Effect of supplemental flax seed oil on learning and memory ability of offspring mice

【Abstract】Objective To investigate the effects of α-linolenic acid (α-LNA)-rich flax seed oil (linseed oil) on brain development and function of offspring mice. Methods Forty-five mature mice were randomly divided into 4 groups. The pregnant female rats were randomly divided into 4 groups and given 2 g (group A), 5 g (group B) and 10 g (group C). /kg body weight and control group (group 0), the pregnant rats were orally administered to the mice for weaning, the mother rats were sacrificed, the male and female rats were divided into cages, and the male rats were immediately sacrificed for brain weight measurement and the brain body ratio was calculated. The female C component was group C and D, and the female rats in groups A, B, and C continued to supplement the flax seed oil at the dose of the mother once a day. Group D did not supplement the flax oil to continue feeding; The mouse was subjected to darkness and water maze experiments, and the brain was taken for protein (TPRO), acetylcholinesterase (AChE) and nitric oxide (NO). Results The body weight of the 1 rat decreased with the increase of the dose, and the decrease of the C group was significant. The difference was statistically significant (P<0.05). There was no significant difference between the brain and the other groups (P>0.05). 2 The results of water maze test showed that the swimming time was shortened and the number of errors decreased in group B and group D compared with the control group (P<0.05). The results of mice avoiding darkness showed that the latency of each dose group was longer than that of the control group, and the number of errors decreased. , but the difference was not statistically significant. 3TPRO increased with the increase of dose, group D was significantly higher than group 0 and group A, the difference was statistically significant (P<0.05); there was no significant difference between groups of Ac hE; NO group A group had more than group C The increase was significantly higher (P<0.05), and the difference was statistically significant compared with the B and D groups (P<0.01). Conclusion The supplementation of flax seed oil in mouse embryonic and infantile stages can increase the protein content in the brain and promote the learning and memory ability of mice, which can cause changes in NO content in the brain.

【Key words】α-linolenic acid; memory; neurotransmitter; linseed oil

Flax seed oil, also known as linseed oil, is the main edible oil in northwestern China and northwestern Shanxi. The data show that the content of α-linolenic acid (α-LNA) in flaxseed oil is 61.27% [1]. of. α-LNA is an n - 3 polyunsaturated fatty acid, which is an essential fatty acid in the body and can be effectively converted into eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in vivo to exert its physiology. Features. Over the years, the role of n - 3 polyunsaturated fatty acids, especially DHA (the main component of fish oil) in promoting brain development and retinal function, has been confirmed by studies; in addition, it is similar to the structure of α-LNA and the content of α-LNA (50%). The above perilla seed oil has been experimentally proven to promote learning and memory in mice. In this experiment, flax seed oil was used as the source of α-LNA. The mice in the flaxseed oil were tested for learning and memory and some neurotransmitters in the brain to observe the flax seed oil on the embryonic and growth-producing mouse brain. The impact of development provides a basis for better development of flaxseed oil.

1 Materials and methods

1.1 Sample source

Flax seed oil: Produced from Wuzhai County, Shanxi Province, flax seed is extracted by supercritical CO2, and the α-LNA content is 50.50%.

1.2 Experimental animals and basic feed

45 mature Kunming mice were selected, 30 females and 15 males; experimental animals and feed were purchased from Experimental Animal Center of Shanxi Medical University.

1.3 Instruments

Mouse water maze instrument, WX-5 mouse passive avoidance condition reflectometer (produced by Institute of Materia Medica, Chinese Academy of Medical Sciences); 1.4 reagents nitric oxide (NO), acetylcholinesterase (AChE) and protein (TPRO) kits All were purchased from Nanjing Jiancheng Bioengineering Research Institute, and the remaining reagents were all commercially available analytical grade.

1.5 Experimental methods

1.5.1 Dose grouping and feeding: 30 females were selected from mature mice, 15 males were caged at 2:1, and sperm was found to be 0 d in the next morning. Females were kept in cages from 0 days after pregnancy. The pregnant rats were randomly divided into 4 groups, namely, A, B, C 3 dose groups and one control group (0 group). A, B, and C were given 2, 5, and 10 g/s from the 0th day of conception. Kg body weight of flaxseed oil, continuous gavage to the weaning of the rats, the newborn rats adjusted the number of litters per litter after the birth of 6 to 9 mice, the weaned rats were separated from male and female, the male rats were immediately sacrificed for brain weight measurement and calculated Brain-to-body ratio; Female C component was the same in the two groups, namely C and D groups. Except for group D, which did not give flax seed oil, the rest continued to be supplemented with maternal seed oil once a day for 5 weeks.

1.5.2 Determination of brain weight: The weaned and 56-day-old mice were weighed separately. After decapitation, the brain tissue was weighed and the brain ratio was calculated.

1.5.3 Mouse water maze test: After 5 weeks of supplementation of flaxseed oil, 10 rats in each group were randomly selected to start training, and the test object was continued during the training, once a day. The labyrinth lane has a water depth of 15 cm and a water temperature of (25 ± 1) °C. During the experiment, the mice were placed at the beginning of the lane, timing was started, the time required to reach the end point, and the number of errors occurred. The mouse training time was limited to 120 s, and the mice that did not reach the end point within 120 s were recorded as 120 s. Trained for 5 days, the last time training from the starting point. Finally, the number of errors in each group of animals, the time to reach the end point, and the number of animals arriving at the end point were calculated.

1.5.4 Mouse darkening test: After 5 weeks of supplementation of flaxseed oil, 10 rats in each group were randomly selected for darkness test, and the time required for each rat to be shocked from the bright room to the dark room was recorded. That is, the incubation period, training for 5 min, and recording the number of electric shocks within 5 min, 24 h after the re-test, recording the incubation period of each animal into the dark room and the number of electric shocks within 5 min, as a result of memory.

1.5.5 Determination of TPRO, NO, AChE: The mice tested in the water maze test were sacrificed, and the brain was homogenized at 4 °C for TPRO, AChE and NO determination. The method was provided by Nanjing Jiancheng Bioengineering Research Institute.

1.6 Statistical analysis

Statistical analysis was performed using statistical software SPSS 10.0, using one-way ANOVA, Poisson distribution and nonparametric statistical methods.

2 results

2.1 Brain to body ratio: See Table 1. The results in Table 1 showed that there was no significant difference in body weight and brain body between the groups during weaning (P > 0.05), while the weight of the 56 d-day mice decreased significantly with the increase of dose. Group C compared with each dose group. The difference was statistically significant (P <0.05), and the brain volume was also increased compared with the C group, which was statistically significant compared with the control group (P<0.05).

2.2 Mouse learning and memory experiments: See Table 2. From Table 2, the results showed that the mice swam out of the labyrinth. The B group and the D group shortened, and the number of errors decreased. The difference was statistically significant (P<0.05). The mice in the dark-avoidance dose group had a prolonged trend, and the number of errors decreased, but there was no significant difference compared with the control group (P>0.05).

2.3 The results of TPRO, NO and AChE in mouse brain tissue showed significant dose-response relationship. Group D was significantly higher than group 0 and group A. The difference was statistically significant. D:0 was P <0.01, D:A was P <0.05; In the determination of NO, the B, C, and D groups were significantly lower than the control group, and the A group was significantly increased. The difference was statistically significant (P<0.05). There was a difference between the A group and the B and D groups. Statistical significance (P <0.01), there was no significant difference between group A and group 0. Although AChE was significantly higher than group B and C, there was no significant difference between groups (3 discussion)

In this experiment, the mother and the rats were supplemented with flax seed oil as the source of α-LNA during the period from the lactation period to the 56 d of the rats. The behavioral test results showed that the flax seed oil promoted the learning and memory of mice. It is consistent with the research report on the ability of perilla oil to promote learning and memory in mice, but its effect is not proportional to the intake, and the effect of group C is weakened, which may be related to the production of lipid peroxidation by excessive intake of unsaturated fatty acids. the study. Moreover, the significant enhancement of learning and memory ability in group D also indicates that even if the α-LNA is terminated after weaning, the sub-mouse can obtain its biological effects by obtaining n- 3 series polyunsaturated fatty acids from the mother. At the critical moment of embryonic and infant brain nervous system development, more n-3 series of polyunsaturated fatty acids need to be supplemented. At this stage, the enzyme system of DHA synthesis in animal brain and liver is not perfect, and the level of desaturase activity is low, in infants. The ability to synthesize DHA using α-LNA is very low, and it is mainly obtained from the mother and milk. It has also been reported that DHA rapidly accumulates in infant tissues during pregnancy and infancy, and is derived from the maternal reserve, that is, directly supplied by the diet during pregnancy or by the synthesis of α-LNA in the diet, and is selectively transported through the placenta. Therefore, supplementation of α-LNA to pregnant mice is beneficial for promoting brain development in the rat.

TPRO, NO and AchE are closely related to brain function. When learning and memory, protein synthesis increases and new proteins or peptides are produced. Protein levels affect the brain development and function of offspring. The protein content in the brain increased with the increase of dose, indicating that α-LNA can affect the synthesis of brain protein and affect brain development and function. NO is a neurotransmitter that has been recognized in recent years. The release of proper amount of NO can promote learning. The ability, and through experiments, found that NO plays a central role in the learning and memory process and memory reproduction of newborn mice. In this experiment, the brain NO test of 56-day-old rats showed an increase or decrease in the different dose groups compared with the 0 group, and the A group was significantly increased, but the difference was not statistically significant compared with the 0 group. There was a difference between the A group and the C group. The significance of learning (P<0.05), the difference between the B and D groups was statistically significant (P<0.01). It indicates that different doses of α-LNA have different effects on NO in the brain, whether it is related to its central regulation and other reasons need to be further explored; acetylcholine is a neurotransmitter in the brain, and high-level nerves such as cognition, learning, memory, etc. Functionally related, the completion of learning and memory tasks is accompanied by changes in acetylcholine in the brain. AChE is a acetylcholine-degrading enzyme whose activity changes indirectly reflect the acetylcholine content in the brain.