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1、 Dan Kong pillow in animal models of dementia anti-apoptosis effect and mechanism of Abstract Objective: To study the hole pillow in the Danish anti-dementia animal model of hippocampal neuronal apoptosis and its mechanism. Methods: 50 Wister rats were randomly divided into normal group, model group
2、, piracetam group, Kong pillow in the Danish low-dose group, high-dose group, 10 in each group. In addition to the normal group, the remaining rats were made from rat model of vascular dementia. After the rats were given gavage, using flow cytometry to detect neuronal apoptosis and bcl-2 expression
3、were observed by immunohistochemistry the expression of p53. Results: The hole in the pillow Dan piracetam able to reduce vascular dementia rat hippocampus neuronal apoptosis rate and bcl-2 expression, increased p53 expression, two groups showed no significant difference. Conclusion: The hole pillow
4、 and Denmark can inhibit neuronal apoptosis, the mechanism may be related to increased p53 expression and lower bcl-2 expression. Keywords: hole pillow Denmark; vascular dementia; apoptosis; bcl-2; p5 Abstract Objective: To observe antiapoptotic effect of Kongsheng Zhenzhong pill on hippocampus neur
5、on of rats with vascular dementia. Methods: 50 Wister rats were randomly divided into normal group, model group, Naofukang treatment group, Kongsheng Zhenzhong pill treatment group 1 (low dosage), group 2 (high dosage). The apoptosis rate of hippocampus neuron and the expression of bcl-2 were measur
6、ed by flow cytometry, the expression of p53 was detected by immunohistochemical staining. Results: Compared with the model group, the apoptosis rate of hippocampus neuron and the expression of bcl-2 were decreased, the expression of p53 was increased in Naofukang group and Kongsheng Zhenzhong pill g
7、roup, but there is no significant difference between Naofukang group and Kongsheng Zhenzhong pill group. Conclusions: Kongsheng Zhenzhong pill can alleviate apoptosis rate of hippocampus neuron through upregulating the expression of p53 and decreasing the expression of bcl-2. Keywords: Kongsheng Zhe
8、nzhong pill; apoptosis; bcl-2; p53; vascular dementia Vascular dementia (Vascular Dementia, VD) is ischemic or hemorrhagic, and acute and chronic hypoxic-ischemic cerebral vascular disease caused by cognitive impairment to memory impairment, facial expressions indifferent, sluggish, or personality c
9、hange as the main clinical manifestations, its incidence related to apoptosis 1 2. In this study, after the intervention of the traditional Chinese medicine model of rat hippocampal neuronal apoptosis and bcl-2, p53 protein expression has been studied, are reported below. 1 Materials and methods 1.1
10、 Experimental Materials 1.1.1 Experimental Animal Clean-level pure Wister male rats were 50 body weight (250 50) g, from the Animal Experimental Center of Shandong Medical University to provide qualified证号 : D20041021. Sub-cage rearing of animals, free drinking water consumption, the standard feed,
11、feeding, feed from the Experimental Animal Center of Shandong Province. 1.1.2 Drugs and reagents Kong pillow in the preparation of Dan: with water as the basic solvent, the turtle shell, keel broken Xian Jian 30 min, then add other drugs to boil, concentration, made of 100% concentration (equivalent
12、 to 1 g crude drug / mL), placed in 4 refrigerator stored until required. Western medicine for the control of brain rehabilitation (Piracetar), produced by Tianjin Jin Pharmaceutical Co., Ltd., batch number: 20,030,302, before the experiment by 0.28 g / kg body weight dubbed suspension spare. Apopto
13、sis kit Annexin V-FITC Cat.NO.51-6587, PI (Cat No 51-66211E); Bcl-2 kit Fluorescein (FITC) anti-mouse and rat Bcl-2, ebioscience, Cat No.11 - 6992; p53 antibodies, santa cruz, Cat No: sc-6243. 1.1.3 Experimental apparatus FACSCaliber flow cytometry, the U.S. BD company (Becton Dickinson), Image pro-
14、plus medical image processing system, Olympus microscope. 1.2 Experimental Methods 1.2.1 Grouping The rats were randomly divided into normal group, model group, piracetam group, Kong pillow in the Danish high-dose group, low-dose group, 10 in each group. 1.2.2 In addition to modeling, and administra
15、tion in rats with reference normal group, the rest of the improved preparation of four-vessel occlusion rat cerebral ischemia-reperfusion model, 10% chloral hydrate 3 mL / kg (body weight) intraperitoneal anesthesia, taking supine bit, the neck incision, isolation of bilateral common carotid artery,
16、 threading back, using a micro-clip closure of bilateral common carotid arteries arteries 10 min, reperfusion 10 min, repeated three times, withdrawal line, sewing 3. The normal group and model group fed with normal saline, daily one time; piracetam group fed western Piracetam 10 mL / kg, Kong Dan p
17、illow in high-and low-dose group, respectively, to holes in the pillow Dan l3. 5 g / kg, 9.0 g / kg, gavage, daily one time, a continuous 14 d. The first 15 d with 4% paraformaldehyde were decapitated after perfusion fixation of brain reserve. 1.2.3 Indicators Observation 1.2.3.1 neuronal apoptosis
18、rate were detected in rats brain tissue taken quickly after decapitation the hippocampus was prepared by single-cell suspension, adjusting the cell number of 1 * 106/mL. Drawing 100 L cell suspension, add 5 L Annexin V-FITC and 5 L PI, the full mixing cell, dark 25 stained 15 min, using flow cytomet
19、ry apoptosis. 1.2.3.2 bcl-2 protein expression in cell suspensions prepared with the 1.2.3.1. By intracellular protein staining by adding 20 L FITC labeled bcl-2 antibodies, dark stained 30 min, using flow cytometry the expression of bcl-2 protein fluorescence value. 1.2.3.3 p53 protein expression w
20、as detected using immunohistochemistry. Normal brain tissue fixed, embedded, sliced, gradient dewaxing, dehydration, hydrogen peroxide at room temperature inactivation of endogenous enzymes, citric acid salt solution by microwave antigen restoration after the closure of serum antigen, dropping the a
21、ppropriate diluted primary antibody, 4 overnight. Dropping biotinylated secondary antibody, 37 20 min, DAB color, dehydration, transparent, Feng Pian. Select the hippocampus G1 area, image analyzer detected an average of optical density value. 1.3 Statistical Methods Statistics with SAS software for
22、 processing single-factor analysis of variance, measurement information and data to mean standard deviation ( s) indicated. 2 Results 2.1 Hole pillow in Dan on dementia in rat neuronal apoptosis rate of the results in Table 1. Can be seen from Table 1, administration of brain Rehabilitation Unit Kon
23、g pillow in the Danish low-dose group, high-dose group compared with model group, hippocampal neuronal apoptosis was significantly reduced (P <0.05), but among the three groups showed no significant difference. 2.2 Hole pillow Dan on dementia in rat neuronal cells bcl-2, p53 expression result
24、s in Table 2. From Table 2 we can see that compared with normal group, model group in hippocampal neurons significantly decreased bcl-2 expression, p53 expression was significantly increased; Compared with model group, piracetam group, Kong Dan pillow in the low and high dose group Hippocampus bcl -
25、2 expression was significantly increased, p53 expression was significantly reduced. However, no difference among the 3 groups was significant. Reposted elsewhere in the paper for free download 3 Discussion Apoptosis in the bodys growth and development and maintenance of a stable internal environment
26、 plays an important role in apoptosis related to pathological cell death and with the pathogenesis of many diseases 4. People in the past that the nerve cell death after cerebral ischemia in the form of necrosis, but in recent years an increasing number of studies have shown that ischemia-induced ne
27、uronal death but also neuronal apoptosis 5. Ischemic brain injury resulting from a large number of free radicals, excitatory amino acid, NO accumulation and lead to intracellular calcium overload and induce the occurrence of apoptosis is the possible mechanism of dementia 6. Apoptosis in the infarct
28、 formation, delayed neuronal death (DND) plays an important role in the process. Therefore, neuronal apoptosis after cerebral ischemia research to better understand the mechanism of ischemic brain injury, and open new avenues for its treatment. In recent years, studies have found that the occurrence
29、 of apoptosis and bcl-2 gene expression, and its decision to the survival of neurons after ischemia play an important role in 7 8. bcl-2 gene family is the apoptosis suppressor, inhibiting cell death in cerebral ischemia and its possible mechanisms involved in phosphorylation-dependent regulation, f
30、ree radical products of the partial inhibition, the regulation of mitochondrial permeability trans-shipment and other relevant, suggesting that bcl-2 expression in the increase in ischemic brain injury may have a protective effect. Overexpression of bcl-2 transgenic animals have confirmed the bcl-2
31、neuroprotective effect 9. p53 as a transcription factor regulating the transcription of multiple genes involved in cell cycle regulation, involved in DNA replication and damage repair involved in inducing apoptosis and so on. Has been a large number of studies have shown that: the existence of ische
32、mic cerebrovascular disease DNA damage and apoptosis. Thus, p53 and its related genes in cerebral ischemia in the expression and regulation has become a mechanism of ischemic neuronal death in a key part. Although different models of ischemia, at different times there may be different in different p
33、arts of the regulatory mechanism of gene expression, but p53 is recognized as the most common pro-apoptotic gene, by activating a series of downstream genes play a role in apoptosis 10. Kong pillow from Denmark Preparation of emergency Qian Jin Yao Fang Fang sentient flesh and blood of the turtle sh
34、ell products, good supplement essence and blood, can Zi-yin Qian-yang, kidney health bones, fill Anshen; Shichangpu exemption for resuscitation Xingshen sputum of medicine, Polygalaceae Ning Anshen, expectorant resuscitation, keel Shibuya Gan convergence can Zhenjing soothe the nerves. Four drug com
35、bination, expectorant resuscitation, kidney soothe the nerves, the clinical treatment of vascular dementia is a commonly used herbs 11. Our results show that neuronal apoptosis in rats of model group was significantly enhanced, while the brain rehabilitation and bore pillow Denmark neuronal apoptosi
36、s in rats compared with model group significantly reduced, indicating Kong pillow in Denmark have antagonistic The role of apoptosis in brain cells of Alzheimer rat brain and learning and memory are closely related to neurons in the hippocampus played a protective effect. Of apoptosis regulatory gen
37、es p53, bcl-2 expression in the test results show that Sino-Danish Kong pillow can reduce dementia caused by cerebral ischemia-reperfusion in rat hippocampal CA1 area of the expression of p53 and enhanced the expression of bcl-2, indicating Kong pillow and Denmark by reducing p53, increased bcl-2 in
38、 neuronal cells and to inhibit neuronal apoptosis. Therefore, the antagonist-ischemic encephalopathy in patients with dementia caused by neuronal apoptosis may be holes pillow and Denmark to improve the symptoms of dementia a major role in the mechanism of anti-apoptosis in combination with other me
39、ans of combination therapy will become the main content of the treatment of dementia. References 1 Zhao Lin, Li Dongpei. Vascular dementia rat hippocampal neurons ultrastructure study J. Anatomical Science, 2000,6 (2): 161. 2 Rosenbanm DM, Damore J, Liena J, et al. Pretreatment with intra-vertricula
40、r aurintricarboxrylic acid desease infarct size by inhibiting apoptosis following tran sientsize by inhibiting apoptosis following transient global ischemia ingerbils J. Ann Neurol, 1998 (43) : 654. 3 Tsutomu A, Norio T, Midori N. The effects of monobromobimane on neuronal cell death in the hippocam
41、pus after transient global cerebralischemia in rats J. Neuroscience Letters, 2004,357 (3) :227-231. 4 Yang E, Korsmeyer S. Molecular thanatopsis: a discourse on thebcl-2 family and cell death J. Blood, 1996,88 (2) :386-401. 5 Leqqio MG, Neri P, Graziano A, et al. Cerebellar contribution tospatial ev
42、ent processing: characterization of procedural learning J. Exp Brain Res, 1999,127 (1) :1-11. 6 Nakamura M, Raghupathi R, Merry DE, et al. Overexpression ofbcl-2 is neuroprotective after experimental brain injury in transgenic mice J. J Comp Neurology, 1999,412 (4): 681. 7 Gao Yun-liang, Kau. Cerebr
43、al neurons after ischemia-reperfusion and endothelial cell apoptosis and bcl-2 and Bax Expression in the relevant departments J. Anatomy reported 2003,33 (2) :151-155. 8 Gao Pei, Gu Ling, Tang Tai-Kun, et al. Rats with focal cerebral ischemia on the cerebral cortex Bax, bcl-2 gene expression J. Kunm
44、ing Medical Journal, 2002 (3) :26-27 . 9 Chopp M, Li Y. Apoptosis in focal cerebral ischemia J. Acta Neu-rochir Suppl, 1996 (66) :21-26. 10 Xiao R, Qiao JT, Zhao HF, et al. Sodium selenite induces apoptosis in cultured cortical neurons with special concomitant changes in expression of the apoptosis-related genes J. Neurotoxicology, 2006,27 (4): 478 - 484. 11 Yan Li-Ping, Zhang Huiping. Flavored Kong pillow in the treatment of stroke, dementia, 36 cases of Denmark J. Journal of Medicine, 2000 (1): 32. Reposted elsewhere in the paper for free download 12