A composition for improving intestinal barrier injury and use thereof

Abstract

This invention relates to a composition for improving intestinal barrier damage and its application. The composition comprises three or more of 2'-FL, LNT, 3'-SL, and zinc gluconate, wherein zinc gluconate is included in the composition. The composition provided by this invention can safely and effectively improve intestinal barrier damage.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a composition for improving intestinal barrier damage and its application. Background Technology

[0002] Damage to the intestinal barrier can lead to malabsorption of nutrients, affecting the growth and development of infants and young children. Due to a decreased ability of the intestines to absorb nutrients such as protein, fat, vitamins, and minerals, infants and young children may experience problems such as failure to thrive and developmental delays. Simultaneously, a damaged intestinal barrier cannot effectively block pathogens and harmful substances, allowing them to easily enter the bloodstream and trigger systemic infections and inflammatory responses, increasing the risk of respiratory infections, urinary tract infections, and other diseases in infants and young children. Furthermore, damage to the intestinal barrier can also lead to intestinal flora imbalance, with a decrease in the number of beneficial bacteria and an overgrowth of harmful bacteria, further disrupting the intestinal microecological balance, exacerbating symptoms, and creating a vicious cycle.

[0003] Currently, clinical treatment for intestinal barrier damage caused by intestinal diseases in infants and young children mostly adopts symptomatic treatment methods. These include correcting dehydration and electrolyte imbalances with oral rehydration salts or intravenous fluids, and using antibiotics to treat bacterial infectious diarrhea. However, the overuse of antibiotics can lead to side effects such as intestinal flora imbalance. Furthermore, existing treatments primarily focus on relieving diarrhea symptoms and have limited effectiveness in repairing the damaged intestinal barrier, failing to fundamentally address the series of problems caused by intestinal barrier damage.

[0004] CN114287629A discloses a composition containing human milk oligosaccharides and Bifidobacterium animalis Bifidobacterium lactis subsp., which can increase transmembrane resistance levels and is associated with improving intestinal barrier function, because increased transmembrane resistance levels are beneficial to maintaining intestinal barrier integrity. However, it mainly focuses on improving intestinal immunity, and there is insufficient research on its direct and targeted improvement of intestinal barrier damage.

[0005] During an infant's growth, gut health is a key factor affecting their overall development, and the intestinal barrier, as an important defense structure of the gut, is of great significance in maintaining infant health. However, the infant's gut is in a developmental stage and is very vulnerable to intestinal diseases, especially bacteria and viruses, which can severely damage the structure and function of the intestinal barrier and cause a series of health problems. This situation highlights the urgent need to develop targeted intervention measures.

[0006] Given the high incidence of intestinal diseases in infants and young children and the limitations of existing treatments, it is crucial to find a safe and effective way to improve intestinal barrier damage. Summary of the Invention

[0007] The problem the invention aims to solve

[0008] In view of this, the object of the present invention is to provide a composition containing human milk oligosaccharides and zinc gluconate, and the use of the composition in the preparation of a treatment or prevention of intestinal barrier damage.

[0009] Solution for solving the problem

[0010] A first aspect of the present invention provides a composition for improving intestinal barrier damage, the composition comprising three or more of 2'-FL, LNT, 3'-SL and zinc gluconate, wherein the composition contains zinc gluconate.

[0011] Preferably, the composition comprises 2'-FL, LNT, 3'-SL and zinc gluconate.

[0012] Preferably, the mass ratio of 2'-FL, LNT, 3'-SL and zinc gluconate is (10-200):(5-100):(1-20):1.

[0013] Preferably, the mass ratio of 2'-FL, LNT, 3'-SL and zinc gluconate is (30-150):(15-100):(1-15):1.

[0014] Preferably, the mass ratio of 2'-FL, LNT, 3'-SL and zinc gluconate is (40-120):(20-80):(3-10):1.

[0015] Preferably, the mass ratio of 2'-FL, LNT, 3'-SL and zinc gluconate is 43:29:5:1.

[0016] A second aspect of the present invention provides a pharmaceutical product, nutritional product, or health product comprising any of the compositions described above.

[0017] A third aspect of the invention provides the use of the composition according to any one of the foregoing claims or the pharmaceutical, nutritional or health product described above in the preparation of a product for the prevention and / or treatment of intestinal barrier damage.

[0018] Preferably, the product is used to reduce the content of diamine oxidase.

[0019] Preferably, the product is used to reduce the content of lipopolysaccharides.

[0020] Preferably, the product is used to reduce the content of D-lactic acid.

[0021] Preferably, the product is used to upregulate the expression of Claudin-1, Occludin, and / or ZO-1.

[0022] The effects of the invention

[0023] The composition provided by this invention possesses multiple physiological functions, including regulating intestinal flora, reducing the overgrowth of pathogenic bacteria, enhancing intestinal immune function, and promoting intestinal epithelial cell repair, thus treating or preventing inflammatory bowel disease (IBD). It offers a new direction for improving intestinal barrier damage, particularly suitable for infants and young children. In the composition of this application, HMOs and zinc gluconate have a synergistic effect to enhance intestinal immune function, exhibiting excellent efficacy in improving intestinal barrier damage. Furthermore, the composition provided by this application has advantages such as safety and no side effects, avoiding the development of drug resistance, and is suitable for long-term use (e.g., added to infant formula or functional foods). Attached Figure Description

[0024] Figure 1 Schematic diagram of HE staining results of ileal tissue sections from mice with different combinations of human milk oligosaccharides;

[0025] Figure 2 This is a schematic diagram showing the expression levels of diamine oxidase (DAO), lipopolysaccharide (LPS), and D-lactic acid in mouse serum in different combinations of human milk oligosaccharides.

[0026] Figure 3 This diagram illustrates the expression levels of the core proteins (Claudin-1, Occludin, and ZO-1) in the mouse gut in different combinations of human milk oligosaccharides.

[0027] Figure 4 This diagram illustrates the expression levels of mouse cytokines (IL-1β, IL-6, and TNF-α) in different combinations of human milk oligosaccharides. Detailed Implementation

[0028] To make the technical solution and beneficial effects of the present invention more apparent and understandable, a detailed description is provided below by listing specific embodiments. The accompanying drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly show the details of the local features; unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application pertains.

[0029] Human milk oligosaccharides (HMOs) are unique functional components of breast milk. As the third largest solid nutrient, their content and structure exhibit significant individual variability and dynamic changes. HMOs regulate gut health through a dual mechanism: on the one hand, they selectively promote the proliferation of beneficial bacteria such as Bifidobacteria as prebiotics, while competitively inhibiting pathogen colonization; on the other hand, they strengthen the intestinal mucosal barrier function by regulating goblet cell mucin secretion and enhancing tight junction protein expression.

[0030] Patients with inflammatory bowel disease (IBD) experience an imbalance in peripheral blood zinc homeostasis. Statistics show that approximately 15% to 40% of IBD patients have hypozincemia. Even after zinc supplementation, zinc deficiency can persist due to reduced zinc absorption and increased zinc loss caused by intestinal inflammation. The severity of hypozincemia in IBD patients is related to disease activity and type, and is associated with mucosal barrier disruption and delayed mucosal healing.

[0031] Based on this, the inventors, through extensive experimental research, have provided a composition containing 2'-FL, LNT, 3'-SL, and zinc gluconate, which can reduce the expression levels of DAO, LPS, and D-lactate, upregulate claudin, occludin, and ZO-1, reduce the levels of inflammatory factors, synergistically inhibit intestinal epithelial cell apoptosis (reducing LPS entry into the bloodstream), regulate gut microbiota metabolism (reducing endotoxin synthesis), and enhance the intestinal physical barrier function. Based on this, the present invention has been completed.

[0032] A first aspect of the present invention provides a composition for improving intestinal barrier damage, the composition comprising three or more of 2'-FL, LNT, 3'-SL and zinc gluconate, wherein the composition contains zinc gluconate.

[0033] In one embodiment, the composition comprises 2'-FL, LNT, 3'-SL and zinc gluconate.

[0034] In one embodiment, the mass ratio of 2'-FL, LNT, 3'-SL and zinc gluconate is (10-200):(5-100):(1-20):1, for example 10:10:1:1, 20:10:1:1, 30:10:1:1, 40:10:1:1, 50:10:1:1, 60:10:1:1, 40:15:1:1, 40:20:1:1, 40:30:1:1, 40:40:1:1, 40:50:1:1, 40:20:2:1, 40:20:4:1, 40:20:6:1, 40:20:8:1, 40:20:10:1, 40:20:12:1, etc.

[0035] In one embodiment, the mass ratio of 2'-FL, LNT, 3'-SL and zinc gluconate is (30-150):(15-100):(1-15):1.

[0036] In one embodiment, the mass ratio of 2'-FL, LNT, 3'-SL and zinc gluconate is (40-120):(20-80):(3-10):1.

[0037] In one embodiment, the mass ratio of 2'-FL, LNT, 3'-SL and zinc gluconate is (40-110):(20-70):(3-10):1.

[0038] In one embodiment, the mass ratio of 2'-FL, LNT, 3'-SL and zinc gluconate is (40-50):(25-35):(3-6):1.

[0039] In one embodiment, the mass ratio of 2'-FL, LNT, 3'-SL and zinc gluconate is 43:29:5:1.

[0040] A second aspect of the present invention provides a pharmaceutical product, nutritional product, or health product comprising any of the compositions described above.

[0041] A third aspect of the invention provides the use of the composition according to any one of the foregoing claims or the pharmaceutical, nutritional or health product described above in the preparation of a product for the prevention and / or treatment of intestinal barrier damage.

[0042] In one embodiment, the product is used to reduce the content of diamine oxidase.

[0043] In one embodiment, the product is used to reduce the content of lipopolysaccharide.

[0044] In one embodiment, the product is used to reduce the content of D-lactic acid.

[0045] In one embodiment, the product is used to upregulate the expression of Claudin-1, Occludin, and / or ZO-1.

[0046] In the following examples, all ingredients used are conventional commercially available products, which can be obtained from commercially available products or synthesized according to known methods. For example, fucoidosyllactose and sialylated lactose were produced by Suzhou Yixi Biotechnology Co., Ltd.

[0047] The present invention will be described in more detail below through examples. Where specific experimental steps or conditions are not specified in the examples, they should be performed according to conventional practices or conditions in the art.

[0048] The materials and reagents used in the following examples are shown in Table 1, and the instruments and equipment are shown in Table 2.

[0049] Table 1 Main Materials and Reagents

[0050]

[0051] Table 2 Main Instruments and Equipment

[0052]

[0053] Example 1

[0054] The following combined mixtures (H1~H3) were prepared respectively.

[0055] Take 2'-FL, LNT, and 3'-SL respectively, and mix them in a mass ratio of 10:6:1 to form composition H1.

[0056] 2'-FL, LNT, 3'-SL and zinc gluconate were respectively mixed in a mass ratio of 100:60:9:1 to form composition H2.

[0057] 2'-FL, LNT, 3'-SL and zinc gluconate were respectively mixed in a mass ratio of 43:29:5:1 to form composition H3.

[0058] Example 2

[0059] Four-week-old male BALB / c mice aged 6-8 weeks were selected. All mice lived in a controlled environment with free access to standard laboratory food and water. The experimental environment was set with a 12-hour light-dark cycle, and the temperature and humidity were maintained between 22±2℃ and 45±5%. After one week of acclimatization feeding, the mice were randomly divided into 9 groups of 5 mice each for the formal experiment. Mouse models were constructed according to different groups. The negative control group was administered an equal volume of PBS by gavage, while the other groups were infected with E. coli to induce intestinal barrier loss once daily for one week. Subsequently, 2'-FL, LNT, 3'-SL, and zinc gluconate were administered by gavage. The negative control group and the model group were administered an equal volume of PBS by gavage once daily for one week (experimental groups and gavage dosages are shown in Table 3).

[0060] Table 3 Experimental Groups and Doses

[0061]

[0062] Based on the dosage of 2'-FL in breast milk and the dosage stipulated by domestic and international standards for addition to infant products, the required dosage for infants was determined to be 1.5 g / L. The dosage for mice was calculated using the body surface area conversion method (BSA method) to be 1700 mg / kg. The total amount of HMOs used in the mixed treatment groups was equivalent to that of other treatment groups, and the sugar ratio was set based on breast milk, combined with domestic standards and EU regulations. The following experiments were then conducted on the mice in each group:

[0063] (1) HE staining: Fixed jejunal and lung tissue samples were dehydrated in ethanol, embedded in paraffin, and sectioned into 4 μm thin sections. Standard hematoxylin-eosin (HE) staining was performed, and histological images were taken using an optical microscope.

[0064] (2) ELISA method for determining intestinal permeability: The levels of diamine oxidase (DAO), LPS and D-lactic acid in mouse serum were determined using an enzyme-linked immunosorbent assay kit according to the manufacturer’s instructions.

[0065] (3) RT-qPCR detection of tight junction expression: Total RNA was extracted from tissue samples, and cDNA was synthesized by reverse transcription before PCR reaction. -ΔΔCt Calculate the relative expression levels of the target genes occludin, claudin1, and ZO-1 mRNA.

[0066] (4) ELISA assay for cytokines: The levels of IL-6, IL-1β, TNF-α and IL-10 in mouse serum were determined using an enzyme-linked immunosorbent assay kit according to the manufacturer’s instructions.

[0067] (5) Perform one-way ANOVA on the data results and use Tukey's multiple comparison test to analyze the significance of differences. Inconsistent letters indicate significant differences.

[0068] The results are as follows:

[0069] (1) Effects of different combinations of human milk oligosaccharides on pathological parameters of mouse ileum tissue

[0070] HE staining was used to reflect the histopathological changes in the colon of mice in each experimental group, and this was used to evaluate the effects of HMOs on ileal tissue structure and villi. Figure 1 As shown, the ileum tissue structure of the treated mice was intact, with no obvious structural damage between the mucosa, submucosa, smooth muscle layer, and serosa. No ulcers or erosions, hemorrhage, or inflammatory cell infiltration were observed on the ileum mucosa surface. No obvious aggregation of lymphocytes, neutrophils, or plasma cells or follicle formation was observed. Moreover, pathological analysis of the ileum tissue of the treated mice showed that it was in a normal state, without any obvious abnormal structures or pathological changes. However, the tissue structure of the MC group was incomplete, with obvious structural damage between the mucosa, submucosa, smooth muscle layer, and serosa.

[0071] (2) Effects of different combinations of human milk oligosaccharides on intestinal permeability in mice

[0072] Intestinal barrier permeability was assessed by quantifying serum diamine oxidase (DAO), lipopolysaccharide (LPS), and D-lactic acid levels. Figure 2DAO, as a marker of intestinal epithelial cell damage, reflects increased cell shedding when elevated; D-lactic acid, produced by intestinal bacteria metabolism, indicates disruption of the intestinal mucosal physical barrier when its serum concentration is elevated; LPS levels characterize the systemic translocation of intestinal microorganisms and their metabolites. Compared with the model control group (MC group), the levels of DAO, LPS, and D-lactic acid were significantly reduced in all intervention groups, indicating that HMOs and zinc gluconate can improve intestinal barrier function through multiple pathways. In the combined intervention group, H3 showed the best barrier repair capacity, with its DAO, LPS, and D-lactic acid levels decreasing by 33.3%, 33.6%, and 24.66% respectively compared to the MC group, significantly better than other intervention groups.

[0073] Structural-specific synergy: 2'-FL reduces intestinal epithelial inflammation by inhibiting the TLR4 / NF-κB pathway; LNT, as a fucosylation receptor agonist, directly upregulates Claudin-1 expression to enhance tight junction integrity; 3'-SL promotes mucin secretion by activating PPARγ signaling, forming a physical-immune dual barrier. Zinc can recognize cellular signals and increase intestinal stem cell activity by regulating the Wnt / β-catenin and mammalian target of rapamycin complex 1 (mTORC1) signaling pathways in the stem cell microenvironment, promoting intestinal epithelial development and post-injury repair. The four components, when used in combination, can simultaneously block inflammatory damage, strengthen the junctional complex, and enhance mucus layer protection, achieving multidimensional regulation.

[0074] Microbe-host interaction: The sialic acid structure of 3'-SL may competitively bind to the adhesion sites of intestinal pathogens, reducing bacterial translocation, while 2'-FL selectively promotes the proliferation of Bifidobacteria, indirectly reducing D-lactic acid production.

[0075] LPS regulation differences: Although all intervention groups reduced LPS levels, the H3 group showed the most significant effect, which may be related to the dual mechanisms of synergistic inhibition of intestinal epithelial cell apoptosis (reducing LPS entry into the blood) and regulation of microbial metabolism (reducing endotoxin synthesis).

[0076] In summary, the H3 combination improves intestinal permeability through multi-target and multi-level synergistic effects. Its effect is positively correlated with the number and structural diversity of components, providing a theoretical basis for developing intestinal barrier repair strategies based on HMOs and zinc gluconate combination formulations.

[0077] (3) Effects of human milk oligosaccharides on intestinal barrier function in mice

[0078] Maintaining the integrity of the intestinal barrier function is a key physiological mechanism for ensuring efficient nutrient absorption by the host and resisting the invasion of pathogenic microorganisms. Experimental results show ( Figure 3Escherichia coli infection significantly downregulated the expression levels of core proteins in intestinal tight junctions (including Claudin-1, Occludin, and ZO-1), and all treatment groups effectively reversed this protein expression inhibition. Further analysis showed that the treatment groups using human milk oligosaccharides in combination, zinc gluconate alone, and in combination with HMOs exhibited superior protein expression upregulation, especially the H3 group (zinc gluconate combined with HMOs), whose regulatory effect was significantly better than other intervention groups. Human milk oligosaccharides and zinc gluconate can maintain the integrity of the intestinal epithelial tight junction structure through multi-target synergistic effects. Their protective mechanism may involve enhancing the intestinal physical barrier function by regulating the claudin / occludin / ZO-1 signaling pathway, thereby providing systemic intestinal health protection for the host.

[0079] (4) Effects of different combinations of human milk oligosaccharides on inflammatory factors in mice

[0080] This study confirms that intervention with HMOs and zinc gluconate can significantly improve the imbalance of intestinal cytokines in bacterial-infected mice. Figure 4 It can be seen that the levels of pro-inflammatory factors IL-1β, IL-6, and tumor necrosis factor-α (TNF-α) in the experimental group were significantly lower than those in the model group (P<0.05). The effect of single-component HMOs in reducing IL-1β and IL-6 was far less than that of the HMOs combination group and the intervention combined with zinc gluconate. In particular, the HMOs combined with zinc gluconate groups H2 and H3 showed the most significant effect in reducing IL-6 compared to other intervention groups. Single HMOs groups could not significantly improve TNF-α levels, but HMOs combinations and the combination with zinc gluconate significantly reduced TNF-α levels, especially group H3, which showed a significant difference compared to the single HMOs group. The multi-component combination groups had comparable effects on IL-10 improvement, both superior to the single-component groups. Mechanistic studies suggest that HMOs and zinc gluconate may inhibit the overactivation of the TLR4 / NF-κB signaling pathway through multi-component synergistic effects, thereby reducing the release of pro-inflammatory mediators and promoting the re-establishment of intestinal mucosal immune homeostasis. This discovery provides crucial experimental evidence for the development of targeted HMOs and zinc gluconate combination formulations, and has significant translational value for precise nutritional intervention in intestinal barrier damage.

[0081] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations included in the claims. Various modifications and changes can be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments can be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only illustrate several implementations of the present invention and do not limit the scope of protection of this patent.

Claims

1. A composition for improving intestinal barrier impairment, characterized by, The composition consists of 2'-FL, LNT, 3'-SL and zinc gluconate, and the mass ratio of the 2'-FL, LNT, 3'-SL and zinc gluconate is (40-50):(25-35):(3-6):

1.

2. The composition of claim 1, wherein, The mass ratio of the 2'-FL, LNT, 3'-SL and zinc gluconate is 43:29:5:

1.

3. A pharmaceutical product containing the composition of any one of claims 1-2.

4. Use of the composition of any one of claims 1-2 or the pharmaceutical product of claim 3 in the preparation of a product for preventing and / or treating intestinal barrier damage.

5. Use according to claim 4, characterized in that, The product is used for reducing the content of diamine oxidase; and / or, the product is used for reducing the content of lipopolysaccharide; and / or, the product is used for reducing the content of D-lactic acid.

6. Use according to claim 4, characterized in that, The product is used for up-regulating the expression of Claudin-1, Occludin and / or ZO-1.

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