Application of nutritional composition helpful for improving social ability

Abstract

The invention relates to application of a nutritional composition helpful for improving social ability, and belongs to the field of nutrient substance research. According to the breast milk oligosaccharide composition containing the lactose-N-neotetraose and the 2 '-fucosyllactose provided by the invention, through the synergistic effect of the lactose-N-neotetraose and the 2'-fucosyllactose, the reduction of the social behavior ability can be improved, and the reduction of the social behavior ability is caused by abnormal activation of maternal immunity. Furthermore, the composition can improve the activity of neurons by inhibiting the transition activation of brain microglial cells, so that the improvement of nerve injury is realized, and help is provided for further improvement of social behavior ability. And a new thought is provided for the development of products for improving the reduction of the social behavior capability caused by maternal immune activation.

Description

Technical Field

[0001] This invention relates to the use of a nutritional composition that helps improve social skills, and belongs to the field of nutrient research. Background Technology

[0002] Social interaction is a complex behavior present in many species, observable from simple, primitive single-celled microorganisms (such as myxococci) to complex humans. For humans, social interaction refers to the interactions between people in society; it is the way people exchange information and ideas to achieve certain goals. Social communication ability is an individual's ability to perceive the emotions and thoughts of others when interacting with them. Social interaction is a ubiquitous social phenomenon, a unique ability of humans as social animals, and an important component of human survival activities. It also promotes economic and cultural exchange. In social interaction, individuals are no longer isolated entities but are closely connected with others; the establishment and development of any interpersonal relationship requires effective social communication. Research shows that children lacking social skills lack eye contact in social situations, do not seek interaction, do not share with others, do not respond to nonverbal cues, ignore surrounding information, and are unaware of social rules and etiquette in various situations.

[0003] Human milk oligosaccharides (HMOs) are the third most abundant solid component in breast milk, after lactose and fat, and are even more abundant than proteins. HMOs are a class of complex sugars with diverse structures. Based on their molecular structure, HMOs can be divided into two main categories: neutral fucosylated HMOs, neutral non-fucosylated HMOs, and acidic sialylated HMOs. Due to their unique structure, HMOs help infants establish a healthy gut microbiota, strengthen immune system development, and promote brain and cognitive development. For example, cited reference 1 describes an HMO composition containing 2'-FL and LNnT, which discloses that the HMO composition, as a prebiotic, can reduce the risk of intestinal inflammation, protect the intestinal barrier, and have potential anti-colon cancer effects. Cited reference 2 discloses an HMO composition containing 2'-FL and LNnT, which can specifically increase the abundance of Akkermansia muciniphila in the gut, and can be used to prevent or treat various metabolic, inflammatory, and neurological diseases.

[0004] However, none of the existing technologies mention improving the social ability impairments in offspring caused by maternal immune activation, nor do they mention specific improvement pathways at the genetic level.

[0005] References:

[0006] Reference 1: CN110650635A

[0007] Reference 2: CN111683665A Summary of the Invention

[0008] The problem the invention aims to solve

[0009] The effects of maternal immune activation on offspring development begin in the fetal period. The resulting damage not only occurs earlier but is also often more persistent and severe compared to other external injuries encountered after birth. Currently, there are no clear intervention strategies for the decline in social behavior in offspring caused by maternal immune activation. Therefore, this invention develops a breast milk-like nutritional composition suitable for use from birth through childhood and beyond, effectively improving the decline in social behavior and brain damage in offspring caused by maternal immune activation. This composition can be consumed by offspring from birth, improving their social abilities as early as possible.

[0010] It should be noted that the decline in social behavior and brain nerve damage mentioned in this invention refer to non-disease states in offspring that are not fully healthy due to maternal immune activation.

[0011] Solution for solving the problem

[0012] [1]. Use of a human milk oligosaccharide composition in the preparation of a product for improving a decline in social behavior, wherein the decline in social behavior is caused by abnormal activation of the maternal immune system;

[0013] The human milk oligosaccharide composition contains the following essential active ingredients: lactose-N-neotetrasaccharide and 2'-fucosylated lactose.

[0014] [2]. According to the use described in [1], wherein in the human milk oligosaccharide composition, the mass ratio of lactose-N-neotetrasaccharide and 2'-fucosylated lactose is 1:(1~10), preferably 1:(1~6), more preferably 1:(2~3.5), and even more preferably 1:(2~3.4).

[0015] [3]. According to the use described in [1] or [2], wherein the improvement of the decline in social behavior ability includes: increasing the social preference index, increasing the social novelty index and / or increasing the total social time.

[0016] [4]. The use according to any one of [1] to [3], wherein the improvement of social behavior decline includes: inhibiting the overactivation and proliferation of microglia in the cerebral cortex and / or regulating neuronal activity.

[0017] [5]. According to the use described in [4], wherein the regulation of neuronal activity includes increasing the expression level of BDNF in the cerebral cortex and / or increasing the expression level of the MECP2 gene in the cerebral cortex.

[0018] [6]. Use of human milk oligosaccharide composition in the preparation of products that improve brain nerve damage, wherein the brain nerve damage includes microglia overactivation, decreased BDNF expression and / or decreased MECP2 gene expression, and the brain nerve damage is caused by abnormal activation of the maternal immune system;

[0019] The human milk oligosaccharide composition contains the following essential active ingredients: lactose-N-neotetrasaccharide and 2'-fucosylated lactose.

[0020] [7]. According to the use described in [6], wherein in the human milk oligosaccharide composition, the mass ratio of lactose-N-neotetrasaccharide and 2'-fucosylated lactose is 1:(1~10).

[0021] [8]. According to the use described in [6] or [7], wherein,

[0022] The microglia overactivation includes increased expression of IBA-1 in the cerebral cortex; and / or

[0023] The decrease in BDNF expression includes a decrease in BDNF expression in the cerebral cortex; and / or

[0024] The reduced MECP2 gene expression includes a decrease in MECP2 gene expression in the cerebral cortex.

[0025] [9]. The use according to any one of [1] to [8], wherein the product includes food, and the food is a beverage, milk and dairy products, baked goods or confectionery.

[0026]

[10] . The use according to any one of [1] to [8], wherein the product is an oral preparation; the oral preparation includes at least one of tablets, pills, granules, powders, teas, capsules and oral liquids.

[0027] The effects of the invention

[0028] The composition comprising 2'-fucosylated lactose and lactose-N-neotetrasaccharide provided by this invention, through the synergistic effect of 2'-fucosylated lactose and lactose-N-neotetrasaccharide, especially lactose-N-neotetrasaccharide and 2'-fucosylated lactose, can significantly amplify each other's improving effect on the decline in social behavior caused by maternal immune activation, mainly manifested in the increase of social preference index, social novelty index, and total social time. Simultaneously, this invention has found that the composition can improve neural damage by inhibiting the overactivation of microglia in the brain and improving neuronal activity, thereby further contributing to the improvement of social behavior. Attached Figure Description

[0029] Figure 1 The social preference index of mice in different groups is shown.

[0030] Figure 2 The social novelty index of mice in different groups is shown. Detailed Implementation

[0031] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0032] Furthermore, to better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the present invention.

[0033] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0034] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0035] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0036] In this specification, "optional" and "optionally" mean that the events or circumstances described below may or may not occur, and the description includes both cases where the events or circumstances occur and cases where the events or circumstances do not occur.

[0037] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0038] As used herein, the term “and / or” covers all combinations of items connected by the term and should be regarded as if each combination had been listed separately herein. For example, “A and / or B” covers “A,” “A and B,” and “B.” For example, “A, B, and / or C” covers “A,” “B,” “C,” “A and B,” “A and C,” “B and C,” and “A and B and C.”

[0039] In addition, unless otherwise defined, other technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0040] This invention is mainly based on the following insights:

[0041] Human milk oligosaccharides (HMOs) have been shown to improve inflammation and neurological disorders. However, these conditions are acquired due to lifestyle habits and environmental factors. In contrast, the effects of maternal immune activation on offspring development begin in the fetal stage, causing damage that is not only earlier but also often more persistent and severe compared to other postnatal injuries. Currently, there are no reports on the decline in social skills in offspring caused by maternal immune activation, and it is not easy to determine whether or how this decline can be improved. This invention established a mouse model of maternal immune homeostasis dysregulation and, using offspring mice born to this model as research subjects, unexpectedly discovered that HMOs can improve the decline in social behavior in offspring caused by maternal immune activation.

[0042] I. Human milk oligosaccharide composition

[0043] Human milk oligosaccharides are a class of structurally complex non-digestible sugars, mainly composed of 3 to 10 monosaccharides (such as glucose, galactose, N-acetylglucosamine, fucose, and sialic acid). In some specific embodiments of the present invention, the human milk oligosaccharide is one or two of 2'-fucosylated lactose and lactose-N-neotetrasaccharide.

[0044] In some specific embodiments, the human milk oligosaccharide composition comprises the following essential active ingredients: lactose-N-neotetrasaccharide and 2'-fucosylated lactose.

[0045] The 2'-fucosyllactose (2'-FL) described in this invention is a neutral trisaccharide composed of L-fucose, D-galactose, and D-glucose units, wherein the monosaccharide L-fucose is linked to the disaccharide D-lactose via an α (1→2) bond. Its molecular formula is C2. 18 H 32 O 15 Its molecular weight is 488.439 g / mol.

[0046] The lactose-N-neotetrasaccharide (LNnT) described in this invention is a tetrasaccharide composed of β-D-galactose-(1→4)-β-D-glucose NAc-(1→3)-β-D-galactose-(1→4)-D-glucose units linked by glycosidic bonds. Its molecular formula is C2. 26 H 45 NO 21 Its molecular weight is 707.63 g / mol.

[0047] This invention does not particularly limit the source of the 2'-fucosyllactose and lactose-N-neotetrasaccharide, which can be, for example, from natural sources, synthetic sources, or microbial fermentation sources. Typically, 2'-fucosyllactose can be synthesized through steps such as glycosylation reaction between lactose acceptor and fucose donor; alternatively, it can be synthesized using exogenously added lactose as a substrate and 5'-guanine diphosphate nucleoside-fucose disodium salt formed through the microbial metabolic pathway as a precursor, under the action of fucosyltransferase. For lactose-N-neotetrasaccharide, it can be synthesized stepwise from lactose through a protecting group and a deprotection reaction via chemical reaction; it can also be synthesized from lactose and UDP-N-acetylglucosamine as substrates via β-1,3-N-acetylglucosamine transferase (LgtA); or it can be synthesized from LNT and UDP-galactose as substrates via β-1,4-galactosyltransferase (GalT).

[0048] In some embodiments, the mass content of 2'-fucosylated lactose and lactose-N-neotetraose may be 60% or more, preferably 80% or more, more preferably 90% or more, or any other content, relative to the total mass of the products from each source of 2'-fucosylated lactose and lactose-N-neotetraose.

[0049] In some embodiments, the human milk oligosaccharide composition comprises an active ingredient (a component that performs a specific physiological function, such as a component that improves a decline in social behavior) and an inactive ingredient (a component that does not perform a specific physiological function, such as a substance that does not improve a decline in social behavior). Exemplarily, the inactive ingredient may be other nutrients, any food-acceptable excipient, and / or substances that are generated during the production or acquisition of the active ingredient and cannot be effectively separated from the active ingredient or do not require separation. In some embodiments, the human milk oligosaccharide composition consists of the active ingredient and the inactive ingredient.

[0050] In some optional embodiments, the mass ratio of 2'-fucosylated lactose to lactose-N-neotetrasaccharide in the human milk oligosaccharide composition is 1:(1~10), preferably 1:(1~6), more preferably 1:(2~6), even more preferably 1:(2~3.5), and even more preferably 1:(2~3.4). For example, it can be 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, or 1:1. 8, 1:1.9, 1:2.0, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3.0, 1:3.1, 1:3.2, 1:3.3, 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9, 1:4.0, 1:4.1, 1:4.2, 1:4.3, 1:4.4, 1:4.5 1:4.6, 1:4.7, 1:4.8, 1:4.9, 1:5.0, 1:5.1, 1:5.2, 1:5.3, 1:5.4, 1:5.5, 1:5.6, 1:5.7, 1:5.8, 1:5.9, 1:6.0, 1:6.1, 1:6.2, 1:6.3, 1:6.4, 1:6.5, 1:6.6, 1:6.7, 1:6.8, 1:6.9, 1:7.0, 1:7.1, 1:7.2, 1: 7.3, 1:7.4, 1:7.5, 1:7.6, 1:7.7, 1:7.8, 1:7.9, 1:8, 1:8.1, 1:8.2, 1:8.3, 1:8.4, 1:8.5, 1:8.6, 1:8.7, 1:8.8, 1:8.9, 1:9.0, 1:9.1, 1:9.2, 1:9.3, 1:9.4, 1:9.5, 1:9.6, 1:9.7, 1:9.8, 1:9.9, 1:10.0.

[0051] II. Products

[0052] The product of this invention contains or uses the aforementioned human milk oligosaccharides, especially 2'-fucosylated lactose and lactose-N-neotetrasaccharide. The product of this invention can be a food product.

[0053] This invention does not particularly limit the specific form of the food. At room temperature, the edible nutrient can be solid, semi-solid, or liquid. Exemplarily, it may include a drinkable composition, a powder or granule composition, a gel, or a frozen or partially frozen composition. The food may optionally be part of a capsule filler, or may optionally be part of a beverage, dairy product, non-dairy cream, sauce, or baked goods.

[0054] In some specific implementations, the food may be powdered reconstituted food (solid beverages, instant coffee, cereal powder, nut powder or lotus root powder, etc.), baked goods (bread, cake or biscuit baked goods, etc.), beverages (carbonated beverages, fruit and vegetable juice beverages, functional beverages, tea beverages, milk beverages or alcoholic beverages, etc.), candy (gel candy, hard candy, compressed candy, etc.), milk and dairy products (fresh milk derived from raw cow (sheep) milk, milk powder, whey powder, fermented milk, cheese or condensed milk, etc.), etc.

[0055] In other specific embodiments, the food described in this invention may also be a health food, such as various types of oral preparations, including but not limited to tablets, pills, granules, powders, teas, capsules, or oral liquids.

[0056] This invention does not specifically limit the absolute content of 2'-fucosyllactose and lactose-N-neotetrasaccharide in food, as long as it meets the requirements of local food-related laws and regulations. In some embodiments, the mass content of 2'-fucosyllactose and lactose-N-neotetrasaccharide relative to the total mass of the food is at least 0.05%, preferably at least 0.1%, more preferably at least 1%, and even more preferably at most 12%.

[0057] In addition to the 2'-fucosylated lactose and lactose-N-neotetrasaccharide mentioned above, other ingredients may be included, such as proteins / amino acids, carbohydrates, fats, vitamins, minerals, and other components commonly found in foods. Furthermore, depending on the type of food and the end-user's needs, in some embodiments, the food described in this invention may also contain one or more of the following ingredients: plant-based ingredients, animal dairy ingredients, animal meat ingredients, functional additives, and any acceptable excipients.

[0058] Examples of plant-based ingredients include fruits such as fig, pomegranate, kiwi, orange, tangerine, pineapple, strawberry, apple, banana, grape, pear, cherry, blueberry, blackberry, blackcurrant, cranberry, raspberry, melon, amla, and bilberry, or their extracts; vegetables such as onion, cucumber, tomato, cauliflower, carrot, spinach, kale, Brussels sprouts, garlic, basil, and oregano, or their extracts; and rice (indica rice). Grains or their extracts, including japonica rice, glutinous rice, cereals (wheat, barley, oats, rye), corn, sorghum, millet, sorghum, yellow millet, buckwheat, soybeans, broad beans, peas, mung beans, red beans, kidney beans, etc.; nuts or their extracts, including walnuts, pistachios, cashews, hazelnuts, almonds, apricot kernels, pine nuts, peanuts, sunflower seeds, chestnuts, macadamia nuts, ginkgo nuts, etc.; coffee or its extracts; and some medicinal and edible plant-based Chinese medicinal materials or their extracts.

[0059] Animal dairy product ingredients can include fresh milk from mammals such as cows, sheep, and camels, as well as reprocessed dairy products such as whole milk powder, skim milk powder, whey protein concentrate, desalted whey powder, whey protein powder, and hydrolyzed whey protein powder.

[0060] Examples of animal meat product ingredients include those from pork, beef, mutton, seafood, or poultry.

[0061] Examples of functional additives include vitamin supplements, mineral supplements, nucleotide supplements, dietary fiber, and functional polyunsaturated fatty acid supplements.

[0062] Any acceptable excipients may include solvents, antioxidants, antibacterial agents, thickeners, diluents, cosolvents, stabilizers, emulsifiers, fillers, disintegrants, lubricants, coating materials, anti-caking agents, flavoring agents, sweeteners, food flavorings, and food colorings.

[0063] III. Applications to improve the decline in social behavior in offspring caused by abnormal activation of the maternal immune system.

[0064] This invention discovers that combining lactose-N-neotetrasaccharide and 2'-fucosylated lactose amplifies the effect of 2'-fucosylated lactose on improving the decline in offspring's social behavior caused by abnormal maternal immune activation. Similarly, 2'-fucosylated lactose also amplifies the effect of lactose-N-neotetrasaccharide on improving the decline in offspring's social behavior caused by abnormal maternal immune activation. Thus, the synergistic amplification effect between the two ensures that the composition containing both significantly improves the decline in offspring's social behavior caused by abnormal maternal immune activation.

[0065] The improvement of offspring's social behavior caused by abnormal maternal immune activation described in this invention is not intended to treat or prevent disease. Furthermore, the decline in social behavior described in this invention does not reach the level that can be identified as a disease.

[0066] In some implementations, the decline in social behavior includes a decrease in the social preference index, the social novelty index, and the total social time.

[0067] In some implementations, the improvement in the decline in offspring's social behavior caused by abnormal maternal immune activation includes: inhibiting microglia activation and proliferation and / or regulating neuronal activity.

[0068] In some specific implementations, the improvement of the decline in offspring's social behavior caused by abnormal maternal immune activation includes: inhibiting the activation and proliferation of microglia.

[0069] In some specific implementations, the improvement of the decline in offspring's social behavior caused by abnormal activation of the maternal immune system includes: regulating neuronal activity.

[0070] In some specific implementations, the improvement of the decline in offspring's social behavior caused by abnormal maternal immune activation includes: inhibiting the activation and proliferation of microglia, and regulating neuronal activity.

[0071] Furthermore, the inhibition of microglia activation includes reducing the expression level of IBA-1 in the cerebral cortex; the regulation of neuronal activity includes increasing the expression level of BDNF in the cerebral cortex and / or increasing the expression level of the MECP2 gene in the cerebral cortex.

[0072] In some specific implementations, the improvement of the decline in offspring's social behavior caused by abnormal activation of the maternal immune system includes reducing the expression of IBA-1 in the cerebral cortex.

[0073] In some specific implementations, the improvement of the decline in offspring's social behavior caused by abnormal activation of the maternal immune system includes: increasing the expression level of BDNF in the cerebral cortex.

[0074] In some specific implementations, the improvement of the decline in offspring's social behavior caused by abnormal activation of the maternal immune system includes: increasing the expression level of the MECP2 gene in the cerebral cortex.

[0075] In some specific implementations, the improvement of the decline in offspring's social behavior caused by abnormal activation of the maternal immune system includes: increasing the expression level of BDNF in the cerebral cortex and increasing the expression level of the MECP2 gene in the cerebral cortex.

[0076] In some specific implementations, the improvement of the decline in offspring's social behavior caused by abnormal activation of the maternal immune system includes: reducing the expression level of IBA-1 in the cerebral cortex and increasing the expression level of BDNF in the cerebral cortex.

[0077] In some specific implementations, the improvement of the decline in offspring's social behavior caused by abnormal maternal immune activation includes: reducing the expression level of IBA-1 in the cerebral cortex and increasing the expression level of the MECP2 gene in the cerebral cortex.

[0078] In some specific implementations, the improvement of the decline in offspring's social behavior caused by abnormal maternal immune activation includes: reducing the expression level of IBA-1 in the cerebral cortex, increasing the expression level of BDNF in the cerebral cortex, and increasing the expression level of the MECP2 gene in the cerebral cortex.

[0079] In some embodiments, when lactose-N-neotetrasaccharide and 2'-fucosylated lactose are combined in a mass ratio of 1:(1~10), preferably 1:(1~6), more preferably 1:(2~6), 1:(2~3.5), and even more preferably 1:(2~3.4), it has a better effect on improving the decline in social behavior ability.

[0080] In some implementations, the improvement of the decline in offspring's social behavior caused by abnormal maternal immune activation includes enhancing social behavior for non-therapeutic purposes.

[0081] IV. Uses to improve brain nerve damage

[0082] The present invention unexpectedly discovered that when the human milk oligosaccharide composition is applied to offspring mice with decreased social abilities due to maternal immune activation, it can significantly improve, for example, the excessive activation and proliferation of microglia in the cerebral cortex, the reduced expression of BDNF in the cerebral cortex, and the reduced expression of the MECP2 gene in the cerebral cortex caused by maternal immune activation. Furthermore, when lactose-N-neotetrasaccharide and 2'-fucosylated lactose in the human milk oligosaccharide composition are combined in a mass ratio of 1:(1~10), preferably 1:(1~6), more preferably 1:(2~6), 1:(1~3.5), and more preferably 1:(1~3.4), they exhibit a synergistic effect. Lactose-N-neotetrasaccharide can amplify the effect of 2'-fucosylated lactose on improving brain nerve damage in offspring caused by abnormal maternal immune activation. This brain nerve damage includes excessive activation of microglia, decreased BDNF expression, and decreased MECP2 gene expression. Similarly, 2'-fucosylated lactose can also amplify the effect of lactose-N-neotetrasaccharide on improving brain nerve damage in offspring caused by abnormal maternal immune activation. Thus, the mutual amplification effect between the two can further enhance the improvement effect of their combination on brain nerve damage.

[0083] In some implementations, microglia overactivation is a state in which microglia in the brain, especially in the cerebral cortex, undergo morphological changes and oxidative stress compared to a healthy state.

[0084] Therefore, the present invention provides the use of a human milk oligosaccharide composition comprising lactose-N-neotetrasaccharide and 2'-fucosylated lactose in the preparation of a product for improving brain nerve damage, said brain nerve damage including microglia overactivation in the cerebral cortex, decreased BDNF expression, and decreased MECP2 gene expression.

[0085] In some specific embodiments, the present invention provides the use of a human milk oligosaccharide composition comprising lactose-N-neotetrasaccharide and 2'-fucosylated lactose in the preparation of a product for improving microglia overactivation in the cerebral cortex, wherein said microglia overactivation in the cerebral cortex includes a significant increase in IBA-1 expression in the cerebral cortex.

[0086] In some specific embodiments, the present invention provides the use of a human milk oligosaccharide composition comprising lactose-N-neotetrasaccharide and 2'-fucosylated lactose in the preparation of a product for improving reduced BDNF expression in the cerebral cortex.

[0087] In some specific embodiments, the present invention provides the use of a human milk oligosaccharide composition comprising lactose-N-neotetrasaccharide and 2'-fucosylated lactose in the preparation of a product for improving reduced MECP2 gene expression in the cerebral cortex.

[0088] In some specific embodiments, the present invention provides the use of a human milk oligosaccharide composition comprising lactose-N-neotetrasaccharide and 2'-fucosylated lactose in the preparation of a product for improving microglia overactivation and reduced BDNF expression in the cerebral cortex, wherein the microglia overactivation includes a significant increase in IBA-1 expression in the cerebral cortex.

[0089] In some specific embodiments, the present invention provides the use of a human milk oligosaccharide composition comprising lactose-N-neotetrasaccharide and 2'-fucosylated lactose in the preparation of a product for improving microglia overactivation in the cerebral cortex and reducing MECP2 gene expression in the cerebral cortex, wherein the microglia overactivation in the cerebral cortex includes a significant increase in IBA-1 expression in the cerebral cortex.

[0090] In some specific embodiments, the present invention provides the use of a human milk oligosaccharide composition comprising lactose-N-neotetrasaccharide and 2'-fucosylated lactose in the preparation of a product for improving reduced MECP2 gene expression and reduced BDNF expression in the cerebral cortex.

[0091] In some specific embodiments, the present invention provides the use of a human milk oligosaccharide composition comprising lactose-N-neotetrasaccharide and 2'-fucosylated lactose in the preparation of a product for improving microglia overactivation in the cerebral cortex, reduced MECP2 gene expression in the cerebral cortex, and reduced BDNF expression in the cerebral cortex, wherein the microglia overactivation in the cerebral cortex includes a significant increase in IBA-1 expression in the cerebral cortex.

[0092] In some implementations, the improvement of brain nerve damage is not intended to prevent or treat disease. Similarly, the improvement of microglia overactivation, the improvement of BDNF expression reduction, and the improvement of MECP2 gene expression reduction in this invention are not intended to prevent or treat disease. Furthermore, the brain nerve damage, microglia overactivation, BDNF expression reduction, and MECP2 gene expression reduction described in this invention do not reach a level that can be considered a disease.

[0093] Example

[0094] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0095] Example 1: Construction of the model and human milk oligosaccharide intervention experiment involved in this invention

[0096] 1. Construction of a model of maternal immune homeostasis disorder

[0097] 150 female mice and 30 male mice were selected, primarily for offspring breeding. When grouping them together, healthy mice with shiny fur were chosen, and a female-to-male ratio of 2:1 was maintained. The weight of the female mice was measured regularly over the following week. Once the female mice showed significant weight gain, they were separated and injected intraperitoneally with 20 mg / kg poly(I:C) solution to establish a maternal immune homeostasis dysregulation model.

[0098] 2. Offspring Intervention

[0099] In this experiment, male offspring rats were used as experimental subjects. Three weeks after the female offspring gave birth, the male offspring were weaned and fed separately. When the male offspring were four weeks old, they were administered either PBS solution or HMOs solutions of different proportions via gavage for six weeks. One week before the end of the gavage period, when the offspring were nine weeks old, animal behavior tests were conducted to analyze the effect of the HMOs combination on the social behavior of offspring activated by maternal immunity.

[0100] 3. Intervention grouping of offspring animals

[0101] The experiment consisted of 11 groups, with 12 mice in each group: a control group, a model group, and 9 HMO intervention groups. "PBS mice" referred to control mice born after their mothers were injected with PBS solution during pregnancy, while "MIA mice" referred to offspring born after their mothers were injected with Poly(I:C) solution during pregnancy. During the experiment, each mouse was housed in a standard cage under a 12-hour light / dark cycle, with humidity of 50 ± 15% and temperature of 22 ± 2℃. Mice had free access to food and water.

[0102] 4. Monitoring of basic growth indicators

[0103] During the 6-week intervention, the mice's body weight, food intake, and water intake were recorded weekly.

[0104] 5. Design of intervention program for human milk oligosaccharides

[0105] In this invention, two human milk oligosaccharides, 2'-FL and LNnT, were selected and combined at different single doses or in different ratios to investigate the effects of different dosage combinations on improving social skills. The experimental design is shown in Table 1. According to the literature, the feeding dose of LNnT was fixed at 250 mg / kg body weight / day, while the dose of 2'-FL was selected at three gradients: 250 mg / kg body weight / day, 500 mg / kg body weight / day, 850 mg / kg body weight / day, and 1500 mg / kg body weight / day. These five single-factor intervention doses of the two substances were used as comparative examples. Four different ratios of LNnT:2'-FL (1:1, 1:2, 1:3.4, and 1:6) were then used as four examples.

[0106] Table 1: Experimental Intervention Design

[0107]

[0108] Example 2: Evaluation of Basal Growth Indicators in Mice

[0109] Table 2 shows the weight changes of mice in the control group, model group, and 9 intervention groups over 6 weeks of intervention. As can be seen from the table, the weight of mice in each group was not significantly different on day 0 of the intervention, with the model group having the lowest average weight of 11.85g. However, after significance analysis, no significant difference was found in the baseline average weight of mice in the control group, model group, 5 comparative groups, and 4 example groups (p > 0.05). With the extension of the intervention time, the average weight of mice in each group continuously increased. Weight monitoring and recording were conducted on days 7, 14, 21, 28, 35, and 42. The final analysis showed no significant difference in the average weight of mice in the 11 groups at each time point (p > 0.05). Furthermore, the weight gain of mice in each group after 42 days of intervention was approximately 9g. However, after significance analysis, it was found that the intervention of different HMOs (monomeric or monomeric) groups did not affect the normal weight gain of mice in all 11 groups, including the model group and different HMOs, and there was no significant difference between groups (p > 0.05).

[0110] Table 2: Weight changes (g) of 9 groups of mice during 6 weeks of intervention.

[0111]

[0112] Meanwhile, the food intake and water consumption of the 11 groups of mice were recorded during the intervention period, and the results are shown in Table 3. The table shows that the food intake of the 11 groups of mice during the entire intervention period varied by approximately 3 g / day. However, after significance analysis, it was found that, except for Comparative Examples 1, 4, and 5, where the average daily food intake was significantly higher than that of the control group (p < 0.05), the food intake of the other groups was not significantly different from that of the control group (p > 0.05). Water consumption was also recorded and compared among the groups, and the table shows that there was no significant difference in water consumption among the 11 groups (p > 0.05).

[0113] Table 3: Comparison of average daily food intake and water consumption of mice in each group during the intervention period

[0114]

[0115] Example 3: Assessment of Social Behavioral Competence

[0116] This invention uses a mouse model to simulate human social behavior in order to screen for human milk oligosaccharide nutritional compositions that can help improve social behavior.

[0117] 1. Social Interaction Experiment

[0118] The social interaction test measures the social behavior of mice through simple social interaction tests. This invention uses a social behavior model to examine the basic social interaction abilities of mice in different groups. In this model, the changes in the basic social abilities of mice after modeling and intervention with breast milk oligosaccharides are determined by examining the total time of interaction between mice and other mice of the same species, such as sniffing, following, grooming, and using the mouse as a mount. The specific procedure is as follows: The test mouse is placed alone in a monitoring box for 15 minutes to allow it to become accustomed to the interaction. A strange mouse of the same sex and age as the test mouse is placed in the lower right corner, and video recording is performed for 10 minutes. Simultaneously, a stopwatch is used to record the total time of the test mouse's active interaction with the strange mouse, including active sniffing, following, grooming, and using the mouse as a mount. The environment must be kept quiet throughout the experiment, and the box should be cleaned with 75% alcohol between mouse changes.

[0119] result:

[0120] Table 4 shows the total interaction time between each group of mice and their peers. Figure 1The results showed that the interaction time between the model group mice and their peers was significantly reduced (p < 0.0001), indicating that the neurodevelopment of the model group mice was impaired and their basic social behavior was reduced. However, after intervention with different doses of human milk oligosaccharide LNnT and 2'-FL (Comparative Examples 1-4), the basic social ability of the mice was improved. Significant difference analysis revealed that Comparative Examples 1 and 2 were significantly different from the model group (P < 0.05), but Comparative Examples 3, 4, and 5 were not significantly different from the model group (p > 0.05). Furthermore, when LNnT and 2'-FL were combined in four different ratios (1:1, 1:2, 1:3.4, and 1:4), the total social interaction time between the four groups of mice in Examples 1-4 was significantly higher than that of the model group (p < 0.0001), and all four groups reached the level of the control group. This means that compared with untreated healthy mice, the social behavior of the neurodevelopmentally impaired mice could be restored to the level of untreated healthy mice after the four different ratios of LNnT and 2'-FL. Figure 1 It can be seen that the overall social time of mice in Example 2 was higher than that of the other three examples, but after the significance analysis, no significant difference was found among the four examples (p>0.05).

[0121] Table 4: Effects of HMOs Combination Intervention on Basic Social Skills in Offspring Rats

[0122]

[0123] 2. Three-room social experiment

[0124] Furthermore, the inventors employed a three-compartment socialization experiment to examine the social preferences and social novelty of the mice in each group. Three-compartment socialization is a typical model experiment for assessing abnormal social behavior. The test area was divided into three interconnected compartments (left, middle, and right).

[0125] During the first 10 minutes, the mice were allowed to freely explore the entire test area and familiarize themselves with the environment.

[0126] Social preference was measured in the second 10 min: Before the test, an empty wire mesh cup was placed in the left chamber and a wire mesh cup containing a strange mouse (mouse 1, same sex, group and age) was placed in the right chamber. The time spent by the test mouse with the empty cup and in the left chamber (smelling, crawling, and time and number of times entering the chamber) was recorded, as well as the time spent by the test mouse with the strange mouse and in the right chamber (smelling, crawling, and time and number of times entering the chamber). The social preference index was calculated according to formula (I).

[0127] Social preference index = (interaction time with mouse 1 - interaction time with empty wire cup) / (interaction time with mouse 1 + interaction time with empty wire cup) (I)

[0128] In the third 10-minute period, a social novelty experiment was conducted: a new, unfamiliar mouse (mouse 2, same sex, group, and age) was placed in the previously empty cup, with the right ventricle set up the same as in the second 10-minute period. The time the test mouse spent in both the left and right ventricles (interaction time) and the interaction time with the two mice were recorded. The social novelty index was then calculated using formula (II).

[0129] Social novelty index = (interaction time with mouse 2 - interaction time with mouse 1) / (interaction time with mouse 2 + interaction time with mouse 1) (II)

[0130] Results: The social preference results are shown in Table 5. Table 5 shows that the average interaction time between normal, untreated healthy mice and unfamiliar mice in the right ventricle was 117.7 seconds, while the average interaction time with an empty cup in the left ventricle was 46 seconds. This indicates that the interaction time with unfamiliar mice was significantly longer than the interaction time with an empty cup, with a social preference index of approximately 0.44. However, after modeling, the interaction time between mice and unfamiliar mice on the right side decreased to 27.3 seconds, and the interaction time with an empty cup on the left side was approximately 25.9 seconds. This indicates successful modeling and a rapid decrease in the mice's social abilities (social preference index approximately 0.039). Further comparison of the degree of recovery of social abilities in mice after intervention with human milk oligosaccharides 2'-FL and LNnT shows in Table 6 that the interaction time between mice in Comparative Examples 1-5 and Examples 1-4 (a total of 9 groups) and unfamiliar mice was longer than the interaction time with an empty cup, and the social preference index was improved (e.g., ...). Figure 1 As shown in the figure, the social preference index of comparative examples 1-5 ranged from 0.21 to 0.24, while the social preference index of examples 1-4 reached between 0.44 and 0.57. Furthermore, after significant difference analysis, it was found that the social preference index of the four examples was significantly higher than that of the five comparative examples, and that the combination of LNnT and 2'-FL had a synergistic effect. This indicates that the combination of the two human milk oligosaccharides 2'-FL and LNnT in different ratios of 1:1, 1:2, 1:3.4, and 1:6 can better enhance the social ability of mice.

[0131] Table 5: Social Preference Performance of Mice in Each Group

[0132]

[0133] Table 5-1: Significant differences in social preference index among different groups of mice

[0134]

[0135] The results of social novelty assessment in mice are shown in Tables 6 and 6-1. The tables show that the control group (untreated healthy mice) exhibited stronger curiosity towards a new, unfamiliar mouse of the same species after the empty cup in the left ventricle was replaced with one, and the interaction time with this new mouse was significantly longer than with the old mouse in the right ventricle. However, the interaction time between the modeled mice and mice in both the left and right ventricles was significantly reduced, and the interaction time with the new mouse was slightly shorter than that with the old mouse. Intervention with human milk oligosaccharides (HMOs) increased the interaction time between mice in all groups with both new and old mice, with the interaction time with the new mouse being significantly longer than that with the old mouse. This indicates that intervention with LNnT and 2'-FL improved social novelty in mice. Social novelty indices (such as...) were also compared. Figure 2 As shown in the figure, the social novelty indices of Comparative Examples 1-5 and Examples 1-4 were significantly higher than those of the model group. The social novelty indices of Comparative Examples 1-5 ranged from 0.11 to 0.21, while those of Examples 1-4 were all above 0.4. This indicates that the combination of LNnT and 2'-FL is more effective than the two oligosaccharide monomers in promoting social novelty, demonstrating a synergistic effect. The inventors also analyzed the interaction time between the experimental mice and the new mice in the left cup. The results showed that the interaction time between the mice in Examples 1-4 and the new mice was significantly longer than that in Comparative Examples 1-5 (p < 0.0001), further validating that the combination of the two oligosaccharides can significantly enhance the social novelty of the offspring mice, demonstrating a synergistic effect. Furthermore, difference analysis revealed that the social novelty index of Example 2 was higher than that of the other three examples.

[0136] Table 6: Comparison of Social Novelty Indicators among Different Groups of Mice

[0137]

[0138] Table 6-1 Analysis of significant differences in social novelty among mice in each group

[0139]

[0140] Table 6-2: Significant differences in interaction time between offspring mice and new mice in the left cup among different groups

[0141]

[0142] The above experiments used a mouse model to simulate social behavioral disorders. The experiments showed that after modeling, mice with normal basic growth and development and normal food and water intake exhibited significant social disorders, specifically a decline in basic social skills and a decrease in social preferences and novelty. However, after intervention for 42 days with two human milk oligosaccharides, LNnT and 2'-FL, in different ratios (1:1, 1:2, 1:3.4, 1:6), their social behaviors were improved.

[0143] Example 4: Research on the Mechanism of Social Behavior Improvement

[0144] The behavioral experiments in the above embodiments showed that the combination of two human milk oligosaccharides, 2'-FL and LNnT, significantly improved the social behavior of offspring mice. This invention investigates the changes in brain neural tissue at the cellular and gene levels behind the impairment of basic social abilities in offspring caused by maternal immune homeostasis dysregulation, and the effects of human milk oligosaccharide intervention on these indicators, thereby clarifying the deep-seated mechanisms of social behavior improvement.

[0145] First, this embodiment uses immunofluorescence to analyze the effects of maternal immune activation on microglia and neurons in offspring brain tissue.

[0146] The specific steps are as follows:

[0147] 1. Brain tissue collection

[0148] Mice were fasted for 12 hours before sacrifice. They were anesthetized with an intraperitoneal injection of 1.25% tribromoethanol solution at a dose of 0.2 mL / 10 g bw, and euthanized by cervical dislocation. Brain tissue samples were then collected by dissection on ice, wrapped in aluminum foil, rapidly frozen in liquid nitrogen, and transferred to a -80°C freezer. Tissue samples for pathological sections were placed in 4% (v / v) paraformaldehyde solution for subsequent experiments.

[0149] 2. Immunofluorescence staining to measure IBA-1 and BDNF in the cerebral cortex

[0150] (1) Preparation of paraffin sections

[0151] The hemibrain was fixed in 4% (v / v) paraformaldehyde / PBS fixative for 24 h. After dehydration, it was embedded in paraffin and cut into 5 μm thin slices using a microtome. The slices were then flattened on a 42℃ water surface, retrieved using APES-coated slides, inserted into a slide holder, and dried in a 37℃ oven.

[0152] (2) Immunofluorescence staining

[0153] After drying overnight in a 37°C oven, the tissue sections were dewaxed and rehydrated using xylene I for 10 min, xylene II for 10 min, 100% ethanol I for 5 min, 100% ethanol II for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, and 70% ethanol for 5 min. They were then washed three times with PBS for 5 min each time. The permeabilization buffer was poured into a beaker, the tissue sections were placed inside, and permeabilized at room temperature for 15 min. They were then washed three times with PBS for 5 min each time. Antigen retrieval was performed using the boiling method: the retrieval buffer was poured into a container, and the sections were boiled in a microwave oven on medium heat for 8 min, or in an induction cooker for 10 min. They were then washed three times with PBS for 5 min each time. The tissue was dried with absorbent filter paper, and one drop of 3% H2O2 deionized water was added to the tissue. The tissue was then placed in a humidified chamber and incubated at room temperature for 10 min to block endogenous peroxidase. Finally, the tissue was washed three times with PBS for 5 min each time. Add one drop of 10% goat serum to the tissue, place it in a humidified chamber, and incubate at room temperature for 60 min. After incubation, discard the goat serum, add the corresponding primary antibody (rabbit anti-IBA-1 antibody for IBA-1; mouse anti-BDNF antibody for BDNF), and incubate overnight at 4°C.

[0154] Tissue sections were removed and allowed to warm to room temperature for 30 min. They were then washed 10 times with PBS, 3 min each time. The tissue was dried with absorbent filter paper, and secondary antibody solutions corresponding to the primary antibody species were added (IBA-1 secondary antibody: anti-rabbit IgG - Alexa Fluor 488 green fluorescence; BDNF secondary antibody: anti-mouse IgG - Alexa Fluor 594 red fluorescence). The sections were placed in a humidified chamber and incubated at room temperature for 2 h. Afterward, the sections were washed 10 times with PBS, 3 min each time. The tissue was dried, mounted with a DAPI-containing anti-fluorescence attenuation mounting medium, and observed and photographed under a fluorescence microscope (Olympus, Tokyo, Japan). Fluorescence microscope channel selection: Alexa Fluor 488: excitation 488 nm, emission 525 nm (green); Alexa Fluor 594: excitation 594 nm, emission 617 nm (red); DAPI: excitation 358 nm, emission 461 nm (blue).

[0155] result:

[0156] 1. Ionized calcium-binding adapter molecule 1 (IBA-1) is a calcium-binding protein commonly used as a marker for microglia. This invention analyzed the expression levels of IBA-1 in the cerebral cortex of offspring mice from the control group, model group, and intervention group to examine the activation of microglia in the brain tissue of each group. The experimental results are shown in Tables 7 and 7-1. The tables show that after maternal immune homeostasis activation during pregnancy, microglia in the cerebral cortex of offspring mice in the model group were significantly activated, and the fluorescence intensity of the cell marker IBA-1 increased significantly (p < 0.0001). However, after 42 days of intervention with breast milk oligosaccharides, the fluorescence intensity of IBA-1 in the model mice showed a decreasing trend. Furthermore, One-Way Anova differential analysis revealed that the expression levels of IBA-1 in the cerebral cortex of mice in all nine groups (Comparative Examples 1-5 and Examples 1-4) were significantly lower than those in the model group (p < 0.0001). This indicates that intervention with human milk oligosaccharides can significantly inhibit excessive activation of microglia.

[0157] Furthermore, the inventors discovered that the fluorescence intensity of IBA-1 in the cerebral cortex after LNnT and 2'-FL were combined in ratios of 1:1, 1:2, 1:3.4, and 1:6 was lower than that of LNnT and 2'-FL monoclonal intervention. After significant difference analysis, it was found that Examples 1-4 were significantly lower than Comparative Examples 1-5 (p < 0.0001, p < 0.001, p < 0.01, or p < 0.05, Tables 7 and 7-1). This indicates that the combination of LNnT and 2'-FL in ratios of 1:1, 1:2, 1:3.4, and 1:6 has a synergistic effect and can better inhibit the activation of microglia in the cerebral cortex. Microglia, as macrophages resident in the central nervous system, play a major role in the inflammatory response of the central nervous system.

[0158] Table 7: IBA-1 fluorescence intensity in the cerebral cortex of mice in each group

[0159]

[0160] Table 7-1: Significant differences in quantitative expression of IBA-1 in the cerebral cortex of mice in different groups

[0161]

[0162] 2. BDNF is a neurotrophic protein that plays an important role in the survival, differentiation, growth, and development of neurons. In this invention, it was found that after intraperitoneal injection of poly(I:C) solution into pregnant mice to construct a maternal immune homeostasis dysregulation model, the activity of offspring neurons was affected, and the fluorescence intensity of the neurotrophic factor BDNF in the cerebral cortex was significantly reduced (p < 0.0001) (as shown in Tables 8 and 8-1). After intervention with human milk oligosaccharides (Comparative Examples 1-5, Examples 1-4), the fluorescence intensity was increased. However, after significant difference analysis, it was found that when LNnT was used alone (Comparative Example 1) and when 2'-FL was used alone at a low dose (Comparative Example 2), there was no significant difference between the two groups (p > 0.05). However, when 2'-FL was used at medium and high doses (Comparative Examples 3-5), the fluorescence intensity was significantly higher than that of the model group (p < 0.05). This indicates that the effect of human milk oligosaccharides alone on improving neurotrophic factors is limited. Further comparison by the inventors revealed that the BDNF production level (0.11-0.15) of the combination of LNnT and 2'-FL was higher than that of Comparative Examples 1-5 (0.060-0.079), and that the two exhibited a synergistic effect. A comparison of the four examples showed that the amount of BDNF in the cerebral cortex of the offspring mice in Example 2 was higher than that in Examples 1, 3, and 4. This indicates that LNnT and 2'-FL have a synergistic effect on neuronal damage caused by maternal immune activation, and can further enhance the activity of brain neurons, especially in Example 2.

[0163] Table 8: Fluorescence intensity of BDNF quantitative expression in the cerebral cortex of mice in each group

[0164]

[0165] Table 8-1: Significant differences in quantitative expression of BDNF in the cerebral cortex of mice in different groups

[0166]

[0167] Second, based on the above, we further analyzed the expression of specific alleles related to impairment of basic social skills at the gene level.

[0168] The MECP2 allele is a key gene located on the human X chromosome, encoding methyl-CpG-binding protein 2. This protein plays a crucial role in epigenetic regulation, particularly in the development of the nervous system. Studies have shown that the MECP2 gene plays an important role in maintaining neuronal connections, synaptic plasticity, and neurotransmitter homeostasis during brain development. MECP2 deletion or mutation can lead to social impairments. Furthermore, research has found that maternal immune activation may cause methylation of certain fetal genes, impair the normal function of infectious transcription factors, or affect the structure and modification of chromatin, thereby increasing or decreasing gene transcription activity and ultimately affecting mRNA expression levels.

[0169] This invention used PCR technology to study the expression level of the MECP2 gene in the cerebral cortex of mice in each group, specifically:

[0170] Quantitative real-time polymerase chain reaction (qRT-PCR) technology is mainly used to analyze changes in the gene transcription levels of specific molecules in the cortex and colon. The specific operation is as follows:

[0171] (1) Tissue RNA extraction

[0172] TRIzol was used to extract mRNA from the tissue, following the instructions. A certain amount of mouse cerebral cortex tissue was taken, added to grinding beads, and a tissue homogenate was prepared. Then, 1 mL of TRIzol was added, and the homogenate was lysed. Subsequently, the RNA was obtained by layering (chloroform extraction), precipitation (isopropanol), and washing (75% ethanol) according to the instructions. The RNA was then dissolved in DEPC water and stored at -80°C.

[0173] (2) Reverse transcription (cDNA synthesis)

[0174] The extracted mRNA was reverse transcribed using the PrimeScript™ RTMaster Mix Reverse Transcription Kit (TaKaRa PrimeScript RTMasterMix, Dalian), following the instructions in the product manual.

[0175] (3) Indicator Measurement

[0176] According to the instructions for the Takara Premix Ex Taq™ II RR036A kit, the PCR amplification system was prepared as follows: 1 μL upstream primer, 1 μL downstream primer, 6 μL ddH2O, 10 μL Premix Ex Taq II, and 2 μL cDNA template. Primers for each detection index are shown in Table 9.

[0177] The standard two-step PCR amplification procedure is as follows: 95°C for 30 s, one cycle; 95°C for 3 s, 60°C for 30 s, 40 cycles; then proceed to the melting curve stage. Finally, GAPDH mRNA is used as an internal control, based on 2... -△△Ct The relative expression level of genes is calculated.

[0178] Table 9: Gene Primer Sequences

[0179]

[0180] The results are shown in Tables 10 and 10-1. The tables show that the mRNA expression level of the MECP2 allele in the cerebral cortex of mice in the model group was significantly reduced after maternal immune activation (p < 0.0001). However, after timely intervention with two human milk oligosaccharides, LNnT and 2'-FL, in early life, the mRNA expression level of MECP2 was enhanced, possibly through epigenetic regulation (such as DNA methylation or histone modification). Specifically, the difference analysis between Comparative Examples 1-5 and Examples 1-4 and the model group shows that the mRNA expression level of MECP2 in the intervention group was significantly higher than that in the model group (p < 0.0001 or p < 0.001 or p < 0.01).

[0181] Further comparison revealed that LNnT and 2'-FL, when combined in ratios of 1:2, 1:3.4, and 1:6, exhibited a synergistic effect, with the expression level of Mecp2 gene mRNA in the cerebral cortex of the treated mice significantly higher than that in comparative examples 1-5 (p < 0.0001 or p < 0.001). Further comparison of the four examples showed that Example 2 was significantly higher than Example 1 (p < 0.001), Example 3 (p < 0.001), and Example 4 (p < 0.001).

[0182] Table 10: MECP2 mRNA expression levels in the cerebral cortex of mice in each group

[0183]

[0184] Table 10-1: Significant differences in MECP2 mRNA expression levels in the cerebral cortex of mice from different groups

[0185]

[0186] The above analysis reveals that maternal immune activation leads to excessive activation of microglia, the immune cells in the offspring's brain. Simultaneously, the stress response caused by immune activation affects neuronal activity in the offspring, significantly reducing BDNF levels. Furthermore, at the gene level, the mRNA expression of the MECP2 allele, which is associated with neuronal proliferation, migration, synapse formation, and neural network stability, is significantly reduced. When offspring were immediately treated with a combination of human milk oligosaccharides (LNnT and 2'-FL in different ratios of 1:1, 1:2, 1:3.4, and 1:4) after birth, the activation level of microglia in the offspring's brain was significantly reduced, neuronal activity was improved, BDNF expression was significantly increased, and the mRNA expression of the MECP2 allele was significantly increased. This resulted in significant improvements in basic social behavior, social preferences, and social novelty in the offspring, particularly in Example 2.

Claims

1. The use of a human milk oligosaccharide composition in the preparation of products that improve declining social behavior, characterized in that, The decline in social behavior ability is caused by abnormal activation of the maternal immune system; The human milk oligosaccharide composition contains the following essential active ingredients: lactose-N-neotetrasaccharide and 2'-fucosylated lactose.

2. The use according to claim 1, characterized in that, In the human milk oligosaccharide composition, the mass ratio of lactose-N-neotetrasaccharide and 2'-fucosylated lactose is 1:(1~10).

3. The use according to claim 1 or 2, characterized in that, The improvement of declining social behavior ability includes: increasing the social preference index, increasing the social novelty index, and / or increasing the total social time.

4. The use according to any one of claims 1 to 3, characterized in that, The improvement of declining social behavior includes: inhibiting the overactivation and proliferation of microglia in the cerebral cortex and regulating neuronal activity.

5. The use according to claim 4, characterized in that, The regulation of neuronal activity includes increasing the expression level of BDNF in the cerebral cortex and / or increasing the expression level of the MECP2 gene in the cerebral cortex.

6. The use of a human milk oligosaccharide composition in the preparation of products that improve brain nerve damage, characterized in that, The brain nerve damage includes microglia overactivation, decreased BDNF expression and / or decreased MECP2 gene expression, and the brain nerve damage is caused by abnormal activation of the maternal immune system; The human milk oligosaccharide composition contains the following essential active ingredients: lactose-N-neotetrasaccharide and 2'-fucosylated lactose.

7. The use according to claim 6, characterized in that, In the human milk oligosaccharide composition, the mass ratio of lactose-N-neotetrasaccharide and 2'-fucosylated lactose is 1:(1~10).

8. The use according to claim 6 or 7, characterized in that, The microglia overactivation includes increased expression of IBA-1 in the cerebral cortex; and / or The decrease in BDNF expression includes a decrease in BDNF expression in the cerebral cortex; and / or The reduced MECP2 gene expression includes a decrease in MECP2 gene expression in the cerebral cortex.

9. The use according to any one of claims 1 to 8, characterized in that, The products include food, which includes beverages, milk and dairy products, baked goods, or confectionery.

10. The use according to any one of claims 1 to 8, characterized in that, The product is an oral preparation; the oral preparation includes at least one of tablets, pills, granules, powders, teas, capsules, and oral liquids.

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