Application of 2 '-fucosyllactose in preparation of synaptic injury medicine or food

Abstract

The invention relates to 2 '-fucosyllactose, in particular to application of 2'-fucosyllactose in preparation of drugs or food for synaptic injury, and the synaptic injury comprises the following steps: reducing the length and width of density of a compact after progeny brain synapses activated by maternal immune response or induced by virus infection; animal experiments find that the length and width of the density of a compact after maternal immunity activates and induces synapses of the brain of a filial generation mouse are remarkably reduced, expression of PSD-95 of the brain of the mouse is inhibited, but dietary supplement of 2 '-fucosyllactose can protect the filial generation mouse from synaptic injury; dietary supplement of 2 '-fucosyllactose can improve neurodevelopment problems such as social and cognitive disorders and repetitive behaviors related to filial generation autism activated and induced by maternal immune response.

Description

Technical Field

[0001] The invention relates to 2'-fucosyllactose, in particular to the application of 2'-fucosyllactose in preparing synaptic injury medicine or food. Background Art

[0002] Studies have found that activation of maternal immune response (MIA) by maternal infection during pregnancy can have adverse effects on the neurodevelopment of offspring.

[0003] In response to this technical problem, the prior art adopts the method of administering human milk oligosaccharides to pregnant mothers in order to reduce the degree of neurological damage caused by maternal immune response to unborn offspring. For example, a Chinese patent discloses an HMO mixture for improving the microbiota of pregnant women, publication number CN114761024A, which records: a human milk oligosaccharide (HMO) for supplementing the diet of pregnant women: for promoting the development of a balanced immune system in unborn babies, for promoting the normal development of the brain in unborn babies, for promoting the development of a healthy gastrointestinal system in babies subsequently born by the pregnant women, for reducing the severity and / or incidence of immune-related diseases in babies subsequently born by the pregnant women, for reducing the severity and / or incidence of neurodevelopmental diseases in babies subsequently born by the pregnant women, and / or for reducing the severity and / or incidence of functional gastrointestinal diseases in babies subsequently born by the pregnant women.

[0004] The limitation of the existing technology is that the premise of administering human milk oligosaccharides is that the pregnant woman has been found to have activated the maternal immune response during pregnancy, and it must be absorbed by the pregnant woman and indirectly delivered to the fetus (offspring); that is to say, if the existing technology fails to intervene during pregnancy, it will not have a positive effect on the fetus near the due date or the baby that has already been born. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides an application of 2′-fucosyllactose in the preparation of synaptic injury drugs or foods, which is used to improve the significant decrease in the length and width of the density of postsynaptic density in the brain of offspring induced by maternal immune response activation or viral infection, the inhibition of brain PSD-95 expression, and the social cognitive disorders related to autism in offspring.

[0006] The technical solution of the present invention is:

[0007] The present invention provides an application of 2′-fucosyllactose in the preparation of synaptic injury medicines or foods, wherein the synaptic injury comprises: a significant decrease in the length and width of the density of postsynaptic density in the brain of offspring induced by maternal immune response activation or viral infection.

[0008] Furthermore, the synaptic damage also includes: inhibition of PSD-95 expression in the offspring brain induced by maternal immune response activation.

[0009] Furthermore, the synaptic damage also includes: social and cognitive disorders and repetitive behaviors related to autism in offspring induced by maternal immune response activation.

[0010] Furthermore, the medicine refers to any tablet, capsule, oral tincture, pill, granule, powder or granule containing an effective dose of 2′-fucosyllactose.

[0011] Furthermore, the food refers to any milk and dairy products, fats / oils and emulsified fat products, frozen drinks, fruits / vegetables, beans / edible fungi / algae / nuts and seeds, cocoa products / chocolate and chocolate products and candies, grains and grain products, baked goods, meat and meat products, aquatic products and their products, eggs and egg products, sweeteners, condiments, special nutritional foods, beverages, alcoholic beverages, jellies, tea, coffee, collagen casings, yeast products, fried foods and puffed foods that contain an effective dose of 2′-fucosyllactose.

[0012] Furthermore, the medicine or food is administered to the offspring born after the immune response of the mother is activated.

[0013] Furthermore, the administration method includes nasogastric tube, jejunal tube, oral, sublingual, anal plug or rectal feeding.

[0014] The beneficial effects of the present invention are as follows: 2′-fucosyllactose was studied, and animal experiments showed that maternal immune activation induced a significant decrease in the length and width of the density of postsynaptic density in the brains of offspring mice, and inhibited the expression of PSD-95 in the brains of mice, but dietary supplementation with 2′-fucosyllactose could protect offspring mice from synaptic damage;

[0015] In addition, it was found that dietary supplementation with 2′-fucosyllactose can improve neurodevelopmental problems such as social interaction, cognitive impairment, and repetitive behaviors associated with autism in offspring induced by maternal immune response activation;

[0016] Experiments have shown that 2′-fucosyllactose improves neurodevelopmental problems in mammal offspring caused by activation of maternal immune response (MIA) during pregnancy due to infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Illustration of the mouse grouping for Experiments 1 and 2.

[0018] Figure 2The figures show the experimental results of the length and width of the postsynaptic density in the mouse brain and the expression level of PSD-95 in the mouse brain; A is a representative picture of synaptic ultrastructure, B is a statistical graph of the length of the synaptic density, C is a statistical graph of the width of the synaptic density, D is a brain section image of PSD-95 immunofluorescence staining, EH are statistical graphs of the expression levels of each gene in the brain, E is GluN2B, F is GluA2, G is PSD-95, and H is FXR1; all data are expressed as mean ± standard error, *p<0.05 and **p<0.01 compared with mPBS-oCD, #p<0.05 and ##p<0.01 compared with mMIA-oCD.

[0019] Figure 3 The results of social and cognitive impairment and repetitive behavior experiments are shown in the figure; AD in the figure is the results of the three-chamber interaction test, E is the results of the buried bead test, F is the results of the open field test, and G is the results of the novel object test. The behavioral test diagram is drawn by FigDraw (ID: OAWSRc75a4). All data are expressed as mean ± standard error, *p<0.05 and **p<0.01 compared with mPBS-oCD, #p<0.05 and ##p<0.01 compared with mMIA-oCD.

[0020] Figure 4 The results of the co-cage experiment; A in the figure is an illustration of the mouse grouping, B is a figure showing the weight measurement results, C is a figure showing the results of the three-chamber interaction test, D is a figure showing the results of the open field test, E is a figure showing the results of the novel object test, and F is a figure showing the results of the buried bead test. All data are expressed as mean ± standard error, *p<0.05 and **p<0.01, compared with mPBS-oCD, #p<0.05 and ##p<0.01, compared with mMIA-oCD. DETAILED DESCRIPTION

[0021] The specific implementation of the present invention will be further described below in conjunction with the accompanying drawings:

[0022] 1. Experiment on the length and width of postsynaptic density in mouse brain and the expression level of PSD-95 in mouse brain

[0023] 1. Grouping of mice

[0024] like Figure 1As shown, pregnant mice (C57BL / 6J mother mice) were divided into two groups. On 12.5 days of pregnancy, pregnant mice in the experimental group were injected with poly(I:C) (maternal immune activation group, hereinafter referred to as mMIA group), and pregnant mice in the control group were injected with PBS (hereinafter referred to as mPBS group). The offspring of the above two groups were fed with normal breast milk after birth, weaned after 3 weeks, and the offspring of the mPBS group were divided into mPBS-oCD group (blank control group) and mPBS-o2′-FL group (2′-fucosyllactose control group), and the offspring of the mMIA group were divided into mMIA-oCD group (blank experimental group) and mMIA-o2'-FL group (2′-fucosyllactose experimental group). Among them, 800 mg / kg bw of 2′-fucosyllactose (2′-FL) was added to the diet of the mPBS-o2′-FL group and the mMIA-o2′-FL group. After 5 weeks of feeding, the mice were euthanized for physical and chemical experiments.

[0025] 2. Transmission electron microscope sampling, sample preparation and observation

[0026] (1) Mouse tissue collection: After the mouse was anesthetized, the hippocampus of the brain was quickly removed at 4°C and the tissue was quickly cut into 1 mm pieces using a scalpel. 3 Small pieces on the left and right (at least one cross section 1mm 2 ). Quickly immerse in glutaraldehyde fixative and store in a 4°C refrigerator. Mouse tissues were fixed with 2.5% glutaraldehyde for more than 6 hours; the tissues were washed once with 0.1M / L PBS at 5min, 10min, 15min, 20min, 25min, and 30min; then fixed with 1% osmium acid in a 4°C refrigerator in the dark for 1.5h; then the tissues were washed again with PBS, and the washing method was as above. Then the tissue samples were dehydrated with gradient concentrations of ethanol: 30%, 50%, 70%, 80%, and 90%, each for 15min, and finally dehydrated twice with 100% ethanol (absolute ethanol), each for 30min; after dehydration, the tissues were placed in 50% white glue ethanol solution at room temperature overnight; then the tissue samples were infiltrated with white glue at room temperature twice, the first time for 6h and the second time for 3h; finally, the samples were embedded in white glue capsules and baked in a 60°C oven for 48h. Finally, the tissue was sliced ​​ultrathinly using an ultramicrotome and floated on 3% uranyl acetate aqueous solution for 15 min, then restained with 4% lead solution for 10 min. All sections were observed under a JEM-1230 transmission electron microscope at 80 kV to measure the length and width of the synaptic density.

[0027] 3. Immunofluorescence

[0028] (1) Sample preparation: After killing the mice, half of the brain tissue was quickly fixed in 4% paraformaldehyde for 48 h (4°C), embedded in paraffin, sliced, and placed at 4°C; (2) Immunofluorescence staining: The slices were removed and rewarmed at room temperature for 30 min. The tissue slices were placed in xylene I for 10 min, xylene II for 10 min, 100% ethanol I for 5 min, 100% ethanol II for 5 min (that is, soaked in xylene twice and anhydrous ethanol twice), 90% ethanol for 5 min, 80% ethanol for 5 min, and 70% ethanol for 5 min for dewaxing and rehydration, and washed with PBS for 4 min, 5 times; the slices were placed in the permeabilization solution X-100 Triton for 15 min, and then washed with PBS for 5 min, 3 times; the repair solution was poured in, and the slices were boiled on medium heat in a microwave for 8 min, and then placed in a 100°C water bath and boiled for 15 min for antigen repair, and washed with PBS for 5 min, 6 times; then placed in serum for blocking for 2 h, and incubated with primary antibody at 4°C overnight. The sections were rinsed with PBS for 5 min, 6 times, and incubated with fluorescent secondary antibody for 2 h in the dark after drying. The sections were rinsed with PBS for 10 min, 6 times, and finally sealed with anti-fluorescence attenuation sealing medium containing DAPI and observed under a fluorescence microscope.

[0029] 4. Nucleic acid extraction, quantification, reverse transcription and qRT-PCR

[0030] (1) RNA extraction

[0031] Take out the mouse tissue sample from the -80°C refrigerator, quickly weigh about 0.1 g of tissue and put it into an RNase-free centrifuge tube containing 1 mL of Trizol reagent, and extract RNA according to the instructions for extracting RNA.

[0032] (2) Nucleic acid quantification

[0033] The concentration of mRNA in the extracted sample was determined using a nucleic acid quantifier, and the sample was diluted to the same concentration using enzyme-free water. The purity of the nucleic acid in the sample was evaluated based on the OD 260 / 280nm value and the OD 260 / 230nm value to determine whether the sample was contaminated by protein, sugar or organic solvent.

[0034] (3) Reverse transcription

[0035] Reverse transcription was performed according to the instructions of the reverse transcription kit (PrimeScript RT Master Mix), and the samples were reverse transcribed into cDNA and stored in a -80°C refrigerator for future use.

[0036] (4) qRT-PCR

[0037] According to the SYBR kit configuration system, the ABIQuant Studio5 system was used to detect the CT value of the sample gene, with β-acting as the internal reference and 2- △△CTThe relative gene expression was calculated by the primers shown in the following table.

[0038]

[0039] 5. Results, see Figure 2 .

[0040] Depend on Figure 2 It can be seen that the length and width of synaptic density, as well as the expression levels of genes such as GluN2B, GluA2, PSD-95, and FXR1 in the brain of the mMIA-o2′-FL group were significantly increased compared with the mMIA-oCD group, almost reaching the same level as the mPBS-oCD group and the mPBS-o2′-FL group. Therefore, it can be inferred that 2′-fucosyllactose can be used to treat the significant decrease in the length and width of the postsynaptic density of the brain and the inhibition of the expression of PSD-95 in the brain induced by the activation of maternal immune response.

[0041] Second, social and cognitive impairments in mice and repeated behavioral experiments

[0042] 1. Grouping of mice

[0043] like Figure 1 As shown, pregnant mice (C57BL / 6J mother mice) were divided into two groups. On the 12.5th day of pregnancy, pregnant mice in the experimental group were injected with poly(I:C) (maternal immune activation group, hereinafter referred to as mMIA group), and pregnant mice in the control group were injected with PBS (hereinafter referred to as mPBS group). The offspring of the above two groups were fed with normal breast milk after birth, weaned after 3 weeks, and the offspring of the mPBS group were divided into the mPBS-oCD group (blank control group) and the mPBS-o2′-FL group (2′-fucosyllactose control group), and the offspring of the mMIA group were divided into the mMIA-oCD group (blank experimental group) and the mMIA-o2′-FL group (2′-fucosyllactose experimental group). Among them, 800 mg / kg bw of 2′-fucosyllactose (2′-FL) was added to the diet of the mPBS-o2′-FL group and the mMIA-o2′-FL group. Animal experiments were carried out after 5 weeks of feeding, and the mice were killed by euthanasia.

[0044] 2. Three-chamber interaction test

[0045] The three-chamber interaction test is used to evaluate social preference and social novelty preference. The test area is evenly divided into three interconnected chambers (left, middle, and right). In the first 5 minutes, the mice to be tested freely explore the entire test area and familiarize themselves with the environment. The time they enter each chamber is recorded. Social preference is measured in the second 5 minutes: before the test, an empty wire mesh cup is placed in the left chamber, and a wire mesh cup containing a strange mouse (mouse 1, of the same sex, group, and age) is placed in the right chamber. The time spent by the test mouse with the empty cup and in the left chamber (sniffing, climbing, and the time and number of times it enters the chamber) is recorded, as well as the time spent by the test mouse with the strange mouse and in the right chamber (sniffing, climbing, and the time and number of times it enters the chamber). The social preference index is calculated according to formula (1-1). In the third 5 minutes, a social novelty preference experiment is conducted: a new strange mouse (mouse 2, of the same sex, group, and age) is placed in the previous empty cup. The right chamber is set up the same as the second 5 minutes, and the interaction time between the test mouse and the two mice in the left and right chambers is also recorded. And calculate the social novelty preference index according to formula (1-2).

[0046] Social novelty preference index = (communication time with mouse 1 - communication time with the empty wire cup) / (communication time with mouse 1 + communication time with the empty wire cup) (1-1)

[0047] Social novelty preference index = (communication time with mouse 2 - communication time with mouse 1) / (communication time with mouse 2 + communication time with mouse 1) (1-2)

[0048] 3. Buried bead test

[0049] Each mouse was placed in a standard and familiar experimental cage with a sawdust bedding layer at a depth of 4 cm. Twenty glass marbles (15 mm diameter, arranged in 4 × 5) were lightly spread on the sawdust bedding and the mice were allowed to explore for 30 min. The number of marbles buried (two-thirds or more) was recorded.

[0050] Bead burying index = (number of beads buried by mice / 20) × 100%

[0051] 4. Open field test

[0052] After acclimation for at least 10 min, mice were introduced into a blue movement monitoring box (40 cm x 40 cm x 40 cm) to facilitate unrestricted movement for 5 min. After each test, the box was cleaned with 75% ethanol. The total distance moved was recorded.

[0053] 5. Novel Object Test

[0054] Day 1: Mice were placed individually in an empty square box (40 cm × 40 cm × 40 cm) for a 5-minute free exploration period. Day 2: Two identical objects were placed in the northeast and southwest corners of the box. The test mice were placed in the center of the box and allowed to explore freely for 10 minutes. Day 3: A familiar object and a novel object were placed in the box, maintaining the same relative positions as during training. Mice were allowed to explore them freely for 10 minutes. The behavioral software recorded the exploration time of the familiar and novel objects.

[0055] Discrimination index = (time spent exploring novel objects - time spent exploring familiar objects) / total exploration time.

[0056] 6. Results, see Figure 3 .

[0057] Depend on Figure 3 AD showed that in terms of sociality and social novelty, the mMIA-o2′-FL group was significantly improved compared with the mMIA-oCD group, and was close to the mPBS-oCD group and the mPBS-o2′-FL group. Figure 3 EG shows that the number of buried beads in the mMIA-o2′-FL group is significantly reduced compared with the mMIA-oCD group, which means that the mMIA-o2′-FL group is not prone to anxiety; the total moving distance and discrimination index of the mMIA-o2′-FL group are higher than those of the mMIA-oCD group and close to the mPBS-oCD group and mPBS-o2′-FL group. Therefore, it can be inferred that 2′-fucosyllactose can be used to treat social, cognitive disorders and repetitive behaviors related to autism in offspring induced by maternal immune response activation.

[0058] III. Co-cage treatment experiment

[0059] 1. Grouping of mice

[0060] like Figure 4As shown in A, pregnant mice (C57BL / 6J female mice) were divided into three groups. On 12.5 days of pregnancy, the pregnant mice in the experimental group were injected with poly(I:C) (maternal immune activation group, hereinafter referred to as mMIA group), the pregnant mice in the control group were injected with PBS (hereinafter referred to as mPBS group), and the pregnant mice in the treatment group were injected with poly(I:C) (hereinafter referred to as mMIA+2′-FL group). The above three groups of offspring were fed with normal breast milk after birth, weaned after 3 weeks, and the offspring of the mPBS group were divided into the mPBS-oCD group (blank control group), the offspring of the mMIA group were divided into the mMIA-oCD group (blank experimental group) and the mMIA-oCD (Co-housing) group (co-cage experimental group), and the offspring of the mMIA+2′-FL group were divided into the mMIA+2′-FL-oCD (Co-housing) group (co-cage treatment group) and the mMIA+2′-FL-oCD group (2′-FL treatment group), and supplemented with 800 mg / kgbw of 2′-fucosyllactose in the diet. The mMIA-oCD (Co-housing) group and the mMIA+2′-FL-oCD (Co-housing) group were co-caged. Animal experiments were performed after 5 weeks of feeding, and the mice were killed by euthanasia.

[0061] 2. Co-cage treatment experiment

[0062] Co-cage treatment is an intervention method that co-cages mice with different treatments. It uses the coprophagy of mice to achieve the purpose of mutual colonization of intestinal flora between co-caged mice. After co-cage treatment, three-chamber interaction test, bead burying test, open field test and novel object test were carried out. The experimental methods are the same as those of "social and cognitive disorders and repetitive behavior experiments in mice".

[0063] 3. Results, see Figure 4 .

[0064] Depend on Figure 4 BE showed that although the data of mMIA-oCD (co-housing) group and mMIA+2′-FL-oCD (co-housing) group were behind those of mPBS-oCD group, and even behind those of mMIA+2′-FL-oCD group, they were significantly improved compared with mMIA-oCD group. Figure 4 F shows that the number of buried beads in the mMIA-oCD (co-housing) group and the mMIA+2′-FL-oCD (co-housing) group was significantly less than that in the mMIA-oCD group. This means that co-housing can improve the social, cognitive impairment and repetitive behavior associated with autism in offspring induced by maternal immune response activation.

[0065] The above embodiments and descriptions are only for illustrating the principles and best embodiments of the present invention. Without departing from the spirit and scope of the present invention, the present invention may be subject to various changes and improvements, all of which fall within the scope of the present invention to be protected.

Claims

1. A use of 2′-fucosyllactose in the preparation of synaptic injury medicines or foods, characterized in that: The synaptic damage includes: a decrease in the length and width of postsynaptic density in the brains of offspring induced by maternal immune response activation or viral infection.

2. The use of 2′-fucosyllactose in the preparation of synaptic injury medicines or foods according to claim 1, characterized in that: The synaptic damage also includes: inhibition of PSD-95 expression in the brain of offspring induced by maternal immune response activation.

3. The use of 2′-fucosyllactose in the preparation of synaptic injury medicines or foods according to claim 1, characterized in that: The synaptic damage also includes: social and cognitive disorders and repetitive behaviors related to autism in offspring induced by maternal immune response activation.

4. The use of 2′-fucosyllactose in the preparation of synaptic injury medicine or food according to claim 1, characterized in that: The medicine refers to tablets, capsules, oral tinctures, pills, granules, powders or granules containing an effective dose of 2′-fucosyllactose.

5. The use of 2′-fucosyllactose in the preparation of synaptic injury medicines or foods according to claim 1, characterized in that: The food refers to milk and dairy products, fats / oils and emulsified fat products, frozen drinks, fruits / vegetables, beans / edible fungi / algae / nuts and seeds, cocoa products / chocolate and chocolate products, as well as candy, grains and grain products, baked goods, meat and meat products, aquatic products and their products, eggs and egg products, sweeteners, condiments, special nutritional foods, beverages, alcohol, jellies, tea, coffee, collagen casings, yeast products, fried foods and puffed foods that contain an effective dose of 2′-fucosyllactose.

6. The use of 2′-fucosyllactose in the preparation of synaptic injury medicines or foods according to claim 1, characterized in that: The medicine or food is administered to the mother to produce offspring after the immune response is activated.

7. The use of 2′-fucosyllactose in the preparation of synaptic injury medicine or food according to claim 6, characterized in that: The administration methods include nasogastric tube, jejunal tube, oral, sublingual, anal plug or rectal feeding.

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