Abstract: Objective: To explore the mechanism of the effect of Chaihu injection on learning and memory behavior. METHODS: Mice were given intraperitoneal injections of different doses of Bupleurum injection twice a day for 5 days. The effects of Bupleurum injection on learning and memory behavior in mice were studied by behavioral observation and biochemical detection, including dose-effect. Relationship and time-effect relationships and changes in superoxide dismutase (SOD), malondialdehyde (MDA) and NO levels in the brain. RESULTS: In addition to 50 mg / kg, the other 4 doses of Bupleurum injection were significant in the 5 doses (50, 100, 200, 400, and 800 mg / kg) compared with the saline-injected control group. Improve the ability of mouse Y-maze to distinguish learning and 24 h memory retention rate, 400 mg / kg dose is the best; the optimal dose of Bupleurum injection facilitating memory is significant 24 to 72 h after training The enhancement (P < 0.01) was most significant at 24 h; the optimal dose of Bupleurum injection significantly increased the activity of SOD in the brain of mice after behavioral training (P < 0.01) and decreased the level of lipid peroxidation MDA (P < 0.01) and increase the NO content (P < 0.01). Conclusion: The appropriate dose of Bupleurum injection can improve the learning and memory ability of mice in a certain period of time. This effect may be related to the increase of antioxidant capacity and NO content in the brain tissue.
Key words: Bupleurum; learning and memory; superoxide dismutase; malondialdehyde; nitric oxide
Learning and memory are an important field in neuroscience research. There is increasing evidence that excessive free radical production in the body is closely related to learning and memory. In recent years, studies have found that Bupleurum has liver and kidney damage caused by ischemia-reperfusion. Protection, its mechanism may be related to the inhibition of oxygen free radical formation, but its impact on learning and memory has not been reported. Therefore, this study explored the role and mechanism of Bupleurum in improving learning and memory by observing the learning and memory behaviors and biochemical indicators in Bupleurum mice.
1 Materials and methods
1.1 Materials
Kunming mice, male and female, weighing 18 ~ 22 g, were provided by the Animal Experimental Center of the School of Public Health, Southeast University, and entered the experiment after 2 days of adaptive feeding. Bupleurum Injection (1 g per 1 ml of Bupleurum crude drug) is produced in Xi'an Yabao Pharmaceutical Factory, Shaanxi Province, batch number 20030404; Brain Superoxide Dismutase (SOD) Assay Kit (batch number 20031003), malondialdehyde (MDA) The assay kit (batch number 20031003), the NO assay kit (batch number 20031003) and the Coomassie brilliant blue protein assay kit (batch number 20031003) were all provided by Nanjing Jiancheng Bioengineering Co., Ltd. All other reagents were of analytical grade.
1.2 Methods
1.2.1 Grouping and administration methods
140 mice were randomly divided into control group, saline control group, 50 mg / kg Bupleurum group, 100 mg / kg Bupleurum group, 200 mg / kg Bupleurum group, 400 mg / kg Bupleurum group and 800 mg / kg Chaihu group. Each group of mice was intraperitoneally injected at a concentration of 0.2 ml / 10 g, 2 times a day, 8 hours apart, for 5 days, and the normal saline control group was intraperitoneally injected with the same volume of normal saline. 1.2.2 Detection method of Y-maze discrimination learning The detection of mouse discrimination learning ability is carried out according to the literature [5] method. The mice were continuously trained in sections for 1 day, and the mice were rested for 10 minutes. The mice in the two sections were rested for 1 min. There were 9 correct responses (ie, 90% of the correct response rate) in 10 reactions until the standard of learning. Record the number of times the mice need to reach the standard of training, and the fewer the number, the stronger the learning ability. The number of trainings required to give Bupleurum-treated mice to the standard of learning is compared with the number of times the normal mice reach the required standard for training, and the learning improvement rate is calculated to reflect changes in the learning ability of the mice. The correct number of mice in 10 training sessions was measured at 24 h intervals, representing the 24 h memory scores of the mice. The 24 h memory retention rate was calculated by comparing the 24 h memory score of the mice with the standard of the study. The same method was used to detect the memory retention rate of mice at 48 and 72 h. The higher the retention rate, the better the memory of the animal. 1.2.3 Determination of SOD activity, MDA content and NO content in mouse brain tissue SOD activity After the mouse behavior test, the brain was immediately decapitated, the brain was taken on ice, and 10% brain tissue homogenate was prepared with physiological saline, and centrifuged at 4 ° C. The supernatant was taken, and the optical density value was measured at a wavelength of 550 nm using a Model 721 spectrophotometer, and the activity of SOD was calculated according to the formula. Determination of MDA content After the behavioral test of mice, the mice were immediately decapitated, the brain was taken on ice, 2% brain tissue homogenate was prepared with physiological saline, and the supernatant was centrifuged at 4 °C. The 721 spectrophotometer was used at 532 nm. The optical density value was measured, and the amount of MDA production was determined according to the formula. Determination of NO content After the experiment, the mice were immediately decapitated, the brain was separated on ice, the olfactory bulb and cerebellum were removed, the blood was washed with ice physiological saline, dried with filter paper, and the balance was weighed by electron analysis. According to brain tissue: normal saline 0.5 g: 1 ml of homogenate, centrifuged at 3 000 r / min, 4 ° C for 15 min, the supernatant was taken and frozen for testing. The professional technicians strictly follow the operating instructions.
1.2 Statistical methods
The suspected values ​​of the experimental data were selected by the Grubb method. The data were expressed as mean ± standard deviation (x ± s ), and the t test was used for comparison between groups. P < 0.05 was considered significant.
2 results
2.1 Dose-effect relationship of Bupleurum injection on learning and memory in mice Y-maze behavior test results show that different doses of Bupleurum injection have different effects on the learning ability and memory retention of mice. As shown in Table 1, compared with the saline control group, the effect of the 50 mg / kg dose of Chaihu injection group on the learning ability of mice was not statistically significant, and the other 4 doses of Bupleurum injection Both significantly enhanced the learning ability of the mice (P < 0.01), showing a significant dose-effect relationship; for the percentage of memory retention, except for the 50 mg / kg dose, the other four agents of Bupleurum injection were obvious The role of facilitating memory. Compared with the saline control group, the doses of 100-800 mg / kg significantly increased the memory retention rate of 24 h, of which 400 mg / kg had the best effect and the memory retention rate increased by 34.45%.
2.2 Time-effect relationship of the effect of Bupleurum injection on memory in mice The above-mentioned dose-effect relationship indicates that the effect of 400 mg / kg of Chaihu injection on the 24 h memory retention of mice is best. This experiment further used this dose to observe the effect of Bupleurum injection on memory retention at 48 and 72 h of Y-maze differential learning in mice. The results showed that the effect of 400mg / kg Bupleurum injection on memory was significantly increased at 24 ~ 72 h after training (P < 0.01), the most significant at 24 h, although it decreased at 48 ~ 72 h, but still significantly Better than the physiology of this phase.
2.3 The effect of Chaihu injection on SOD activity and MDA production in brain tissue of mice: To explore the mechanism of Chaihu injection to improve the learning ability of mice and enhance memory, blank control group, saline control group and optimal dose ( 400 mg / kg) The activity of SOD and the content of MDA in brain tissue were measured after the behavior of mice in the Chaihu injection group. This dose of Bupleurum injection can significantly increase the SOD activity of brain tissue and reduce the production of MDA (P < 0.01).
2.4 Effect of Chaihu injection on NO content in mouse brain The experiment found that Chaihu injection 400 mg / kg can significantly increase the content of NO in the brain of normal mice (P < 0.01).
3 Discussion
Learning memory is the most advanced function of the brain and an important indicator of animal development. At home and abroad, the method of establishing conditioned reflex is used to detect animal behavior and advanced brain function. The Y-maze method is widely used. The results of this experiment show that: Bupleurum injection can significantly improve the learning ability and memory influence of mice in the Y-maze in a certain dose range; the effect of facilitating memory is best at 24 h after training, although 48 ~ 72 h decreased, but still significantly better than the saline control group of the phase, in which the optimal dose group mice were accompanied by an increase in SOD activity and a decrease in MDA levels in brain tissue. SOD is an important enzyme in the body's anti-lipid peroxidase enzymatic defense system, which specifically removes superoxide anion radicals produced by biooxidation. Free radicals can initiate chain reaction of lipid peroxidation. MDA, the end product of decomposition of lipid peroxide, crosslinks and combines to form lipofuscin, which damages cell membranes, causes dysfunction of the nervous system, and acts as brain function in behavior. decline. Therefore, this study suggests that Bupleurum can improve the learning and memory function of mice with the increase of SOD activity, thereby enhancing the role of SOD in terminating the free radical chain reaction and indirectly reducing the production of MDA, but the specific mechanism needs further investigation. In this experiment, it was observed that the content of NO in the brain tissue of the best dose group of Chaihu injection was significantly higher than that of the saline group (P <0.01), suggesting that the effect of Bupleurum on improving learning and memory may increase the NO in brain tissue. The content is closely related. It is generally believed that long-term potentiation (LTP) of hippocampal synaptic effects may be a synaptic mechanism of learning and memory, and NO acts as a reverse messenger involved in the formation and maintenance of LTP. In the process of animal learning and memory, the NO level in different brain regions increased, and the results of this experiment are consistent with this. It is true that studies have also found that the content of NO in the hippocampus of mice with spatial learning disorders increases, and NO mediates neurotoxicity, thereby impairing learning and memory. The authors speculate that this may be related to the "double-edged sword-like" effect of NO with neurotoxicity and neurotrophy. When the NO content fluctuates within a reasonable range, it may mediate neurotrophic effects. When the NO content is too low or too high, It is manifested as a neurotoxic effect. In summary, the appropriate dose of Bupleurum injection has the effect of improving the learning and memory ability of mice in a certain period of time. This effect may be related to the increase of antioxidant capacity and NO content in brain tissue, and its mechanism deserves further investigation.
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