Application of 2 '-fucosyllactose in preparation of immune balance medicine or food

Abstract

The invention relates to 2 '-fucosyllactose, in particular to application of the 2'-fucosyllactose to preparation of immune balance drugs or food. The immune balance comprises regulation and control of the ratio of Th17 cells to Treg cells of Th17 / Treg immune imbalance offspring activated by maternal immune or induced by virus infection; according to the application disclosed by the invention, the fact that filial generations generated after the maternal body of a mammal generates immune reaction activation can regulate and control the proportion of Th17 and Treg cells through the influence of 2 '-fucosyllactose on intestinal flora is found, so that Th17 and Treg immune balance is realized; in addition, social and cognitive disorders and repetitive behaviors related to filial generation autism induced by Th17 / Treg immune imbalance can be improved.

Description

Technical Field

[0001] The invention relates to 2'-fucosyllactose, in particular to the application of 2'-fucosyllactose in the preparation of immune balancing medicines or foods. Background Art

[0002] The balance between Th17 cells and Treg cells is an important component of the normal function of the immune system. When TH17 cells are overactivated or Treg cell function is impaired, it may lead to an imbalance in the immune system, thereby triggering inflammatory and autoimmune diseases. Intestinal Th17 and Treg cells are regulated by the intestinal microbiota, while brain-related Th17 and Treg cells maintain immune homeostasis to control neuroinflammation, microglial activation, astrocyte activation, and brain development in offspring during pregnancy. It is worth noting that the imbalance between Th17 and Treg cells, or the abnormal ratio of Th17 / Treg cells, plays a key role in psychiatric disorders such as depression. It is easy to understand that when maternal immune activation (MIA) occurs, it can induce Th17 / Treg immune imbalance in offspring, which in turn leads to autism-related social and cognitive disorders and repetitive behaviors in offspring.

[0003] The existing technology usually uses effective ingredients or extracts of traditional Chinese medicine such as evodiamine, astragaloside, naringin or inulin to regulate Th17 / Treg immune imbalance, but there is currently no application of 2′-fucosyllactose in the preparation of medicines or foods for treating Th17 / Treg immune imbalance induced by maternal immune activation. In addition, the existing technology generally treats or regulates pregnant women or adult individuals, and there is currently no product for the offspring of Th17 / Treg immune imbalance induced by maternal immune activation. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present invention provides an application of 2′-fucosyllactose in the preparation of immune balance medicines or foods, which is used to regulate the ratio of Th17 and Treg cells in offspring with Th17 / Treg immune imbalance induced by maternal immune activation or viral infection.

[0005] The technical solution of the present invention is:

[0006] Application of 2′-fucosyllactose in the preparation of immune balance medicines or foods, wherein the immune balance includes regulating the ratio of Th17 and Treg cells in offspring with Th17 / Treg immune imbalance induced by maternal immune activation or viral infection.

[0007] Specifically, the regulation of the ratio of Th17 and Treg cells includes upregulating the mRNA expression levels of TGR5 and VDR in the brain, and downregulating the mRNA expression level of FXR in the brain.

[0008] Specifically, the regulation of the ratio of Th17 and Treg cells also includes upregulating the mRNA expression level of FOXP3 in the brain and the expression level of serum IL-10.

[0009] Specifically, regulating the ratio of Th17 and Treg cells also includes downregulating the expression level of serum IL-17A.

[0010] Specifically, the regulation of the ratio of Th17 and Treg cells also includes downregulating the mRNA expression levels of IL-1β, IL-6, TNF-α, RORγT and STAT3 in the brain.

[0011] Specifically, the Th17 / Treg immune imbalance also includes: social and cognitive disorders and repetitive behaviors related to autism in offspring.

[0012] Specifically, the medicine refers to any tablet, capsule, oral tincture, pill, granule, powder or granule containing an effective dose of 2′-fucosyllactose.

[0013] Specifically, the food refers to any milk and dairy products, fats / oils and emulsified fat products, frozen drinks, fruits / vegetables, beans / edible fungi / algae / nuts and seeds, cocoa products / chocolate and chocolate products and candy, grains and grain products, baked goods, meat and meat products, aquatic products and their products, eggs and egg products, sweeteners, condiments, special nutritional foods, beverages, alcohol, jellies, tea, coffee, collagen casings, yeast products, fried foods and puffed foods that contain an effective dose of 2′-fucosyllactose.

[0014] Specifically, the medicine or food is administered to a mammalian mother to produce offspring born after an immune response is activated.

[0015] Specifically, the administration method includes nasogastric tube, jejunal tube, oral, sublingual, anal plug or rectal feeding.

[0016] The beneficial effects of the present invention are as follows: the present invention finds that the offspring born after the immune response of the mammalian mother is activated can regulate the ratio of Th17 and Treg cells through the intestinal flora under the influence of 2′-fucosyllactose, thereby achieving Th17 / Treg immune balance; in addition, it can also improve the social, cognitive disorders and repetitive behaviors related to autism in the offspring induced by Th17 / Treg immune imbalance. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1Schematic diagram and results of the co-cage treatment experiment; A is a diagram illustrating the grouping of mice, B is a diagram showing the weight measurement results, C is a diagram showing the results of the three-chamber interaction test, D is a diagram showing the results of the open field test, E is a diagram showing the results of the novel object test, and F is a diagram showing the results of the buried bead test. All data are expressed as mean ± standard error. * p<0.05 and ** p<0.01, compared with mPBS-oCD, # p<0.05 and ## p<0.01, compared with mMIA-oCD.

[0018] Figure 2 Figure 1 is the result of Th17 / Treg immune balance experiment; A is the statistical graph of FXR expression, B is the statistical graph of TGR5 expression, C is the statistical graph of VDR expression, D is the statistical graph of IL-17A expression, E is the statistical graph of IL-1β expression, F is the statistical graph of IL-6 expression, G is the statistical graph of TNF-α expression, H is the statistical graph of IL-10 expression, I is the statistical graph of FOXP3 expression, J is the statistical graph of RORγT expression, and K is the statistical graph of STAT3 expression; all data are expressed as mean ± standard error. * p<0.05 and ** p<0.01, compared with mPBS-oCD, # p<0.05 and ## p<0.01, compared with mMIA-oCD. DETAILED DESCRIPTION

[0019] The specific implementation of the present invention will be further described below in conjunction with the accompanying drawings:

[0020] Co-housing experiment (e.g. Figure 1 )

[0021] 1. Grouping of mice

[0022] like Figure 1As shown in A, pregnant mice (C57BL / 6J female mice) were divided into three groups. At 12.5 days of pregnancy, pregnant mice in the experimental group were injected with poly(I:C) (maternal immune activation group, hereinafter referred to as mMIA group), pregnant mice in the control group were injected with PBS (hereinafter referred to as mPBS group), and pregnant mice in the treatment group were injected with poly(I:C) and supplemented with an effective dose of 2′-fucosyllactose in the diet (hereinafter referred to as mMIA+2′-FL group). The offspring of the above three groups were fed with normal breast milk after birth, weaned after 3 weeks, and the offspring of the mPBS group were changed to the mPBS-oCD group (blank control group), the offspring of the mMIA group were changed to the mMIA-oCD group (blank experimental group), and the offspring of the mMIA+2′-FL group were changed to the mMIA+2′-FL-oCD group, and the offspring of the mMIA-oCD and mMIA+2′-FL-oCD groups were treated in the same cage. The mice were divided into five groups: mPBS-oCD group; mMIA-oCD group; mMIA-oCD (co-housing) group; mMIA+2′-FL-oCD (co-housing) group; and mMIA+2′-FL-oCD group. Animal experiments were performed after 5 weeks of feeding, and the mice were then euthanized.

[0023] 2. Co-cage treatment test

[0024] Co-housing is an intervention method that co-housing mice with different treatments. It uses the coprophagy of mice to achieve the purpose of mutual colonization of intestinal flora between co-housing mice. After co-housing, three-chamber interaction test, buried bead test, open field test and novel object test were carried out.

[0025] 2-1. Three-chamber interaction test

[0026] The three-chamber interaction test is used to evaluate social preference and social novelty preference. The test area is evenly divided into three interconnected chambers (left, middle, and right). In the first 5 minutes, the mice to be tested freely explore the entire test area and familiarize themselves with the environment. The time they enter each chamber is recorded. Social preference is measured in the second 5 minutes: before the test, an empty wire mesh cup is placed in the left chamber, and a wire mesh cup containing a strange mouse (mouse 1, of the same sex, group, and age) is placed in the right chamber. The time spent by the test mouse with the empty cup and in the left chamber (sniffing, climbing, and the time and number of times it enters the chamber) is recorded, as well as the time spent by the test mouse with the strange mouse and in the right chamber (sniffing, climbing, and the time and number of times it enters the chamber). The social preference index is calculated according to formula (1-1). In the third 5 minutes, a social novelty preference experiment is conducted: a new strange mouse (mouse 2, of the same sex, group, and age) is placed in the previous empty cup. The right chamber is set up the same as the second 5 minutes, and the interaction time between the test mouse and the two mice in the left and right chambers is also recorded. And calculate the social novelty preference index according to formula (1-2).

[0027] Social novelty preference index = (communication time with mouse 1 - communication time with the empty wire cup) / (communication time with mouse 1 + communication time with the empty wire cup) (1-1)

[0028] Social novelty preference index = (communication time with mouse 2 - communication time with mouse 1) / (communication time with mouse 2 + communication time with mouse 1) (1-2)

[0029] 2-2. Buried bead test

[0030] Each mouse was placed in a standard and familiar experimental cage with a sawdust bedding layer at a depth of 4 cm. Twenty glass marbles (15 mm diameter, arranged in 4 × 5) were lightly spread on the sawdust bedding and the mice were allowed to explore for 30 min. The number of marbles buried (two-thirds or more) was recorded.

[0031] Bead burying index = (number of beads buried by mice / 20) × 100%

[0032] 2-3. Open field test

[0033] After acclimation for at least 10 min, mice were introduced into a blue movement monitoring box (40 cm x 40 cm x 40 cm) to facilitate unrestricted movement for 5 min. After each test, the box was cleaned with 75% ethanol. The total distance moved was recorded.

[0034] 2-4 Novel Object Test

[0035] Day 1: Mice were placed individually in an empty square box (40 cm × 40 cm × 40 cm) for a 5-minute free exploration period. Day 2: Two identical objects were placed in the northeast and southwest corners of the box. The test mice were placed in the center of the box and allowed to explore freely for 10 minutes. Day 3: A familiar object and a novel object were placed in the box, maintaining the same relative positions as during training. Mice were allowed to explore them freely for 10 minutes. The behavioral software recorded the exploration time of the familiar and novel objects.

[0036] Discrimination index = (time spent exploring novel objects - time spent exploring familiar objects) / total exploration time.

[0037] 3. Results, see Figure 1 .

[0038] Depend on Figure 1 BE showed that although the data of mMIA-oCD (co-housing) group and mMIA+2′-FL-oCD (co-housing) group were behind those of mPBS-oCD group, and even behind those of mMIA+2′-FL-oCD group, they were significantly improved compared with mMIA-oCD group. Figure 1F shows that the number of buried beads in the mMIA-oCD (co-housing) group and the mMIA+2′-FL-oCD (co-housing) group was significantly less than that in the mMIA-oCD group. This means that co-housing can improve the social, cognitive impairment and repetitive behavior associated with autism in offspring induced by Th17 / Treg immune imbalance.

[0039] Th17 / Treg immune balance experiment (such as Figure 2 )

[0040] 1. Grouping of mice

[0041] like Figure 1 As shown in A, pregnant mice (C57BL / 6J female mice) were divided into three groups. At 12.5 days of pregnancy, pregnant mice in the experimental group were injected with poly(I:C) (maternal immune activation group, hereinafter referred to as mMIA group), pregnant mice in the control group were injected with PBS (hereinafter referred to as mPBS group), and pregnant mice in the treatment group were injected with poly(I:C) and supplemented with an effective dose of 2′-fucosyllactose in the diet (hereinafter referred to as mMIA+2′-FL group). The offspring of the above three groups were fed with normal breast milk after birth, weaned after 3 weeks, and the offspring of the mPBS group were changed to the mPBS-oCD group (blank control group), the offspring of the mMIA group were changed to the mMIA-oCD group (blank experimental group), and the offspring of the mMIA+2′-FL group were changed to the mMIA+2′-FL-oCD group, and the offspring of the mMIA-oCD and mMIA+2′-FL-oCD groups were treated in the same cage. The mice were divided into five groups: mPBS-oCD group; mMIA-oCD group; mMIA-oCD (co-housing) group; mMIA+2′-FL-oCD (co-housing) group; mMIA+2′-FL-oCD group. The diets of the mPBS-o2′-FL group and the mMIA-o2′-FL group were supplemented with 800 mg / kg bw of 2′-fucosyllactose (2′-FL). After 5 weeks of feeding, the mice were euthanized and subjected to physical and chemical experiments.

[0042] 2. Nucleic acid extraction, quantification, reverse transcription and qRT-PCR

[0043] (1) RNA extraction

[0044] Take out the mouse tissue sample from the -80°C refrigerator, quickly weigh about 0.1 g of tissue and put it into an RNase-free centrifuge tube containing 1 mL of Trizol reagent, and extract RNA according to the instructions for extracting RNA.

[0045] (2) Nucleic acid quantification

[0046] The concentration of mRNA in the extracted sample was determined using a nucleic acid quantifier, and the sample was diluted to the same concentration using enzyme-free water. The purity of the nucleic acid in the sample was evaluated based on the OD 260 / 280nm value and the OD 260 / 230nm value to determine whether the sample was contaminated by protein, sugar or organic solvent.

[0047] (3) Reverse transcription

[0048] Reverse transcription was performed according to the instructions of the reverse transcription kit (PrimeScript RT Master Mix), and the samples were reverse transcribed into cDNA and stored in a -80°C refrigerator for future use.

[0049] (4) qRT-PCR

[0050] According to the SYBR kit configuration system, the ABIQuant Studio5 system was used to detect the CT value of the sample gene, with β-acting as the internal reference and 2- △△CT The relative gene expression was calculated by the primers shown in the following table.

[0051] The primers are shown in the table below.

[0052]

[0053] 3. Serum physiological indicators (IL-17A, IL-10)

[0054] The mice were sampled and serum was collected. The samples were stored in a -80°C refrigerator. The blood samples were equilibrated at room temperature and centrifuged at 3000 r / min for 15 min to obtain serum. The expression levels of IL-17A and IL-10 in serum were detected using ELISA kits according to the manufacturer's instructions.

[0055] 4. Results, see Figure 2 .

[0056] from Figure 2 It can be seen that compared with the mMIA-oCD group, the mRNA expression levels of TGR5, VDR, and FOXP3 in the mMIA-oCD (co-housing) group and the mMIA+2′-FL-oCD (co-housing) group were upregulated and close to those in the mPBS-oCD group, while the mRNA expression levels of FXR, IL-1β, IL-6, TNF-α, RORγT, and STAT3 were downregulated and close to those in the mPBS-oCD group; the expression level of serum IL-10 was upregulated and close to that in the mPBS-oCD group, while the expression level of serum IL-17A was downregulated and close to that in the mPBS-oCD group.

[0057] The occurrence of ASD (Autism Spectrum Disorder) is often accompanied by inflammation and the involvement of various immune cells. Specifically, T cells, including specific subtypes such as Th17 and Treg cells, are believed to play a key role in the pathogenesis of ASD and are regulated by gut microbiota-derived metabolites, including bile acid metabolites. Therefore, we evaluated the expression levels of characteristic transcription factors and typical cell markers in Th17 and Treg cells.

[0058] It can be inferred from the experimental data that dietary supplementation with an effective dose of 2′-fucosyllactose can regulate the ratio of Th17 and Treg cells in offspring with Th17 / Treg immune imbalance induced by maternal immune activation; that is, it can treat Th17 / Treg immune imbalance induced by maternal immune activation. In addition, it was unexpectedly found that co-cage treatment can also allow offspring with Th17 / Treg immune imbalance induced by maternal immune activation to maintain Th17 / Treg immune balance through the influence of 2′-fucosyllactose on the intestinal flora.

[0059] The above embodiments and descriptions are only for illustrating the principles and best embodiments of the present invention. Without departing from the spirit and scope of the present invention, the present invention may be subject to various changes and improvements, all of which fall within the scope of the present invention to be protected.

Claims

1. Application of 2′-fucosyllactose in the preparation of immune balancing medicines or foods, characterized in that: The immune balance includes regulating the ratio of Th17 and Treg cells in offspring with Th17 / Treg immune imbalance induced by maternal immune activation or viral infection.

2. The use of 2′-fucosyllactose according to claim 1 in the preparation of immune balancing medicines or foods, characterized in that: The regulation of the ratio of Th17 and Treg cells includes up-regulating the mRNA expression levels of TGR5 and VDR in the brain, and down-regulating the mRNA expression level of FXR in the brain.

3. The use of 2′-fucosyllactose according to claim 1 in the preparation of immune balancing medicines or foods, characterized in that: The regulation of the ratio of Th17 and Treg cells also includes upregulating the mRNA expression level of FOXP3 in the brain and the expression level of serum IL-10.

4. The use of 2′-fucosyllactose according to claim 1 in the preparation of immune balancing medicines or foods, characterized in that: The regulating the ratio of Th17 and Treg cells also includes downregulating the expression level of serum IL-17A.

5. The use of 2′-fucosyllactose according to claim 1 in the preparation of immune balancing medicines or foods, characterized in that: The regulation of the ratio of Th17 and Treg cells also includes downregulating the mRNA expression levels of IL-1β, IL-6, TNF-α, RORγT and STAT3 in the brain.

6. The use of 2′-fucosyllactose according to claim 1 in the preparation of immune balancing medicines or foods, characterized in that: The Th17 / Treg immune imbalance also includes: social and cognitive disorders and repetitive behaviors related to autism in offspring.

7. The use of 2′-fucosyllactose according to claim 1 in the preparation of immune balancing medicines or foods, characterized in that: The medicine refers to any tablet, capsule, oral tincture, pill, granule, powder or granule containing an effective dose of 2′-fucosyllactose.

8. The use of 2′-fucosyllactose according to claim 1 in the preparation of immune balancing medicines or foods, characterized in that: The food refers to any milk and dairy products, fats / oils and emulsified fat products, frozen drinks, fruits / vegetables, beans / edible fungi / algae / nuts and seeds, cocoa products / chocolate and chocolate products and candy, grains and grain products, baked goods, meat and meat products, aquatic products and their products, eggs and egg products, sweeteners, condiments, special nutritional foods, beverages, alcohol, jellies, tea, coffee, collagen casings, yeast products, fried foods and puffed foods that contain an effective dose of 2′-fucosyllactose.

9. The use of 2′-fucosyllactose according to claim 1 in the preparation of immune balancing medicines or foods, characterized in that: The medicine or food is administered to a mammalian mother to produce offspring after immune response activation.

10. The use of 2′-fucosyllactose according to claim 9 in the preparation of immune balancing medicines or foods, characterized in that: The administration methods include nasogastric tube, jejunal tube, oral, sublingual, anal plug or rectal feeding.

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