Conditional Fear Experiment-Mao Dongqing Improves Learning and Memory in Mice with Vascular Dementia

Mao Dongqing Improves Learning and Memory in Mice with Vascular Dementia

Summary:

OBJECTIVE : To observe the effect of Maodongqing extract on learning and memory ability of vascular dementia (VD) model mice, and to explore its potential mechanism.

METHODS : A mouse model of dementia was prepared by repeated clamping, recanalization of bilateral common carotid arteries and tail vein bleeding. The mice were then randomly divided into sham operation group, model group, and nimodipine control group (30 mg/kg). Maodongqing low, medium and high dose group (5, 10, 20 g / kg), each group of corresponding drugs 10 mL / kg, for 35 consecutive days, from the 30th day to conduct behavioral testing, after the last dose Brain samples were collected and pathological changes, immunohistochemistry and Weest Blot were used to detect the expression of Bcl-2/Bax protein by HE staining. Results Compared with the sham operation group, the fear memory time of the model group was significantly shortened, the neuronal cell lesions in the hippocampus were severe, and the ratio of Bcl-2/Bax protein expression in the hippocampus was decreased, and it was mainly distributed in the cytoplasm. Compared with the model group, the fear memory time of the mice in each drug group was significantly prolonged, the tissue lesions in the hippocampus of the brain were improved, and the ratio of Bcl-2/Bax protein expression in the hippocampus was significantly increased.

Conclusion : Maodongqing extract may improve the learning and memory function of mice with vascular dementia by up-regulating Bcl-2 protein expression, down-regulating B ax protein expression, reducing apoptosis and protecting nerve cells.

Key words: Maodongqing extract; vascular dementia; learning and memory ability; apoptosis

With the emergence of global aging, chronic disease dementia has attracted more and more attention. Among them, vascular dementia is the second most common cause of senile dementia after Alzheimer's disease Czl, and it is currently the only type of degenerative brain disease that can be prevented. Therefore, it is of great significance to develop drugs that effectively prevent and treat VD. Mao Dongqing is the dried root or leaf of the holly genus Maodong, which is a common Chinese medicine in the south of China. It has the functions of promoting blood circulation, relieving swelling and relieving pain, clearing away heat and detoxifying. A large number of studies have shown that various preparations of Mao Dongqing are suitable for coronary heart disease. Cerebral thrombosis, thrombotic vasculitis and other cardiovascular and cerebrovascular diseases have significant effects. In this study, we investigated the effects of Maodongqing extract on learning and memory, pathological changes of hippocampus and expression of apoptosis-related proteins in vascular dementia mice, and explored the possible mechanism of Maodongqing in improving learning and memory of vascular dementia. Experimental basis.

1 Materials and methods

1.1 Animal KM mice, male, SPF grade, body weight 32-35 g, provided by Guangdong Experimental Animal Center, animal license number: SCXK (Guangdong) 2013-0002. Experimental animal feeding environment: temperature 23-25 ​​° C; relative humidity (50 ± 5)%, conventional feed.

1.2 Drugs and main reagents Maodongqing Pieces were purchased from Kangmei Pharmaceutical Co., Ltd. and Guangzhou Daxiang Pharmaceutical Co., Ltd., respectively, and identified as Mao Dongqing plant Ilex pubescens Hook. et. Arn. by Professor Huang Haibo of the Department of Chinese Medicine Identification, Guangzhou University of Traditional Chinese Medicine. 300g of Maodongqing Decoction pieces, pulverized into coarse powder, added with 60% ethanol and refluxed for 2 times, and then extracted with 6 times and 4 times of solvent respectively for 1.5 h and 1 h. After filtration, the drug solution was combined and concentrated to 150 under reduced pressure. m L, that is, the PHRE stock solution is diluted with distilled water to the desired concentration.

Nimodipine tablets, Bayer HealthCare, Ltd., batch number: BJ24208; SABC immunohistochemical staining kit, Dr. De Bio, batch number: 11C29C. P-actin antibody, b iow orld, lot number: AP0060. Bcl-2 antibody, abeam, lot number: ab182858. B ax antibody, abeam, batch number: ab32503. Horseradish peroxidase-labeled goat anti-rabbit factory lg (H 10 L), Beijing Dingguo Changsheng Biotechnology Co., Ltd., batch number: III-0011;

1.3 Instruments

AUM 220D electronic analytical balance, Japan S h in ad zu company; ultra-low temperature refrigerator, American Thermo company; ice machine, Japan Sanyo company products; high-speed refrigerated centrifuge, China Zhongke Zhongjia Technology Instrument Co., Ltd.; multi-function microplate reader , USA erk iti E lm er company; CM 1950 frozen slicer, A SP200S dewatering machine, EG 1160 embedding machine, RM 2135 slicer, ST5020 dyeing machine, DC 300 optical microscope, all of Germany Leica company; mouse conditions Fear test box, XR-XC404 conditional fear analysis system, Shanghai Xinsoft Technology Co., Ltd.

1.4 grouping, model preparation and administration

Male KM mice were used to prepare a classic VD model by common carotid artery ligation and repeated ischemia-reperfusion. The mice were intraperitoneally injected with 3.5% chloral hydrate (10 mL} kgl) and the bilateral common carotid arteries were isolated. The arterial clip blocked its blood flow for 30 min, loosened the arterial clip and restored the blood flow for 10 min, repeated 3 times. After the first blood flow was blocked for 5 min, the mice bleed and bleed for about 0.3 m L, and the blood was condensed to stop bleeding. 30 min after the third blood flow reperfusion, the mice were observed for breathing and heartbeat, and the skin was sutured after being normal. In the sham-operated group, the skin was sutured only by exposing the bilateral common carotid arteries for the same time. On the 2nd day after model establishment, the model mice were randomly divided into 5 groups except the sham operation group, the model group, the nimodipine control group (30 mg/kg), and the PHRE low, medium and high dose groups (including The drug dose was 5, 10, 20 g/kg }, 12 rats in each group. Each group of mice was intragastrically administered at l0mL/kg for 35 days, once a day. The sham operation group and the model group were given the same volume of distilled water. .

1.5 Behavioral testing

1.5.1 The rod climbing experiment was carried out 2 hours after the 30th day of the rod climbing experiment. A bamboo pole 50 cm long and 1.5 cm thick was fixed on a foam box. The bamboo raft was wrapped with gauze to prevent slipping. The mouse was placed face down on the top of the bamboo pole, and the time required for the mouse to climb the length of the rod was recorded. Before the official record score, the mice were trained to climb for 3 days, once a day, to ensure that each mouse learned to climb. According to the time of climbing time, if the mouse falls 7 points, no comment is 6 points, 40 s U-turn is 5 points, 20 s U-turn is 4 points, and climbing is 31-40 s. 3 points, 21-30 s is rated 2 points, 11 to 20 s for 1 point, 0 to 11 s for 0 points.

1.5.2 Conditional fear experiment

After 34 days of administration, the conditional fear test was carried out. The experiment was divided into two stages. The first stage was the fear training stage. After 2 hours of administration on the 34th day, the mice were placed in a fear box and the sound stimulation was given from 20 s. (75 dB , 2000 H ) for 10 s, giving light stimulation from 25 s for 5 s, starting from 28 s for foot electrical stimulation ((0.8 m A) 2 s. A total of 4 cycles, each at intervals 15 s. The second stage is the test phase, that is, after 2 hours of administration on the 35th day, the mouse is placed in the fear box, and the scene is consistent with the scene of the training stage, providing the same sound and light suggestive signals, but no electric shock is given. Animals will still exhibit a fear response, reflecting the animal's ability to remember fear by observing the animal's stiffness time.

1.6 sample collection

Two hours after the end of the fear experiment, 6 mice were randomly selected from each group, and 3.5% chloral hydrate (10 mL/kg} was anesthetized by intraperitoneal injection. Brain tissue was fixed by perfusion of 4% paraformaldehyde. After perfusion, brain tissue was taken. It was fixed in 10% neutral formalin solution for pathological sectioning. The other mice were killed by decapitation, the brain was quickly removed, and the hippocampus was isolated for immunohistochemistry and Wesenl Blot detection.

1.7HE staining observation

After the fixed brain tissue was trimmed, dehydrated, embedded, sliced, HE stained, and mounted, the tissue structure of the hippocampus CA 1 region was observed using an optical microscope.

1.8 immunohistochemistry

Distribution and expression of Bcl-2, Bax protein The mouse brain was embedded in T issue 0 C T-Freeze Medium and frozen sections were prepared. Strictly follow the SA BC immunohistochemical staining kit instructions for sequential fixation, blocking, primary antibody incubation, secondary antibody incubation, SA BC incubation, DAB color development, xylene transparent, neutral gum seal, microscopic observation, and digital medical image analysis The system performs digital photography and image analysis of the distribution and expression of Bcl-2 and Bax proteins.

1.9 Western Blot assay for expression of Bcl-2 and Bax proteins

The hippocampus tissue of the mouse brain was isolated, and total protein was extracted and quantified by BCA kit. Take 50 ug protein sample and electrophoresis with 15% polypropylene hydrazine gel for 80 V, 20 m in, then convert to 120 V, 80 min; then transfer to 300 m A, 1 h by wet transfer method; TB ST Wash the membrane 10 m in , 3 times; 5% B SA was blocked at room temperature for 4 h; 40 °C was incubated overnight; TBST was washed 10 m in 3 times; 37 0C was incubated for 1 h; TBST was washed 10 min, Three times; ECL was used for luminescence, and the image of Wesenl B lot was analyzed by ltu age L ab 3.0 software, and the relative expression values ​​of each group of proteins and p-actin were calculated.

1.10 statistical processing method

Statistical analysis was performed using SPSS 17.0 software, and the data was expressed as x s". One-way analysis of variance was used for comparison between groups, LSD test was used for homogeneity of variance, and D unnett's test was used for variance.

2 results

2.1 Climbing scores of each group of mice The climbing rod experiment was used to evaluate the difference in limb coordination ability between mice. The results showed that there was no significant difference between the sham operation group, the model group and each drug-administered group (P > 0.05), indicating that the VD modeling method used in this study did not affect the exercise and balance ability of the mice, see Table 1. ;

2.2PHRE impact on the conditions of fear memory in mice compared with sham group, the percentage of stiff time model mice was significantly shorter ((P <0.09, showed that VD model to replicate the success, and memory in mice. Compared with the model group, The percentage of stiffness in the nimodipine group and the PH RE medium and high dose group was significantly prolonged, and the difference was statistically significant (P<0.09, indicating that the PH RE in the range of 10-20 g/kg was able to remember the memory of VD mice. There is a significant improvement, as shown in Figure 1.

Effect of Maodongqing extract on the morphology of brain neurons in VD mice

Histopathological examination showed that the morphology and distribution of nerve cells in the cerebral cortex and hippocampus of the sham operation group were not abnormal. Compared with the sham operation group, the model group mice had neuronal interstitial edema, nuclear staining and pyknosis, and the cortical nerve cells were significantly reduced, accompanied by a large number of glial cell hyperplasia, multiple accumulations in the heap, and the hippocampal CA 1 region. The pyramidal cells are largely deficient, degenerated, and necrotic, and the dentate granule cells are denatured into vacuoles. Compared with the model group, the brain neuron lesions in the PH RE low, medium and high dose groups and the nimodipine group were significantly improved, which was reflected in the decrease of glial cell proliferation and the reduction of pyramidal cell lesions in the hippocampal CA 1 region. , see Figure 2;

2.4 immunohistochemical detection

The distribution and expression of Bcl-2 and Bax in the hippocampal CA1 region of VD mice showed that Bcl-2 and Bax showed discontinuous circular or flaky distribution, mainly in cytoplasmic expression. Compared with the sham operation group, the expression of Bc}2 and Bax protein in the model group was significantly increased. Compared with the model group, the expression of Bcl-2 was increased and the expression of Bax was decreased in the middle and high dose groups of nimodipine and PH RE. It indicates that the neuronal cells in the hippocampal CA1 region of VD model mice are apoptotic, but the apoptosis phenomenon is reduced under the intervention of nimodipine or PH RE, suggesting that Maodongqing extract can reduce hippocampal CA1 neurons in VD model mice. The occurrence of apoptosis. See Figure 3-5;

2.5 Western Blot detection of Bcl-2 in the hippocampus of VD mice, the expression of Bax protein increased and the decrease of Bax protein showed that the resistance of cells to apoptosis was enhanced, which is a marker for the protective effect of drugs. The greater the ratio of Bcl-2 and Bax, the stronger the anti-apoptotic ability. The results showed that compared with the model group, Maodongqing extract can increase the expression level of Bc}2 protein in hippocampus of VD mice, and decrease the expression of Bax protein, which significantly increases the ratio of Bcl-2 and Bax, indicating Mao Dongqing. The extract can enhance the anti-apoptotic ability of cells in the hippocampus of VD mice, thereby protecting neurons in the hippocampus of VD mice. See Figure 6 ;

3 Discussion

In this study, the mouse VD model was replicated by blood flow blockade combined with bloodletting. The model was successfully tested by mouse intelligent test and pathological examination. The experimental results showed that the fear memory time of the VD model group was shortened, and the neurons in the hippocampal CA 1 area of ​​the brain were damaged, while the Maodongqing extract could significantly increase the fear memory time of VD mice and reduce the neuronal cell damage of VD mice. The extent of this, combined with its effect on the apoptotic proteins Bcl-2 and Bax, can be inferred that Maodongqing exerts neuroprotective effects by inhibiting apoptosis in the hippocampal CA1 region.

Damage to the hippocampus of the brain can cause vascular dementia, leading to cognitive dysfunction. The hippocampus is associated with brain memory and is sensitive to cerebral ischemia. A large number of experimental results indicate that the death of vertebral neurons in the hippocampal CA1 region causes cognitive dysfunction in rodents, primates and humans. Therefore, pyramidal cells in the hippocampal CA1 region are critical for learning and memory, whereas the CA1 region is particularly sensitive to cerebral ischemia and can cause severe learning and memory deterioration. The results of pathological experiments showed that the pyramidal cells in the hippocampal CA 1 region of the VD model group were largely deficient, degenerated and necrotic, and the dentate granule cells were degenerated into vacuoles. Maodongqing extract can prevent the loss of pyramidal cells in CA1 region, thereby protecting nerve cells in hippocampus and improving learning and memory in VD mice.

Apoptosis plays an important role in ischemia-reperfusion injury. Bcl-2 and Bax belong to the Bcl-2 family of proteins and regulate apoptosis. Bc}2 is an inhibitory protein that prevents cytochrome C from releasing from the mitochondria to the cytosol. B ax is a pro-apoptotic protein that promotes apoptosis by translocating to the mitochondrial membrane and promotes the release of cytochrome C. Bc}2 maintains mitochondrial structure stability by balancing Bcl-2. The results showed that compared with the sham operation group, the expression of Bcl-2 and Bax protein in the hippocampus of the model group was increased, which may increase the expression of proapoptotic protein Bax and induce cells after intermittent ischemia in mice. Apoptosis, and due to the cell's own protective mechanism, the anti-apoptotic protein Bc}2 expression is up-regulated and plays a role in inhibiting apoptosis. Due to the ratio of Bcl-2 and Bax, whether the cells undergo apoptosis after being stimulated by apoptotic signals, the expression of Bax protein is dominant in the VD mouse model, which leads to apoptosis of hippocampal neurons and hippocampal morphology. Impaired, memory impediment; compared with the model group, Maodongqing extract can increase the expression of Bcl-2, reduce the expression of Bax, thereby significantly increasing the ratio of Bc}2 ax, and causing apoptosis of hippocampal CA1 cells. inhibition. Therefore, Maodongqing extract can improve the learning and memory ability of VD mice by regulating the expression of Bcl-2 and Bax in CA1 region of hippocampus and inhibiting apoptosis in CA1 region.

Mao Dongqing has been widely used in the treatment of cardiovascular diseases, and the curative effect is exact. In recent years, some scholars have also in-depth discussion on the protective effect of Mao Dongqing on brain tissue damage. The results of this experiment showed that the effect of Maodongqing extract on learning and memory impairment in VD mice was similar to that of nimodipine at high dose, which could improve learning and memory ability by inhibiting apoptosis of hippocampus in the brain of VD mice. It is expected to develop into a new drug for the treatment of vascular dementia.

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