Application of vitamin D and derivative thereof in preparation of product for preventing and / or treating intestinal aging

Abstract

The invention provides an application of vitamin D and a derivative thereof in preparation of a product for preventing and / or treating intestinal aging, which proves that the vitamin D has the effect of preventing and / or treating intestinal aging, including reducing the expression of a cyclically dependent kinase inhibitor 1; the permeability of the intestinal tract is improved; the intestinal villus height is increased; the colon crypt depth is increased; the integrity of the intestinal tissue structure is improved; the expression of tight junction protein in intestinal tracts is improved; partial immunity related to T cells is activated; and the like.

Description

Technical Field

[0001] The present invention belongs to the field of food science and technology, and specifically relates to the application of vitamin D and its derivatives in the preparation of products for preventing and / or treating intestinal aging. Background Art

[0002] Aging is a degenerative change that occurs in the body during aging. Along with the aging process, multiple tissues and organ functions in the body undergo degenerative changes. Nervous system decline, cardiovascular aging, intestinal dysfunction and imbalance of microbiota homeostasis are common manifestations in the elderly.

[0003] Many existing theories believe that aging begins in the intestine. Due to its immune and nutrient intake functions, the intestine is considered an important organ for regulating the body and prolonging life. Aging can lead to an imbalance in intestinal homeostasis, which is manifested in decreased digestion and absorption function of the small intestine, damaged intestinal mucosal barrier (for example, decreased number of intestinal villi, shortened length, and decreased number of crypts), weakened intestinal motility, insufficient secretion of digestive enzymes, increased levels of inflammation, tissue degenerative changes, decreased function of intestinal stem cell regeneration and differentiation, and intestinal flora disorders. Therefore, it is of great significance to restore intestinal homeostasis, delay the aging process, and improve the quality of life of the elderly.

[0004] Intestinal aging involves many of the above aspects. There is currently no truly effective drug for prevention and / or treatment, and long-term use of drugs will have other side effects. Summary of the invention

[0005] The present invention verifies the application of vitamin D in the preparation of products for preventing and / or treating intestinal aging, can provide a new strategy for the prevention of clinical diseases, and has good application prospects in the prevention and / or treatment of intestinal aging.

[0006] According to a first aspect of the present invention, there is provided a use of vitamin D in the preparation of a product for preventing and / or treating intestinal aging.

[0007] Vitamin D is a micronutrient, and the main sources of vitamin D in the human body are synthesised in the skin and ingested through the diet. However, vitamin D2 and vitamin D3 ingested from the skin or diet are biologically inactive and need to be converted into 1,25-dihydroxyvitamin D3 with biological functions after two cycles in the liver and kidneys. There are many factors that affect the level of vitamin D in the human body, including ultraviolet radiation, dietary supplements, age and racial factors. At present, it is known that the main function of vitamin D is to regulate the absorption of calcium and phosphorus and promote muscle growth and bone development. More and more studies have found that in addition to being related to bone diseases, vitamin D also has a certain correlation with other tissues and (or) system diseases, including muscle-related diseases, cardiovascular diseases, autoimmune diseases and tumors, etc.

[0008] The present invention verifies for the first time that vitamin D can effectively improve the problem of intestinal aging. Specifically, after relevant model experimental verification and data detection and analysis, vitamin D can reduce the expression of cyclin-dependent kinase inhibitor 1; improve intestinal permeability; increase the height of small intestinal villi; increase the depth of colonic crypts; improve the integrity of intestinal tissue structure; increase the expression of tight junction proteins in the intestine; activate some immunity related to T cells, etc. That is, vitamin D can alleviate intestinal aging at the cellular and genetic levels, improve the health of the intestine, and promote the health of the human intestine and other aspects.

[0009] As mentioned above, vitamin D is an essential nutrient for the human body. It has many functions such as promoting calcium and phosphorus absorption, promoting new bone formation, promoting skin cell growth, and regulating immune function. It is a nutrient that most people, especially the elderly, need to supplement on a daily basis through nutritional supplements or health care products. Taking vitamin D as a daily nutritional supplement or health care product to prevent and / or treat intestinal aging will not bring additional economic burden, psychological burden or other side effects and adverse effects to the human body. It has a wide range of use and safety, and the cost is relatively low, so it is worthy of vigorous promotion.

[0010] Preferably, vitamin D and its derivatives include one or more of 1α,25-dihydroxyvitamin D2, 1α-hydroxyvitamin D2, 1α,24-dihydroxyvitamin D2, 1α,24,25-trihydroxyvitamin D2, 1α,25-dihydroxyvitamin D3, 1α-dihydroxyvitamin D3, and 24-hydroxyvitamin D2.

[0011] Preferably, vitamin D and its derivatives are 1α,25-dihydroxyvitamin D3.

[0012] Vitamin D is a family of molecules with the same A, B, C, and D ring structures but different side chains. The structures of the A, B, C, and D rings are derived from the cyclopentahydrophenanthrene ring structure of steroids. Vitamin D has multiple forms such as D2, D3, D4, D5, D6, and D7 depending on the side chain structure. Only two active forms, D2 (ergocalciferol) and D3 (cholecalciferol), really play a role in nutrition.

[0013] Preferably, the product is a medicine, a health product or a food.

[0014] Preferably, the product is a health care product, and the health care product is at least one of tablets, granules, capsules, powders, oil drops, and solutions.

[0015] Preferably, the health care product is used orally.

[0016] Preferably, vitamin D and its derivatives are 1α, 25-dihydroxyvitamin D3, and the dosage of 1α, 25-dihydroxyvitamin D3 as an active ingredient is 0.1-100 μg / / day.

[0017] Preferably, the dosage of 1α,25-dihydroxyvitamin D3 as an active ingredient is 2-15 μg / day.

[0018] Preferably, the prevention and / or treatment of intestinal aging includes at least one of the following:

[0019] (1) Reduce the expression of cyclin-dependent kinase inhibitor 1;

[0020] (2) Improve intestinal permeability;

[0021] (3) Increase the height of the small intestinal villi;

[0022] (4) increase the depth of colonic crypts;

[0023] (5) Improve the integrity of intestinal tissue structure;

[0024] (6) Increase the expression of tight junction proteins in the intestine;

[0025] (7) Activate T cell-related partial immunity.

[0026] Preferably, the partial immunity associated with activation of T cells includes at least one of the following:

[0027] (1) Activate gene expression in Th1 and Th2 differentiation pathways;

[0028] (2) Activate the expression of T cell receptor-related pathways.

[0029] Among them, the gene expression in the activation of Th1 and Th2 differentiation pathways includes one or more of the following genes: IL-12, STAT1, STAT4, T-Bet, CD3E, CD3G, CD3D, ZAP70, LAT, IL2R; the expression of the activation of T cell receptor-related pathways includes one or more of the following genes: CD8A, CD8B, GADS, LPC2.

[0030] On the other hand, the present application also provides a food, medicine or health product for preventing and / or treating intestinal aging, including vitamin D or its derivatives and a food or medicine acceptable carrier.

[0031] Compared with the prior art, the present invention discloses a new use of vitamin D, namely, the use of vitamin D in the preparation of medicines, health products or foods for preventing and / or improving intestinal aging, which has the following advantages:

[0032] (1) The present invention has discovered new medicinal value for known vitamin D, which has good efficacy in preventing and / or improving intestinal aging, has no toxic side effects, and has opened up a new field for the application of vitamin D;

[0033] (2) The series of experimental studies of the present invention have proved that vitamin D has preventive and ameliorative effects on gene or cell structure changes related to intestinal aging, including reducing the expression of cyclin-dependent kinase inhibitor 1; improving intestinal permeability; increasing the height of small intestinal villi; increasing the depth of colonic crypts; improving the integrity of intestinal tissue structure; increasing the expression of tight junction proteins in the intestine; activating partial immunity related to T cells, etc.

[0034] (3) The vitamin D of the present invention has small toxic and side effects, good safety, high acceptance compared to other drugs or health products, can be taken for a long time, and has good application prospects;

[0035] (4) The vitamin D of the present invention can be prepared into various dosage forms, with a small dosage, convenient use, and easy promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1A and Figure 1B The effects of vitamin D on the gene expression levels of P21 in the small intestine and P21 in the colon of D-gal model intestinal aging mice are shown respectively.

[0037] Figure 2 The effect of vitamin D on intestinal permeability in D-gal model intestinal aging mice is shown.

[0038] Figure 3 The effect of vitamin D on the height of small intestinal villi in D-gal model intestinal aging mice was shown.

[0039] Figure 4 The effect of vitamin D on colonic crypt depth in D-gal model intestinal aging mice is shown.

[0040] Figure 5 The effect of vitamin D on the integrity of intestinal tissue structure in D-gal model intestinal aging mice was shown.

[0041] Fig. 6A and 6B The effect of vitamin D on the expression of intestinal tight junction proteins in D-gal model intestinal aging mice is shown.

[0042] Figure 7 The effect of vitamin D on the expression of genes in the Th1 and Th2 differentiation pathways of D-gal model intestinal aging mice is shown.

[0043] Figure 8The effect of vitamin D on the gene expression of T cell receptor-related pathways in D-gal model intestinal aging mice is shown. DETAILED DESCRIPTION

[0044] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only embodiments of a part of the present invention, rather than all embodiments.

[0045] Aging models can be divided into two categories: natural aging models and accelerated aging models. Natural aging models are time-consuming, labor-intensive, and expensive, so accelerated aging models are favored because of their ease of application, short research time, and high animal survival rate during the experiment. There are various types of accelerated aging models, including in vitro studies induced by basal vein treatment and D-galactose, as well as in vivo studies induced by radiation, jet delay, senescence-accelerated mice, Klotho mice, thymus removal, and D-galactose. Among these aging models, the D-galactose-induced aging model is the most popular because of its convenience, minimal side effects, and high survival rate throughout the experiment.

[0046] D-galactose is a reducing aldohexose. When taken in high doses, it can be converted into aldose and hydrogen peroxide under the catalysis of galactose oxidase, thereby generating ROS. The increase of ROS may lead to oxidative stress, inflammation, mitochondrial dysfunction and cell apoptosis. In vitro studies have confirmed that different types of senescent cell models induced by D-galactose can produce oxidative stress and inflammatory responses.

[0047] 1. Animal experiment design

[0048] 8-week-old male SPF C57BL / 6 mice, weighing 23±1g / mouse, were purchased from Vitallife. The breeding conditions were room temperature 22±2℃, light and dark cycle 12h each, and they were adapted for one week after purchase.

[0049] Experimental drug preparation:

[0050] aD-galactose: D-galactose powder (Sigma, A3134) was accurately weighed and the model was established at a dose of 500 mg / kg / day according to the weight of the mouse, and dissolved in distilled water. The intraperitoneal injection volume for each mouse was 100 μL.

[0051] Animal Grouping:

[0052] Eight-week-old male SPF C57BL / 6 mice were randomly divided into three groups, with 8 mice in each group, namely the VD Suff+PBS group (fed with vitamin D-sufficient feed and intraperitoneal injection of PBS every day), the VD Suff+D-Gal group (fed with vitamin D-sufficient feed and intraperitoneal injection of D-galactose every day), and the VD Def+D-Gal group (fed with vitamin D-deficient feed and intraperitoneal injection of D-galactose every day). The normal control group was intraperitoneally injected and gavaged with normal saline every day, and the other groups were intraperitoneally injected with D-galactose every day for 4 months. During the experiment, the mice in each group had free access to food and water, and the mice were weighed regularly every week.

[0053] Test materials:

[0054] After the last oral administration to mice, relevant mouse behavioral experiments were immediately carried out the next day. After the test, the colon, ileum, duodenum, etc. of the mice were obtained and their lengths were measured. Part of the tissue was preserved in tissue fixative, and the other part was quickly frozen and placed in a -80°C refrigerator for use.

[0055] 2. Evaluation of Pathological Indicators

[0056] 1) Preliminary preparation

[0057] Collection: Use a sharp instrument to remove intestinal tissue from the animal.

[0058] Fixation: The removed intestinal tissue was immediately immersed in cold PBS, and then the colon was divided into several sections by length using dissecting scissors and marked. The sections used for pathological staining were fixed with 4% paraformaldehyde (PFA) or 10% formalin solution for 24 hours.

[0059] Flushing: Use PBS to flush out the intestinal contents and keep the intestine moist.

[0060] 2) H&E staining of colon tissue

[0061] 2.1 Preparation of intestinal paraffin sections

[0062] Dehydration and wax immersion: Dehydrate the tissue using gradient ethanol (70%, 80%, 90%, 100%), then use xylene for transparent treatment, immerse the dehydrated tissue block in melted paraffin for 30 minutes, and repeat 3 times.

[0063] Tissue embedding: first inject melted paraffin into the mold, carefully remove the tissue with heated tweezers, and quickly place it into the mold with the cross section facing upwards. Wait for the paraffin to solidify slightly and cover the base of the embedding box. Continue to add wax until the base of the embedding box is submerged, and leave it to cool at room temperature to solidify.

[0064] Sectioning and spreading: Take out the embedded wax block from the mold, place it on the biological tissue freezing table, and fix it on the paraffin slicer after the wax block cools down. Adjust the slice thickness and trim the slice first. When complete muscle tissue appears, start continuous sectioning. The thickness of each slice is 5 μm. Remove the slice with tweezers, spread it flat on the water surface of the spreader, and use curved tweezers to spread the folds. The spreading temperature is 42°C.

[0065] Fishing and baking: When the slices are fully unfolded, quickly pick them up with a glass slide, absorb excess moisture with filter paper, mark them with numbers, and place them on a baking machine. Bake at 42°C for 2 hours. After baking, place the slices in a 60-70°C oven overnight to prevent them from falling off.

[0066] At this point the sections can be dewaxed and rehydrated.

[0067]

[0068] 2.2 Pathological H&E staining

[0069] ① After hydration, place the slices in hematoxylin stain for 10 minutes to stain the cell nuclei. Rinse with tap water for 5 minutes;

[0070] ②Differentiate with 1% hydrochloric acid and ethanol for 15 seconds, and rinse with tap water for 3 minutes;

[0071] ③ Then put it in eosin staining solution for 5 minutes to stain the cytoplasm;

[0072] ④ Soak in gradient ethanol (70% ethanol, 80% ethanol, 95% ethanol, anhydrous ethanol) for 5 minutes each to dehydrate the slices. Then soak the slices in xylene for 10 minutes to make them transparent;

[0073] ⑤Finally, seal the slide with neutral resin.

[0074] 3) Intestinal permeability test

[0075] After fasting for 6 hours, mice were gavaged with 100 mg / mL FITC-dextran solution. The final concentration was 500 mg (amount of FITC-dextran) / kg (mouse weight). The gavage volume was calculated based on the weight of mice after 6 hours of fasting. 100-120 μl of blood was collected from the eye sockets 4 hours after gavage. The blood was placed in a coagulant tube and kept away from light for 2 hours. After centrifugation at 4°C for 15 minutes, serum was collected. The mouse serum gavaged with the same dose of phosphate buffer was used as a negative control. The serum fluorescence value was determined by an enzyme-labeled instrument under the conditions of excitation wavelength / emission wavelength -485 nm / 528 nm, and a standard curve was drawn to quantify the fluorescence signal in each serum sample. The whole process should be done in a dark environment.

[0076] 4) Detection of small intestinal villus height and colonic crypt depth

[0077] Place the HE-stained sections under an optical microscope for observation. Generally, an eyepiece with a micrometer or a microscope system connected to image analysis software is used for measurement. For the height of the small intestinal villi, measure the vertical distance from the top of the villi to the base of the villi (the junction with the lamina propria); for the depth of the colonic crypt, measure the vertical distance from the crypt opening to the bottom of the crypt. In each sample, it is usually necessary to measure multiple villi and crypts (such as 10-20), and then calculate the average value to reduce errors.

[0078] 5) Detection of expression levels of aging markers, intestinal tight junction proteins, genes in Th1 and Th2 differentiation pathways, and genes in T cell receptor-related pathways

[0079] Intestinal tissue was collected and ground with Trizol solution to extract total RNA. After reverse transcription, cDNA was obtained. Then, β-actin was selected as the internal reference gene to analyze the cyclin-dependent kinase inhibitor 1 (P21) in the small intestine and colon; tight junction proteins (ZO-1, Occludin, Claudn-1); IL-12 (interleukin-12), STAT1 (signal transducer and activator of transcription 1), STAT4 (signal transducer and activator of transcription 4), T-Bet (T-Bet) in the Th1 and Th2 differentiation pathways. x gene family), CD3E (T cell receptor), CD3G (T cell receptor), CD3D (T cell receptor), ZAP70 (ζ chain associated kinase 70), LAT (T-cell activation junction protein), IL2R (interleukin 2 receptor); the expression of CD8A (type I transmembrane glycoprotein on the surface of T lymphocytes), CD8B (type I transmembrane glycoprotein on the surface of lymphocytes), GADS (downstream adapter protein), and LPC2 (Lipocalin-2) genes in the T cell receptor-related pathway.

[0080] Example 1: Effect of vitamin D on the gene expression levels of P21 in the small intestine and P21 in the colon of D-gal model intestinal aging mice.

[0081] Cyclin-dependent kinase inhibitor 1 (P21) is a negative regulator of the cell cycle. P21 inhibits the cylin-CDK complex, causing the cell cycle to arrest at the G1 phase. It is highly expressed in senescent cells. As shown in Figure 1, compared with the control group, after vitamin D intervention in D-gal modeled intestinal aging mice, the cell senescence marker gene P21 in the small intestine and colon was significantly decreased.

[0082] Example 2: Vitamin D improves intestinal permeability in D-gal model intestinal aging mice

[0083] Intestinal permeability can be evaluated by detecting the entry of fluorescently labeled macromolecular compounds into the blood after intestinal absorption. Figure 2As shown in the figure, compared with the control group, the intestinal permeability of the mice in the model group increased significantly, indicating that long-term continuous intake of D-galactose can cause changes in intestinal permeability. Compared with the model group, after vitamin D intervention, the intestinal permeability of mice decreased significantly, indicating that vitamin D supplementation can alleviate the changes in intestinal barrier permeability caused by D-galactose.

[0084] Example 3: Effect of vitamin D on colon tissue morphology of D-gal model intestinal aging mice

[0085] The height of the intestinal villi has an important impact on intestinal function. The height and shape of the villi directly affect its ability to absorb nutrients. The longer the villi, the larger the surface area, thus being able to absorb more nutrients. Figure 3 as well as Figure 5 The results of H&E staining of the intestines of each group of mice showed that the height of the intestinal villi of the D-gal aging group mice was significantly reduced compared with the normal control group mice, which is a clear feature of intestinal aging. The height of the intestinal villi of the colon of the vitamin D intervention group mice was significantly longer than that of the D-gal aging group mice.

[0086] like Figure 4 as well as Figure 5 The results of H&E staining of the intestines of each group of mice showed that the crypt depth of the colon of the D-gal aging group mice was significantly reduced and the arrangement was sparse and loose compared with the normal control group mice, which is an obvious feature of intestinal aging, which will lead to the decline of intestinal peristalsis. The crypt depth of the colon of the vitamin D intervention group mice was significantly longer than that of the D-gal aging group mice, and the arrangement was also more compact and neat.

[0087] like Figure 5 As shown, the colon tissue structure of the control group was normal, the intestinal glands in the lamina propria were arranged neatly and tightly, the glandular structure was clear and complete, and there was no deformation or atrophy; the colon gland structure of the model group was changed, the intestinal glands were loosely arranged, the glands were deformed and atrophied, and the gaps were widened; the glands of the colon of the mice in the vitamin D intervention group were arranged more neatly, and the destruction of the intestinal gland structure was alleviated to a certain extent.

[0088] Example 4: Effect of vitamin D on the expression of intestinal tight junction proteins in D-gal model intestinal aging mice

[0089] Tight junction proteins are composed of cytoplasmic attachment proteins, transmembrane proteins and cytoskeletal proteins. Tight junctions mainly exist in the junctional complexes between epithelial cells and endothelial cells, which bring adjacent cell membranes close together to form a physical barrier structure surrounding the cells. Among them, transmembrane proteins include the following three integral membrane proteins: junctional adhesion molecules (JAM), occludin, and claudin. It is currently believed that occludin and claudin play the main role in transmembrane proteins, especially claudin, which is the main protein that forms tight junctions.

[0090] ZO-1 protein can interact with other tight junction proteins (such as claudin and occludin) to form a complex that mediates tight junctions and signal transduction between cells. Its functions involve multiple aspects such as cell polarity differentiation, cell adhesion and cell movement.

[0091] Intestinal tight junction proteins are important molecules that form tight junctions between intestinal cells and play a key role in maintaining the integrity and permeability of the intestinal mucosal barrier. When the expression of intestinal tight junction proteins is abnormal, the integrity of the intestinal mucosal barrier is damaged and the permeability increases.

[0092] like Fig. 6A and 6B As shown in the figure, the expression levels of intestinal tight junction protein genes (ZO-1, Occludin, Claudn-1) in the D-gal aging group were significantly downregulated, while after vitamin D intervention, the expression levels of each tight junction protein decreased to a lesser extent.

[0093] Example 5: Effect of vitamin D on immunity of D-gal model intestinal aging mice

[0094] The use of D-galactose-induced aging model research can not only successfully simulate the aging process but also show relatively ideal pathological characteristics. These changes include oxidative stress, inflammation, apoptosis, mitochondrial dysfunction, and up-regulation of P53 and P21 gene expression, etc., which are characteristic manifestations consistent with natural aging. This experiment used D-galactose-induced aging mice as research subjects to study the effect of vitamin D on intestinal inflammation.

[0095] like Figure 7 and Figure 8 As shown, compared with the normal control group, the use of vitamin D can increase the expression of genes in the Th1 and Th2 differentiation pathways and T cell receptor-related pathways. Enhanced systemic inflammation in mice is associated with accelerated aging and increased risk of death, while anti-inflammatory intervention can rescue premature aging in mice.

[0096] In summary, it can be seen that the use of vitamin D can improve the expression of intestinal tight junction protein genes and immune-related genes, inhibit the expression of intestinal aging marker genes, and help delay intestinal structural aging.

[0097] The above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention is described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the technical solutions of the present invention can be modified or equivalently replaced, but these modifications or replacements are all within the protection scope of the present invention.

Claims

1. Application of vitamin D and its derivatives in the preparation of products for preventing and / or treating intestinal aging.

2. The use as claimed in claim 1, wherein vitamin D and its derivatives include one or more of 1α,25-dihydroxyvitamin D2, 1α-hydroxyvitamin D2, 1α,24-dihydroxyvitamin D2, 1α,24,25-trihydroxyvitamin D2, 1α,25-dihydroxyvitamin D3, 1α-dihydroxyvitamin D3, and 24-hydroxyvitamin D2.

3. The use according to claim 2, wherein vitamin D and its derivatives are 1α,25-dihydroxyvitamin D3.

4. The use according to claim 1, wherein the product is a medicine, a health product or a food.

5. The use according to claim 4, wherein the product is a health care product, and the health care product is at least one of tablets, granules, capsules, powders, oil drops, and solutions.

6. The use according to claim 5, wherein the health product is taken orally.

7. The use according to claim 5, wherein: Vitamin D and its derivatives are 1α, 25-dihydroxyvitamin D3, and the dosage of 1α, 25-dihydroxyvitamin D3 as an active ingredient is 0.1-100 μg / / d. Preferably, the dosage of 1α, 25-dihydroxyvitamin D3 as an active ingredient is 2-15 μg / d.

8. The use according to claim 1, wherein: The prevention and / or treatment of intestinal aging comprises at least one of the following: (1) Reduce the expression of cyclin-dependent kinase inhibitor 1; (2) Improve intestinal permeability; (3) Increase the height of the small intestinal villi; (4) increase the depth of colonic crypts; (5) Improve the integrity of intestinal tissue structure; (6) Increase the expression of tight junction proteins in the intestine; (7) Activate T cell-related partial immunity.

9. The use according to claim 1, wherein: The activated T cell-related partial immunity includes at least one of the following: (1) Activate gene expression in Th1 and Th2 differentiation pathways; (2) Activate the expression of T cell receptor-related pathways.

10. A food, medicine or health product for preventing and / or treating intestinal aging, characterized in that: It comprises vitamin D or its derivatives and a carrier acceptable to food or medicine.

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