Application of inulin with different polymerization degrees in prevention and treatment of intestinal injury

Abstract

The invention discloses application of inulin with different polymerization degrees in prevention and treatment of intestinal injury, and belongs to the technical field of biological prevention and treatment, and in the application, the inulin with different polymerization degrees comprises inulin with a low polymerization degree and inulin with a high polymerization degree. The low-polymerization-degree inulin is a short chain of 2-9, and the high-polymerization-degree inulin is a long chain of 10 or more. According to the application, the inulin with different polymerization degrees is found to be capable of reducing small intestine inflammation infiltration and intestinal barrier damage caused by radiation and promoting small intestine epithelial cell proliferation and anti-inflammatory factor secretion by verifying the effect of the inulin with different polymerization degrees on prevention and treatment of acute radiation intestinal injury of mice; and the inulin with different polymerization degrees is high in safety when being used for preventing and treating the symptoms, and has no obvious adverse reaction and toxic and side effects. Particularly, compared with the inulin with high polymerization degree, the inulin with low polymerization degree has a better effect of preventing and treating acute radiation intestinal injury.

Description

Technical Field

[0001] The present invention relates to the technical field of biological control, and particularly to the application of inulins with different degrees of polymerization in preventing and treating intestinal injuries. Background Art

[0002] Radiation intestinal injury (RII) is an inevitable complication caused by radiotherapy for abdominal and pelvic tumors, and the severity of the injury depends on the sensitivity to radiation and the dose. According to pathological staging, characteristics and clinical manifestations, radiation intestinal injury can be divided into acute and chronic radiation intestinal injury, generally with 3 to 6 months as the boundary. Acute radiation intestinal injury is mainly manifested as intestinal mucosal inflammatory reaction, while the pathological manifestation of chronic radiation intestinal injury is progressive intestinal wall ischemia and fibrosis. The clinical manifestations of radiation intestinal injury almost cover all possible digestive tract symptoms and signs. The main clinical manifestations of acute radiation intestinal injury are diarrhea, hematochezia, abdominal pain, etc.; the main clinical manifestations of chronic radiation intestinal injury are intestinal obstruction, intestinal fistula, diarrhea, digestive tract bleeding, anemia and malnutrition. The treatment of acute radiation intestinal injury mainly focuses on symptomatic treatment and nutritional support treatment. The first-choice treatment drugs for diarrhea in acute radiation intestinal injury are loperamide or compound diphenoxylate. The treatment of chronic radiation intestinal injury includes medical treatment, nutritional support treatment and surgical treatment, etc.

[0003] In summary, radiation intestinal injury seriously affects the quality of life of radiotherapy patients, and there is currently no good prevention and treatment drug. In acute radiation intestinal injury, the destruction of the intestinal barrier is one of the earliest pathological changes, and enhancing the intestinal barrier function is beneficial to the prevention and treatment of acute radiation intestinal injury. The intestinal barrier includes a biochemical barrier, a physical barrier and an immune barrier. The biochemical barrier is composed of mucus secreted by intestinal epithelial cells, digestive juices, antibacterial substances secreted by normal bacteria, mucosal flora and intestinal lumen flora. The physical barrier is also called the mechanical barrier, and its physiological structure basis is the mucosal epithelium, lamina propria and muscularis mucosa. The connections between cells are composed of tight junctions, adherens junctions and desmosomes, effectively blocking the entry of bacteria, viruses and endotoxins. The immune barrier is composed of gut-associated lymphoid tissue and diffuse immune cells, and has functions such as resisting the invasion of pathogenic microorganisms, anti-allergic reactions and inhibiting immune responses.

[0004] Inulin belongs to a group of indigestible carbohydrates called fructans, and chicory root is considered the richest source of inulin. Inulin has an appearance similar to wheat flour and is in the form of a white powder. Inulin is classified into short-chain (low-degree polymerization inulin) with a degree of polymerization of 2-9 and long-chain (high-degree polymerization inulin) with a degree of polymerization above 10. The degree of polymerization affects aspects such as the solubility, water-holding capacity, texture characteristics, and food processing stability of inulin. As recorded in patent publication number CN106617081A, research shows that long-chain inulin in the diet can relieve the symptoms of patients with necrotizing colitis, and a diet of low-fermentation oligosaccharides, disaccharides, and polyols can effectively reduce the functional gastrointestinal symptoms of patients with irritable bowel syndrome. This indicates that inulin has a certain regulatory effect on inflammation while providing nutrition.

[0005] Therefore, it is of great significance to explore the preventive and therapeutic effects of inulins with different degrees of polymerization on radiation-induced intestinal injury. Summary of the Invention

[0006] For this purpose, the present application provides the use of inulins with different degrees of polymerization in the prevention and treatment of acute radiation-induced intestinal injury, mainly aiming at the problem that the treatment plan for acute radiation-induced intestinal injury is not comprehensive enough or there is no suitable treatment method for some of the pathogenic causes, and at the same time to study new medical uses of inulin.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] According to the first aspect of the present invention, the present invention provides the use of inulins with different degrees of polymerization in the prevention and treatment of intestinal injury.

[0009] Among them, the intestinal injury is acute radiation-induced intestinal injury.

[0010] Among them, the acute radiation-induced intestinal injury is inflammatory infiltration and intestinal barrier disruption.

[0011] Among them, the inulins with different degrees of polymerization include low-degree polymerization inulin and high-degree polymerization inulin; the low-degree polymerization inulin is a short chain with a degree of polymerization of 2-9, and the high-degree polymerization inulin is a long chain with a degree of polymerization above 10.

[0012] According to the second aspect of the present invention, there is provided the use of low-degree polymerization inulin in the preparation of a drug for the prevention and treatment of acute radiation-induced intestinal injury.

[0013] Among them, the use of low-degree polymerization inulin in the preparation of a drug for reducing small intestine inflammatory infiltration and intestinal barrier disruption.

[0014] Among them, the low-degree polymerization inulin is a short chain with a degree of polymerization of 2-9.

[0015] Among them, the drug includes a pharmaceutically acceptable excipient; the pharmaceutically acceptable excipient refers to any diluent, adjuvant, and / or carrier that can be used in the pharmaceutical field.

[0016] The inulins with different degrees of polymerization of the present invention can be used in combination with other active ingredients as long as they do not produce other adverse effects, such as allergic reactions.

[0017] Among them, the drug is formulated into several dosage forms, including oral preparations, injectable preparations, and topical preparations, which contain excipients in the pharmaceutical field. For example, oral preparations (such as tablets, capsules, solutions or suspensions); injectable preparations (such as injectable solutions or suspensions, or injectable dry powders that can be used immediately after adding water for injection before injection); topical preparations (such as ointments or solutions).

[0018] In one embodiment of the present invention, the carriers for the pharmaceutical compositions of the present invention are common types available in the pharmaceutical field, including: binders, lubricants, disintegrants, solubilizers, diluents, stabilizers, suspending agents, colorless agents, flavoring agents, etc. for oral preparations; preservatives, solubilizing agents, stabilizers, etc. for injectable preparations; matrices, diluents, lubricants, preservatives, etc. for topical preparations. The pharmaceutical preparations can be administered orally or parenterally (such as intravenously, subcutaneously, intraperitoneally or topically). If some drugs are unstable under gastric conditions, they can be formulated into enteric-coated tablets.

[0019] According to the third aspect of the present invention, there is provided the use of inulin with a low degree of polymerization in the preparation of a drug for promoting the proliferation of small intestinal epithelial cells and the secretion of anti-inflammatory factors.

[0020] The present invention has the following advantages:

[0021] The present application discloses the use of inulins with different degrees of polymerization in the prevention and treatment of intestinal injuries. Using C57 mice as experimental subjects, the effects of inulins with different degrees of polymerization on acute radiation-induced intestinal injuries were studied. It was found that inulins with different degrees of polymerization can reduce small intestinal inflammatory infiltration and intestinal barrier damage, and promote the proliferation of small intestinal epithelial cells and the secretion of anti-inflammatory factors. Moreover, when inulins with different degrees of polymerization are used for the prevention and treatment of the above symptoms, they are highly safe, and no obvious adverse reactions or toxic and side effects are observed. In particular, compared with inulin with a high degree of polymerization, inulin with a low degree of polymerization has a better effect on the prevention and treatment of acute radiation-induced intestinal injuries. This study also overcomes some biases regarding the uses of inulin and explores its new pharmaceutical uses. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only exemplary, and for those of ordinary skill in the art, without creative efforts, other implementation drawings can be obtained based on the provided drawings.

[0023] The structures, ratios, sizes, etc. shown in this specification are only used to match the content disclosed in the specification for those familiar with this technology to understand and read, and are not used to limit the implementation conditions of the present invention. Therefore, they do not have substantial technical significance. Any modification of the structure, change in the ratio relationship, or adjustment of the size, without affecting the effects that the present invention can produce and the purposes that can be achieved, should still fall within the scope covered by the technical content disclosed in the present invention.

[0024] Figure 1 It is an experimental flow chart.

[0025] Figure 2 It is a result graph of different degrees of polymerization of inulin alleviating the reduction of mouse body weight caused by irradiation. Among them, Figure 2 A is the curve of the percentage change in the body weight of mice in the control group and the group sacrificed 3.5 days after irradiation after 17 Gy abdominal irradiation after 2 weeks of intervention with different degrees of polymerization of inulin; Figure 2 B is the curve of the percentage change in the body weight of mice in the group sacrificed 7 days after irradiation after 17 Gy abdominal irradiation after 2 weeks of intervention with different degrees of polymerization of inulin; Figure 2 C is a bar graph of the percentage change in body weight on the day of sacrifice of the control group, the group sacrificed 3.5 days after irradiation, and the group sacrificed 7 days after irradiation. Among them, Inulin-O and Inulin-M are both short-chain inulins, and Inulin-H is a long-chain inulin. *: P < 0.05, **: P < 0.01, ***: P < 0.005, ****: P < 0.001.

[0026] Figure 3 It is the effect of different degrees of polymerization of inulin intervention on the blood picture indexes of mice; among them, Figure 3 A is the statistical result of the white blood cell count in the blood routine of mice in different groups, Figure 3 B is the statistical result of the neutrophil count in the blood routine of mice in different groups. *: P < 0.05, **: P < 0.01.

[0027] Figure 4 It is a result graph of different degrees of polymerization of inulin improving the intestinal pathological damage caused by radiation; Figure 4 A is the H&E staining image of the ileum tissue of mice in different groups, Figure 4 B is the statistical result graph of the villus length of the ileum tissue of mice in different groups, Figure 4 C is the statistical result graph of the crypt depth of the ileum tissue of mice in different groups. *: P < 0.05, **: P < 0.01, ***: P < 0.005, ****: p < 0.001.

[0028] Figure 5 It is a result graph of different degrees of polymerization of inulin improving the reduction of the expression level of the intestinal tight junction protein ZO-1 caused by irradiation.

[0029] Figure 6 Results showing the improvement of the decrease in the expression level of the tight junction protein Occludin in the small intestine caused by irradiation by inulins with different degrees of polymerization.

[0030] Figure 7 WB detection results showing the effects of inulins with different degrees of polymerization on the expression of tight junction proteins ZO-1 and Occludin in the ileum tissues of mice after irradiation.

[0031] Figure 8 Results showing the improvement of the inhibition of intestinal cell proliferation in mice caused by irradiation by inulins with different degrees of polymerization.

[0032] Figure 9-10 Results showing the alleviation of DNA damage repair in small intestinal epithelial cells caused by irradiation by fecal metabolites of C57 mice fed with inulins with different degrees of polymerization; Figure 9 A. Immunofluorescence images showing the effects of mouse intestinal flora metabolites from inulins with different degrees of polymerization on the formation of γH2AX foci in mouse small intestinal epithelial cell line MODE-K at 6 h after 4 Gy irradiation. Figure 9 B. Statistical results of the number of γH2AX foci in each cell of different groups. Figure 10 A. Immunofluorescence images showing the effects of mouse intestinal flora metabolites from inulins with different degrees of polymerization on the formation of γH2AX foci in mouse small intestinal epithelial cell line MODE-K at 12 h after 4 Gy irradiation. Figure 10 B. Statistical results of the number of γH2AX foci in each cell of different groups. ***: P < 0.005, ****: P < 0.001.

[0033] Figure 11-13 Results showing the improvement of small intestinal epithelial vitality by metabolites of C57 mice fed with inulins with low degrees of polymerization; Figure 11 A. Visible light microscope images showing the effects of mouse intestinal flora metabolites from inulins with different degrees of polymerization on the growth status of mouse small intestinal epithelial cell line MODE-K at 24 h after non-irradiation, 4 Gy irradiation, and 8 Gy irradiation. Figure 11 B. Detection of the effects of mouse intestinal flora metabolites from inulins with different degrees of polymerization on the cell viability of mouse small intestinal epithelial cell line MODE-K at 24 h after non-irradiation, 4 Gy irradiation, and 8 Gy irradiation using a CCK8 kit. Figure 12 A. Fluorescence images showing the effects of intestinal flora metabolites from inulins with different degrees of polymerization on the cell proliferation of mouse small intestinal epithelial cell line MODE-K under non-irradiated conditions or at 24 h after 4 Gy irradiation using an EdU assay, Figure 12 B. Statistical results of the percentage of EdU-positive cells in each group. Figure 13Figure A shows the statistical results of the effects of intestinal flora metabolites from inulin with different degrees of polymerization on the cell cycle of mouse small intestinal epithelial cell line MODE-K under non-irradiation conditions. Figure 13 Figure B shows the statistical results of the effects of intestinal flora metabolites from inulin with different degrees of polymerization on the cell cycle of mouse small intestinal epithelial cell line MODE-K at 48 h after 4 Gy irradiation. *: P<0.05, **: P<0.01, ***: P<0.005.

[0034] Figure 14 This shows the effects of intestinal flora metabolites from inulin with different degrees of polymerization on the expression levels of tight junction proteins ZO-1 and Occludin in mouse small intestinal epithelial cell line MODE-K under non-irradiation or 8 Gy irradiation conditions. Detailed implementation manners

[0035] The following specific embodiments illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0036] The present application provides the application of inulin with different degrees of polymerization in the prevention and treatment of acute radiation-induced intestinal injury, mainly aiming at the problem that the treatment plan for acute radiation-induced intestinal injury is not comprehensive enough or there is no suitable treatment method for some of its pathogenic causes. At the same time, it is also for studying new medical uses of inulin, such as Figure 1 shown in the roadmap of this study.

[0037] According to the first aspect of the present invention, the present invention provides the application of inulin with different degrees of polymerization in the prevention and treatment of intestinal injury.

[0038] Among them, the intestinal injury is acute radiation-induced intestinal injury.

[0039] Among them, the acute radiation-induced intestinal injury is inflammation infiltration and intestinal barrier disruption.

[0040] Among them, the inulin with different degrees of polymerization includes inulin with low degrees of polymerization and inulin with high degrees of polymerization; the inulin with low degrees of polymerization is short chains of 2-9, and the inulin with high degrees of polymerization is long chains of 10 or more.

[0041] According to the second aspect of the present invention, there is provided the application of inulin with low degrees of polymerization in the preparation of drugs for the prevention and treatment of acute radiation-induced intestinal injury.

[0042] Among them, the application of inulin with low degrees of polymerization in the preparation of drugs for reducing small intestinal inflammation infiltration and intestinal barrier disruption.

[0043] Among them, the inulin with low degree of polymerization is a short chain of 2-9.

[0044] Among them, the drug includes a pharmaceutically acceptable excipient; a pharmaceutically acceptable excipient refers to any diluent, adjuvant and / or carrier that can be used in the pharmaceutical field.

[0045] Among them, the drug is formulated into several dosage forms, including oral preparations, injectable preparations, and topical preparations, which contain excipients in the pharmaceutical field.

[0046] According to the third aspect of the present invention, there is provided the use of inulin with low degree of polymerization in the preparation of a drug for promoting the proliferation of small intestinal epithelial cells and the secretion of anti-inflammatory factors.

[0047] The present invention will be further described below in conjunction with the accompanying drawings and embodiments. The equipment, materials, reagents, etc. used in the present invention can be obtained through commercial channels unless otherwise specified.

[0048] 1. Drugs

[0049] The inulin with different degrees of polymerization in this application includes inulin with low degree of polymerization and inulin with high degree of polymerization. Among them, the inulin with low degree of polymerization is a short chain of 2-9, and the inulin with high degree of polymerization is a long chain of 10 or more. Specifically, in the embodiments of this application, the inulin with low degree of polymerization further adopts two degrees of polymerization. Among them, Inulin-O adopts short-chain inulin with a degree of polymerization of 2, Inulin-M adopts short-chain inulin with a degree of polymerization of 5, and Inulin-H adopts long-chain inulin with a degree of polymerization of 18.

[0050] 2. Experimental animals and feeding methods

[0051] (1) C57 mice, male, weighing 18g-22g, growing for 8-10 weeks. Divide them into 12 groups according to body weight, including non-irradiated (4 groups, 5 mice in each group), the group for sampling 3.5 days after irradiation (4 groups, 6 mice in each group), and the group for sampling 7 days after irradiation (4 groups, 6 mice in each group).

[0052] (2) Feed them with water, inulin with low degree of polymerization, and inulin with high degree of polymerization respectively. Dissolve the two kinds of inulin with low degree of polymerization in the drinking water of mice (2g dissolved in 200mL of autoclaved ddH2O), and change the drinking water every three days. Among them, the inulin with high degree of polymerization is insoluble in water and is administered by gavage (1g dissolved in 10mL of autoclaved ddH2O, gavaged 6 days a week), and each gavage is 300uL / mouse.

[0053] (3) After feeding the mice for 7 days, collect the feces of the mice fed with water, low and high degree of polymerization inulin every day respectively. Do not collect the feces of the mice after irradiation, and store them in a -40°C refrigerator.

[0054] (4) During the feeding process, the body weights of the mice in each group were recorded every 3 days, and the body weights of the mice were recorded daily after irradiation.

[0055] (5) After two weeks of feeding, irradiation (17 Gy abdominal irradiation) or non-irradiation was performed. The irradiated groups were sampled at 3.5 days or 7 days respectively, and the non-irradiated group and the group sampled at 3.5 days after irradiation were sampled together. Irradiation conditions: Using a 137Cs radiation source with a dose rate of 0.8215 Gy / min.

[0056] 3. Experimental methods

[0057] 3.1 Establishment of a mouse model of acute radiation-induced intestinal injury

[0058] (1) Anesthetize with sodium pentobarbital;

[0059] (2) Using a 137Cs radiation source with a dose rate of 0.8215 Gy / min, perform 17 Gy irradiation;

[0060] (3) Euthanize the mice at 3.5 days or 7 days after modeling and conduct various evaluations.

[0061] 3.2 Tissue fixation, paraffin embedding, and sectioning

[0062] (1) Fix the samples with 4% paraformaldehyde (PFA);

[0063] (2) Wash away PFA: After fixation, rinse with running water 3 times, 5 minutes each time;

[0064] (3) Gradient dehydration with alcohol: 50%-70%-80%-95%-95% alcohol for 2 hours each; 100% ethanol for 40 minutes, 2 times;

[0065] (4) Clearing: 50% ethanol + 50% xylene for 5 - 30 minutes. Observe the tissue morphology in time during the clearing process to adjust the clearing time. Observe whether the tissue is successfully cleared under light. Appropriate clearing is sufficient;

[0066] (5) Wax infiltration: At 60°C, first soft wax then hard wax, 2 times each, 50 minutes each time;

[0067] (6) Embedding: Quickly place the tissue into the mold containing wax liquid, with the required tissue section parallel to the bottom to prevent the wax liquid from solidifying in the cold environment;

[0068] (7) Sectioning: The thickness of each section is 4 μm.

[0069] 3.3 H&E staining

[0070] (1) Bake the slides at 60°C for 2 - 4 hours;

[0071] (2) Dewaxing: Xylene Ⅰ for 15 min -- Xylene Ⅱ for 15 min -- 100% ethanol for 5 min -- 95% ethanol for 5 min -- 85% ethanol for 5 min -- 75% ethanol for 5 min -- Wash with ddH2O to remove alcohol;

[0072] (3) Hematoxylin staining for 35 s, wash with tap water;

[0073] (4) Eosin staining for 5 min, wash with tap water;

[0074] (5) Dehydration: 75% alcohol for 2 s - 85% alcohol for 2 s - 95% alcohol for 2 s - 100% alcohol for 2 s - 100% alcohol for 2 s - Xylene Ⅰ for 3 min - Xylene Ⅱ for 3 min;

[0075] (6) Mount with neutral balsam.

[0076] 3.4 Immunohistochemical staining method

[0077] (1) Place the sections in an oven and bake at 60 °C for 2 - 4 h;

[0078] (2) Dewax with xylene and dehydrate with gradient alcohol;

[0079] Xylene Ⅰ for 30 min -- Xylene Ⅱ for 30 min -- 100% ethanol for 10 min -- 100% ethanol for 10 min -- 95% ethanol for 5 min -- 85% ethanol for 5 min -- 75% ethanol for 5 min -- Wash with ddH2O;

[0080] (3) Antigen retrieval: Add 400 mL of antigen retrieval solution (prepared by diluting 4 mL of 0.01 M sodium citrate buffer with 396 mL of ddH2O at a ratio of 1:100) to a plastic box containing tissue sections, and repair with a microwave oven for 20 min (after boiling), switch between high, medium, and low heat after boiling, do not keep boiling violently;

[0081] (4) Place the plastic box with tissue sections in cold water and air dry naturally for 1 h, wash with PBS 3 times, 5 min each time;

[0082] (5) Block endogenous peroxidase: Drain the tissue sections, circle the tissue with a histochemical pen, drop peroxidase to cover the circled area (about 200 μL per section), place in a wet box (add water to the wet box to prevent dry sections); Incubate at 37 °C for 15 min, wash with PBS 3 times, 5 min each time;

[0083] (6) Block with 5% BSA (prepared with PBS) for 40 min;

[0084] (7) Prepare the primary antibody, drain after blocking, drop the primary antibody, and incubate overnight at 4 °C;

[0085] Take out the tissue sections after overnight incubation at 4℃, let them stand at room temperature for 30 min, and wash them 3 times with PBS for 5 min each time.

[0086] (9) Incubate with the enhancement solution: for 20 min at room temperature, wash 3 times with PBS for 5 min each time, flick off the liquid, and place on a wet box.

[0087] (10) Incubate with the secondary antibody: Incubate with the enhanced enzyme-labeled goat anti-mouse / rabbit IgG polymer for 20 min at room temperature, and wash 3 times with PBS for 5 min each time.

[0088] (11) Stain with DAB for 2 min 30 s and observe under the microscope.

[0089] (12) Stain with hematoxylin for 1 min 30 s, observe under the microscope, and counterstain for 30 s.

[0090] (13) Dehydrate with alcohol: 75% ethanol for 3 min - 85% ethanol for 3 min - 95% ethanol for 3 min - 100% ethanol for 3 min - xylene for 3 min.

[0091] (14) Mount the sections with neutral resin glue.

[0092] 3.5 Immunofluorescence

[0093] 3.5.1 Tissue immunofluorescence

[0094] (1) Place the tissue sections in an oven and bake at 60℃ for 2 - 4 h.

[0095] (2) Deparaffinize with xylene and dehydrate with gradient alcohol.

[0096] Xylene for 30 min - xylene (fresh) for 30 min - 100% ethanol for 10 min - 100% ethanol for 10 min - 95% ethanol for 5 min - 85% ethanol for 5 min - 75% ethanol for 5 min - wash with ddH2O.

[0097] (3) Antigen retrieval: Add 400 mL of the antigen retrieval solution (prepared by mixing 4 mL of 0.01 M sodium citrate buffer with 396 mL of ddH2O at a ratio of 1:100) to the plastic box containing the tissue sections, and repair with a microwave for 20 min (after boiling), switch between high, medium, and low heat after boiling, and do not keep boiling violently.

[0098] (4) Place the plastic box containing the tissue sections in cold water, air-dry naturally for 1 h, and wash 3 times with PBS for 5 min each time.

[0099] (5) Block with 5% bovine serum albumin (BSA) (prepared with PBS) for 40 min.

[0100] (6) Prepare the primary antibody, drain the blocking solution after blocking, add the primary antibody, and incubate overnight at 4°C;

[0101] (7) Take out the tissue sections after overnight incubation at 4°C, incubate at room temperature for 30 min, and wash with PBS 3 times, 5 min each time;

[0102] (8) Incubate with the secondary antibody (40 min - 1 h), and wash with PBS three times, 5 min each time;

[0103] (9) Mount with DAPI.

[0104] 3.5.2 Cellular Immunofluorescence

[0105] (1) Take out the cells from the incubator, and wash with PBS 3 times, 5 min each time;

[0106] (2) Fixation: Fix in 4% paraformaldehyde for 10 min, and wash with PBS 3 times, 5 min each time;

[0107] (3) Permeabilization: Add 0.3% Triton and incubate at room temperature for 10 min, and wash with PBS 3 times, 5 min each time;

[0108] (4) Blocking: Block with 1% bovine serum albumin (BSA) for 2 h;

[0109] (5) Incubate with the primary antibody: Incubate overnight at 4°C, and wash with PBS 3 times, 5 min each time;

[0110] (6) Incubate with the secondary antibody: Incubate for 40 min - 1 h, and wash with PBS 3 times, 5 min each time;

[0111] (7) Mount with DAPI.

[0112] 3.6 Immunoblotting (Western blot)

[0113] (1) Harvest the protein and store it frozen at -80°C;

[0114] (2) Measure the concentration;

[0115] (3) Electrophoresis:

[0116] ① Experimental preparation: Prepare the electrophoresis buffer and take out the marker;

[0117] ② Heat the extracted protein: 55°C for 5 min;

[0118] ③ Loading: Pull out the comb, add from left to right, add 4 μL of the marker and the corresponding volume of the protein sample;

[0119] ④ Electrophoresis: About 1 h at 70 V, and adjust the voltage to 110 V after the electrophoresis passes through the stacking gel.

[0120] (4) Transfer membrane:

[0121] ① Experimental preparation: Ice, methanol, transfer membrane buffer, sponge filter paper soaked with TBST in advance, and membrane soaked with methanol;

[0122] ② Preparation of transfer membrane buffer: 750 mL of water, 150 mL of methanol, 100 mL of transfer membrane buffer;

[0123] ③ Take out the gel: Pry open the glass plate with a scraper, scrape off the upper layer, take out the gel, which can be washed with TBST, and directly place the gel on the black surface by translation;

[0124] ④ Place the membrane: After the gel is placed, take out the membrane, wash it in TBST, then take it out and place it on the gel, and use a scraping blade to add water to scrape out all the air bubbles;

[0125] ⑤ Place it in ice water: After the gel and the membrane are fixed, put them into the electrophoresis tank, place the electrophoresis tank on ice water, and fill it with wet transfer solution;

[0126] ⑥ Electrophoresis: 300 mA for 2 h.

[0127] (5) Blocking:

[0128] ① Prepare 1% BSA: Use an electronic scale, zero it with a weighing paper before use, and dissolve it with TBST;

[0129] ② Cut the membrane: After taking out the membrane, place it in TBST solution for cutting. The side next to the gel is the front side, with the front side facing up, and cut a small notch in the upper left corner to distinguish the sample loading order;

[0130] ③ Shake: At room temperature for 2 h.

[0131] (6) Incubate with primary antibody overnight at 4°C, wash three times with TBST, 5 min each time;

[0132] (7) Incubate with secondary antibody for 2 h, wash three times with TBST, 5 min each time;

[0133] (8) Exposure.

[0134] 3.7 Flow cytometry - Cell cycle detection

[0135] (1) Harvest cells: Digest with 500 μL of trypsin, terminate digestion with 500 μL of 10% FBS, transfer to a 1.5 mL EP tube, centrifuge at 2500 rpm for 5 min at 4°C;

[0136] (2) Discard the supernatant, resuspend with 1 mL of pre-cooled PBS;

[0137] (3) Centrifuge at 2500 rpm for 5 min at 4°C, discard PBS, leave 50 μL of PBS, resuspend the cells by flicking to avoid cell clumping;

[0138] (4) Fix the cells, add 1 mL of pre-cooled 75% ethanol, gently blow and mix evenly, and fix at 4°C for 2 h;

[0139] (5) PI staining:

[0140] Take out the cells, centrifuge at 3000 rpm at 4°C for 10 min, discard the ethanol, leave 50 μL of 75% ethanol to resuspend the cells, add 1 mL of pre-cooled PBS to resuspend the cells. Centrifuge at 4000 rpm at 4°C for 4 min, discard the upper layer of PBS, and leave 50 μL of PBS to resuspend the cells;

[0141] Preparation of 1 mL of PI staining solution:

[0142] 10* sodium citrate PI staining solution (red, light-proof) RNAseA ddH2O

[0143] 100 μL 50 μL 2 μL 850 μL

[0144] (6) Add 500 μL of PI staining solution to each tube and slowly resuspend the precipitate;

[0145] (7) Incubate in a water bath at 37°C in the dark for 10 min;

[0146] (8) Run on the flow cytometer;

[0147] (10) Analyze data with FLOWJO.

[0148] 3.8 Cell viability detection

[0149] 3.8.1 CCK-8

[0150] 3.8.2 EDU (EDU kit)

[0151] 4. Result analysis

[0152] 4.1 Body weight

[0153] The recorded results of body weight after irradiation showed that on the 3.5th day after irradiation, there was no significant difference in the change of body weight among the groups sampled at 3.5 days after irradiation. On the 7th day after irradiation, the degree of decrease in the body weight of the mice fed with low- and high-degree-of-polymerization inulin slowed down ( Figure 2 ).

[0154] 4.2 Blood item indexes

[0155] The statistical results of the blood routine of the mice showed that among the groups sampled at 3.5 days after irradiation, the numbers of white blood cells and neutrophils in the mice fed with low-degree-of-polymerization inulin were significantly higher than those in the control group. In the group sampled at 7 days after irradiation, the numbers of white blood cells and neutrophils in the mice fed with low-degree-of-polymerization inulin were significantly lower than those in the control group ( Figure 3 ).

[0156] 4.3 Pathological sections

[0157] The results of H&E staining showed that Figure 4 ), the length of small intestinal villi was statistically analyzed. The results showed that among the non-irradiated groups, the lengths of small intestinal villi in mice fed with low- and high-degree of polymerization inulin were significantly higher than those in the control group. Among the groups sampled 3.5 days after irradiation, the small intestinal villi in mice fed with low- and high-degree of polymerization inulin were significantly higher than those in the control group, and the small intestinal villi in mice fed with low-degree of polymerization inulin were longer. Among the groups sampled 7 days after irradiation, the length of small intestinal villi in mice fed with low-degree of polymerization inulin was significantly higher than that in the control group, and the length of small intestinal villi in mice fed with low-degree of polymerization inulin was longer. The depth of small intestinal crypts was statistically analyzed. The results showed that among the non-irradiated groups, the depths of small intestinal crypts in mice fed with low- and high-degree of polymerization inulin were significantly higher than those in the control group. Among the groups sampled 3.5 days after irradiation, the crypt depths in mice fed with low- and high-degree of polymerization inulin were significantly higher than those in the control group, and the crypt depth in mice fed with low-degree of polymerization inulin was deeper. Among the groups sampled 7 days after irradiation, the depth of small intestinal crypts in mice fed with low-degree of polymerization inulin was significantly higher than that in the control group, and the depth of small intestinal crypts in mice fed with low-degree of polymerization inulin was deeper.

[0158] 4.4 Tissue immunofluorescence

[0159] The detection results of the expression of ZO-1 and Occludin in small intestinal tissues showed that in the non-irradiated groups, there was no significant difference in the protein expression levels of ZO-1 and Occludin among the groups. In the group sampled 3.5 days after irradiation, the protein expression level of ZO-1 in the small intestine of mice fed with low-degree of polymerization inulin was significantly higher than that in the control group, and there was no significant change in the protein expression level of ZO-1 in the small intestine of mice fed with high-degree of polymerization inulin compared with the control group. In the group sampled 7 days after irradiation, the protein expression levels of ZO-1 and Occludin in the small intestine of mice fed with low-degree of polymerization inulin were significantly higher than those in the control group, and there was no significant change in the protein expression levels of ZO-1 and Occludin in the small intestine of mice fed with high-degree of polymerization inulin compared with the control group Figure 5 , Figure 6 ).

[0160] 4.5 WB detection of the expression of intestinal barrier tight junction proteins

[0161] The WB detection results of the tight junction proteins ZO-1 and Occludin in small intestinal tissues showed that Figure 7) In the group sampled 3.5 days after irradiation, ZO-1 in the small intestine tissue of the simple irradiation group was significantly decreased, while the expression of ZO-1 in the groups treated with inulin of different polymerization degrees combined with irradiation was basically not affected by irradiation; at 3.5 d after irradiation, Occludin in all irradiation groups was hardly detectable. In the group sampled 7 days after irradiation, the expression of ZO-1 protein in the two groups treated with low-polymerization-degree inulin combined with irradiation was significantly higher than that in the simple irradiation group, while there was no significant difference in the expression of ZO-1 protein between the group treated with high-polymerization-degree inulin combined with irradiation and the simple irradiation group; the detection results of Occludin protein showed that the two low-polymerization-degree inulins could alleviate the decrease in the level of Occludin protein caused by irradiation, while the high-polymerization-degree inulin did not have this ability.

[0162] 4.6 Proliferation of small intestinal stem cells

[0163] The results of immunohistochemistry (Ki67) showed that there was no significant difference in the proliferation of small intestinal stem cells among the non-irradiated groups; 3.5 days after irradiation, the proliferation effect of small intestinal stem cells in the mice fed with low-polymerization-degree inulin was better than that in the simple irradiation group. 7 days after irradiation, the proliferation effect of small intestinal stem cells in the mice fed with low-polymerization-degree inulin was significantly better than that in the simple irradiation group ( Figure 8 ).

[0164] The following are all cell experiments

[0165] Mouse small intestinal epithelial cells (MOED-K) were treated with the metabolites of mice (dissolved in PBS), and were divided into five groups, namely the PBS group, the metabolites of mice drinking water only (MET-C), the metabolites of mice fed with the first low-polymerization-degree inulin (MET-O), the metabolites of mice fed with the second low-polymerization-degree inulin (MET-M), and the metabolites of mice fed with high-polymerization-degree inulin (MET-H).

[0166] 4.7 DNA damage repair of small intestinal epithelial cells (MODE-K)

[0167] Cell immunofluorescence was used to fluorescently label the DNA damage sites, and high-content analysis was used to count the DNA damage sites. The results showed that 6 h after cell irradiation, the number of DNA damage sites in the MET-O group and the MET-M group was significantly less than that in the MET-C group. The number of DNA damage sites in the MET-H group was significantly more than that in the MET-C group ( Figure 9 ). 12 h after cell irradiation, the number of DNA damage sites in the MET-O group and the MET-M group was significantly less than that in the MET-C group. The number of DNA damage sites in the MET-H group was significantly more than that in the MET-C group ( Figure 10 ). The above results prove that the metabolites of the two low-polymerization-degree inulins have a significant effect of reducing the DNA damage of mouse small intestinal epithelial cells MODE-K caused by irradiation.

[0168] 4.8 Proliferation effect of small intestinal epithelial cells (MODE-K)

[0169] The results of CCK-8 showed that after irradiation with 4 Gy or 8 Gy, the cell viability of the MET-O group was significantly higher than that of the MET-C group; the cell viability of the MET-M group was lower than that of the MET-C group; there was no significant difference in the cell viability between the MET-H group and the MET-C group ( Figure 11 ). The results of EDU showed that after irradiation with 4 Gy, the proliferation effect of the MET-O group was significantly higher than that of the MET-C group; there was no significant difference in the proliferation effects between the MET-M group, the MET-H group and the MET-C group ( Figure 12 ). The results of cell cycle flow cytometry showed that after irradiation with 8 Gy for 48 h in MODE-K cells, the G2 / M phase arrest in the MET-O group was significantly lower than that in the MET-C group; there was no significant difference in the G2 / M phase arrest effects between the MET-M group, the MET-H group and the MET-C group ( Figure 13 ). The above results showed that the small intestinal epithelial proliferation effect of the MET-O group was better.

[0170] 4.9 Expression of tight junction proteins in small intestinal epithelial cells

[0171] The results of Western blotting showed that when MODE-K cells were not irradiated, the expression level of ZO-1 in the MET-O group was significantly higher than that in the MET-C group; there was no significant change in the expression levels of ZO-1 in the MET-M group and the MET-H group compared with the MET-C group; there was no significant difference in the Occludin expression among the non-irradiated groups. After irradiation of MODE-K cells, the protein expression levels of ZO-1 and Occludin in the MET-O group and the MET-M group were significantly higher than those in the MET-C group; there was no significant difference in the protein expression levels of ZO-1 and Occludin in the MET-H group compared with the MET-C group ( Figure 14 ).

[0172] Based on the results of animal experiments and cell experiments, we found that inulin could promote the expression of genes related to intestinal barrier proteins, and intestinal barrier proteins included at least one of tight junction proteins, claudin, mucin, and cadherin, promote the proliferation of intestinal epithelial cells, promote the DNA damage repair of intestinal epithelial cells, and reduce the level of intestinal inflammatory infiltration. At the same time, it was found that compared with high-degree-polymerization inulin, low-degree-polymerization inulin had a better effect on preventing and treating acute radiation-induced intestinal injury. At the same time, it was known from the experimental data that the first low-degree-polymerization inulin (Inulin-O) also had a better effect than the second low-degree-polymerization inulin (Inulin-M).

[0173] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made thereto based on the present invention, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of the present invention claimed.

Claims

1. Application of inulins with different degrees of polymerization in preventing and treating intestinal injury.

2. The application according to claim 1, characterized in that The intestinal injury is acute radiation-induced intestinal injury.

3. The application according to claim 2, characterized in that, The acute radiation-induced intestinal injury is characterized by inflammatory infiltration and intestinal barrier disruption.

4. The application according to claim 1, characterized in that, The inulins with different degrees of polymerization include inulins with low degrees of polymerization and inulins with high degrees of polymerization; Among them, the inulins with low degrees of polymerization are short chains of 2-9, and the inulins with high degrees of polymerization are long chains of more than 10.

5. Application of inulins with low degrees of polymerization in the preparation of drugs for preventing and treating acute radiation-induced intestinal injury.

6. The application according to claim 5, characterized in that, Application of inulins with low degrees of polymerization in the preparation of drugs for reducing small intestine inflammatory infiltration and intestinal barrier disruption.

7. The application according to claim 5, characterized in that The inulins with low degrees of polymerization are short chains of 2-9.

8. The application according to claim 5, characterized in that, The drug includes a pharmaceutically acceptable excipient; the pharmaceutically acceptable excipient refers to any diluent, adjuvant, and / or carrier that can be used in the pharmaceutical field.

9. The application according to claim 5, characterized in that The drug is formulated into several dosage forms, including oral preparations, injectable preparations, and topical preparations, which contain excipients in the pharmaceutical field.

10. Application of inulins with low degrees of polymerization in the preparation of drugs for promoting small intestinal epithelial cell proliferation and anti-inflammatory factor secretion.

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