Nutritional composition for regulating brain immunity and application thereof

Abstract

The invention belongs to the field of nutrient substance research, and particularly relates to a nutrient composition and application thereof. The nutritional composition provided by the invention comprises the following two necessary components: i) osteopontin and ii) hyaluronic acid or salt thereof, and in the nutritional composition, the mass ratio of the i) osteopontin to the ii) hyaluronic acid or salt thereof is 1: (0.05-10). Research finds that compared with osteopontin monomers or hyaluronic acid or salt monomers thereof, after osteopontin and hyaluronic acid or salt thereof are combined according to a certain proportion, the osteopontin and the hyaluronic acid or salt thereof have a synergistic effect of regulating brain immunity.

Description

Technical Field

[0001] The present invention belongs to the field of nutrient research, and relates to a nutrient composition and the use of the composition, and specifically relates to a nutrient composition and the use of the composition in preparing food for regulating brain immunity, or in preparing food for improving the health of the central nervous system for non-therapeutic purposes. Background Art

[0002] Microglia, as resident immune cells of the central nervous system (CNS), originate from yolk sac precursors and maintain their number through self-renewal. They account for 5% to 12% of all CNS-specific cells and are the most abundant immune cells in the CNS. During brain development, microglia are involved in controlling neurogenesis, the fate of oligodendrocyte progenitor cells, and the formation of myelin, and can indirectly affect synaptic and neuronal function by regulating the activation of microglia and the activity of other non-neuronal cells.

[0003] After activation, microglia can be transformed into different phenotypes (such as M1 and M2). M1 phenotype microglia mainly release pro-inflammatory cytokines, such as tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β) and chemical signal nitric oxide (NO), which can easily cause excessive inflammatory response in the CNS, leading to neuronal damage and eventually death; M2 phenotype microglia mainly promote damage repair and play a neuroprotective role by secreting anti-inflammatory cytokines, such as interleukin 10 (IL-10).

[0004] Microglia play an important role in neuroinflammation, which is a complex immune response in which nerve tissue clears pathogens, damaged cells, and harmful substances. Excessive neuroinflammatory response can affect the health of the CNS.

[0005] Osteopontin (OPN) was originally found in bones and has a relatively high concentration in human milk. It is a highly glycosylated and phosphorylated acidic protein containing an arginine-glycine-aspartic acid sequence. 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. OPN is a matrix protein that has cytokine-like effects on a variety of cells and is a secretory extracellular matrix protein between cells. In the field of aneurysmal subarachnoid hemorrhage, OPN plays a neuroprotective role in early brain injury (EBI) and delayed cerebral ischemia by inhibiting neuronal apoptosis, destroying the blood-brain barrier and / or cerebral vasoconstriction, while excessive and prolonged secretion of OPN can cause harm through the occurrence of chronic hydrocephalus.

[0006] Currently, there have been studies on the physiological functions of osteopontin or compositions containing osteopontin, such as:

[0007] Reference 1 discloses a nutritional composition for promoting neurodevelopment, which includes 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.

[0008] Reference 2 discloses a nutritional composition comprising 2'-fucosyllactose and osteopontin. Studies have found that the nutritional 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.

[0009] Reference 3 discloses a nutritional composition comprising 2'-fucosyllactose, docosahexaenoic acid and osteopontin. Studies have found that the nutritional 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 4 discloses a nutritional component for improving intelligence, which contains milk fat globule membrane protein and osteopontin. Studies have found that the nutritional component can be added to food and health products to promote brain development, improve and enhance memory and learning ability, especially to promote brain development in infants and young children, and improve and enhance the memory and learning ability of students, office workers and Alzheimer's patients.

[0011] The study cited in reference 5 found 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.

[0012] Sodium hyaluronate (SH) is a high molecular weight linear mucopolysaccharide produced by polymerization reaction of glucuronic acid and acetylhexose as disaccharides. Its chemical formula is (C 14 H 20 NO 11 Na) nSH is a substance that exists in the human body. It is widely present in human tissues and body fluids, such as joint fluid, cartilage, vitreous body, skin and umbilical cord. SH is easily hydrolyzed into hyaluronic acid (HA) in water. HA constitutes the most abundant extracellular matrix component in the brain development process. It only quickly becomes a secondary component after birth and remains in specific areas in adulthood. SH and HA have similar functions, but SH has a smaller molecular weight and can be absorbed by deep tissues of the human body. At the same time, SH has the functions of lubricating joints, beautifying and nourishing the skin, and immune and anti-inflammatory.

[0013] At present, there have been studies on the physiological functions of hyaluronic acid or its derivatives, such as:

[0014] The study in reference 6 found that hyaluronan extracellular matrix surrounds developing excitatory synapses, which regulates synapse formation and the balance between excitatory and inhibitory signaling.

[0015] Reference 7 found that hyaluronic acid hydrogel can protect the spinal cord from inflammation and reduce secondary injury.

[0016] Reference 8 discloses that the natural antioxidant gallic acid-modified hyaluronic acid (HGA) macromolecule is introduced into tyramine-hyaluronic acid (HT) to construct a new, in situ injectable, antioxidant hydrogel. Compared with HT hydrogel, HT / HGA hydrogel has significantly enhanced scavenging ability of DPPH and hydroxyl free radicals (p<0.05), and has significantly improved protection against H2O2-induced cell oxidative damage (p<0.05), and reduces the accumulation of reactive oxygen species (ROS) in cells. Studies on a mouse model of moderate traumatic brain injury (TBI) have found that in situ transplantation of HT / HGA hydrogel can inhibit oxidative stress by activating the Nrf2 / HO-1 pathway, promote the polarization of M2 phenotype microglia and regulate inflammatory response, thereby improving the harsh microenvironment of the injury site and improving the movement, learning and memory functions of TBI mice.

[0017] References:

[0018] Reference 1: CN114586983A

[0019] Reference 2: CN115413789A

[0020] Reference 3: CN115316660A

[0021] Reference 4: CN114586986A

[0022] Reference 5: 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.

[0023] Reference 6: Emily Wilson, Warren Knudson, Karen Newell-Litwa. Hyaluronanregulates synapse formation and function in developing neural networks [J]. Scientific Reports, 2020, 10: 16459.

[0024] Cited literature 7: Kushchayev SV, Giers MB, Hom Eng D, et al. Hyaluronic acidscaffold has aneuroprotective effect in hemisection spinal cord injury[J]. JNeurosurg Spine, 2016, 25(1):114-124.

[0025] Reference 8: Zhang Dan. Study on the neurological function recovery effect of antioxidant hyaluronic acid hydrogel on brain-injured mice[D]. Henan: Zhengzhou University, 2022. Summary of the invention

[0026] Problem that the invention aims to solve

[0027] At present, for hyaluronic acid or its derivatives, the research direction of the prior art is mostly aimed at using it as a hydrogel composite scaffold, carrying other functional components in a medium manner to play a role in damage repair. For osteopontin or a composition containing it, the research direction of the prior art is mostly aimed at promoting neurodevelopment, but while the brain is developing neurons, neuroprotection is also an important aspect. Prior art studies have found that the administration of osteopontin can inhibit the polarization of microglia M1 in SAH rats and thus inhibit the inflammatory response, but this does not mean that osteopontin can play the same role in combination with any substance or under any conditions. Similarly, although prior art studies have found that gallic acid-modified hyaluronic acid has an improvement effect on mouse brain injury models, this does not mean that hyaluronic acid itself or in combination with other substances can still play the same role. Therefore, there is still room for further research on the physiological function of the combination of osteopontin and hyaluronic acid or its salt.

[0028] After extensive research, the present invention unexpectedly discovered that osteopontin and hyaluronic acid or its salts combined in a specific ratio have a synergistic effect in regulating brain immunity, and the brain immunity regulation 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.

[0029] Therefore, the object of the present invention is to provide a nutritional composition comprising osteopontin and hyaluronic acid or its salt in a specific ratio, for use in preparing foods for regulating brain immunity, or for preparing foods for non-therapeutic purposes to assist in improving the health of the central nervous system, etc.

[0030] Solutions for solving problems

[0031] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0032] [1]. A nutritional composition, wherein the nutritional composition comprises the following two essential components:

[0033] i) osteopontin,

[0034] ii) hyaluronic acid or a salt thereof;

[0035] Furthermore, in the nutritional composition, the mass ratio of i) osteopontin to ii) hyaluronic acid or a salt thereof is 1:(0.05-10).

[0036] [2] Use of the nutritional composition according to [1] in the preparation of food for regulating brain immunity.

[0037] [3] The use according to [2], wherein the regulation of brain immunity is achieved by regulating microglial M1 / M2 polarization.

[0038] [4]. The use according to [3], wherein the regulation of microglial M1 / M2 polarization includes at least one 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 [4], wherein the regulation of microglial M1 / M2 polarization has one or more of the following manifestations (I) to (IV):

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

[0040] (II) Promote the expression of CD206;

[0041] (III) promotes the release of anti-inflammatory cytokine IL-10;

[0042] (IV) Inhibit the release of proinflammatory cytokines TNF-α, IL-1β and / or NO.

[0043] [6]. The use according to any one of [2] to [5], wherein the food is candy, a beverage, a dairy product or a baked product.

[0044] [7]. The use according to [6], 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.

[0045] [8]. The use according to any one of [2] 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.

[0046] [9]. Use of the nutritional composition according to [1] in the preparation of food for assisting in improving the health of the central nervous system.

[0047]

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

[0048] Effects of the Invention

[0049] The present invention has found that osteopontin and hyaluronic acid or its salt combination, especially after combining according to a certain ratio, have synergistic effect of regulating brain immunity and assisting in improving the health of the central nervous system. In vitro cell experiment data show that compared to osteopontin monomer or hyaluronic acid or its salt monomer, after BV2 cells are treated with a combination of osteopontin and hyaluronic acid or its salt, the expression of its 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, osteopontin and hyaluronic acid or its salt have a good synergistic effect between the two, which can play an effective role in regulating brain immunity and assisting in improving the health of the central nervous system. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 : Schematic diagram of the experimental process of studying the regulatory effect of functional nutrients on microglia in the present invention.

[0051] 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.

[0052] Description of reference numerals:

[0053] Figure 2 Group 1 was the LPS model group, group 2 was the blank control group, group 3 was the OPN medium-dose group, group 4 was the SH medium-high-dose group, group 5 was the OPN medium-dose + SH low-dose group (OPN:SH was 1:0.1), and group 6 was the OPN medium-dose + SH high-dose group (OPN:SH was 1:5). * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. DETAILED DESCRIPTION

[0054] 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.

[0055] 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.

[0056] In this specification, unless otherwise specified, the word "multiple" in "multiple", "multiple", "plurality", etc. means a numerical value of 2 or more.

[0057] 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.

[0058] In the present specification, a numerical range expressed using "above" or "below" means a numerical range including the number.

[0059] In this specification, the word "may" means both performing a certain process and not performing a certain process.

[0060] 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.

[0061] In this specification, unless otherwise specified, the "normal temperature" used generally refers to the temperature of 23±2°C.

[0062] In this specification, the unit names used are all international standard unit names, and unless otherwise stated, the "%" used indicates weight or mass percentage.

[0063] 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.

[0064] 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.

[0065] The technical solution of the present invention is mainly based on the following insights:

[0066] 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 explores functional nutrients (such as LPS) that are safe (non-cytotoxic) and have obvious effects on regulating brain immunity and maintaining the health of the nervous system. Figure 1 The present invention has found through research that the combination of osteopontin and hyaluronic acid or its salts can inhibit the expression of iNOS, promote the expression of CD206, promote the release of anti-inflammatory cytokine IL-10, and reduce the release of pro-inflammatory cytokines TNF-α, IL-1β and / or NO after intervening in microglia stimulated by LPS.

[0067] The technical solution of the present invention is further described in detail below:

[0068] <First aspect>

[0069] The first aspect of the present invention provides a nutritional composition comprising the following two essential components: i) osteopontin (hereinafter sometimes referred to as "OPN") and ii) hyaluronic acid or a salt thereof, and, in the nutritional composition, the osteopontin and the hyaluronic acid or a salt thereof are combined in a specific mass. Compared with osteopontin or hyaluronic acid or a salt thereof alone, the composition provided by the present invention can more effectively regulate brain immunity.

[0070] Osteopontin

[0071] Osteopontin (OPN) is a glycosylated protein that is widely present in the extracellular matrix. It was originally believed that OPN is an important bone matrix protein that is closely related to the formation and development of bones. It plays an important physiological role in immune activation, wound repair, angiogenesis, and bone remodeling. Common nutrients rich in OPN include concentrated whey protein powder rich in osteopontin, osteopontin powder, etc.

[0072] At present, the production methods of osteopontin mainly include natural raw material extraction, enzyme method and microbial fermentation method, etc. The present invention does not specifically limit the source and extraction process of osteopontin. Typically, it can be prepared by microbial fermentation method, enzyme method, or extracted from osteopontin-rich substances such as animal milk. Finished osteopontin can also be obtained commercially.

[0073] In some embodiments, the osteopontin of the present invention includes one or more of osteopontin from bovine milk, osteopontin from horse milk, osteopontin from goat milk, and osteopontin from camel milk; preferably, osteopontin from bovine milk. Accordingly, osteopontin can be isolated from bovine milk, or bovine milk osteopontin can be synthesized by microbial fermentation or other means.

[0074] The present invention does not impose any particular limitation on the addition form of the osteopontin, as long as it complies with the requirements of relevant laws and regulations. Within the permitted range, it can be added as a single component, or other components that can be added containing the component can be used to provide the desired osteopontin in the form of use.

[0075] Hyaluronic acid or its salt

[0076] In the present invention, hyaluronic acid or its salt is compounded with the above-mentioned osteopontin as an essential component to achieve the technical effect of the present invention. In principle, there is no particular restriction on the type of hyaluronate, and a common metal salt of hyaluronic acid or a mixture of multiple metal salts can be used. Preferably, an alkali metal salt of hyaluronic acid, such as sodium salt (hereinafter also referred to as sodium hyaluronate) can be used.

[0077] Sodium hyaluronate (hereinafter sometimes referred to as "SH") is also known as sodium hyaluronate. Its dried product is white granules or powder and has no odor. It is a sodium salt of glycosaminoglycan composed of D-glucuronic acid and N-acetyl-D-glucosamine disaccharide units and is a linear macromolecular polysaccharide. Sodium hyaluronate can be used as a new food raw material. For example, the recommended consumption is ≤200 mg / day. Sodium hyaluronate is soluble in water and forms a viscous and elastic solution in water with physiological pH and ionic strength. Its molecular form is variable and it belongs to a non-drug neutral medium.

[0078] There is no particular restriction on the source of sodium hyaluronate in the present invention. For example, it can be produced by fermentation of Streptococcus equi subsp. zooepidemicus using glucose, yeast powder, peptone, etc. as culture medium, or it can be artificially extracted by the fermentation liquid of cockscomb. Since hyaluronic acid is commonly found in animals and humans, such as skin, muscle, cartilage, brain, cockscomb, arterial and venous walls, etc., commonly used raw materials for extraction include cockscomb, umbilical cord, bull's eye, etc. In order to facilitate actual production and application, commercial sodium hyaluronate products can also be directly used.

[0079] Furthermore, in the present invention, there is no particular limitation on the usage of sodium hyaluronate. Preferably, it can be prepared as an aqueous solution for use.

[0080] In some embodiments, the osteopontin and hyaluronic acid or its salt are the main active ingredients in the nutritional composition; that is, the nutritional composition mainly relies on the osteopontin and hyaluronic acid or its salt contained therein to exert specific physiologically active functions, such as regulating brain immunity by regulating microglia M1 / M2 polarization.

[0081] In some embodiments, the nutritional composition may further include other components or other nutrients in addition to osteopontin and hyaluronic acid or its salt. There are no particular limitations on other components or other nutrients that may be used in the nutritional composition of the present invention. Without prejudice to 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.

[0082] The nutritional composition of the present invention may be in a liquid state, a semi-solid state, or a solid state at room temperature.

[0083] In some embodiments, the nutritional composition consists of i) osteopontin and ii) hyaluronic acid or a salt thereof.

[0084] In some embodiments, in the nutritional composition, the mass ratio of the osteopontin to the hyaluronic acid or its salt is 1:(0.05-10); for example, it can be 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, etc.; preferably, it is 1:(0.1-10), more preferably, it is 1:(0.1-5), and even more preferably, it is 1:(0.2-5).

[0085] The present invention finds that when the mass ratio of osteopontin and hyaluronic acid or its salt 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 improving neuroinflammation for non-therapeutic purposes.

[0086] <Second Aspect>

[0087] A second aspect of the present invention provides use of a nutritional composition containing osteopontin and hyaluronic acid or a salt thereof in preparing a food.

[0088] The present invention does not specifically limit the specific types of food. For example, the food may be candy (such as hard candy, hard sandwich candy, jelly candy, polished candy, gum-based candy, aerated candy, compressed candy, etc.), beverage (such as carbonated beverage, fruit-flavored beverage, tea beverage, coffee, etc.), dairy product (such as milk powder, whey powder, fermented milk, cheese, condensed milk, etc.) or baked food (such as biscuits, bread, cake, 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] Animal meat ingredients include but are not limited to meat ingredients from pigs, cattle, sheep, aquatic animals or poultry.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] Polyunsaturated fatty acid supplements include, but are not limited to, docosahexaenoic acid, eicosatetraenoic acid, eicosapentaenoic acid, and the like.

[0097] 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.

[0098] In addition, food may also contain functional ingredients such as probiotics and prebiotics to meet the dietary needs of different groups of people.

[0099] In some embodiments, the food is added with or uses the nutritional composition so that the mass ratio of osteopontin to hyaluronic acid or a salt thereof in the food is 1:(0.05-10).

[0100] The present invention has no particular restrictions on the absolute content of osteopontin and hyaluronic acid or its salt in food, as long as it complies with the requirements of relevant laws and regulations. In some embodiments, the content of osteopontin in food is 0.005% to 0.5%, preferably 0.01% to 0.2%; the content of hyaluronic acid or its salt in food is 0.001% to 1%, preferably 0.01% to 0.2%.

[0101] The food of the present invention has the function of regulating brain immunity. The "regulating brain immunity" of 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 interaction between immune cells and immune molecules and other systems such as the neuroendocrine system. At the same time, the purpose of regulating brain immunity is not to prevent and treat diseases.

[0102] In some embodiments, the regulation of brain immunity is achieved by regulating microglial M1 / M2 polarization; that is, the present invention provides the use of the nutritional composition in preparing a food that achieves regulation of brain immunity by regulating microglial M1 / M2 polarization.

[0103] In some specific embodiments, 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 (e.g., increasing the number of M2 phenotype cells, reducing the number of M1 phenotype cells, and increasing the ratio of M2 / M1). In some specific embodiments, the regulation of microglial M1 / M2 polarization has one or more of the following manifestations (I) to (IV):

[0104] (Ⅰ) Inhibit the expression of iNOS;

[0105] (II) Promote the expression of CD206;

[0106] (III) promotes the release of anti-inflammatory cytokine IL-10;

[0107] (IV) Inhibit the release of proinflammatory cytokines TNF-α, IL-1β and / or NO.

[0108] <Third Aspect>

[0109] The third aspect of the present invention provides the use of a nutritional composition containing osteopontin and hyaluronic acid or a salt thereof in preparing a health food. The food has the function of assisting in improving the health of the central nervous system and is used for non-therapeutic purposes.

[0110] In some embodiments, the auxiliary improvement of central nervous system health includes improving neuroinflammation, and such neuroinflammation does not reach the level of disease. In some embodiments, the central nervous system health is affected by neuroinflammation, and the above-mentioned food assists in improving central nervous system health by improving neuroinflammation.

[0111] 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.

[0112] Example

[0113] 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.

[0114] 1. Materials and Methods

[0115] 1.1 Raw materials

[0116] Osteopontin (OPN): Arla Foods Ingredients Group P / S, Denmark, Lacprodan OPN-10;

[0117] Sodium hyaluronate (SH): Bloomage Biotech Co., Ltd.

[0118] 1.2 Cells

[0119] Mouse microglial cell line BV2 cells were purchased from Shanghai Enzyme Research Biotechnology (CC-Y2022).

[0120] 1.3 Reagents

[0121] Table 1 Main reagents for the experiment

[0122] 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

[0123] 1.4 Instruments

[0124] Table 2 Main instruments and equipment for the experiment

[0125] 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.

[0126] 1.5 Cell culture

[0127] 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.

[0128] 1.6 Nutrient cytotoxicity assay

[0129] CCK-8 kit was used to detect the cytotoxicity of nutrients.

[0130] 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.

[0131] BV2 cells were seeded into 96-well plates at 4 × 10 4cells, 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.

[0132] 1.7 Establishment of microglial activation model and experimental grouping

[0133] 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 15 groups, namely blank control group, LPS model group, and 13 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.

[0134] Table 3 Experimental groups and doses

[0135] Corresponding experiment Group LPS (μg / mL) OPN (μg / mL) SH(μg / mL) OPN:SH Blank control Blank control group - - Positive Control LPS model group 1 - Experimental Example 1 OPN high dose group 1 200 Experimental Example 2 OPN medium dose group 1 100 Experimental Example 3 OPN low dose group 1 50 Experimental Example 4 SH high dose group 1 500 Experimental Example 5 SH medium and high dose group 1 200 Experimental Example 6 SH medium dose group 1 100 Experimental Example 7 SH low dose group 1 10 Experimental Example 8 OPN high + SH high 1 200 500 1:2.5 Experimental Example 9 OPN high + SH medium high 1 200 200 1:1 Experimental Example 10 OPN high + SH medium 1 200 100 1:0.5 Experimental Example 11 OPN medium + SH high 1 100 500 1:5 Experimental Example 12 OPN medium + SH low 1 100 10 1:0.1 Experimental Example 13 OPN low + SH low 1 50 10 1:0.2

[0136] 1.8 Detection of NO content in BV2 cell supernatant using NO kit

[0137] 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.

[0138] 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.

[0139] 1.9 Detection of TNF-α, IL-1β and IL-10 in the supernatant of BV2 cell culture by enzyme-linked immunosorbent assay (ELISA)

[0140] 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.

[0141] 1.10 Detection of iNOS and CD206, markers of M1 and M2 phenotypes of BV2 cells

[0142] 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 5 Each 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:

[0143] iNOS:

[0144] Upstream: 5'-GGCTTGCCCCTGGAAGTTT-3' (SEQ ID NO. 1),

[0145] Downstream: 5′-TGCAAGTGAAATCCGATGTGG-3′ (SEQ ID NO. 2);

[0146] CD206:

[0147] Upstream: 5'-TTCAGCTATTGGACGCGAGG-3' (SEQ ID NO. 3),

[0148] Downstream: 5'-GAATCTGACACCCAGCGGAA-3' (SEQ ID NO. 4);

[0149] GAPDH:

[0150] Upstream: 5'-GGTTGTCTCCTGCGACTTCA-3' (SEQ ID NO. 5),

[0151] Downstream: 5'-TGGTCCAGGGTTTCTTACTCC-3' (SEQ ID NO. 6).

[0152] 1.11 Western blot detection of relative expression of proteins in BV2 cells

[0153] 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.

[0154] 1.12 Statistical analysis

[0155] 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.

[0156] 2. Experimental results

[0157] 2.1 Nutrient cytotoxicity assay

[0158] 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.

[0159] Table 4 Effects of different nutrients on cell viability of BV2 cells

[0160] Corresponding experiment Group OPN (μg / mL) SH(μg / mL) OPN:SH Cell survival rate (%) Blank control Blank control group - 100.62±0.31 Positive Control LPS model group - 94.62±0.52 Experimental Example 1 OPN high dose group 200 99.76±0.47 Experimental Example 2 OPN medium dose group 100 97.39±1.47 Experimental Example 3 OPN low dose group 50 96.42±0.51 Experimental Example 4 SH high dose group 500 101.31±1.5 Experimental Example 5 SH medium and high dose group 200 97.83±0.41 Experimental Example 6 SH medium dose group 100 96.7±0.45 Experimental Example 7 SH low dose group 10 96.66±0.47 Experimental Example 8 OPN high + SH high 200 500 1:2.5 101.21±1.6 Experimental Example 9 OPN high + SH medium high 200 200 1:1 100±0.52 Experimental Example 10 OPN high + SH medium 200 100 1:0.5 98.66±0.42 Experimental Example 11 OPN medium + SH high 100 500 1:5 99.14±0.26 Experimental Example 12 OPN medium + SH low 100 10 1:0.1 96.66±1.65 Experimental Example 13 OPN low + SH low 50 10 1:0.2 99.31±0.36

[0161] 2.2 Effects of nutrients on NO production in LPS-stimulated BV2 cells

[0162] 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 SH on NO production of LPS-stimulated BV2 cells.

[0163] Table 5 Effects of different nutrient compositions on NO production in BV2 cells stimulated by LPS

[0164]

[0165] The results of NO research showed that compared with the LPS model group, when OPN was used alone, the medium dose showed a reduction in NO production. When SH was used alone, it showed a reduction in NO production, especially at low and medium doses. When the two were used in combination, especially when the dosage ratio of OPN:SH was controlled in the range of 1:0.2 to 1:5, a significant effect of reducing LPS-induced NO production could be obtained.

[0166] Specifically, by comparing Experimental Example 1, Experimental Example 5 and Experimental Example 9, relative to the LPS model group, cell experiment Experimental Examples 1 and 5 reduced the NO content by 0.67 and 1.03, respectively, while cell experiment Experimental Example 9 reduced NO by 4.12, which is greater than the sum of the first two (0.67+1.03), indicating that there is a synergistic effect between the two components, which can synergistically reduce the NO production of BV2 cells stimulated by LPS.

[0167] 2.3 Effects of nutrients on pro-inflammatory factors in LPS-stimulated BV2 cells

[0168] 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 SH on the production of proinflammatory factors in BV2 cells stimulated by LPS.

[0169] Table 6 Effects of different nutritional compositions on the production of pro-inflammatory factors in BV2 cells stimulated by LPS

[0170]

[0171] The results showed that compared with the LPS model group, OPN alone showed almost no effect on reducing pro-inflammatory factors. However, SH alone showed a reduction in the production of pro-inflammatory factors. When the two were used in combination, and the dosage ratio of OPN:SH was controlled in the range of 1:0.1 to 1:5, a significant effect of reducing the production of LPS-induced pro-inflammatory factors was achieved.

[0172] Specifically, by comparing Experimental Example 1, Experimental Example 5 and Experimental Example 9, relative to the LPS model group, cell experiment Experimental Examples 1 and 5 reduced the TNF-α content by 2.43 and 6.94, and the IL-1β content by 0.58 and 1.02, respectively, while cell experiment Experimental Example 9 reduced TNF-α by 23.95, which was greater than the sum of the first two (2.43+6.94), and reduced IL-1β by 3.06, which was greater than the sum of the first two (0.58+1.02), 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.

[0173] 2.4 Effects of nutrients on anti-inflammatory factors in LPS-stimulated BV2 cells

[0174] 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 SH on the production of anti-inflammatory factors in BV2 cells stimulated by LPS.

[0175] Table 7 Effects of different nutritional compositions on the production of anti-inflammatory factors in BV2 cells stimulated by LPS

[0176]

[0177] The results showed that compared with the LPS model group, when OPN or SH 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 effect of increasing the production of IL-10 was obtained.

[0178] Specifically, by comparing Experimental Example 1, Experimental Example 5 and Experimental Example 9, compared with the single nutritional treatment of Experimental Example 1 and Experimental Example 5, the IL-10 content of the mixed nutritional treatment of the cell experiment Experimental Example 9 was significantly increased (p<0.001), indicating that the two can synergistically increase the production of anti-inflammatory factors.

[0179] 2.5 Effects of nutrients on iNOS and CD206, markers of M1 and M2 phenotypes of BV2 cells stimulated by LPS

[0180] 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.

[0181] 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 populations and M2 cell populations to evaluate the effect of regulating microglial polarization. Table 8 shows the effects of different nutritional composition samples containing OPN and SH on the mRNA expression levels of BV2 cell markers stimulated by LPS.

[0182] Table 8 Effects of different nutritional compositions on the expression levels of BV2 cell marker mRNAs stimulated by LPS

[0183]

[0184] The results showed that compared with the LPS model group, when OPN or SH 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:SH was controlled in the range of 1:0.1 to 1:5, a more obvious effect could be obtained.

[0185] Specifically, by comparing Experimental Example 1, Experimental Example 5 and Experimental Example 9, relative to the LPS model group, cell experiment Experimental Examples 1 and 5 respectively reduced the relative expression of iNOS by 2.9 and 0.89, and increased the relative expression of CD206 by 2.86 and 1.92; while cell experiment Experimental Example 9 reduced the relative expression of iNOS by 6.03, which was greater than the sum of the first two (2.9+0.89), and increased the relative expression of CD206 by 5.15, which was greater than the sum of the first two (2.86+1.92), indicating that there is a synergistic effect between the two components, which can synergistically regulate microglial polarization.

[0186] 2.6 Effects of nutrients on the TLR4 / NF-κB signaling pathway in LPS-stimulated BV2 cells

[0187] 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.

[0188] 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, SH and their combination on the expression of TLR4 / NF-κB signaling pathway proteins in LPS-stimulated BV2 cells.

[0189] 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<0.01, Figure 2 B~2D); Compared with the LPS model group, there was no significant difference in TLR4 expression and NF-κB p65 expression in the OPN group and the SH group, but the combination had a more significant effect in inhibiting the expression of TLR4 and NF-κB p65 (p<0.01, Figure 2 B, 2C). The expression of p-NF-κB p65 in the OPN group, SH group and combination group was inhibited, and the inhibitory effect of the SH group and the combination group was more significant (p<0.0001, Figure 2 D). 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 reduced, 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, there was no significant difference in the expression of IKB-α in the OPN group and the SH group, but the IKB-α expression in the combination group was higher (p<0.05, Figure 2 E); Compared with the LPS model group, the OPN group, SH group and combination group all inhibited the phosphorylation of IKB-α in BV2 cells (p<0.01, Figure 2 F). It can be seen that the combination of OPN and SH alleviates neuroinflammatory response possibly through the TLR4 / NF-κB signaling pathway.

Claims

1. A nutritional composition, characterized in that The nutritional composition comprises the following two essential components: i) osteopontin, ii) hyaluronic acid or a salt thereof; Furthermore, in the nutritional composition, the mass ratio of i) osteopontin to ii) hyaluronic acid or a salt thereof is 1:(0.05-10).

2. Use of the nutritional composition according to claim 1 in preparing food for regulating brain immunity.

3. The use according to claim 2, characterized in that: The regulation of brain immunity is achieved by regulating microglial M1 / M2 polarization.

4. The use according to claim 3, characterized in that The regulation of microglial M1 / M2 polarization includes at least one 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 claim 4, characterized in that The regulation of microglial M1 / M2 polarization has one or more of the following manifestations (I) to (IV): (Ⅰ) Inhibit the expression of iNOS; (II) Promote the expression of CD206; (III) promotes the release of anti-inflammatory cytokine IL-10; (IV) Inhibit the release of proinflammatory cytokines TNF-α, IL-1β and / or NO.

6. The use according to any one of claims 2 to 5, characterized in that: The food is dairy products, beverages, candies, grain products or cakes.

7. The use according to claim 6, 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.

8. The use according to any one of claims 2 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.

9. Use of the nutritional composition according to claim 1 in preparing food for assisting in improving the health of the central nervous system.

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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