Composition for improving intestinal barrier injury and application thereof

Abstract

The invention relates to a composition for improving intestinal barrier injury and application thereof. The composition comprises three or more of 2 '-FL, LNT, 3'-SL and zinc gluconate.The composition comprises the zinc gluconate.The composition can safely and effectively improve intestinal barrier injury.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a composition for improving intestinal barrier damage and application thereof. Background Art

[0002] Intestinal barrier damage can lead to nutrient absorption disorders, impacting the growth and development of infants and young children. Due to the decreased intestinal absorption capacity of nutrients such as protein, fat, vitamins, and minerals, infants and young children may experience weight loss and developmental delays. Furthermore, a damaged intestinal barrier cannot effectively block pathogens and harmful substances, allowing them to easily enter the bloodstream, triggering systemic infection and inflammatory responses, and increasing the risk of infants and young children developing respiratory tract infections, urinary tract infections, and other diseases. Furthermore, intestinal barrier damage can also lead to intestinal dysbiosis, reducing the number of beneficial bacteria and allowing harmful bacteria to proliferate, further disrupting the intestinal microecological balance and exacerbating symptoms, creating a vicious cycle.

[0003] Currently, clinical treatment for intestinal barrier damage caused by intestinal diseases in infants and young children primarily relies on symptomatic treatments. These include oral rehydration salts or intravenous fluids to correct dehydration and electrolyte imbalances, and antibiotics to treat bacterial diarrhea. However, overuse of antibiotics can lead to side effects such as intestinal dysbiosis. Furthermore, existing treatments primarily focus on relieving diarrhea symptoms, with limited effectiveness in repairing the damaged intestinal barrier, making it difficult to fundamentally address the range of issues associated with intestinal barrier damage.

[0004] CN114287629A discloses a composition containing human milk oligosaccharides and animal Bifidobacterium Bifidum lactis subsp. lactis, which can increase the level of transmembrane electrical resistance and is related to improving intestinal barrier function, because the increase in transmembrane electrical resistance is beneficial to maintaining the integrity of the intestinal barrier. However, it mainly focuses on improving intestinal immunity, and there is insufficient direct targeted research on improving intestinal barrier damage.

[0005] During the growth of infants and young children, intestinal health is a key factor affecting their overall development. As a crucial defense structure of the intestine, the intestinal barrier plays a crucial role in maintaining infant health. However, the infant intestine is still in its 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, leading to a range of health problems. This situation has created an urgent need for the development of targeted interventions.

[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 method to improve intestinal barrier damage. Summary of the Invention

[0007] Problems to be solved by the invention

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

[0009] Solutions for solving problems

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

[0011] Preferably, the composition consists of 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 the 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] The second aspect of the present invention provides a medicine, a nutritional product or a health product containing any one of the above-mentioned compositions.

[0017] The third aspect of the present invention provides use of the composition according to any one of the above items or the medicine, nutrient or health product described above in the preparation of a product for preventing and / or treating intestinal barrier damage.

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

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

[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] Effects of the Invention

[0023] The composition provided by the present invention has multiple physiological functions, such as regulating intestinal flora, reducing excessive growth of pathogenic bacteria, enhancing intestinal immune function, and promoting intestinal epithelial cell repair, and can treat or prevent inflammatory bowel disease (IBD). It provides a new direction for improving intestinal barrier damage, especially for intestinal barrier damage in infants and young children. The HMOs and zinc gluconate in the composition of this application have a synergistic effect to enhance intestinal immune function and have an excellent effect on improving intestinal barrier damage. At the same time, the composition provided by this application has the advantages of being safe and having no side effects, can avoid the problem of drug resistance, and is suitable for long-term use (such as adding to infant formula or functional foods). BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of HE staining results of ileum tissue sections of mice in different combinations of human milk oligosaccharide groups;

[0025] Figure 2 Schematic diagram of the expression levels of serum diamine oxidase (DAO), lipopolysaccharide (LPS) and D-lactic acid in mice in different combinations of human milk oligosaccharide groups;

[0026] Figure 3 Schematic diagram of the expression levels of intestinal tight junction core proteins (Claudin-1, Occludin and ZO-1) in mice in different combinations of human milk oligosaccharide groups;

[0027] Figure 4 Schematic diagram of the expression levels of mouse cytokines (IL-1β, IL-6 and TNF-α) in different combinations of human milk oligosaccharide groups. DETAILED DESCRIPTION

[0028] To make the technical solutions and beneficial effects of the present invention more clearly understood, the following detailed description is given by way of specific embodiments. The accompanying drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly illustrate 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 belongs.

[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 intestinal health through dual mechanisms: First, they act as prebiotics, selectively promoting the proliferation of beneficial bacteria such as Bifidobacterium and inhibiting pathogen colonization through competitive exclusion; second, they strengthen 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. Approximately 15% to 40% of IBD patients experience hypozincemia. Zinc deficiency can persist even after zinc supplementation due to decreased 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 developed a composition containing 2'-FL, LNT, 3'-SL, and zinc gluconate, which can reduce the expression levels of DAO, LPS, and D-lactic acid, upregulate claudin, occludin, and ZO-1, reduce the levels of inflammatory factors, synergistically inhibit intestinal epithelial cell apoptosis (reduce LPS entry into the blood), regulate bacterial metabolism (reduce endotoxin synthesis), and enhance the intestinal physical barrier function. Based on this, the present invention was completed.

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

[0033] In a certain embodiment, the composition consists of 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] The second aspect of the present invention provides a medicine, a nutritional product or a health product containing any one of the above-mentioned compositions.

[0041] The third aspect of the present invention provides use of the composition according to any one of the above items or the medicine, nutrient or health product described above in the preparation of a product for preventing and / or treating intestinal barrier damage.

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

[0043] In one embodiment, the product is used to reduce the level 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 commercial products, which can be obtained from commercial products or synthesized according to known methods. For example, fucosyllactose and sialyllactose are produced by Suzhou Yixi Biotechnology Co., Ltd.

[0047] The present invention is described in more detail below by way of examples. Where specific experimental steps or conditions are not specified in the examples, they were performed according to conventional procedures 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 combination mixtures (H1 to H3) were prepared respectively.

[0055] 2'-FL, LNT and 3'-SL were respectively taken and compounded into composition H1 in a mass ratio of 10:6:1.

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

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

[0058] Example 2

[0059] 4-week-old BALB / C male mice were selected for 6-8 weeks. All mice lived in a controlled environment with free access to standard laboratory food and water. The experimental environment was set to a 12-h light-dark cycle, and the temperature and humidity were maintained between 22±2℃ and 45±5%. After a week of adaptive feeding, the mice were randomly divided into 9 groups, 5 in each group, for formal experiments. Mouse models were constructed according to different groups, in which the negative control group mice were gavaged with equal volumes of PBS, and the remaining groups were infected with Escherichia coli to cause intestinal barrier loss, once a day for one week. Subsequently, 2'-FL, LNT, 3'-SL, and zinc gluconate were used for gavage treatment, and the negative control group and model group were gavaged with equal volumes of PBS, once a day for one week (see Table 3 for experimental groups and gavage doses).

[0060] Table 3 Experimental groups and dosages

[0061]

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

[0063] (1) HE staining: The fixed jejunum and lung tissue samples were dehydrated in ethanol, embedded in paraffin, and sliced ​​into 4 μm slices. Standard hematoxylin-eosin (HE) staining was used, and histological images were taken using an optical microscope.

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

[0065] (3) RT-qPCR method to detect tight junction expression: Tissue samples were taken to extract total RNA, reverse transcribed to synthesize cDNA, and then PCR reaction was performed. -ΔΔCt The relative expression levels of target genes occludin, claudin1 and ZO-1 mRNA were calculated.

[0066] (4) Cytokine determination by ELISA: The levels of IL-6, IL-1β, TNF-α, and IL-10 in mouse serum were determined using enzyme-linked immunosorbent assay kits according to the manufacturer's instructions.

[0067] (5) One-way ANOVA was performed on the data, and Tukey's multiple comparison test was used to analyze the significant differences. Inconsistent letters indicate significant differences.

[0068] Here are the results:

[0069] (1) Effects of different combinations of human milk oligosaccharides on mouse ileal histopathological parameters

[0070] HE staining was used to reflect the histopathological changes of the colon of mice in each experimental group and to evaluate the effects of HMOs on the histological structure and villi of the ileum. Figure 1 As shown, the ileum tissue structure of the treated mice was intact, with no significant structural disruption between the mucosa, submucosa, smooth muscle layer, and serosa. No ulcers or erosions were observed on the ileal mucosal surface, nor was there bleeding or inflammatory cell infiltration. No significant aggregation or follicular formation of lymphocytes, neutrophils, or plasma cells was observed. Furthermore, pathological analysis of the ileum tissue of the treated mice showed normal condition, without any obvious abnormal structures or pathological changes. However, the tissue structure of the MC group was incomplete, with significant structural disruption 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 2). DAO is a marker of intestinal epithelial cell damage, and its increase reflects the aggravation of cell shedding; D-lactic acid is produced by intestinal bacterial metabolism, and its increased serum concentration indicates the destruction of the intestinal mucosal physical barrier; LPS level represents 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 in each intervention group were significantly reduced, indicating that HMOs and zinc gluconate can improve intestinal barrier function through multiple pathways. In the combined group, H3 showed the best barrier repair ability, and its DAO, LPS and D-lactic acid levels decreased by 33.3%, 33.6% and 24.66% respectively compared with the MC group, which was significantly better than the other intervention groups.

[0073] Structural-specific synergy: 2'-FL reduces intestinal epithelial inflammatory damage by inhibiting the TLR4 / NF-κB pathway; LNT, a fucosyl receptor agonist, directly upregulates claudin-1 expression to enhance tight junction integrity; 3'-SL promotes mucin secretion by activating PPARγ signaling, forming a dual physical and immune barrier. Zinc recognizes cellular signals and increases 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, in combination, simultaneously block inflammatory damage, strengthen the junctional complex, and enhance the protective mucus layer, achieving multi-dimensional 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 and indirectly reduces D-lactic acid production.

[0075] Differences in LPS regulation: Although all intervention groups reduced LPS levels, the effect was most significant in the H3 group, which may be related to the dual mechanisms of synergistically inhibiting intestinal epithelial cell apoptosis (reducing LPS entry into the blood) and regulating bacterial metabolism (reducing endotoxin synthesis).

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

[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 to ensure the host's nutrient absorption efficiency and resist the invasion of pathogenic microorganisms. Experimental results show that ( Figure 3), Escherichia coli infection can significantly downregulate the expression levels of intestinal tight junction core proteins (including Claudin-1, Occludin and ZO-1), and each treatment group can effectively reverse this protein expression inhibition phenomenon. Further analysis showed that the treatment groups using human milk oligosaccharides in combination, zinc gluconate alone and in combination with HMOs showed excellent protein expression upregulation, especially the H3 group combined with zinc gluconate and HMOs, which had a significantly better regulatory effect 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. The 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 confirmed that HMOs and zinc gluconate intervention can significantly improve the intestinal cytokine imbalance in bacterial-infected mice. Figure 4 As can be seen, levels of the proinflammatory cytokines IL-1β, IL-6, and tumor necrosis factor-α (TNF-α) were significantly lower in the experimental group compared with the model group (P < 0.05). The effect of single-component HMOs on reducing IL-1β and IL-6 was far less pronounced than that of the HMO combination group or the combined intervention with zinc gluconate. In particular, the HMO and zinc gluconate combination groups, H2 and H3, showed the most significant effect on reducing IL-6 compared with the other intervention groups. While single HMOs failed to significantly improve TNF-α levels, the combination of HMOs and zinc gluconate significantly reduced TNF-α levels, particularly in group H3, which showed significant differences compared with the single HMO group. The multi-component combination groups had comparable effects on IL-10, outperforming both single-component groups. Mechanistic studies suggest that HMOs and zinc gluconate may, through a multi-component synergistic effect, inhibit the overactivation of the TLR4 / NF-κB signaling pathway, thereby reducing the release of proinflammatory mediators and promoting the reestablishment of intestinal mucosal immune homeostasis. This discovery provides key experimental evidence for the development of a targeted HMOs and zinc gluconate combination formula, and has important translational value for precise nutritional intervention of intestinal barrier damage.

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

Claims

1. A composition for improving intestinal barrier damage, characterized in that: The composition comprises three or more of 2'-FL, LNT, 3'-SL and zinc gluconate, wherein the composition comprises zinc gluconate.

2. The composition according to claim 1, characterized in that The composition consists of 2'-FL, LNT, 3'-SL and zinc gluconate.

3. The composition according to claim 2, characterized in that The mass ratio of the 2'-FL, LNT, 3'-SL and zinc gluconate is (10-200):(5-100):(1-20):

1.

4. The composition according to claim 3, characterized in that The mass ratio of the 2'-FL, LNT, 3'-SL and zinc gluconate is (30-150):(15-100):(1-15):

1.

5. The composition according to claim 4, characterized in that The mass ratio of the 2'-FL, LNT, 3'-SL and zinc gluconate is (40-120):(20-80):(3-10):

1.

6. The composition according to claim 5, characterized in that The mass ratio of the 2'-FL, LNT, 3'-SL and zinc gluconate is 43:29:5:

1.

7. A medicine, nutritional product or health product containing the composition according to any one of claims 1 to 6.

8. Use of the composition according to any one of claims 1 to 6 or the medicine, nutrient or health product according to claim 7 in the preparation of a product for preventing and / or treating intestinal barrier damage.

9. The use according to claim 8, characterized in that The product is used to reduce the level of diamine oxidase; and / or, the product is used to reduce the content of lipopolysaccharide; And / or, the product is used to reduce the content of D-lactic acid.

10. The use according to claim 8, characterized in that The product is used to upregulate the expression of Claudin-1, Occludin and / or ZO-1.

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