Effect of Naolizhibao on Learning and Memory and Neuronal Apoptosis in Rats with Experimental Cerebral Ischemia

【Abstract】 Objective To study the effects of Naolizhibao on learning and memory function and neuronal apoptosis in experimental cerebral ischemia rats. Methods The cerebral ischemia model was established by bilateral common carotid arteries in rats. Morris water maze method was used to test the learning and memory function. HE staining was used to observe the morphological changes of nerve cells. TUNEL method was used to detect apoptotic cells. Results Brain Zhibao can significantly shorten the escape latency of rats with experimental cerebral ischemia and increase the number of times across the original platform; significantly reduce the number of apoptotic cells in the parietal cortex and hippocampal CA1 region. Conclusion Brain Lizhi has an improved effect on learning and memory dysfunction caused by cerebral ischemic injury, which may be related to its ability to reduce neuronal apoptosis.

[Keywords] brain power wisdom brain ischemia learning memory apoptosis

Brain Intelligence is used to treat mental retardation, cerebral palsy, sequelae of cerebrovascular disease, sequelae of brain trauma, sequelae of encephalitis, senile dementia and other mental and limb dysfunction associated with encephalopathy. Clinical studies have shown that Brain Intelligence can significantly improve the motor, language and mental symptoms of patients with sequelae of cerebral infarction. In vitro cell research indicates that Naozhizhibao and its drug-containing serum may have protective effects on sodium nitroprusside-induced apoptosis of PC12 cells by promoting the expression of Bcl-2 protein and increasing cell membrane fluidity . This experiment observed the effect of Naolizhibao on learning and memory function and neuronal apoptosis in rats with experimental cerebral ischemia, aiming to elucidate the mechanism of Naolizhibao for treating sequelae of cerebral ischemic injury.

1 Materials and methods

1.1 Animal Health Wistar rats weighing 220 ~ 250 g, male and female, Guangzhou University of Traditional Chinese Medicine Experimental Animal Center offers.

1.2 Drugs and Instruments Brain Intelligence is provided by Guangdong Gaoming Encephalopathy Medical and Medical Research Institute, prepared as 1 g of crude drug /ml for test solution, diluted at the required concentration before gavage. Nimium film (nimodipine, nimodipine, Nim ) tablets, Bayer products, Germany, batch number CAGFT3 . Apoptotic cells by TUNEL assay kit, Boehringer Mannheim (BOEHRINGER MANNHEIM) product, Morris water maze (SuperMaze animal behavior analysis system) provided by the Information Technology Co., Ltd. Shanghai Xin soft.

1.3 Preparation of learning and memory impairment model caused by cerebral ischemic injury Rats were anesthetized by intraperitoneal injection of 10% urethane 1 g/kg , fixed in supine position, midline neck incision, bilateral common carotid arteries, ligation with silk, sutured skin . In the sham-operated group, the bilateral common carotid arteries were isolated by the same method, but not ligated.

1.4 Grouping and administration The successful model animals were randomly divided into 4 groups: model group, nimodipine group, brain power Zhibao low-dose group and brain power Zhibao high-dose group, 8 rats in each group , male and female. There were 8 rats in the sham operation group , half male and half female. After waking in rats, nimodipine group were 12 mg / kg oral administration of nimodipine suspension; Naolizhibao different dosage groups were small dose 4 g / kg, a large dose of 8 g / kg administered orally brain Tchibo test solution; sham operation and model groups were given equal volume of saline, 1 / d, 30 consecutive d.

1.5 Morris water maze test Each group of rats began the water maze test after the end of the drug intervention. The water maze consists of a circular pool with a diameter of 130 cm and a height of 50 cm . The water depth is 30 cm and the water temperature is ( 26 ± 1 ) °C . Four water inlet points are indicated on the pool wall , thus dividing the pool into four quadrants, one quadrant. In the middle, put a platform with a diameter of 12 cm and a height of 29 cm . It is not 1 cm underwater and the water surface is covered with foam. The tests included: 1 Positioning navigation test: 6 days , respectively, from 4 different markers into the water point, the rats were placed in the water facing the pool wall, and the time required to find the platform within 120 s (evacuation latency) was recorded . If the platform fails to find the platform within 120 s after entering the water , place it on the platform for 15 s and the escape latency is recorded as 120 s . 2 Space exploration experiment: After the navigation experiment is completed, the platform is removed, and a water inlet point is selected to put the rat into the water in the pool wall, and the number of times across the original platform in 120 s is recorded .

1.6 Morphological observation of brain tissue The rats subjected to the learning and memory test were sacrificed under anesthesia, and the brain was quickly decapitated and immersed in 10% neutral formaldehyde solution. The sections were subjected to HE staining, and the photographs were observed under a light microscope. The number of intact vertebral cells in the 500 μm range of the hippocampal CA1 pyramidal cell layer and the number of intact intact neurons in each of the parietal cortex were counted by ordinary light microscopy at 400× times, and 5 non-overlapping fields were counted for each slice .

1.7 In situ apoptosis detection The sections were dewaxed, washed with water, proteinase K digestion, TUNEL reaction, and routinely operated according to the kit instructions. Film light microscope, the number of apoptotic cells in the parietal cortex and the number of apoptotic cells per high power field within the pyramidal cell layer of 500 μm wavelength range of the hippocampal CA1 region count 400 × light microscopy, each slice count 5 do not overlap Vision.

1.8 Statistical analysis The experimental data were expressed by ± s , and the statistical analysis software SPSS11.0 for windows 11.5 was used for repeated measurement analysis of variance and one-way analysis of variance.

2 results

2.1 Positioning navigation test results In the positioning navigation experiment, with the increase of training times, the average escape latency of each group of rats showed a gradual decline, but the rate of decline was inconsistent. The average escape latency of each group was compared with Figure 1. . 1, 4, 5, 6, model group, the average escape latency was significantly longer than sham group (P <0.01). Compared with the model group, the average escape latency was significantly shortened on the 5th and 6th day of the nimodipine group ( P<0.01 ), and the average escape latency was significantly shortened on the 1st , 4th , 5th and 6th day of the brainpower Zhibao low-dose group ( P<0.05 or P<0.01 ), the average escape latency of the 1st , 2nd , 4th , 5th and 6th day of the brainpower Zhibao group was significantly shorter ( P<0.01 ), but there was no significant difference compared with the sham operation group ( P>0.05 ).

Figure 1 Comparison of average escape latency in each group of rats

2.2 Space exploration experiment results In the space exploration experiment on the 7th day, the rats in the model group mostly swim outside the quadrant where the platform is located, while the movement trajectories of the rats in other groups are mostly located in the quadrant of the original platform, and the rats in each group cross the original platform position. The comparison of the number of times is shown in Table 1 . Compared with the sham operation group, the number of rats across the original platform was significantly reduced in the model group (P<0.01) . The number of rats crossing the original platform in the nimodipine group, the brainpower Zhibao group and the high-dose group was significantly increased compared with the model group. (P<0.01) . Table 1 The number of times each group of rats crossed the original platform position in the space exploration test

2.3 Morphological observation of brain tissue In the model group, a large number of nerve cells were necrotic or severely shrunk, the volume became smaller, the boundary between the nucleus and the cytoplasm was unclear, and the space around the blood vessels, neurons and glial cells was enlarged. The necrotic nerve cells of the rats in each drug-administered group were significantly reduced, and some damaged cells were observed sporadically. The morphology and arrangement of the hippocampal vertebral cells were basically normal. Figure 2 to 9. The comparison of the number of intact neurons in the parietal cortex and hippocampal CA1 region of each group is shown in Table 2 . Compared with the sham operation group, the number of intact neurons in the parietal cortex and hippocampal CA1 region of the model group was significantly reduced ( P<0.01 ). Compared with the model group, nimodipine and brain wisdom were both significantly improved in small doses and large doses. Nerve cell necrosis and loss caused by complete cerebral ischemia ( P<0.05 or P<0.01 ).

2.4 Detection of apoptotic cells by TUNEL method After 30 days of incomplete cerebral ischemic injury , a large number of apoptotic cells appeared in the brain tissue. The positive cells showed brownish yellow color and the negative cells were light blue. Some apoptotic cells are scattered and distributed, and may be necrotic. The results of comparison of the number of apoptotic cells in the parietal cortex and hippocampal CA1 region of each group are shown in Table 3 . Compared with the model group, nimodipine and brain-powered Zhibao could significantly reduce the number of apoptotic cells in the parietal cortex and hippocampal CA1 after incomplete cerebral ischemia ( P<0.05 or P<0.01 ). Table 2 Comparison of the number of intact neurons in the parietal cortex and hippocampal CA1 nuclei of each group of rats . Comparison of the number of apoptotic cells in the parietal cortex and hippocampal CA1 of each group

3 discussion

Permanent ligation of bilateral common carotid arteries in rats can cause learning and memory disorders. It is an easy-to-obtain, ideal animal model for studying neurological damage and mental retardation after cerebral ischemia. Neuronal apoptosis after cerebral ischemia is an important pathophysiological process in ischemic brain injury. It is mainly distributed in the marginal region of cerebral infarction, mostly delayed, and interferes with apoptosis after ischemia. The process helps to treat cerebral ischemic injury.

Brain wisdom treasure is mainly composed of Angelica, safflower, Polygala, Shichangpu, Huangjing, Yizhiren, Tortoiseshell, Scorpion, Walnut and so on. Angelica sinensis, safflower nourishing blood and promoting blood circulation, breaking silt and dispersing; Shichangpu, Yuanzhi Huayu sputum; Huang Jing, Yizhi Renqi spleen; tortoise shell, scorpion, walnut kernel flat kidney yin and kidney yang, beneficial marrow filling. Modern pharmacological research data show that Angelica can inhibit the expression of Bax protein and reduce the occurrence of apoptosis in cerebral ischemic area; safflower can reduce the volume of cerebral infarction, and increase the neuronal apoptosis-related protein bcl- in the acute phase of cerebral ischemic injury . Expression of 2 reduces the expression of Caspase-3 ; Yuanzhi, Huangjing and Yiren can improve learning and memory function; Shichangpu can improve the learning and memory function of dementia rats by enhancing synaptic plasticity, and volatile oil of Shichangpu can effectively inhibit brain deficiency Abnormally elevated levels of excitatory amino acids Glu , Asp , and GABA after blood - reperfusion , inhibiting Bax gene expression in rat cerebral cortex and hippocampal neurons , and enhancing Bcl-X gene expression, thereby inhibiting rat neuronal apoptosis and reducing Damage to nerve cells.

This study showed that after 30 days of bilateral ischemic injury caused by bilateral common carotid artery ligation , the rats in the model group had reduced learning and memory abilities, and a large number of neuronal necrosis and apoptosis. The rats with cerebral ischemic injury treated by Naolizhibao had significantly improved learning and memory function and significantly decreased apoptotic cells. Previous studies have shown that Brain Intelligence can inhibit NO- induced PC12 cell apoptosis through anti-oxidation, Ca2+ antagonism, promotion of Bcl-2 protein expression and regulation of cell membrane fluidity . Therefore, the mechanism of brain force Zhibao antagonizing neuronal apoptosis after cerebral ischemic injury may be related to its inhibition of neurotoxicity of excitatory amino acids and NO , and regulation of expression of apoptosis-related genes. Reducing neuronal apoptosis after cerebral ischemic injury may be one of the mechanisms by which Zhili Zhibao can improve learning and memory impairment in rats with cerebral ischemic injury. This study provides an experimental basis for the use of brain wisdom treasure for the treatment of sequelae of cerebral ischemic injury.