Use of nutritional composition in regulating brain immunity

Abstract

The present invention belongs to the field of nutrient research, and is particularly related to the use of functional nutritional compositions in regulating brain immunity. The present invention provides the use of functional nutritional compositions in the preparation of foods for regulating brain immunity, wherein the functional nutritional compositions include the following two essential components: 3'-sialyllactose and osteopontin, and, in the functional nutritional compositions, the mass ratio of the 3'-sialyllactose and the osteopontin is (0.01-50): 1, the 3'-sialyllactose and osteopontin are endogenous components or exogenous components of the functional nutritional compositions, and the regulation of brain immunity is achieved by regulating microglia M1 / M2 polarization. The present invention has found that, compared to 3'-sialyllactose monomers or osteopontin monomers, after 3'-sialyllactose and osteopontin are combined in a certain ratio, there is a synergistic effect of regulating brain immunity.

Description

Technical Field

[0001] The present invention belongs to the field of nutrient research, and relates to the use of a functional nutrient composition in regulating brain immunity, and specifically relates to the use of a functional nutrient composition containing 3'-sialyllactose and osteopontin in regulating brain immunity and assisting in improving the health of the central nervous system. Background Art

[0002] Microglia are important innate immune components in the central nervous system. Their main function is to remove damaged nerves, cell debris and infectious substances, and to help maintain or promote the health of the central nervous system. Under physiological conditions, microglial cell bodies maintain slender protrusions and continuously detect changes in the brain's internal environment. Once stimulated, microglia are instantly activated, their number surges, and their cell bodies enlarge, and their protrusions decrease and become shorter. When the central nervous system is adversely stimulated, microglial activation leads to changes in cell phenotype and function, and activated microglia usually show one of two different phenotypes, called M1 and M2. M1 microglia can trigger neuroinflammatory responses by releasing cytotoxic substances and inflammatory factors. In severe cases, they can cause neuronal damage, loss of synaptic function, and affect the local microenvironment of the brain. In contrast, M2 microglia play a neuroprotective role by secreting anti-inflammatory components and neurotrophic factors.

[0003] Osteopontin (OPN) is a widely distributed O-glycosylated phosphoprotein that was first discovered in bones. It is expressed in many different types of cells and is therefore present in most tissues and physiological secretions. Its mass concentration in human milk is relatively high. Many studies have also shown that osteopontin plays an important role in organisms, especially in immune activation, bone damage repair, angiogenesis, bone remodeling, and promoting brain development.

[0004] Human milk oligosaccharides (HMO) are the third largest solid component in breast milk and are recognized as the first choice prebiotics for infants. Sialyl HMO is an important type of HMO, accounting for about 13% of the total HMO. 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL) are the two simplest sialylated HMOs, which are abundant throughout the lactation period and have concentrations much higher than those in milk or infant formula. Studies have shown that sialylated HMOs have a variety of health effects, including anti-adhesion and antibacterial ability, antiviral activity, prevention of necrotizing enterocolitis, immunomodulatory activity, regulation of intestinal epithelial cell responses, promotion of brain development, and improvement of cognitive ability. Both HMOs show good prebiotic effects, especially in promoting the proliferation of bifidobacteria and shaping the intestinal flora. Both sialylated HMOs contain a sialic acid moiety, a key molecule required for the normal development of gangliosides and therefore plays a key role in brain development and function.

[0005] At present, there have been studies on the physiological functions of osteopontin, human milk oligosaccharides and compositions containing the two, such as:

[0006] The experimental results in reference 1 show that intranasal administration of OPN can improve neurological dysfunction and cerebral edema in rats with subarachnoid hemorrhage (SAH), inhibit the expression of CD86, iNOS, IL-1β, and IL-6 in the brain, and promote the expression of CD206, Arg-1, IL-10, and IL-13. OPN reduces the inflammatory response after SAH by inhibiting the polarization of microglia M1.

[0007] Reference 2 recruited normally growing children (2-25 months old, average 10 months old) to investigate the relationship between breast milk HMOs and cognitive development. The study found that 3'-SL was positively correlated with language development in infancy.

[0008] Reference 3 discloses a nutritional composition for promoting neurodevelopment, the composition comprising 2′-fucosyllactose and osteopontin. Studies have found that the nutritional composition has a significant synergistic effect in promoting neurodevelopment such as neuronal maturation, synaptogenesis and myelination, especially in promoting the proliferation, maturation and differentiation of oligodendrocyte precursor cells (OPCs) into mature oligodendrocytes (OLs) and / or the myelination characteristics of OLs.

[0009] References 4 and 5 both disclose a nutritional composition comprising 2'-fucosyllactose and osteopontin. Studies have found that such a composition can improve the brain development and intelligence of infants and young children, especially promote neural development such as promoting neuronal maturation, synapse formation and / or myelination.

[0010] Reference 6 discloses an oligosaccharide composition for improving neural development and dysfunction, wherein the oligosaccharide composition includes 6'-sialyllactose and lactose-N-trisaccharide. Studies have found that the oligosaccharide composition can effectively inhibit the accumulation of Aβ protein, thereby playing a role in protecting nerves. In addition, it can also inhibit the activity of acetylcholinesterase, protect the normal development of cholinergic neurons and increase the content of neurotransmitters.

[0011] Reference 7 discloses a composition based on human milk oligosaccharides and its application, wherein the composition includes human milk oligosaccharides and osteopontin, wherein the human milk oligosaccharides include at least one of 2'-fucosyllactose, 3'-sialyllactose and 6'-sialyllactose. Studies have found that the combined use of human milk oligosaccharides and osteopontin can regulate intestinal flora and promote intestinal balance through multiple mechanisms, thereby improving resistance to viruses and pathogenic bacteria such as Salmonella, effectively reducing Salmonella colonization, improving infant diarrhea, and improving immunity.

[0012] Reference 8 discloses a composition comprising osteopontin, the composition comprising osteopontin and at least one human milk oligosaccharide, the human milk oligosaccharide comprising 2'-fucosyllactose, 3-fucosyllactose, lactose-N-tetraose, lactose-N-neotetraose, 3'-sialyllactose, 6'-sialyllactose. Studies have found that the composition comprising osteopontin is used to treat and / or prevent viral infections in mammals, such as humans, particularly infants, toddlers and preschoolers.

[0013] References:

[0014] Reference 1: Chen Ligang, Zou Zheng, Hao Guangzhi, et al. Osteopontin inhibits the polarization of microglia to M1 in rats with subarachnoid hemorrhage[J]. Journal of Neuroanatomy, 2024, 40(3): 373-377.

[0015] Cited literature 2: Seoyoon Cho, Ziliang Zhu, Tengfei Li, et al. Human milk 3'-Sialyllactose is positively associated with language development duringinfancy[J]. The American Journal of Clinical Nutrition, 2021, 114(2):588-597.

[0016] Reference 3: CN114586983A

[0017] Reference 4: CN115413789A

[0018] Reference 5: CN115316660A

[0019] Reference 6: CN118104826A

[0020] Reference 7: CN118160926A

[0021] Reference 8: CN118632706A Summary of the invention

[0022] Problem that the invention aims to solve

[0023] At present, although existing technologies (such as cited document 7) disclose the physiological activity function research of the combination of human milk oligosaccharides (such as 3'-sialyllactose) and osteopontin, these studies mainly focus on regulating intestinal flora, promoting intestinal balance, improving diarrhea, etc., and there is still room for further research on other aspects of the physiological activity function of the combination.

[0024] After a lot of further research, the present invention unexpectedly discovered that 3'-sialyllactose and osteopontin combined in a specific ratio have a synergistic effect in regulating brain immunity, and the regulation of brain immunity in the present invention is achieved by regulating the polarization of microglia M1 / M2; and such a synergistic effect has not been reported so far.

[0025] Therefore, the main purpose of the present invention is to provide a safe and effective functional nutritional composition, which combines the breast milk component 3'-sialyllactose and the long-term edible osteopontin in an appropriate ratio to achieve the non-therapeutic purpose of synergistically regulating brain immunity and assisting in improving the health of the central nervous system.

[0026] Solutions for solving problems

[0027] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0028] [1]. Use of a functional nutritional composition in the preparation of a food for regulating brain immunity, wherein the functional nutritional composition comprises the following two essential components:

[0029] 3′-sialyllactose and osteopontin;

[0030] Furthermore, in the functional nutritional composition, the mass ratio of the 3'-sialyllactose to the osteopontin is (0.01-50):1,

[0031] The 3'-sialyllactose and osteopontin are endogenous components or exogenous components of the functional nutritional composition,

[0032] The regulation of brain immunity is achieved by regulating microglial M1 / M2 polarization.

[0033] [2] The use according to [1], wherein the mass ratio of the 3'-sialyllactose to the osteopontin is (0.01-30):1.

[0034] [3] The use according to [1] or [2], wherein at least part of the osteopontin in the composition is derived from bovine milk.

[0035] [4]. The use according to any one of [1] to [3], wherein the regulation of microglial M1 / M2 polarization includes one or more of inhibiting microglial polarization to M1 phenotype, promoting microglial polarization to M2 phenotype, and inducing microglial transformation from M1 phenotype to M2 phenotype.

[0036] [5] The use according to any one of [1] to [4], wherein the regulation of microglial M1 / M2 polarization has one or more of the following manifestations (I) to (IV):

[0037] (Ⅰ) inhibiting the release of proinflammatory cytokines TNF-α, IL-1β and / or NO;

[0038] (II) Promote the release of anti-inflammatory cytokine IL-10;

[0039] (III) Inhibit the expression of iNOS;

[0040] (IV) Promote the expression of CD206.

[0041] [6]. The use according to any one of [1] to [5], wherein the food is an oral preparation, and the oral preparation includes one or more of tablets, pills, granules, powders, capsules and oral liquids.

[0042] [7]. The use according to any one of [1] to [5], wherein the food is a dairy product, a beverage, a candy or a baked product.

[0043] [8] The use according to any one of [1] to [5], wherein the food contains one or more of the following ingredients: plant product ingredients, animal dairy product ingredients, animal meat product ingredients, vitamin supplements, mineral supplements, nucleotide supplements, polyunsaturated fatty acid supplements and any food acceptable excipients.

[0044] [9]. Use of a functional nutritional composition in the preparation of a drug for assisting in improving the health of the central nervous system, wherein the functional nutritional composition comprises the following two essential components: 3'-sialyllactose and osteopontin, and in the functional nutritional composition, the mass ratio of the 3'-sialyllactose to the osteopontin is (0.01-50):1.

[0045]

[10] . The use according to [9], wherein the auxiliary improvement of central nervous system health includes improving neuroinflammation for non-therapeutic purposes.

[0046] Effects of the Invention

[0047] The present invention has found that the combination of 3'-sialyllactose and osteopontin, especially in a certain ratio, has a synergistic effect on regulating brain immunity. In vitro cell experimental data show that compared with 3'-sialyllactose monomer or osteopontin monomer, after using a combination of 3'-sialyllactose and osteopontin to intervene in microglia stimulated by LPS, the expression of iNOS is reduced, while the expression of CD206 is increased. In addition, the release of proinflammatory cytokines (such as TNF-α, IL-1β, NO, etc.) is also significantly reduced. Moreover, there is a good synergistic effect between 3'-sialyllactose and osteopontin, which can play an effective role in regulating brain immunity.

[0048] In addition, the above combination also provides a non-therapeutic effect of assisting in improving the health of the central nervous system. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 : Schematic diagram of the experimental process of studying the regulatory effect of functional nutrients on microglia in the present invention.

[0050] Figure 2 : Figure 2 A shows the Western blot results of the effects of functional nutrients on the TLR4 / NF-κB signaling pathway in BV2 cells stimulated by LPS; Figure 2B shows the results of Western blot analysis of the effects of functional nutrients on TLR4 protein expression in the TLR4 / NF-κB signaling pathway of BV2 cells stimulated by LPS; Figure 2 C shows the results of Western blot analysis of the effects of functional nutrients on the expression of NF-κB p65 protein in the TLR4 / NF-κB signaling pathway of BV2 cells stimulated by LPS; Figure 2 D shows the results of Western blot analysis of the effects of functional nutrients on the expression of p-NF-κBp65 protein in the TLR4 / NF-κB signaling pathway of BV2 cells stimulated by LPS; Figure 2 E shows the results of Western blot analysis of the effects of functional nutrients on the expression of IKB-α protein in the TLR4 / NF-κB signaling pathway of BV2 cells stimulated by LPS; Figure 2 F shows the results of Western blot analysis of the effects of functional nutrients on the expression of p-IKB-α protein in the TLR4 / NF-κB signaling pathway of BV2 cells stimulated by LPS.

[0051] Description of reference numerals:

[0052] Figure 2 Group 1 was the LPS model group, group 2 was the blank control group, group 3 was the OPN high-dose group, group 4 was the 3'-SL high-dose group, group 5 was the OPN high-dose + 3'-SL low-dose group (OPN:3'-SL was 1:0.1), group 6 was the OPN low-dose + 3'-SL high-dose group (OPN:3'-SL was 1:30), ** p<0.01, *** p<0.001, **** p<0.0001. DETAILED DESCRIPTION

[0053] The following describes the embodiments of the present invention, but the present invention is not limited thereto. The present invention is not limited to the various structures described below, and various changes can be made within the scope of the invention claim, and the embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples are also included in the technical scope of the present invention.

[0054] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints numerical values ​​A and B.

[0055] In this specification, unless otherwise specified, the word "multiple" in "multiple", "multiple", "plurality", etc. means a numerical value of 2 or more.

[0056] In this specification, "comprising", "having", "including" or "containing" may mean inclusive or open-ended, not excluding additional, uncited elements or method steps. At the same time, "comprising", "having", "including" or "containing" may also mean closed, excluding additional, uncited elements or method steps.

[0057] In the present specification, a numerical range expressed using "above" or "below" means a numerical range including the number.

[0058] In this specification, the word "may" means both performing a certain process and not performing a certain process.

[0059] In this specification, the use of "optional" or "optional" indicates that certain substances, components, execution steps, application conditions and other factors are used or not used.

[0060] In this specification, unless otherwise specified, the "normal temperature" used generally refers to the temperature of 23±2°C.

[0061] In this specification, the unit names used are all international standard unit names, and unless otherwise stated, the "%" used indicates weight or mass percentage.

[0062] In this specification, the references to "some specific / preferred embodiments", "other specific / preferred embodiments", "embodiments", etc., mean that the specific elements (e.g., features, structures, properties and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not exist in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0063] In addition, unless otherwise defined, other technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0064] The technical solution of the present invention is mainly based on the following insights:

[0065] Lipopolysaccharide (LPS) is the main component of the outer membrane of Gram-negative bacteria. Studies have shown that LPS can enter the brain through the blood-brain barrier to activate microglia and induce neuroinflammatory reactions, induce the release of proinflammatory cytokines, and cause memory impairment, etc. The present invention uses microglia stimulated by LPS as a microglia activation model to study the regulatory effects of different types and concentrations of nutrients on microglia, and explore functional nutrients (such as LPS) that are safe (non-cytotoxic) and have obvious effects on regulating brain immunity and maintaining or promoting the health of the nervous system. Figure 1 The present invention has found through research that the combination of 3'-sialyllactose and osteopontin can inhibit the expression of iNOS, promote the expression of CD206, promote the release of anti-inflammatory cytokine IL-10, and inhibit the release of pro-inflammatory cytokines TNF-α, IL-1β and / or NO after intervening in microglia stimulated by LPS.

[0066] (Functional nutritional composition)

[0067] The functional nutritional composition of the present invention comprises the following two essential components: 3'-sialyllactose and osteopontin.

[0068] In some embodiments, the 3'-sialyllactose and osteopontin are the main active ingredients in the nutritional composition; that is, the nutritional composition mainly relies on the 3'-sialyllactose and osteopontin it contains to exert specific physiologically active functions, such as regulating brain immunity by regulating microglia M1 / M2 polarization.

[0069] In some embodiments, the nutritional composition may include other components or other nutrients in addition to 3'-sialyllactose and osteopontin. There are no particular limitations on other components or other nutrients that may be used in the nutritional composition of the present invention. Without impairing the effects of the present invention, other edible ingredients, food additives or solvent components commonly used in the art may be used. Other nutrients include one or more of proteins, vitamins, monosaccharides, polysaccharides and plant extracts.

[0070] The nutritional composition of the present invention may be in a liquid state, a semi-solid state, or a solid state at room temperature.

[0071] In some embodiments, the functional nutritional composition consists of the 3'-sialyllactose and osteopontin.

[0072] At present, the production methods of 3'-sialyllactose mainly include chemical synthesis, enzymatic synthesis and bioengineering synthesis. At the same time, these ingredients can also be indirectly introduced from various existing milk ingredients.

[0073] It should be noted that the use of the above-mentioned 3'-sialyllactose in the present invention should comply with the requirements of local laws and regulations. In some cases, if permitted by laws and regulations, the ingredient can be directly introduced into the food composition in the form of a separate raw material; in other cases, if permitted by laws and regulations, it can be indirectly introduced into the composition or food by adding a milk raw material that meets the requirements.

[0074] The present invention does not specifically limit the source of 3'-sialyllactose, and typically, it can be introduced from different milk-containing raw materials, such as animal milk raw materials (such as cow's milk, milk powder, etc.). In the present invention, "animal milk" is used to refer to the liquid obtained from the mammary gland of a mammal in the lactation period. The term "animal milk" should be interpreted broadly and covers both raw milk (i.e., liquid obtained directly from the mammary gland) and standardized dairy products.

[0075] The present invention does not specifically limit the source of the osteopontin, for example, it can be extracted from osteopontin-rich cow milk. In order to facilitate actual production and application, commercial osteopontin products, such as protein powder or whey powder containing osteopontin, can also be directly used.

[0076] In addition, the 3'-sialyllactose and osteopontin components in the functional nutritional composition of the present invention can be endogenous components or exogenous components. The endogenous component refers to the component that is indirectly introduced into the composition through the use of other components of the composition, and the exogenous component refers to the component that can be directly introduced into the composition by adding it separately.

[0077] In some embodiments, in the nutritional composition, the mass ratio of the 3'-sialyllactose to the osteopontin is (0.01-50):1; for example, it can be 0.01:1, 0.02:1, 0.03:1, 0.04:1, 0.05:1, 0.06:1, 0.07:1, 0.08:1, 0.09:1, 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 1:1, more preferably (0.01-20):1, further preferably (0.05-10):1, and even more preferably (0.1-6):1.

[0078] The present invention finds that when the mass ratio of 3'-sialyllactose to osteopontin is within the above range, the two have a synergistic effect of regulating the polarization of microglia M1 / M2 (for example, inhibiting the polarization of microglia to M1 phenotype, promoting the polarization of microglia to M2 phenotype, inducing the transformation of microglia from M1 phenotype to M2 phenotype), thereby achieving the effect of regulating brain immunity, and at the same time, it has the effect of assisting in improving the health of the central nervous system.

[0079] (food)

[0080] The food of the present invention contains or uses the functional nutritional composition, so that the food of the present invention also contains 3'-sialyllactose and osteopontin.

[0081] The present invention does not specifically limit the specific types of food. For example, the food may be dairy products (such as milk powder, whey powder, fermented milk, cheese, condensed milk, etc.), beverages (such as carbonated beverages, fruit-flavored beverages, tea beverages, coffee, etc.), candies (such as hard candies, hard sandwich candies, gel candies, polished candies, gum-based candies, inflated candies, pressed candies, etc.), or baked goods (such as biscuits, bread, cakes, etc.). In addition, the food of the present invention may also be health food, such as various types of oral preparations, including tablets, pills, granules, powders, capsules, oral liquids, etc.

[0082] Depending on the type of food and the final needs of the applicable object, in some embodiments, the food also contains one or more of the following ingredients: plant product ingredients, animal dairy product ingredients, animal meat product ingredients, vitamin supplements, mineral supplements, nucleotide supplements, polyunsaturated fatty acid supplements and any food-acceptable excipients.

[0083] Plant product ingredients include, but are not limited to, fruits such as figs, pomegranates, kiwis, tangerines, oranges, pineapples, strawberries, apples, bananas, grapes, pears, cherries, blueberries, blackberries, black currants, cranberries, raspberries, melons, emblica chinensis and bilberry or their extracts; vegetables such as onions, cucumbers, tomatoes, cauliflower, carrots, spinach, kale, Brussels sprouts, garlic, basil, oregano or their extracts; grains such as rice (indica rice, japonica rice, glutinous rice), wheat (wheat, barley, oats, rye), corn, sorghum, millet, sorghum, yellow rice, buckwheat, soybeans, broad beans, peas, mung beans, red beans, kidney beans or their extracts; nuts such as walnuts, pistachios, cashews, hazelnuts, almonds, apricots, pine nuts, peanuts, melon seeds, chestnuts, macadamia nuts, ginkgo nuts or their extracts; coffee or its extracts; and some botanical Chinese medicinal materials or their extracts that are both medicine and food.

[0084] Animal dairy ingredients include but are not limited to fresh milk from mammals such as cows, sheep, and camels, as well as whole milk powder, skim milk powder, concentrated whey protein powder, desalted whey powder, whey protein powder, hydrolyzed whey protein powder or cheese and other processed dairy products.

[0085] Animal meat ingredients include but are not limited to meat ingredients from pigs, cattle, sheep, aquatic animals or poultry.

[0086] For vitamin supplements, including but not limited to vitamin A, beta-carotene, vitamin D3, vitamin E, vitamin K1, vitamin B1, vitamin B2, vitamin B6, vitamin B 12 , vitamin C, pantothenic acid, folic acid, niacin, choline, inositol, biotin, etc.

[0087] Mineral supplements include, but are not limited to, calcium citrate, calcium L-lactate, calcium hydrogen phosphate, potassium gluconate, sodium citrate, ferrous gluconate, potassium iodide, zinc gluconate, sodium selenite, copper gluconate, chromium sulfate, manganese gluconate, magnesium gluconate, and the like.

[0088] Nucleotide supplements include, but are not limited to, disodium 5'-cytidylate, disodium 5'-uridylate, adenosine 5'-monophosphate, disodium 5'-guanylate, disodium 5'-inosinate, and the like.

[0089] Polyunsaturated fatty acid supplements include, but are not limited to, docosahexaenoic acid, eicosatetraenoic acid, eicosapentaenoic acid, and the like.

[0090] Any food-acceptable excipients include, but are not limited to, solvents, antioxidants, antibacterial agents, thickeners, diluents, cosolvents, stabilizers, emulsifiers, fillers, disintegrants, lubricants, coating materials, anti-caking agents, flavoring agents, sweeteners, edible flavors, edible colorings, etc.

[0091] In addition, food may also contain functional ingredients such as probiotics and prebiotics to meet the dietary needs of different groups of people.

[0092] In some embodiments, in the food, the mass ratio of the 3'-sialyllactose to the osteopontin is (0.01-50):1.

[0093] The present invention has no particular limitation on the absolute content of 3'-sialyllactose and osteopontin in food, as long as it complies with the requirements of relevant laws and regulations. In some embodiments, the content of 3'-sialyllactose in food is 0.001% to 1%, preferably 0.01% to 0.5%; the content of osteopontin in food is 0.005% to 0.5%, preferably 0.01% to 0.2%.

[0094] (Use in regulating brain immunity)

[0095] The present invention unexpectedly discovered that the combination of 3'-sialyllactose and osteopontin can play a role in regulating brain immunity. Moreover, when 3'-sialyllactose and osteopontin are combined in a certain ratio, there is a synergistic effect between the two, which can further enhance the effect of regulating brain immunity. Therefore, the functional nutritional composition containing 3'-sialyllactose and osteopontin described in the present invention and the food added or used with the functional nutritional composition have the effect of regulating brain immunity.

[0096] The "regulating brain immunity" mentioned in the present invention means that the immune activity in the brain is maintained at the most appropriate level in the most appropriate form by regulating the interactions between immune cells and immune molecules and other systems such as the neuroendocrine system. At the same time, the regulation of brain immunity mentioned in the present invention is not intended to prevent and treat diseases.

[0097] Furthermore, the present invention provides use of a functional nutritional composition containing 3'-sialyllactose and osteopontin in preparing food for regulating brain immunity.

[0098] In some embodiments, the regulation of brain immunity is achieved by regulating microglial M1 / M2 polarization. In some specific embodiments, the regulation of microglial M1 / M2 polarization includes inhibiting microglial polarization to M1 phenotype, promoting microglial polarization to M2 phenotype, and inducing microglial transformation from M1 phenotype to M2 phenotype (e.g., increasing the number of M2 phenotype cells, reducing the number of M1 phenotype cells, and increasing the ratio of M2 / M1). One or more.

[0099] In some embodiments, the regulation of microglial M1 / M2 polarization has one or more of the following manifestations (I) to (IV):

[0100] (Ⅰ) inhibiting the release of proinflammatory cytokines TNF-α, IL-1β and / or NO;

[0101] (II) Promote the release of anti-inflammatory cytokine IL-10;

[0102] (III) Inhibit the expression of iNOS;

[0103] (IV) Promote the expression of CD206.

[0104] (Assists in improving the health of the central nervous system)

[0105] The present invention unexpectedly found that the combination of 3'-sialyllactose and osteopontin can play a role in assisting the improvement of the health of the central nervous system, especially can improve neuroinflammation, which has not yet reached the level of the disease. In addition, when 3'-sialyllactose and osteopontin are combined in a certain proportion, there is a synergistic effect between the two, which can further enhance the effect of improving neuroinflammation. Therefore, the functional nutritional composition containing 3'-sialyllactose and osteopontin of the present invention and the food added or using the functional nutritional composition also have the effect of assisting the improvement of the health of the central nervous system and improving the non-therapeutic purpose of neuroinflammation.

[0106] In some embodiments, the aiding in improving central nervous system health comprises improving neuroinflammation. In some embodiments, the central nervous system health is affected by neuroinflammation.

[0107] In some embodiments, the improvement of neuroinflammation is achieved by affecting the TLR4 / NF-κB signaling pathway in microglia. In some specific embodiments, the effect on the TLR4 / NF-κB signaling pathway in microglia includes inhibiting the expression of TLR4, inhibiting the expression of NF-κB p65, inhibiting the phosphorylation of NF-κB p65, increasing the expression of IKB-α and inhibiting the phosphorylation of IKB-α.

[0108] Example

[0109] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0110] 1. Materials and Methods

[0111] 1.1 Raw materials

[0112] Osteopontin (OPN): Arla Foods Ingredients Group P / S, Lacprodan OPN-10;

[0113] 3'-Sialyl lactose (3'-SL): DSM, 3-SL9001.

[0114] 1.2 Cells

[0115] Mouse microglial cell line BV2 cells were purchased from Shanghai Enzyme Research Biotechnology (CC-Y2022).

[0116] 1.3 Reagents

[0117] Table 1 Main reagents for the experiment

[0118] serial number Reagent name factory model 1 DMEM high glucose SkyJet CA0004-500ML 2 0.25% pancreatin solution SkyJet CN0004-100ML 3 Penicillin-Streptomycin Mixture SkyJet CM0001-100ML 4 Special grade fetal bovine serum Vicente 085-150 5 PBS Meilun Biotechnology MA0020 6 Cell cryopreservation medium Meilun Biotechnology MA0405 7 LPS Sigma L2880 8 CCK-8 Kit White Shark BS350A 9 Nitric Oxide Test Kit Blue Sky S0021S 10 IL-10 ELISA Kit Chenglin Biotechnology AD2837Mo 11 IL-β ELISA Kit Chenglin Biotechnology AD3364Mo 12 TNF-α ELISA Kit Chenglin Biotechnology AD3051Mo 13 EASYspinPlus Tissue / Cell RNA Rapid Extraction Kit Adele Biology RN2802 14 Reverse transcription kit Adele Biology PC7002 15 SGExcel FastSYBR Master Mix Bioengineering B532955-0005 16 Primers Bioengineering H520001-0001 / H510001-0002 17 NF-κB p65 Antibody Wuhan Tri-Eagle Biotech 10745-1-AP 18 Phospho-NF-κB p65 Antibody Wuhan Tri-Eagle Biotech 80379-2-RR 19 IKB-α Antibody Wuhan Tri-Eagle Biotech 10268-1-AP 20 Phospho-IKB-α Antibody Wuhan Tri-Eagle Biotech 82349-1-RR 21 GAPDH Antibody Wuhan Tri-Eagle Biotech 10494-1-AP 22 TLR4 antibodies Wuhan Tri-Eagle Biotech 19811-1-AP 23 HRP-conjugated Goat Anti-Rabbit IgG Wuhan Tri-Eagle Biotech SA00001-2

[0119] 1.4 Instruments

[0120] Table 2 Main instruments and equipment for the experiment

[0121] serial number Instrument Name factory 1 VD-1320 clean workbench Beijing Donglian Har Instrument Manufacturing Company 2 Carbon dioxide incubator Thermo Fisher Scientific China Co., Ltd. 3 Motic Microscope Harbin Wayne Trading Co., Ltd. 4 Cell Imaging Microplate Reader Meigu Molecular Instruments Co., Ltd. 5 Constant temperature water bath Quanzhou Yiguan Technology Co., Ltd. 6 Multifunctional ELISA reader Dynatech Corporation 7 Centrifuge Thermo Fisher Scientific China Co., Ltd. 8 Fluorescence quantitative PCR instrument Thermo Fisher Scientific China Co., Ltd. 9 Water bath Quanzhou Yiguan Technology Co., Ltd. 10 Nucleic acid protein analyzer Thermo Fisher Scientific China Co., Ltd. 11 Ordinary PCR instrument Harbin Wayne Trading Co., Ltd. 12 Multifunctional imaging system Wuhan Sewell Biotechnology Co., Ltd. 13 Transfer electrophoresis instrument Wuhan Sewell Biotechnology Co., Ltd.

[0122] 1.5 Cell culture

[0123] Mouse microglial cell line (BV2) was cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37° C. in a 5% CO 2 incubator.

[0124] 1.6 Nutrient cytotoxicity assay

[0125] CCK-8 kit was used to detect the cytotoxicity of nutrients.

[0126] The working principle of the CCK-8 kit is: using the dehydrogenase in the cell mitochondria, in the presence of the electron carrier 1-methoxy-5-methylphenazine dimethyl sulfate, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt is reduced to yellow formazan. Because only mitochondria in living cells can produce dehydrogenase, the production of formazan is directly proportional to the number of living cells, and the OD value is detected at 450nm on a microplate reader.

[0127] BV2 cells were seeded into 96-well plates at 4 × 10 4 cells, 200 μL / well, 3 replicates per group. Place in an incubator and culture for 12 hours. After the cells are completely attached, add the prepared culture medium of each nutrient to the 96-well plate. After 12 hours of culture, add 20 μL CCK-8 solution (protected from light) to each well and continue incubating in the cell culture incubator for 0.5 to 1 hour. Use an enzyme marker to measure the absorbance at 450nm, and then calculate the cell survival rate according to the formula: cell survival rate = [(absorbance of experimental well-absorbance of blank well) / (absorbance of control well-absorbance of blank well)] × 100% (wherein the experimental well contains cells, culture medium, nutrients and CCK-8 solution; the blank well contains culture medium and CCK-8 solution, but does not contain cells and nutrients; the control well contains cells, culture medium and CCK-8 solution, but does not contain nutrients). The CCK8 method was used to detect the viability of BV2 microglia.

[0128] 1.7 Establishment of microglial activation model and experimental grouping

[0129] Mouse microglial cell line (BV2) was cultured in a 5% CO2 incubator (37°C, saturated humidity) using high-glucose DMEM medium (containing 10% fetal bovine serum). BV2 cells in logarithmic growth phase were selected, and cells in logarithmic growth phase and reaching 90% confluence were digested with trypsin. The cell concentration was adjusted, and the cells were inoculated in a suitable cell culture dish, and then cultured for another 24 hours before the cells were grouped. 4 The number of cells / mL was inoculated in a 96-well plate, 200 μL / well, and 3 replicates were added to each group. The experiment was divided into 17 groups, namely blank control group, LPS model group, and 15 experimental groups. (1) Control group: conventional cell culture was used; (2) LPS model group: LPS (1 μg / mL) was used to intervene BV2 cells for 24 hours to establish a microglial activation model; (3) Experimental group: LPS stimulated BV2 cells for 24 hours, and each nutrient was added for intervention for 12 hours.

[0130] Table 3 Experimental groups and doses

[0131] Corresponding experiment Dose Group Description LPS (μg / mL) OPN (μg / mL) 3'-SL (μg / mL) OPN:3'-SL Blank control Blank control group - - - Positive Control LPS model group 1 - - Experimental Example 1 OPN high dose group 1 500 - Experimental Example 2 OPN medium dose group 1 250 - Experimental Example 3 OPN low dose group 1 50 - Experimental Example 4 3'-SL high dose group 1 - 1500 Experimental Example 5 3'-SL medium dose group 1 - 1000 Experimental Example 6 3'-SL medium and low dose group 1 - 250 Experimental Example 7 3'-SL low dose group 1 - 50 Experimental Example 8 OPN high + 3'-SL high 1 500 1500 1:3 Experimental Example 9 OPN High + 3'-SL Medium 1 500 1000 1:2 Experimental Example 10 OPN high + 3'-SL medium low 1 500 250 1:0.5 Experimental Example 11 OPN high + 3'-SL low 1 500 50 1:0.1 Experimental Example 12 OPN Medium + 3'-SL High 1 250 1500 1:6 Experimental Example 13 OPN+3'-SL 1 250 1000 1:4 Experimental Example 14 OPN low + 3'-SL high 1 50 1500 1:30 Experimental Example 15 OPN low + 3'-SL medium 1 50 1000 1:20

[0132] 1.8 NO kit to detect the NO content in the supernatant of BV2 cells

[0133] The BV2 cell experimental grouping and nutrient intervention methods were the same as above. After the culture was completed, the cells were placed in a temperature environment of 4°C and centrifuged at a speed of 1000 rpm for 5 minutes. The supernatant was collected and the NO content in the cell supernatant was determined using the NO kit according to the operating procedures.

[0134] The principle of NO kit to detect NO content is: NO is easily oxidized into nitrite ions (NO2 - ), under acidic conditions, NO2 - It can react with sulfonamide to produce diazo compounds, which can then react with naphthylethylenediamine to produce colored compounds. The concentration of the product of this reaction is similar to that of NO2 - There is a linear relationship between the concentration and the absorption peak at 520-560nm. - The NO content in the liquid was calculated using the standard curve.

[0135] 1.9 Detection of TNF-α, IL-1β and IL-10 in the supernatant of BV2 cell culture by enzyme-linked immunosorbent assay (ELISA)

[0136] The BV2 cell experimental grouping and nutrient intervention methods were the same as above. After the culture was completed, the cells were placed in a 4°C temperature environment and centrifuged at 1000 rpm for 5 min. The supernatant was collected and the amounts of TNF-α, IL-1β and IL-10 in the cell supernatant were detected according to the operating steps in the kit instructions.

[0137] 1.10 Detection of iNOS and CD206, markers of M1 and M2 phenotypes of BV2 cells

[0138] qRT-PCR was used to detect the mRNA expression levels of iNOS and CD206-related genes and then determine the changes in the content of M1 cell population and M2 cell population. The third-generation BV2 microglial cell suspension was inoculated in a 24-well culture plate with a cell density of 4×10 5Each well was divided into groups according to the experimental design. After treatment, the cells were washed twice with PBS, using 200 μL per well. Digested with trypsin for 2.5 min, and digested with 400 μL of culture medium. The cells were blown up with a pipette and collected in enzyme-free centrifuge tubes. Total RNA of each group of cells was extracted. Reverse transcription was performed using a reverse transcription kit according to the operating procedures to obtain cDNA, and the amplified cDNA was mixed with primers, fluorescent probes and qPCR Master Mix. The corresponding PCR cycle program and parameters were set according to the requirements of qPCR, and the corresponding mRNA expression level was analyzed in the real-time PCR detection system. The specific primer information is as follows:

[0139] iNOS:

[0140] Upstream: 5'-GGCTTGCCCCTGGAAGTTT-3' (SEQ ID NO. 1),

[0141] Downstream: 5′-TGCAAGTGAAATCCGATGTGG-3′ (SEQ ID NO. 2);

[0142] CD206:

[0143] Upstream: 5'-TTCAGCTATTGGACGCGAGG-3' (SEQ ID NO. 3),

[0144] Downstream: 5'-GAATCTGACACCCAGCGGAA-3' (SEQ ID NO. 4);

[0145] GAPDH:

[0146] Upstream: 5'-GGTTGTCTCCTGCGACTTCA-3' (SEQ ID NO. 5),

[0147] Downstream: 5'-TGGTCCAGGGTTTCTTACTCC-3' (SEQ ID NO. 6).

[0148] 1.11 Western blot detection of relative expression of proteins in BV2 cells

[0149] Western blot was used to detect the expression of TLR4, NF-κB, p-NF-κB, IKB-α and p-IKB-α, and the metabolic pathways of TLR4, NF-κB, p-NF-κB, IKB-α and p-IKB-α were studied. The third generation BV2 cell suspension was inoculated in a 6-well culture plate with a cell density of 1×10 5 / well, and treated in groups according to the experimental design. After treatment, wash the cells 3 times with 4℃ precooled PBS solution, lyse the cells on ice with RIPA lysis buffer containing protease inhibitors, centrifuge at 4℃ for 15min, take the supernatant, use BCA quantitative kit to detect protein concentration, and quantitatively divide the samples. Add 5 times the concentration of SDS-PAGE protein loading buffer and boil in a 100℃ metal water bath for 5min to fully denature the protein. After equal amounts of each group of proteins are loaded, electrophoresis and transfer are performed, and the membrane is blocked with protein-free rapid blocking solution (1×) for 1h. Take out the PVDF membrane and add the primary antibody (1:1000) and incubate at 4℃ overnight. Recover the primary antibody, wash 3 times with TBST solution, add the corresponding secondary antibody (dilution ratio 1:5000) and incubate at room temperature for 1h. Wash again with TBST solution 3 times, prepare ECL developer (ratio 1:1), expose and develop with chemiluminescence imaging analysis system, and collect images. Image J software was used to perform grayscale analysis on protein bands, and the final results were expressed as the grayscale value of the target protein / grayscale value of the internal reference protein, with GAPDH as the internal reference protein.

[0150] 1.12 Statistical analysis

[0151] All experiments were repeated three times independently, and SPSS22.0 statistical software was used to analyze the experimental data. The experimental results were tested for homogeneity of variance, and the statistical differences between the groups were analyzed using one-way ANOVA, expressed as mean ± standard deviation (x ± SD). p>0.05 indicated no statistical significance, and p<0.05 indicated statistically significant differences.

[0152] 2. Experimental results

[0153] 2.1 Nutrient cytotoxicity assay

[0154] Table 4 shows the effects of different concentrations of nutrients on the cell viability of BV2 cells. The results show that the nutrients have no significant effect on cell viability.

[0155] Table 4 Effects of different nutrients on cell viability of BV2 cells

[0156] Corresponding experiment Dose Group Description OPN (μg / mL) 3'-SL (μg / mL) OPN:3'-SL Cell survival rate (%) Blank control Blank control group - - 100.62±0.44 Positive Control LPS model group - - 94.62±0.73 Experimental Example 1 OPN high dose group 500 - 99.86±0.36 Experimental Example 2 OPN medium dose group 250 - 99.28±0.21 Experimental Example 3 OPN low dose group 50 - 95.77±0.37 Experimental Example 4 3'-SL high dose group - 1500 101.24±0.41 Experimental Example 5 3'-SL medium dose group - 1000 100.72±0.21 Experimental Example 6 3'-SL medium and low dose group - 250 98.00±0.98 Experimental Example 7 3'-SL low dose group - 50 96.32±0.32 Experimental Example 8 OPN high + 3'-SL high 500 1500 1:3 99.14±0.47 Experimental Example 9 OPN High + 3'-SL Medium 500 1000 1:2 98.35±0.10 Experimental Example 10 OPN high + 3'-SL medium low 500 250 1:0.5 97.38±0.16 Experimental Example 11 OPN high + 3'-SL low 500 50 1:0.1 97.11±0.21 Experimental Example 12 OPN Medium + 3'-SL High 250 1500 1:6 96.28±1.24 Experimental Example 13 OPN+3'-SL 250 1000 1:4 98.24±0.83 Experimental Example 14 OPN low + 3'-SL high 50 1500 1:30 98.04±0.37 Experimental Example 15 OPN low + 3'-SL medium 50 1000 1:20 99.48±0.21

[0157] 2.2 Effects of nutrients on NO production in LPS-stimulated BV2 cells

[0158] In order to measure the effects of mixed nutrition or single nutrition treatment on LPS-induced BV2 cells, this experiment measured the secretion of pro-inflammatory chemical signal NO to evaluate the effect of regulating microglial polarization. Table 5 shows the effects of different nutrition composition samples containing OPN and 3'-SL on NO production of LPS-stimulated BV2 cells.

[0159] Table 5 Effects of different nutrient compositions on NO production in BV2 cells stimulated by LPS

[0160]

[0161] The results of the NO study showed that compared with the LPS model group, OPN alone had no effect on reducing NO production. 3'-SL alone showed a reduction in NO production, especially at low and medium doses. When the two were used in combination, especially when the OPN:3'-SL ratio was controlled within the range of 1:0.1 to 1:30, a significant effect of reducing LPS-induced NO production could be obtained.

[0162] Specifically, by comparing Experimental Example 1, Experimental Example 6 and Experimental Example 10, it is illustrated that relative to the LPS model group, cell experiment Examples 1 and 6 reduced the NO content by 0.24 and 1.27, respectively, while cell experiment Example 10 reduced NO by 2.73, which is greater than the sum of the first two (0.24+1.27), indicating that there is a synergistic effect between the two components, which can synergistically reduce the NO production of BV2 cells stimulated by LPS.

[0163] 2.3 Effects of nutrients on pro-inflammatory factors in LPS-stimulated BV2 cells

[0164] In order to measure the effect of mixed nutrition or single nutrition treatment on LPS-induced BV2 cells, this experiment measured the secretion of proinflammatory factors TNF-α and IL-1β to evaluate the effect of regulating microglial polarization. Table 6 shows the effect of different nutrient composition samples containing OPN and 3'-SL on the production of proinflammatory factors in LPS-stimulated BV2 cells.

[0165] Table 6 Effects of different nutritional compositions on the production of pro-inflammatory factors in BV2 cells stimulated by LPS

[0166]

[0167] The results showed that compared with the LPS model group, when OPN or 3'-SL was used alone, the effect of reducing pro-inflammatory factors was not obvious. When the two were used in combination, and the dosage ratio of OPN:3'-SL was controlled in the range of 1:0.1 to 1:30, a significant effect of reducing the production of pro-inflammatory factors induced by LPS was achieved.

[0168] Specifically, by comparing Experimental Example 1, Experimental Example 6 and Experimental Example 10, it is illustrated that relative to the LPS model group, cell experiment Experimental Examples 1 and 6 reduced the TNF-α content by 8.68 and 26.74, and the IL-1β content by 0.27 and 1.25, respectively, while cell experiment Experimental Example 10 reduced TNF-α by 63.89, which is greater than the sum of the first two (8.68+26.74), and reduced IL-1β by 3.13, which is greater than the sum of the first two (0.27+1.25), indicating that there is a synergistic effect between the two components, which can synergistically reduce the production of proinflammatory factors in BV2 cells stimulated by LPS.

[0169] 2.4 Effects of nutrients on anti-inflammatory factors in LPS-stimulated BV2 cells

[0170] In order to measure the effect of mixed nutrition or single nutrition treatment on LPS-induced BV2 cells, this experiment measured the secretion of anti-inflammatory factor IL-10 to evaluate the effect of regulating microglial polarization. Table 7 shows the effect of different nutrition composition samples containing OPN and 3'-SL on the production of anti-inflammatory factors in BV2 cells stimulated by LPS.

[0171] Table 7 Effects of different nutritional compositions on the production of anti-inflammatory factors in BV2 cells stimulated by LPS

[0172]

[0173] The results showed that compared with the LPS model group, when OPN or 3'-SL was used alone, both showed an increase in the production of anti-inflammatory factors. At the same time, when the two were used in combination, a significant increase in the production of IL-10 was achieved.

[0174] Specifically, by comparing Experimental Example 2 (OPN), Experimental Example 5 (3'-SL) and Experimental Example 13 (OPN + 3'-SL), it can be shown that compared with the single nutritional treatment of Experimental Example 2 and Experimental Example 5, the IL-10 content of the mixed nutritional treatment of the cell experiment Experimental Example 13 was significantly increased (p<0.001), indicating that the two can synergistically increase the production of anti-inflammatory factors.

[0175] 2.5 Effects of nutrients on iNOS and CD206, markers of M1 and M2 phenotypes of BV2 cells stimulated by LPS

[0176] After activation, microglia eventually polarize into two cell groups with opposite functions, M1 or M2. M1 phenotype microglia mainly release pro-inflammatory cytokines, such as tumor necrosis factor (TNF-α), interleukin 1β (IL-1β) and chemical signal nitric oxide (NO), which may cause inflammatory response in the CNS, leading to neuronal damage and eventually death; M2 phenotype microglia mainly promote damage repair and exert neuroprotective effects by secreting anti-inflammatory cytokines, such as interleukin 10 (IL-10). The main phenotypic marker of M1 phenotype microglia is iNOS, and the main phenotypic marker of M2 phenotype microglia is CD206.

[0177] In order to measure the effect of mixed nutrition or single nutrition treatment on LPS-induced BV2 cells, this experiment used qRT-PCR to detect the mRNA expression levels of iNOS and CD206-related genes and then measured the changes in the content of M1 cell population and M2 cell population to evaluate the effect of regulating microglial polarization. Table 8 shows the effects of different nutritional composition samples containing OPN and 3'-SL on the mRNA expression levels of BV2 cell markers stimulated by LPS.

[0178] Table 8 Effects of different nutritional compositions on the expression levels of BV2 cell marker mRNAs stimulated by LPS

[0179]

[0180] The results showed that compared with the LPS model group, when OPN or 3'-SL was used alone, the relative expression of CD206 increased and the relative expression of iNOS decreased. At the same time, when the two were used in combination, the dosage ratio of OPN:3'-SL was controlled in the range of 1:0.1 to 1:30, a more obvious effect could be obtained.

[0181] Specifically, by comparing Experimental Example 1, Experimental Example 6 and Experimental Example 10, it is illustrated that relative to the LPS model group, cell experiment Experimental Examples 1 and 6 reduced the relative expression of iNOS by 1.13 and 2.19, respectively, while cell experiment Experimental Example 10 reduced the relative expression of iNOS by 5.01, which is greater than the sum of the first two (1.13+2.19), indicating that there is a synergistic effect between the two components, which can synergistically regulate microglial polarization.

[0182] 2.6 Effects of nutrients on the TLR4 / NF-κB signaling pathway in LPS-stimulated BV2 cells

[0183] LPS induces the activation of Toll-like receptors (TLR4), which in essence activates the NF-κB signaling pathway and accelerates the transcription of inflammatory genes. Under normal conditions, NF-κB is in an inactive form and combines with the inhibitor IKB-α in the cytoplasm to form a dimer. After being stimulated by inflammation, the IKB-α protein is phosphorylated and degraded, releasing NF-κB to transfer to the cell nucleus and participate in the expression of several inflammatory factors such as IL-1β and TNF-α. At the same time, these inflammatory factors are involved in regulating the TLR-4 / NF-κB signaling pathway, thus forming a feedback loop and jointly participating in the process of neuroinflammation.

[0184] In order to explore the pathways that affect mixed nutrients or single nutrient treatment on LPS-induced BV2 cells, we extracted the whole protein of each group of cells and used Western blot to detect the expression of TLR4, NF-κB, p-NF-κB, IKB-α and p-IKB-α. Figure 2 A to 2F show the effects of OPN, 3'-SL and their combination on the expression of TLR4 / NF-κB signaling pathway proteins in LPS-stimulated BV2 cells.

[0185] The results showed that compared with the control group, the protein expressions of TLR4, NF-κB p65 and p-NF-κB p65 (phosphorylated NF-κB p65) in the LPS model group were significantly increased (p<0.0001, Figure 2 B~2D); Compared with the LPS model group, there was no significant difference in TLR4 expression in the high-dose OPN group and the high-dose 3'-SL group, but the inhibitory effect of the combination was more significant (p<0.0001, Figure 2 B). The expressions of NF-κB p65 and p-NF-κB p65 in the high-dose OPN group, high-dose 3'-SL group and combination group were all inhibited, and the inhibitory effect of the combination was more significant (p<0.0001, Figure 2 C, 2D). The phosphorylation degree of IKB-α protein was also determined. The results showed that compared with the control group, the expression of IKB-α protein in the LPS model group was significantly decreased, and the expression of p-IKB-α (phosphorylated IKB-α) protein was significantly increased, and the difference was statistically significant (p<0.0001, Figure 2 E, 2F); Compared with the LPS model group, the high-dose OPN group, high-dose 3'-SL group and the combination group inhibited the phosphorylation of IKB-α in BV2 cells, with significant differences (p<0.0001, Figure 2 F). It can be seen that the combination of OPN and 3'-SL alleviates neuroinflammatory response possibly through the TLR4 / NF-κB signaling pathway.

Claims

1. Use of a functional nutritional composition in preparing a food for regulating brain immunity, characterized in that: The functional nutritional composition comprises the following two essential components: 3′-sialyllactose and osteopontin; Furthermore, in the functional nutritional composition, the mass ratio of the 3'-sialyllactose to the osteopontin is (0.01-50):1, The 3'-sialyllactose and osteopontin are endogenous components or exogenous components of the functional nutritional composition, The regulation of brain immunity is achieved by regulating microglial M1 / M2 polarization.

2. The use according to claim 1, characterized in that The mass ratio of the 3'-sialyllactose to the osteopontin is (0.01-30):

1.

3. The use according to claim 1 or 2, characterized in that: The osteopontin in the composition is at least partially derived from bovine milk.

4. The use according to any one of claims 1 to 3, characterized in that The regulation of microglial M1 / M2 polarization includes one or more of inhibiting microglial polarization to M1 phenotype, promoting microglial polarization to M2 phenotype, and inducing microglial transformation from M1 phenotype to M2 phenotype.

5. The use according to any one of claims 1 to 4, characterized in that: The regulation of microglial M1 / M2 polarization has one or more of the following manifestations (I) to (IV): (Ⅰ) inhibiting the release of proinflammatory cytokines TNF-α, IL-1β and / or NO; (II) Promote the release of anti-inflammatory cytokine IL-10; (III) Inhibit the expression of iNOS; (IV) Promote the expression of CD206.

6. The use according to any one of claims 1 to 5, characterized in that: The food is an oral preparation, which includes one or more of tablets, pills, granules, powders, capsules and oral liquids.

7. The use according to any one of claims 1 to 5, characterized in that The food is dairy products, beverages, candies or baked goods.

8. The use according to any one of claims 1 to 5, characterized in that The food contains one or more of the following ingredients: plant product ingredients, animal dairy product ingredients, animal meat product ingredients, vitamin supplements, mineral supplements, nucleotide supplements, polyunsaturated fatty acid supplements and any food-acceptable auxiliary materials.

9. Use of a functional nutritional composition in preparing a food for assisting in improving the health of the central nervous system, characterized in that: The functional nutritional composition comprises the following two essential components: 3'-sialyllactose and osteopontin, and in the functional nutritional composition, the mass ratio of the 3'-sialyllactose to the osteopontin is (0.01-50):

1.

10. The use according to claim 9, characterized in that The auxiliary improvement of central nervous system health includes the improvement of neuroinflammation for non-therapeutic purposes.

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