Application of human milk oligosaccharide composition in the preparation of medicines

Abstract

The present invention relates to the field of biomedicine technology, and specifically provides the use of a human milk oligosaccharide composition in the preparation of a drug. The present invention provides a composition that has the effect of enhancing immunity by accurately proportioning 2'-fucosyllactose, oligogalactose and short-chain oligofructose, thereby realizing the verification of the medicinal value of the human milk oligosaccharide composition. Specifically, the human milk oligosaccharide composition provided by the present invention can not only enhance the immunity of a normal body, but also can be used to repair the immunoregulatory ability of an immunocompromised or immunocompromised body.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the application of a human milk oligosaccharide composition in the preparation of medicines. Background Art

[0002] Human milk oligosaccharide compositions (HMOs) will not be decomposed by infants' gastric acid and digestive enzymes, and can directly reach the intestines, play a prebiotic role, and promote the growth of beneficial bacteria such as intestinal bifidobacteria, thereby inhibiting intestinal pathogens and regulating the health of the human intestinal microecology. Based on this, human milk oligosaccharide compositions have been widely used in food and health products.

[0003] The prebiotic compositions containing human milk oligosaccharides provided in the prior art are all based on the biological effects of regulating intestinal flora and enhancing immunity under normal immune conditions of the body. However, there are few studies on the conditions where the body's immunity is low or damaged and is in urgent need of protection.

[0004] In an immunocompromised body, the intestinal flora is usually abnormal and the balance between probiotics and harmful bacteria has been disrupted. It is not clear whether prebiotics, as their food, still have the same effect and what effect they have. Summary of the invention

[0005] In existing reports, it is only recorded that the prebiotic composition can be used as food for probiotics. However, the present invention has verified through animal experiments that the prebiotic composition provided in a specific ratio can achieve immune repair in an immune-suppressed body.

[0006] In the animal experiment of the present invention, 80 mg / kg of cyclophosphamide was intraperitoneally injected daily during the animal modeling, resulting in immunosuppression of peripheral immune organs, nonspecific and specific lymphocytes of experimental mice, and an immunosuppressive or immunocompromised mouse animal model was constructed. Compared with normal immunity, the immune response and mechanism of action of the body to exogenous pathogenic substances are greatly different in immunocompromised and immunocompromised. In existing studies, only the effects of prebiotic compositions on normal bodies were studied, and the effects of prebiotic compositions on immune abnormalities were not mentioned at all, and whether the prebiotic compositions have medicinal value was not considered.

[0007] Based on this, the first aspect of the present invention provides the use of a human milk oligosaccharide composition in the preparation of a drug for the treatment of immunosuppression, wherein the purity of 2'-fucosyllactose, galacto-oligosaccharides and short-chain oligofructose is 100%, and the pure weight ratio of 2'-fucosyllactose, galacto-oligosaccharides and short-chain oligofructose in the human milk oligosaccharide composition is 1:3:3.

[0008] In a second aspect, the present invention provides a human milk oligosaccharide composition for use in preparing a drug for treating immunosuppression, wherein the purity of 2'-fucosyllactose, galacto-oligosaccharides and short-chain oligofructose is 100%, and the pure weight ratio of 2'-fucosyllactose, galacto-oligosaccharides and short-chain oligofructose in the human milk oligosaccharide composition is 2:3:3.

[0009] In a third aspect, the present invention provides a human milk oligosaccharide composition for use in preparing a drug for treating immunosuppression, wherein the purity of 2'-fucosyllactose, galacto-oligosaccharides and short-chain oligofructose is 100%, and the pure weight ratio of 2'-fucosyllactose, galacto-oligosaccharides and short-chain oligofructose in the human milk oligosaccharide composition is 1:5:5.

[0010] Based on the fact that 100% pure 2'-fucosyllactose, galacto-oligosaccharides and short-chain oligofructose have not been produced in the art, it is preferred that when preparing the human milk oligosaccharide composition, the purity of the raw material of 2'-fucosyllactose used is not less than 94%, the moisture content does not exceed 6%, and the content of D-lactose does not exceed 3% (on DM). The content of galactobiose to galacto-oligooctasaccharides in the raw material of oligogalactose used is ≥57% (on DM), and the content of lactose is ≤23% (on DM), and the content of anhydrous glucose is ≤22% (on DM). Further, the purity of the raw material of oligogalactose used is not less than 57%. Further, the purity of the raw material of short-chain oligofructose used is not less than 95%, and the moisture content does not exceed 5%. The above "on DM" refers to "dry basis content".

[0011] In the preparation of the human milk oligosaccharide composition of the present invention, if the purity of the 2'-fucosyllactose, galacto-oligosaccharide or short-chain fructo-oligosaccharide used is less than 100%, the ratio of the three can be achieved by increasing the amount of low-purity prebiotics or reducing the amount of high-purity prebiotics.

[0012] The human milk oligosaccharide composition provided by the present invention is used in the preparation of a drug for treating immunosuppression, wherein the immunosuppression refers to abnormal expression of proteins related to the immune response signaling pathway caused by an imbalance between anti-inflammatory and pro-inflammatory effects in an animal.

[0013] The present invention successfully induced an immunosuppressive animal model using cyclophosphamide. Cyclophosphamide can activate the nuclear transcription factor κB (NF-κB) pathway by affecting signal pathways such as dendritic cell-associated c-type lectin-1 (Detin-1), Toll-like receptor 2 (TLR2) and TLR4, thereby affecting the secretion of downstream cytokines. Therefore, the immunosuppressive model constructed using cyclophosphamide can induce an imbalance of Th1 / Th2 cytokines in the body, that is, an imbalance of anti-inflammatory and pro-inflammatory.

[0014] In the embodiments of the present invention, immunocompromised mice supplemented with human milk oligosaccharide compositions also achieved anti-inflammatory-pro-inflammatory balance regulation through the Tolls-like receptor 4 signaling pathway, correcting the Th1 / Th2 imbalance of immunocompromised mice. The research results of the present invention verify the advantages of using an immunocompromised model on the one hand, and on the other hand, verify the specific effect of a specific ratio of human milk oligosaccharide compositions on the immunoregulation of immunocompromised animals. However, the study of this mechanism will be difficult to clearly explain in normal animal models.

[0015] It can be seen that the use of immunodeficiency models for research provides a scientific basis for studying immunity enhancement programs for immunocompromised groups caused by specific disease states, and expands the application scope of scientific technology and prebiotic composition products.

[0016] In a fourth aspect, the present invention provides a drug for the treatment of immunosuppression, wherein the main components of the drug for the treatment of immunosuppression are 2'-fucosyllactose, galacto-oligosaccharides and short-chain fructo-oligosaccharides in a pure weight ratio of 1:3:3; the immunosuppression refers to the abnormal expression of proteins related to the immune response signaling pathway caused by the anti-inflammatory-pro-inflammatory imbalance in the animal body.

[0017] Alternatively, the main components of the immunosuppression therapeutic drug are 2'-fucosyllactose, galacto-oligosaccharide and short-chain fructo-oligosaccharide in a pure weight ratio of 2:3:3.

[0018] Alternatively, the main components of the immunosuppression therapeutic drug are 2'-fucosyllactose, galacto-oligosaccharide and short-chain fructo-oligosaccharide in a pure weight ratio of 1:5:5.

[0019] The immunosuppression therapeutic drug provided by the present invention comprises: adding pharmaceutical excipients to the main components to prepare capsules, tablets, granules, injections, sustained-release preparations, oral liquids or pills.

[0020] The present invention uses a more reliable "low immunity" animal experimental model to demonstrate the immune enhancement effect of different proportions of HMO / GOS / scFOS, expands the application range of human milk oligosaccharide compositions, and provides solutions for infants and children with low immunity while taking into account normal infants and children. In the present invention, human milk oligosaccharide HMO refers to 2'-fucosyllactose (2'-FL), scFOS refers to short-chain oligofructose, and GOS refers to oligogalactose.

[0021] Compared with normal experimental animals, the present invention uses immunocompromised animal models to conduct research, which has the following advantages:

[0022] (1) It makes up for the shortcomings of existing technologies. Existing technologies are mostly based on the study of the functions of human milk oligosaccharides or prebiotic compositions containing human milk oligosaccharides under normal immune conditions, and rarely use immunodeficiency models, resulting in a lack of research and nutritional technology for immunodeficient children who are more vulnerable and in urgent need of immune protection.

[0023] (2) It is more conducive to studying the regulatory mechanism of the immune system: Animal models with low immunity usually have abnormal expressions of some components of the immune system, abnormal immune cell functions, or abnormal signal transduction pathways. These abnormalities provide valuable opportunities for in-depth research on the regulatory mechanism of the immune system and the signal transduction mechanism of immune cells after receiving external stimuli.

[0024] The human milk oligosaccharide composition provided by the present invention has immune repair and therapeutic effects. The present invention is the first to focus on the application prospects of compositions containing human milk oligosaccharides in the field of disease treatment. Specifically, the present invention uses an immunocompromised animal model to verify the efficacy of prebiotic compositions in immune repair and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 The spleen tissue pathological structure diagram of animals in the HMO composition intervention experiment of Example 1, A is the blank control, B is the immunosuppression model group, C is the low-dose group, D is the medium-dose group, and E is the high-dose group.

[0027] Figure 2The diagram is a histopathological structure diagram of spleen tissues of animals in the HMO composition intervention experiment of Example 2, A is a blank control, B is an immunodeficiency model group, F is a low-dose group, G is a medium-dose group, and H is a high-dose group.

[0028] Figure 3 It is a bar graph of the relative expression of ileal proteins. The blue series of columns are the results of Example 1, F1L is the low-dose treatment group of Example 1, F1M is the medium-dose treatment group of Example 1, and F1H is the high-dose treatment group of Example 1; the green series of columns are the results of Example 2, F2L is the low-dose treatment group of Example 2, F2M is the medium-dose treatment group of Example 2, and F2H is the high-dose treatment group of Example 2; CON is the blank control group, and MODEL is the immunosuppression model group. In the figure, Compared with the blank control group, P< 0.05 ; Compared with the blank control group, P<0.01 ; Compared with the immunodeficiency model group, P<0.05 ; Compared with the immunodeficiency model group, P<0.01 . DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0031] The raw material purity of 2'-fucosyllactose used in the present invention is not less than 94%, the water content is not more than 6%, and the content of D-lactose is not more than 3%; the content of galactobiose to galacto-oligooctasaccharide in the raw material of galacto-oligosaccharide used is ≥57% (onDM), and the content of lactose is ≤23% (on DM), the content of anhydrous glucose is ≤22% (on DM), and the purity of the raw material of galacto-oligosaccharide used is not less than 57%; the purity of the raw material of short-chain fructo-oligosaccharide (scFOS) used is not less than 95%, and the water content is not more than 5%.

[0032] The experimental method used in the present invention is as follows:

[0033] 1. Zebrafish experiment

[0034] The zebrafish model was used to evaluate the health effects of the HMO composition in enhancing immunity and protecting intestinal mucosal damage, thereby achieving multi-ratio screening among 2'-FL, GOS, and scFOS in the HMO composition. The zebrafish experiment is suitable for evaluating the HMO compositions of Examples 1-3 and Comparative Examples 1-5.

[0035] Evaluation indicators include the number of T cells, the number of macrophages, the number of goblet cells, and the phagocytic function of macrophages. For the first three indicators, experimental zebrafish 3 days after fertilization were placed in a 6-well plate with a volume of 3 mL per well, and 30 zebrafish were treated in each well. The zebrafish HMO composition was treated with water-soluble method for 2 days, and the modeling reagent was treated for 2 days during this period. For the phagocytic function of macrophages, the age of the experimental zebrafish, the experimental sample treatment cycle, and the modeling treatment cycle were 3 days, 4 days, and 4 days, respectively.

[0036] For the detection of goblet cell number, the samples were stained with Alcian blue after treatment. After the treatment, 10 zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope. NIS-Elements D 3.20 advanced image processing software was used to analyze and collect data to analyze the differences in the effects of different proportions of the composition on various indicators. The zebrafish strains, modeling methods and evaluation indicators of each experiment are detailed in Table 1.

[0037] Table 1 Details of zebrafish strains, modeling methods and evaluation indicators used in each experiment

[0038]

[0039] Three control groups (model control group, positive control group, normal control group) and eight treatment groups with different ratios were set for each indicator. According to the recommended intake dose of HMO composition for humans and the human-zebrafish conversion factor, after preliminary experimental dose range exploration, the amount of HMO composition in each treatment group was finally determined to be 480 μg / mL, but the ratio of 2'-FL / GOS / scFOS in each group was different, namely 1:10: 1, 1:5:5, 1:3:3, 2:3:3, 1:1:1, 3:5:3, 10:1:10 and 10:10:1.

[0040] 2. Mouse Experiment

[0041] The mouse experimental method is used to optimize the ratio of the three components in the composition, which is suitable for testing the composition of Examples 1-2.

[0042] 1) Animal husbandry and grouping

[0043] For each example, 70 healthy SPF-grade weaned female BALB / c mice, weighing 18-22 g, were provided by the Experimental Animal Center of Peking University Health Science Center. The experimental mice were housed in a barrier environment with a temperature controlled at (22±2)°C, a relative humidity of 50%-60%, and a 12h:12h alternating lighting schedule. During the experiment, the animals were free to drink water and eat. Adaptive feeding was performed for 3 days.

[0044] After 3 days of adaptive feeding, the experimental mice were weighed for initial body weight and randomly divided into 5 groups according to body weight, namely: blank control group, immunodeficiency model group, low-dose combination intervention group, medium-dose combination intervention group, and high-dose combination intervention group. Each group was fed with maintenance feed.

[0045] 2) Modeling and intervention

[0046] Modeling: On the 1st to 3rd day of the experiment, except for the blank control group, the animals in other groups were intraperitoneally injected with 80 mg / kg cyclophosphamide every day to establish the immunocompromised mouse model. The control group was intraperitoneally injected with an equal amount of normal saline every day.

[0047] Intervention: Starting from the 4th day, the control group and the model group were given 1% deionized water by gavage every day, and the low-, medium-, and high-dose groups of the combination were given 0.962 g / kgˑbw, 1.924 g / kgˑbw, and 3.847 g / kgˑbw by gavage, respectively. The amount of each gavage was calculated according to the weight of the mice, and the gavage volume was 0.1 mL / 10 g body weight. The gavage was continued for 30 days.

[0048] The basis for setting the low, medium and high doses in the embodiments of the present invention is: according to the human-mouse body surface area conversion, 5 times, 10 times and 20 times the intake of the human milk oligosaccharide composition in the human body are respectively used as the low, medium and high dose groups of the corresponding mouse experiments.

[0049] 3) Detection indicators and methods

[0050] (1) Body weight and immune organ index: The initial and final body weights of the experimental mice were weighed using an electronic balance. After removing the eyeballs and drawing blood, the mice were killed by cervical dislocation, and the spleen and thymus were removed and weighed using an electronic balance to calculate the spleen index and thymus index. Organ index = organ weight (mg) / body weight (g).

[0051] (2) Pathological examination of immune organs: The spleen and thymus were weighed and fixed in 4% formaldehyde solution, embedded in paraffin, sectioned, and stained with hematoxylin-eosin. The histomorphological changes of the spleen and thymus were observed under a microscope.

[0052] (3) Detection of peripheral blood T lymphocyte subsets: Take an appropriate amount of whole blood from each animal and place it in an EDTA anticoagulant tube. Pipette 50 μL of whole blood into a flow cytometer, add three fluorescent dye-conjugated antibodies, and mix by pipetting and flicking. Incubate at room temperature in the dark for 30 minutes, mix once every 5 minutes, then add 1 mL of lysis buffer. After lysis, add 1 mL of PBS. Centrifuge at 1500 r / min for 5 minutes, discard the supernatant, and flick the cells. Add 1 mL of PBS to wash the cells, centrifuge at 350×g for 5 minutes, discard the supernatant, add 200 μL of 2% paraformaldehyde solution to resuspend the cells, and use a FACS Calibur flow cytometer to detect the percentage of CD3+ cells, CD4+ cells, and CD8+ cell subsets.

[0053] (4) Detection of spleen T lymphocyte subsets: Harvest the spleen of mice to prepare a single cell suspension, centrifuge at 350×g for 5 min, discard the supernatant, add 2 mL of 1× lysis buffer to resuspend the cell pellet. Incubate on ice for 5 min, add 2 mL of PBS, centrifuge at 350×g for 5 min, discard the supernatant, and add 1 mL of PBS. Count the cells and adjust the concentration to 1×10 6 / 100μL. Take 100μL of cell suspension in a flow tube, add three fluorescent dye-conjugated antibodies, incubate in the dark for 30min, and mix every 5min. Add 1mL PBS to wash the cells, centrifuge at 350×g for 5min, discard the supernatant, add 300μL 2% paraformaldehyde solution to resuspend the cells, and use FACS Calibur flow cytometer to detect the percentage of CD3+ cells, CD4+ cells and CD8+ cell subsets.

[0054] (5) Determination of peripheral blood immunoglobulin content: The remaining blood of each animal was centrifuged to separate the serum. 200 μL of serum was taken and the immunoglobulin IgG and IgM content was detected using Multiscience EK271 or EK276 kits.

[0055] (6) Determination of serum cytokine levels: Take 200 μL of serum and use the Millipore MTH17MAG-47K kit to determine the levels of cytokines such as ILs, CRP, IFN-γ, and TNF.

[0056] (7) Bone marrow nucleated cell count and DNA content: Femurs of 6 mice were collected from each group for bone marrow nucleated cell count: the muscles and connective tissue attached to the femurs were removed, and the bone marrow fluid was flushed into a centrifuge tube with EDTA added using 1 mL of Hank's solution. 0.38 mL (0.1 mol / L, 10%) of hydrochloric acid solution was drawn into another centrifuge tube, and 20 μL of the mixed bone marrow fluid was added and mixed, and the mixture was allowed to stand for 2-3 minutes to allow the red blood cells to dissolve. 20 μL of the mixture was flushed into a cell counting plate for counting. Femurs of 6 other mice were taken for bone marrow DNA content detection: 10 mL of 5 mmol / L CaCl2 solution was aspirated, and all the bone marrow was flushed into a centrifuge tube, placed at 4°C for 30 min, centrifuged at 2500 r / min for 15 min, the supernatant was discarded, 5 mL of 0.2 mol / L HClO4 solution was added to the precipitate, mixed thoroughly, placed in a 90°C water bath for 15 min, cooled to room temperature, centrifuged at 3500 r / min for 15 min, the supernatant was taken, and the absorbance A260 value was measured at 260 nm using a FLUOstarOmega fully automatic multifunctional microplate reader to calculate the bone marrow DNA content.

[0057] (8) Intestinal protein detection - Western Blot: Western Blot was used to detect the following indicators:

[0058] Intestinal barrier-related proteins: Claudin-1, ZO-1, Occludin, MUC2, etc.;

[0059] Proteins related to immune response signaling pathways: Toll-like receptors (TLRs), p38 mitogen-activated protein (p38), extracellular regulated kinase 1 / 2 (ERK1 / 2), c-Jun N-terminal kinase (JNK1 / 2), NF-κB p65, p-p65.

[0060] 4) Statistical analysis methods:

[0061] All experimental results were continuous variables, expressed as mean ± SD. All data were statistically analyzed using SPSS 25.0 software. One-way ANOVA was used to compare differences between groups. P< 0.05 As the criterion for judging whether the difference is statistically significant, LSD method was used for pairwise comparison when the variance was equal; Tamhane's method was used for pairwise comparison when the variance was unequal.

[0062] Example 1

[0063] In the human milk oligosaccharide composition of this embodiment, the weight ratio of the pure products of 2'-fucosyllactose, galacto-oligosaccharide and short-chain fructo-oligosaccharide is 2:3:3.

[0064] Example 2

[0065] In the human milk oligosaccharide composition of this embodiment, the weight ratio of the pure products of 2'-fucosyllactose, galacto-oligosaccharide and short-chain fructo-oligosaccharide is 1:3:3.

[0066] Example 3

[0067] In the human milk oligosaccharide composition of this embodiment, the weight ratio of the pure products of 2'-fucosyllactose, galacto-oligosaccharide and short-chain fructo-oligosaccharide is 1:5:5.

[0068] Comparative Example 1

[0069] In the human milk oligosaccharide composition of this comparative example, the weight ratio of the pure products of 2'-fucosyllactose, galacto-oligosaccharide and short-chain fructo-oligosaccharide is 1:10:1.

[0070] Comparative Example 2

[0071] In the human milk oligosaccharide composition of this comparative example, the weight ratio of the pure products of 2'-fucosyllactose, galacto-oligosaccharide and short-chain fructo-oligosaccharide is 1:1:1.

[0072] Comparative Example 3

[0073] In the human milk oligosaccharide composition of this comparative example, the weight ratio of the pure products of 2'-fucosyllactose, galacto-oligosaccharide and short-chain fructo-oligosaccharide is 3:5:3.

[0074] Comparative Example 4

[0075] In the human milk oligosaccharide composition of this comparative example, the weight ratio of the pure products of 2'-fucosyllactose, galacto-oligosaccharide and short-chain fructo-oligosaccharide is 10:1:10.

[0076] Comparative Example 5

[0077] In the human milk oligosaccharide composition of this comparative example, the weight ratio of the pure products of 2'-fucosyllactose, galacto-oligosaccharide and short-chain fructo-oligosaccharide is 10:10:1.

[0078] Experimental Example 1 Zebrafish Experiment Results

[0079] Under the experimental conditions of the aforementioned zebrafish immunosuppression model / intestinal mucosal injury model, the compositions of Examples 1-3 and Comparative Examples 1-5 all showed a certain degree of immune enhancement efficacy. Except for the group with a ratio of 10:1:10, the remaining groups all increased the number of intestinal goblet cells, increased the secretion of intestinal mucus, improved intestinal mucosal protection, and thus improved nonspecific intestinal defense capabilities, indicating that the composition has the efficacy of intestinal protection-improving nonspecific intestinal defense capabilities.

[0080] Among them, the ratio of 2'-fucosyllactose, galacto-oligosaccharide and short-chain oligofructose in the composition of 1:3:3 and 2:3:3 was significantly better than that of other groups in increasing immunity and intestinal protection. The ratio of 1:5:5 was also very effective in increasing immunity and intestinal mucosal protection, and was significantly higher than that of the model control group in the four indicators examined.

[0081] The effects of the compositions with other ratios were not as good as those of Examples 1-3. The experimental data indicated that the ratios of 2'-FL, GOS and scFOS had a greater impact on the immune effect, especially the ratio of 2'-FL to GOS and the ratio of GOS to scFOS. When the ratio of 2'-FL to GOS and the ratio of GOS to scFOS differed too much (e.g., 10 times), the effects of immune enhancement and intestinal mucosal protection decreased significantly.

[0082] The different immune effects produced by different proportions of the composition are related to the dominant role of 2'-FL, GOS, and scFOS in immune regulation. The body's immune regulation is a very complex process, which is the result of the combined effects of multiple mechanisms such as strengthening the intestinal mucosal protective barrier, activating immune cell activity, and balancing inflammatory response. 2'-FL, GOS, and scFOS are all prebiotics and play a role in the above three dimensions, but the mechanism and degree of action are different.

[0083] Studies have shown that HMOs such as 2'-FL are more advantageous in promoting the balance of TH1 and TH2 immune cells to maintain inflammatory balance, selectively proliferating probiotics (such as bifidobacteria) to regulate immunity, and directly binding to pathogens to prevent pathogen invasion. GOS has a strong effect on strengthening the intestinal mucosal protective barrier and reducing inflammatory response and oxidative stress. The mechanisms of HMOs and GOS in regulating inflammatory response are different. GOS can regulate the BAs-dependent TGR5-cAMP signaling pathway and reduce intestinal inflammatory response by inhibiting NF-κB activation. HMOs mostly regulate inflammatory response through the TLR (toll-like receptor) pathway. scFOS also has the effect of mucosal protection and regulating inflammatory response, but its effect on regulating intestinal flora is not as strong as GOS, and it is not as targeted as HMOs.

[0084] It can be seen that the biological effects of 2'-FL, GOS, and FOS in regulating immunity are both related and different. If the difference in the ratio of the three is too large, it is not conducive to exerting the advantages of their respective effects, but if the ratio is too close, excessive immune regulation may occur. The goal of immune regulation is to maintain immune balance, and excessive enhancement in one direction is also disadvantageous.

[0085] According to the zebrafish experimental data, it can be observed that when 2'-FL / GOS / scFOS are within a smaller ratio range, and when GOS / scFOS is 1:1 and the amount of GOS or scFOS is greater than 2'-FL, the immune enhancement effect is the best. For example, the compositions of Example 1 (2:3:3), Example 2 (1:3:3), and Example 3 (1:5:5) all showed significant immune repair effects on immunosuppressed zebrafish ( p<0.05 ), and at the same time, it has the effect of significantly increasing the number of intestinal goblet cells and enhancing intestinal mucosal protection in zebrafish with intestinal mucosal damage ( p<0.05 See Table 2 for details.

[0086]

[0087] Even if the ratio of 2'-FL / GOS / scFOS is within a relatively small range, when the ratio of 2'-FL / GOS / scFOS is too close to or equal to 1:1:1, the maximum effect of the composition cannot be exerted, and the effect is reduced, such as in Comparative Example 2 (1:1:1) and Comparative Example 3 (3:5:3). See Table 3 for details.

[0088] On the other hand, when the ratio of 2'-FL to GOS and the ratio of GOS to scFOS are too different (e.g. 10 times), the effects of immune enhancement and intestinal mucosal protection are significantly reduced, such as in Comparative Example 1 (1:10:1) and Comparative Example 4 (10:1:10). See Table 3 for details.

[0089] At the same time, according to the results of zebrafish research, when the contents of 2'-FL and (GOS+scFOS) were fixed, the ratio of GOS to scFOS was different, and the composition had different effects in immune enhancement and intestinal mucosal protection. For example, Comparative Example 4 (10:1:10) and Comparative Example 5 (10:10:1) showed different effects.

[0090] It can be inferred from the experimental results of Comparative Examples 4 and 5 that when the contents of 2'-FL and (GOS+scFOS) are fixed, the higher proportion of GOS has a more obvious protective effect on specific immunity (number of T lymphocytes) and intestinal mucosa compared with scFOS. See Table 3 for details.

[0091]

[0092] To further verify whether the two most effective ratios (1:3:3 and 2:3:3) have the same immune-enhancing effect on higher-level experimental animals, mouse experiments were conducted using the same methods described above.

[0093] Experimental Example 2 Mouse experiment with 2'-FL / GOS / scFOS in a ratio of 2:3:3 (Example 1)

[0094] Each component was compounded into the HMO composition provided in Example 1 according to the following weight: 2'-FL2 kg, GOS 3 kg, scFOS 3 kg, based on a purity of 100%. The prepared HMO composition was used as a sample to carry out a mouse experiment on immune function. The experimental mouse grouping and intervention information are shown in Table 4.

[0095] Table 4 Experimental mouse grouping and intervention information

[0096]

[0097] The indicators of immune organ recovery, enhancement of humoral immunity, cellular immunity and immune response regulation of experimental mice in the blank control group, immunosuppression model group and HMO combination groups were statistically analyzed and compared to determine whether the test substance has the effect of enhancing immunity for immunosuppressed animals. The experimental results are as follows:

[0098] (1) Body weight and immune organ index: As shown in Table 5, there was no statistical difference in the initial and final body weights of mice in each group ( P>0.05 The body weight of mice in the model group, low-dose group, medium-dose group, and high-dose group decreased significantly after modeling compared with the control group ( P<0.05 There was no statistical difference in the thymus index among the mice in each group ( P>0.05 The spleen index of mice in the low-dose group was significantly higher than that in the control group ( P<0.05 ).

[0099]

[0100] (2) Immune organ recovery: Figure 1 This is a diagram of the spleen tissue pathological structure of animals in the HMO composition intervention experiment of Example 1. As can be seen from the figure, compared with the immunosuppression model group, the morphological structure of immune cells in the spleen and thymus of the high, medium and low dose HMO composition treatment groups have been restored to a certain extent, and the morphology is close to that of the blank control group, which is most obvious in the low and medium dose groups.

[0101] (3) Humoral immune function: As shown in Table 6, compared with the immunosuppression model group, the peripheral blood immunoglobulin IgG and IgM levels of mice in the HMO combination treatment group showed an increasing trend, with the IgG content in the medium-dose group increasing the most. Although this improvement has not yet reached a statistically significant level, there is no significant difference in IgG content between the medium- and low-dose groups and the blank control group, indicating that it has also been improved to a certain extent.

[0102]

[0103] (4) Immune response regulation: As shown in Table 7, the IL-6 concentration in the high-dose group was significantly reduced compared with the immunodeficiency model group ( P<0.05 ). This suggests that the HMO combination may enhance the regulation of immune response by suppressing the levels of some pro-inflammatory cytokines in the serum of immunocompromised mice, thereby achieving the effect of enhancing immunity. The intervention of this ratio combination had no significant effect on the levels of other inflammatory factors ( P>0.05 ).

[0104]

[0105] (5) Cellular immunity: Compared with the immunosuppression model group, there was no significant difference in the percentage of peripheral blood CD3+, CD4+, and CD8+ T lymphocytes and CD4+ / CD8+ in each intervention dose group.

[0106] As shown in Table 8, compared with the immunodeficiency model group, the percentages of splenic CD3+, CD4+, and CD8+ T lymphocytes were significantly restored in the medium- and high-dose HMO composition treatment groups ( P<0.05 ). CD4+ / CD8+ was also significantly increased in the medium-dose group. This suggests that HMO combination intervention can improve the proliferation capacity of T lymphocytes in immunocompromised experimental mice.

[0107]

[0108] (6) Bone marrow DNA concentration: As shown in Table 9, there was no significant difference in bone marrow DNA concentration between each dose group and the immunodeficiency model group ( P>0.05 ).

[0109] Table 9 DNA concentration in bone marrow of experimental mice (n=12)

[0110]

[0111] (7) Analysis of the mechanism of the composition regulating immune function:

[0112] Compared with the immunosuppression model group, the intervention of the composition of Example 1 significantly increased the expression of intestinal barrier-related protein Claudin-1, suggesting that the intestinal mucosal barrier function has a certain improvement effect, but has no significant effect on the expression of ZO-1, Occludin, and MUC2 proteins.

[0113] At the same time, the composition increased the expression of proteins related to the immune response signaling pathway (TLRs, p38, ERK1 / 2, JNK1 / 2, NF-κB p65, p-p65) to a certain extent.

[0114] Experimental Example 3 Mouse experiment with 2'-FL / GOS / scFOS in a ratio of 1:3:3 (Example 2)

[0115] The components were compounded into a composition according to the following weights: 1 kg of 2'-FL, 3 kg of GOS, and 3 kg of scFOS, based on a purity of 100%. The HMO composition of Example 2 was used as a sample to carry out an immune function experiment. The experimental mouse grouping and intervention information are shown in Table 10.

[0116] Table 10 Experimental mouse grouping and intervention information

[0117]

[0118] (1) Body weight and immune organ index: There was no statistical difference in the initial body weight of mice in each group ( P>0.05 The body weight of mice in the model group, low-dose group, medium-dose group, and high-dose group decreased significantly after modeling compared with the control group ( P<0.05 The final body weight of mice in the medium-dose group was significantly higher than that in the control group ( P<0.05 There was no statistical difference in the thymus index and spleen index of each group of mice ( P>0.05 ).

[0119] (2) Immune organ recovery: Figure 2 This is a diagram of the spleen tissue pathological structure of animals in the HMO composition intervention experiment of Example 2. As can be seen from the figure, compared with the immunosuppression model group, the morphological structure of the spleen and thymus in the high, medium and low dose groups of the HMO composition treatment group was restored to a certain extent.

[0120] (3) Humoral immune function: As shown in Table 11, compared with the immunosuppression model group, the peripheral blood immunoglobulin IgM content of mice in the high- and medium-dose HMO combination treatment groups tended to increase (but did not reach statistical significance).

[0121]

[0122] (4) Immune response regulation: As shown in Table 12, compared with the immunosuppressive model group, the IL-6 levels in the low- and medium-dose HMO composition-treated groups were significantly reduced, while the concentrations of IL-4 and IL-10 in the high-dose group were significantly increased. This suggests that the HMO composition of Example 2 not only regulates the levels of some pro-inflammatory cytokines in the serum of immunosuppressive mice by inhibiting them, but also promotes the levels of anti-inflammatory cytokines in the serum, thereby comprehensively improving the regulation of immune responses and achieving the effect of enhancing immunity.

[0123]

[0124] (5) Cellular immunity: Compared with the immunosuppression model group, there was no significant difference in the percentage of peripheral blood CD3+, CD4+, and CD8+ T lymphocytes and CD4+ / CD8+ in each intervention dose group.

[0125] As shown in Table 13, compared with the immunodeficiency model group, the percentages of splenic CD3+, CD4+, and CD8+ T lymphocytes were significantly restored in the low-, medium-, and high-dose HMO composition treatment groups ( P<0.05 ).

[0126]

[0127] (6) Bone marrow DNA concentration: As shown in Table 14, although it did not reach statistical significance, the DNA concentration in the high-dose group showed an increasing trend compared with the immunosuppression model group, suggesting that the HMO composition of Example 2 has certain potential in restoring bone marrow immune function.

[0128] Table 14 DNA concentration in bone marrow of experimental mice (n=12)

[0129]

[0130] (7) Analysis of the mechanism of the composition regulating immune function:

[0131] pass Figure 3 The relative expression of ileum proteins shown in the figure shows that compared with the immunosuppression model group, the low, medium and high dose groups of the composition of Example 2 significantly increased the expression of intestinal barrier-related proteins Claudin-1 and ZO-1, and the medium dose group significantly increased the expression of MUC2 protein. The results show that the composition of Example 2 can significantly improve the intestinal mucosal barrier function. The intestinal barrier is an important line of defense for maintaining the balance of intestinal microecology and resisting the invasion of pathogenic microorganisms, among which tight junction proteins and mucins play an important role in filling the gaps between epithelial cells and forming a leaky barrier.

[0132] At the same time, the HMO composition of Example 2 significantly increased the expression of proteins related to the immune response signaling pathway (TLRs, p38, ERK1 / 2, JNK1 / 2, NF-κB p65, p-p65). Both the medium and high dose groups showed high expression of TLR4, which was significantly higher than that of the immunosuppression model group. The up-regulation of TLR4 expression can activate the MAPK / NF-κB (p65) signaling pathway, promote the secretion of cytokines and NO by macrophages RAW264.7 to regulate immune activity, improve Th1 / Th2 cytokine imbalance, mediate immune response, and antagonize cyclophosphamide-induced immunosuppression.

[0133] From the above results, it can be seen that the present invention proposes a more precise dosage of human milk oligosaccharides and a scientific ratio of human milk oligosaccharides and other prebiotics, that is, the weight ratio of 2'-fucosyllactose, galacto-oligosaccharides and short-chain fructo-oligosaccharides is 2:3:3 or 1:3:3. The human milk oligosaccharide composition formula has the best synergistic effect of immunoregulation and immune repair.

[0134] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a human milk oligosaccharide composition in the preparation of a drug for treating immunosuppression, characterized in that: Taking the purity of 2'-fucosyllactose, galacto-oligosaccharides and short-chain oligofructose as 100%, the pure weight ratio of 2'-fucosyllactose, galacto-oligosaccharides and short-chain oligofructose in the human milk oligosaccharide composition is 1:3:

3.

2. Use of a human milk oligosaccharide composition in the preparation of a drug for treating immunosuppression, characterized in that: Taking the purity of 2'-fucosyllactose, galacto-oligosaccharides and short-chain oligofructose as 100%, the pure weight ratio of 2'-fucosyllactose, galacto-oligosaccharides and short-chain oligofructose in the human milk oligosaccharide composition is 2:3:

3.

3. Use of a human milk oligosaccharide composition in the preparation of a drug for treating immunosuppression, characterized in that: Taking the purity of 2'-fucosyllactose, galacto-oligosaccharides and short-chain fructo-oligosaccharides as 100%, the pure weight ratio of 2'-fucosyllactose, galacto-oligosaccharides and short-chain fructo-oligosaccharides in the human milk oligosaccharide composition is 1:5:

5.

4. The use according to any one of claims 1 to 3, characterized in that: Immunosuppression refers to the abnormal expression of proteins related to the immune response signaling pathway caused by the imbalance of anti-inflammatory and pro-inflammatory factors in animals.

5. A drug for treating immunosuppression, characterized in that: The main components of the immunosuppression therapeutic drug are 2'-fucosyllactose, galacto-oligosaccharides and short-chain fructo-oligosaccharides in a pure weight ratio of 1:3:3; immunosuppression refers to the abnormal expression of proteins related to the immune response signaling pathway caused by the anti-inflammatory-pro-inflammatory imbalance of the animal body.

6. A drug for treating immunosuppression, characterized in that: The main components of the immunosuppression therapeutic drug are 2'-fucosyllactose, galacto-oligosaccharides and short-chain fructo-oligosaccharides in a pure weight ratio of 2:3:3; immunosuppression refers to the abnormal expression of proteins related to the immune response signaling pathway caused by the anti-inflammatory-pro-inflammatory imbalance of the animal body.

7. A drug for treating immunosuppression, characterized in that: The main components of the immunosuppression therapeutic drug are 2'-fucosyllactose, galacto-oligosaccharides and short-chain fructo-oligosaccharides in a pure weight ratio of 1:5:5; immunosuppression refers to the abnormal expression of proteins related to the immune response signaling pathway caused by the anti-inflammatory-pro-inflammatory imbalance of the animal body.

8. The immunosuppression therapeutic drug according to any one of claims 5 to 7, characterized in that: The immunosuppression treatment drug comprises: adding pharmaceutical excipients to the main components to prepare capsules, tablets, granules, injections, sustained-release preparations, oral liquids or pills.

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