removed, the escape latencies and route of swim in model group are much longer than those in sham group. The escape latencies and route of swim have been shorted and the times of passing through the platform correctly have been increased in the effective fraction of Rhizoma Gastrodiae 1210 group. After the platform removed, the escape latencies and route of swim in 1210 group are much shorter than those in model group.Conclusion: There is obviously learning memory injury of MACO and chronic lower reperfusion vascular dementia rat two models. The effective fraction 1210 of Rhizoma Gastrodiae can improve the ability of learning memory of vascular dementia model.1.2 Protection of nerve injury of the effective fraction of Rhizoma Gastrodiae 1210 on vascular dementia model.Objective: to estimate the effective fraction of Rhizoma Gastrodiae 1210 with pathological change of MACO and chronic lower reperfusion vascular dementia rat two models.Method: to observe whole injury region of cerebral ischemia with HE staining in two models rats and take count of pyramidal cell in CA1 and CA3 region of hippocampus. To examine the change of nissl bodies with nissl staining integrated combined image pattern analysis technology and judge the ability of nerve cell’s synthesized protein and biological enzyme. To observe the change of GFAP which is the maker of the activation and proliferation of astrocyte using immunochemistry combined image pattern analysis technology. To judge the degree of injury of white matter of chronic lower reperfusion vascular dementia model rats with immunochemistry combined image pattern analysis technology.Result: Nissl bodies of nerve cell decreased and a lot of nerve cell lost and the expression of GFAP increased in MCAO model. The effective fraction of Rhizoma Gastrodiae 1210 can alleviate the degree denaturalization of hippocampus and increase the number of pyramidal cell in CA1 region and decrease the reaction f astrocyte in MCAO model. There exists abnormity of hippocampus and denaturalization and neurosis of parts of neuron, pyramidal cell lessened and the expression of GFAP upregulated and MBP in internal capsule obviously decreased in chronic lower reperfusion vascular dementia model. The effective fraction of Rhizoma Gastrodiae 1210 can protect the nerve cell in hippocampus area and reduce the reaction of astrocyte in hippocampus area as well as obviously increase the expression of MBP in internal capsule in chronic lower reperfusion vascular dementia model.Conclusion: There exists obviously ischemia injury in MACO and chronic lower reperfusion vascular dementia rat two models. The effective fraction of Rhizoma Gastrodiae 1210 can protect the nerve cell in hippocampus area and reduce the reaction of astrocyte in hippocampus area as well as obviously increase the expression of MBP in internal capsule.2 Study of mechanism of the effective fraction of Rhizoma Gastrodiae 1210 on prevention and treatment of vascular dementia2.1 Effects of the effective fraction of Rhizoma Gastrodiae 1210 on metabolism of free oxygen radical in vascular dementia model.Objective: to estimate the effective fraction of Rhizoma Gastrodiae 1210 on decomposition of lipid peroxid and the contents of antioxidation enzyme with chronic lower reperfusion vascular dementia model.Method: to estimate the contents of GSH-PX and SOD as well as MDA with biochemistry technology.Results: The content of MDA in the hippocampus area is higher and that of SOD and GSH-PX is lower .The content of MDA in the hippocampus area decreased and that of SOD increased after using the effective fraction of Rhizoma Gastrodiae 1210 group. The content of GSH-PX also increased after using the effective fraction of Rhizoma Gastrodiae 1210 group(6.4mg/kg).Conclusion: The effective fraction of Rhizoma Gastrodiae 1210 can increase the activation of antioxidation enzyme and lower the content of MDA and improve the metabolism of free oxygen radical.2.2Effects of the effective fraction of Rhizoma Gastrodiae 1210 on cholinergic fibers and structure of synapse.Objective:to estimate effects of the effective fraction of Rhizoma Gastrodiae 1210 on cholinergic system with qualitative and half-quantitative analysis of positive ChAT—acetylcholine synthesized enzyme. To judge the medicine on structure protein of neuron process with half-quantitative analysis of expression of GAP-43 and SYN1 and MAP-2.Method: to embed with paraffin after paraformaldehyde heart reperfusion and then observe the change of ChAT, GAP-43,SYN1and MAP-2 with immunochemistry staining combined image pattern analysis technology.Result: positive expression of ChAT obviously decreased in CA1 and CA3 areas of hippocampus and dentation in MCAO model. The effective fraction of Rhizoma Gastrodiae 1210 (1.6 mg/kg)can increase positive expression of ChAT in CA3 area and the dose of 1210 (3.2、6.4 mg/kg) can obviously increase thepositive expression of ChAT in CA1 and CA3 areas. Immunoreaction of SYN obviously decreased in model group and the effective fraction of Rhizoma Gastrodiae 1210 can upregulate the expression of SYN. There is no obvious difference of expression of GAP-43 between the model group and sham group. The expression of GAP-43 increased in the effective fraction of Rhizoma Gastrodiae 1210 group. The expression of MAP-2 decreased in model group and increased in 1210 group. Conclusion: the effective fraction of Rhizoma Gastrodiae 1210 can upregulate the expression of ChAT,SYNI,GAP-43 and MAP2 protein.2.3 The effects of the effective fraction of Rhizoma Gastrodiae on Glu/GABA system of vascular dementia rat model in hippocampus region.Objective: to study the molecular mechanism of Glu/GABA system of vascular dementia rats model and the pharmacological function and molecular target of the effective fraction of Rhizoma Gastrodiae.Method: to give medicine after replication model 24h and last 60d using the chronic lower perfusion vascular dementia rats model and then examine the following index: To examine the contents of Glu/GABA of vascular dementia rat model hippocampus with ELISA method; to observe the expression of GABA and Glu neuron of hippocampus and GAD ,NR1,NR2A and NR2B protein expression with immunochemistry combined image pattern analysis technology ; to examine EAAC1 and GAT1mRNA expression using Real time RT-PCR method and protein expression of EAAC1 and GAT1 with Western blot method.Results: There is no difference of the contents of Glu in hippocampus tissue between the model group and the sham group. But the contents of GABA in hippocampus tissue of model group is lower and the ratio of Glu and GABA is markedly higher .After using the effective fraction 1210 of Rhizoma Gastrodiae, the contents of GABA in hippocampus tissue is raised and the ratio of Glu and GABA is decreased. Inhibition neuron GABA in the hippocampus CA1 area and dentation area is selected deficiency and Glu neuron immunologic competence increased in the model group. Immunoreactivity of Glu neuron in the hippocampus CA1 area is decreased and expression intensity of GABA neuron is raised in the effective fraction of Rhizoma Gastrodiae 1210(1.6、3.2 mg/kg)group. Positive expression of GAD65 in the hippocampus CA1 area is markedly decreased in the model group. Immunointensity of GAD65 in the hippocampi CA1 and CA3 area in the effective fraction of Rhizoma Gastrodiae 1210(3.2 mg/kg)group. Immunointensity of GAD65 in the hippocampus CA1 area in the effective fraction of Rhizoma Gastrodiae 1210(1.6mg/kg)group is also higher than that of the model group. Expression of EAAC1 protein is lower and that of EAAC1mRNA is not markedly different in the model group. Expression of EAAC1mRNA is not markedly changeable in the effective fraction of Rhizoma Gastrodiae 1210 group and that of EAAC1 protein is raised to some extent in the effective fraction of Rhizoma Gastrodiae 1210 group. Especially there is a statistics difference of expression of EAAC1 protein between the model group and in the effective fraction of Rhizoma Gastrodiae 1210(3.2、6.4 mg/kg) group .Expression of GAT1 is not markedly changeable in the model group. There is not a statistics difference of expression of GAT1 protein and expression of GAT1mRNA between the model group and in the effective fraction of Rhizoma Gastrodiae 1210 group. Functional subunit of NMDA receptor and modulatory subunit is decreased to some extent in the model group. Expression of NR1 is markedly lower in CA1 area and expression of NR2A in CA3 area is also decreased. But expression of NR2B in CA1 and CA3 as well as dentation area is also decreased in the model group. Expression of NR1 is markedly raised in CA1 area and expression of NR2A in CA3 area is also raised. Immunoreactivity of NR2A in dentation area is markedly raised in the effective fraction of Rhizoma Gastrodiae 1210 group(6.4 mg/kg). Immunointensity of NR2B in the hippocampi is also raised in the effective fraction of Rhizoma Gastrodiae 1210 group(3.2、6.4 mg/kg).Conclusion: the effective fraction of Rhizoma Gastrodiae 1210 can efficiently ameliorate learning and memory of the vascular dementia rat model and its main courses are the following :To rebuild the homeostasis between the Glu and GABA through activation of immunoactivation and increasing suppression of medium neuron. To accelerate the synthesize of GABA through increasing the expression of GAD65 protein.To accelerate Glu transmitter entering into cell recycle through improving the expression of EAAC1.To improve the ability of learning and memory through upregulating functional subunit of receptor of NMDA and regulating the expression of subunit of receptor of NMDA.ConclusionThe effective fraction of Rhizoma Gastrodiae 1210 can efficiently increase spatial learning and memory and decrease the pathological injury of neuron in the vascular dementia model and its main courses are the following: to ameliorate the metabolism of free oxygen radical. To effectively induce the synthesize of growing correlation protein of neuron in cerebral repair course and suppress the proliferation of asrtocyte and accelerate the regenesis of neuron process.To upregulate the expression of the structure protein of synapse and functional protein of hippocampus–CHAT. To rebuild the homeostasis between the Glu and GABA.The innovation of the researchPay attention to the effects of medicine on excitatory synapse meanwhile study the GABA neuron and synthesize of GABA as well as retake of GABA transmitter in order to supply the new molecular target and pharmacological information in clinical medicament intervention and treatment of vascular dementiaThere is no report of the change of EAAC1 and GAT1 with vascular dementia model in literature.

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