Probiotic gel microspheres and application thereof in radiation damage protection

Abstract

According to the probiotic gel microspheres disclosed by the invention, probiotics are wrapped by the gel microspheres, so that the probiotics are protected from being absorbed or the influence of gastric acid and intestinal digestive juice is reduced, and the release of the probiotics in intestinal tracts is promoted. When the probiotics adopt functional coatings, after the probiotics gel microspheres reach the intestinal tract and release the probiotics, the coating layers on the surfaces of the probiotics can promote the probiotics to adhere to the intestinal mucosa, and the problem that traditional probiotics are difficult to colonize in the intestinal tract is solved. The probiotic gel microspheres disclosed by the invention have an obvious radiation injury protection effect after being orally taken, and can be used for relieving whole-body radiation injury or local radiation injury (such as radioactive intestinal injury) caused by whole-body radiation or local radiation (such as abdominal radiotherapy).

Description

Technical Field

[0001] The invention relates to the fields of food and biomedicine, and in particular to probiotic gel microspheres and applications thereof in protecting against radiation damage. Background Art

[0002] Ionizing radiation refers to the general term for radiation that has enough energy to ionize electrons in atoms or molecules, including α, β, γ, X-rays, neutron radiation, etc. It is currently mainly used in medical imaging diagnosis, radiotherapy, industrial non-destructive testing, and environmental sewage treatment. Ionizing radiation can directly cause DNA breakage, induce structural changes and loss of activity of biological macromolecules. Ionizing water molecules in the body and destroying the structure of mitochondria lead to a continuous increase in reactive oxygen species (ROS), producing a large number of oxygen free radicals, hydroxyl free radicals and superoxide anions, further causing protein and lipid peroxidation, and then causing cell apoptosis and necrosis, chronic inflammation and tissue damage. According to the radiation sensitivity of different parts, acute radiation syndrome is divided into bone marrow hematopoietic system syndrome, gastrointestinal syndrome and brain syndrome. Bone marrow hematopoietic system syndrome is the most common and is also the main cause of death after exposure to high-dose ionizing radiation. Ionizing radiation can lead to a decrease in the number of hematopoietic stem cells, inhibition or loss of proliferation and division, aging and destruction of hematopoietic stem cell niches, severe hematopoietic dysfunction, and lead to lymphocytopenia, thrombocytopenia and anemia.

[0003] Local radiation damage mainly comes from complications after radiotherapy for cancer and other diseases. Radiation enteritis often occurs as a complication of radiotherapy for abdominal and pelvic malignancies such as bladder cancer, cervical cancer, and rectal cancer. Clinical manifestations include gastrointestinal bleeding, bacterial infection, anorexia, diarrhea, nausea and vomiting, and endotoxemia. After irradiation, intestinal epithelial cell proliferation is inhibited and apoptosis occurs, the tight junctions of intestinal epithelial cells are destroyed, the intestinal villi fall off, the mucosal barrier is destroyed, and the intestinal flora is imbalanced, causing gastrointestinal inflammation.

[0004] At present, drugs used to prevent and treat ionizing radiation damage, such as the aminothiol compound amifostine, can scavenge free radicals, but they are rapidly metabolized in the body and have many side effects, which limits the scope of clinical application. Cytokines such as granulocyte colony stimulating factor (G-CSF) can induce the proliferation and differentiation of hematopoietic cells. Active substances in the body, such as melatonin, are used for radiation protection of various organs such as the hematopoietic system and small intestine through free radical scavenging and immune regulation, but they have defects such as dose dependence and short half-life. Most studies are in the early stages, and the dosage and timing of administration are not studied in depth. At present, the prevention and treatment of radiation damage in clinical practice is mainly symptomatic and supportive treatment such as anti-infection, nutritional support, mucosal protection, antidiarrhea, and intestinal flora regulation, and there is a lack of special radiation prevention and treatment drugs. Therefore, the development of effective, low-toxic and safe products for the prevention and treatment of ionizing radiation damage is an urgent problem to be solved.

[0005] Probiotics are a type of active microorganisms that are beneficial to the host by colonizing in the human body and changing the composition of the flora in a certain part of the host. They promote nutrient absorption and maintain intestinal health by regulating the host mucosal and systemic immune functions or by regulating the balance of intestinal flora, thus producing single microorganisms or mixed microorganisms with clear composition that are beneficial to health. Summary of the invention

[0006] The invention discloses a probiotic gel microsphere which has obvious protective effect against ionizing radiation damage after being taken orally.

[0007] The probiotic gel microspheres contain probiotics and gel microsphere materials.

[0008] The probiotics in the present invention are selected from a single type of probiotic microorganism, a mixed microorganism or a microbial community composed of multiple types of probiotics. The probiotics are selected from the original naked probiotics and coated probiotics, preferably coated probiotics. The coated probiotics contain probiotics and coating materials. The coating material is selected from sodium alginate, chitosan, tannic acid, polylysine, polydopamine, and whey protein, preferably sodium alginate, chitosan, and tannic acid. The preparation method of the coated probiotics is selected from the layer-by-layer self-assembly method and the coacervation method. The coating layer of the coated probiotics is selected from a single layer and multiple layers. The multiple layers are selected from the same coating material and different coating materials. When double-layer coating is selected for probiotic coating, the coating material is chitosan and tannic acid. Chitosan is a cationic polysaccharide after chitin is partially acetyl-removed, which can interact with compounds of opposite charge through electrostatic forces and has good biocompatibility and degradability. Tannic acid is a natural polyphenolic compound. As an emerging prebiotic, it can be used by intestinal bacteria to promote the proliferation of probiotics. Its catechol group can form chemical bonds with different substrates and has a certain degree of adhesion. It has been approved for use by the US FDA and has good biosafety. As prebiotics, polysaccharides and polyphenolic compounds can be selectively metabolized by intestinal probiotics and play a regulatory role on intestinal flora.

[0009] The probiotics in the present invention can be selected from various probiotics sold on the market and their combinations, and the probiotics in research. Probiotics can be purchased from the market, or obtained from a microbial collection that collects probiotic strains. Probiotic types are selected from Lactobacillus, Bifidobacterium, Enterococcus, Streptococcus, Bacillus, Clostridium, Saccharomyces, and Akkermansia. Lactobacillus is selected from Lactobacillus delbrueckii, Lactobacillus brevis, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus reuteri. Bifidobacterium is selected from Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, and Bifidobacterium breve. Enterococcus is selected from Enterococcus faecalis and Enterococcus faecium. Streptococcus is selected from thermophilic Streptococcus and lactic Streptococcus. Bacillus is selected from Bacillus subtilis, Bacillus cereus, Bacillus licheniformis and Bacillus coagulans. Clostridium is selected from Clostridium butyricum and Clostridium butyricum. Saccharomyces is Saccharomyces boulardii. Akkermansia is Akkermansia muciniphila (Akk). Specifically, probiotics are selected from Lactobacillus rhamnosus (LGG), Lactobacillus reuteri (LR), Akkermansia muciniphila, Clostridium butyricum and Bifidobacterium, preferably from Lactobacillus rhamnosus and Lactobacillus reuteri. Probiotics are common health supplements with good oral safety.

[0010] The gel microsphere material in the present invention is selected from sodium alginate, konjac glucomannan, and chitosan, and sodium alginate is preferred. Sodium alginate is an anionic polysaccharide composed of β-D-mannuronic acid and α-L-guluronic acid. Its aqueous solution has high viscosity and pH sensitivity and can react with Ca 2+ Ion exchange occurs under mild conditions to obtain water-insoluble calcium alginate.

[0011] The preparation method of probiotic gel microspheres is selected from microfluidics, photolithography, electrospraying, centrifugation and gas shearing. The shape, size and monodispersity of the gel microspheres can be controlled by adjusting the process parameters and the adopted prescriptions of these methods.

[0012] When naked probiotics are used, the probiotic gel microspheres are prepared using the following steps:

[0013] (1) Mixing the probiotics and the sodium alginate solution uniformly;

[0014] (2) The sodium alginate solution containing probiotics is sprayed into fine droplets under the action of an electrostatic field and dropped into a CaCl2 solution to obtain probiotic gel microspheres after surface solidification.

[0015] When coated probiotics are used, the probiotic gel microspheres are prepared by the following steps:

[0016] (1) mixing the probiotics with a chitosan solution to coat the probiotics with chitosan, then adding a tannic acid solution to electrostatically adsorb the chitosan to form a second coating, and adding sodium tripolyphosphate to cross-link the chitosan to promote the stable attachment of the double-layer coating to the surface of the strain;

[0017] (2) mixing the coated probiotics and the sodium alginate solution evenly;

[0018] (3) The sodium alginate solution containing the coated probiotics is sprayed into fine droplets under the action of an electrostatic field and dropped into a CaCl2 solution to solidify the surface to obtain probiotic gel microspheres.

[0019] The particle size of the probiotic gel microspheres is selected from 1 micron to 1000 microns, preferably from 10 microns to 500 microns, and further preferably from 100 microns to 300 microns.

[0020] After oral administration, naked probiotics need to experience the acidic environment in the stomach and the digestive fluid environment in the intestines. Most probiotics cannot withstand the harsh gastrointestinal environment and lose their activity. The small amount of surviving probiotics is difficult to colonize in the intestines, and their residence time in the intestines is short, making it difficult to exert their effects.

[0021] The probiotic gel microspheres of the present invention protect the probiotics from being absorbed or reduce the influence of gastric acid and intestinal digestive juice by encapsulating the probiotics in the gel microspheres, thereby promoting the release of the probiotics in the intestine. In particular, when the probiotics are coated with functional coatings, after the probiotic gel microspheres reach the intestines and release the probiotics, the coating layer on the surface of the probiotics can promote the probiotics to adhere to the intestinal mucosa, thus overcoming the problem that traditional probiotics are difficult to colonize in the intestines.

[0022] The probiotic gel microspheres of the present invention have an obvious radiation damage protection effect after oral administration, reducing the whole body radiation damage or local radiation damage (such as radiation intestinal damage) caused by whole body radiation or local radiation (such as abdominal radiotherapy). The dosage of the probiotic gel microspheres can be selected from 10 8 ~10 12 CFU / time, preferably from 109 to 10 11 CFU / time. The radiation damage protection mechanism of probiotics includes eliminating inflammation, eliminating ROS, and secreting short-chain fatty acids to protect the body.

[0023] The probiotic gel microspheres are used for protection against ionizing radiation damage, and the timing of administration is selected from before the whole body or abdomen is exposed to ionizing radiation, after the whole body or abdomen is exposed to ionizing radiation, or a combination of before and after the whole body or abdomen is exposed to ionizing radiation. Preferably, it is before the whole body or abdomen is exposed to ionizing radiation, and more preferably, it is a combination of before and after the whole body or abdomen is exposed to ionizing radiation.

[0024] When the probiotic gel microspheres are taken orally before the whole body or abdomen is exposed to ionizing radiation, the probiotic gel microspheres are taken orally 1 to 3 times a day before the whole body or abdomen is exposed to ionizing radiation. The time for taking the probiotic gel microspheres orally before the whole body or abdomen is exposed to ionizing radiation is selected from 2 to 14 days before the ionizing radiation until the day of the ionizing radiation, preferably 14 days before the ionizing radiation until the day of the ionizing radiation, further preferably 7 days before the ionizing radiation until the day of the ionizing radiation, and more preferably 2 days before the ionizing radiation until the day of the ionizing radiation.

[0025] When the whole body or abdomen is exposed to ionizing radiation, the probiotic gel microspheres are taken orally 1 to 3 times a day. The time for taking the probiotic gel microspheres orally after the whole body or abdomen is exposed to ionizing radiation is selected from the day of ionizing radiation until 3 to 14 days after ionizing radiation, preferably from the day of ionizing radiation until 3 days after ionizing radiation, more preferably from the day of ionizing radiation until 7 days after ionizing radiation, and more preferably from the day of ionizing radiation until 14 days after ionizing radiation.

[0026] When the oral administration of probiotic gel microspheres is selected before and after the whole body or abdomen is exposed to ionizing radiation, the oral administration of probiotic gel microspheres is 1 to 3 times a day. The time of oral administration of probiotic gel microspheres before and after the whole body or abdomen is exposed to ionizing radiation is selected from 2 to 14 days before the ionizing radiation to 2 to 14 days after the ionizing radiation, preferably from 14 days before the ionizing radiation to 14 days after the ionizing radiation, further preferably from 7 days before the ionizing radiation to 7 days after the ionizing radiation, and more preferably from 2 days before the ionizing radiation to 7 days after the ionizing radiation. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 . Transmission electron microscopy images of LGG, LGG@CS, and LGG@CT (A) and changes in Zeta potential (B).

[0028] Figure 2 LGG@CT@CA microspheres appearance (A), scanning electron microscopy (B), EcN gfp Microscopic morphology of @CT@CA microspheres (C).

[0029] Figure 3.Gastrointestinal tolerance of LGG@CT@CA microspheres.

[0030] Figure 4 .LGG@CT@CA particle size distribution.

[0031] Figure 5 .EcN gfp @CA and EcN gfp Confocal 3D scanning imaging of @CT@CA microspheres.

[0032] Figure 6 .EcN gfp , EcN gfp @CA and EcN gfp @CT@CA distribution and retention in the cecum and colon after oral administration (A) and EcN distribution in the colon gfp Quantification curve of strain number (B).

[0033] Figure 7 .Colonization of LGG strain delivered by LGG@CT@CA in the mouse intestine.

[0034] Figure 8 Oral administration of LGG@CT@CA prevents and treats weight changes (A) and survival rate (B) in a mouse model of radiation-induced intestinal injury.

[0035] Fig. 9 .H&E and Ki67 immunohistochemical staining results of small intestinal tissue in a mouse model of radiation-induced intestinal injury after oral administration of LGG@CT@CA.

[0036] Fig.10 .Oral administration of LGG@CT@CA improves the antioxidant capacity of small intestinal tissue in mice with radiation enteritis.

[0037] Fig.11 Oral administration of LGG@CT@CA increased the gene expression levels of IL-10 and GPX4 in the small intestine of mice, and decreased the gene expression level of SLC39A14. (A) IL-10 gene expression level; (B) SLC39A14 gene expression level; (C) GPX4 gene expression level.

[0038] Fig.12 .Effect of oral administration of LR@CT@CA on body weight in a mouse model of radiation-induced hematopoietic system damage.

[0039] Fig.13 Effects of oral administration of LR@CT@CA on peripheral blood WBC, RBC, and PLT in a mouse model of radiation-induced hematopoietic system damage. (A) WBC; (B) RBC; (C) PLT.

[0040] Fig.14.Oral administration of LR@CT@CA increases the number of bone marrow nucleated cells and Ki67-positive cells in the mouse model of radiation-induced hematopoietic system damage.

[0041] Fig.15 .Oral administration of LR@CT@CA improves the protective effect and Ki67-positive cell count in the spleen of a mouse model of radiation-induced hematopoietic system injury. DETAILED DESCRIPTION

[0042] Example 1. Probiotic gel microspheres

[0043] Chitosan was added to physiological saline containing 2% acetic acid to swell, and the pH was adjusted to 6 with NaOH solution to obtain a 2 mg / mL chitosan solution; Lactobacillus rhamnosus LGG was cultured anaerobically, and the bacteria in the logarithmic growth period were collected, washed with physiological saline and collected by centrifugation; LGG was dispersed in the above chitosan solution, stirred at room temperature for 30 minutes, centrifuged and washed with physiological saline to obtain LGG@CS; LGG@CS was dispersed in physiological saline, and 2 mg / mL tannic acid solution was added dropwise with stirring, and stirring was continued for 10 minutes after the addition was completed, and 5 mg / mL sodium tripolyphosphate solution was continued to be added dropwise, and stirring was continued for 10 minutes after the addition was completed; the product was collected by centrifugation The dispersion was then washed with physiological saline to obtain LGG@CT; sodium alginate was added into physiological saline to swell and dissolve to obtain a 3% sodium alginate solution; LGG@CT was re-dispersed with physiological saline, an equal volume of 3% sodium alginate solution was added, mixed, and the bubbles were removed; the dispersion was loaded into a syringe, the syringe was installed in an electrospinning instrument, and the liquid was pushed at a rate of 0.1 mL / h. Under an 8 kV electrostatic field, the dispersion sprayed out micron-sized spherical droplets, which entered into a 3 mg / mL calcium chloride solution, and the outer layer solidified to form gel microspheres; the gel microspheres were collected, passed through a 200-mesh sieve, and washed with physiological saline to obtain probiotic gel microspheres LGG@CA@CT.

[0044] Replace Lactobacillus rhamnosus LGG with probiotic Escherichia coli EcN expressing green fluorescent protein GFP gfp , Lactobacillus reuteri LR, prepared according to the above method, can respectively obtain bacteria-loaded gel microspheres EcN gfp @CT@CA and LR@CT@CA. The corresponding probiotic gel microspheres can be obtained by replacing Lactobacillus rhamnosus with other probiotics.

[0045] Experimental Example 1. Investigation of the properties of coated bacteria and bacteria-loaded gel microspheres

[0046] Materials: chitosan, tannic acid, sodium tripolyphosphate, sodium alginate, calcium chloride, pepsin, trypsin, LGG@CS, LGG@CT, LGG@CT@CA, EcN prepared according to Example 1 gfp @CT@CA.

[0047] Methods: The morphological changes of the strains before and after coating were detected. 10 μL of LGG, LGG@CS, and LGG@CT suspensions were dropped onto the copper mesh. After the liquid dried, they were detected using a transmission electron microscope (TEM). The Zeta potential of the samples was detected using a Malvern laser particle size analyzer.

[0048] After drying LGG@CT@CA at 4°C, its surface morphology was observed using a scanning electron microscope (SEM). In order to characterize the morphology of the strain in the microsphere, the fluorescent strain EcN gfp Building EcN gfp @CT@CA, the morphology of the microspheres was observed under a bright field microscope, and a 3D scan was performed using a fluorescence confocal microscope to observe the distribution of the strains in the microspheres.

[0049] To test the gastrointestinal tolerance and swelling level of the bacteria-loaded gel microspheres, appropriate amounts of microspheres were placed in artificial gastric juice (pH 1.2), artificial small intestinal juice (pH 6.8), and artificial colonic juice (pH 7.8), incubated at 37°C with shaking at 80 rpm, and 0.2 mL was taken from the above solutions every 0.5 h to measure the absorbance at 600 nm.

[0050] LGG@CT@CA microspheres were prepared in large quantities, and the particle size distribution of the collected microspheres was measured by a wet particle size analyzer.

[0051] Results: TEM images showed that the LGG strain was rod-shaped; under the action of electrostatic force, a layer of chitosan particles was adsorbed on the surface of LGG@CS; after adding tannic acid and sodium tripolyphosphate, tannic acid and chitosan were cross-linked, increasing the cross-linking and adhesion between strains ( Figure 1 A). Zeta potential diagram shows that the surface of LGG strain is negatively charged, with a Zeta potential of -10.25mV. After coating with cationic polysaccharide chitosan, the potential flips to +9.08mV. When negatively charged tannic acid is added, the Zeta potential flips again to -7.2mV ( Figure 1 B). LGG@CT@CA is milky white gel microspheres observed by naked eyes ( Figure 2 A) Observation of EcN under a microscope gfp @CT@CA microspheres have a smooth morphology and contain strong green fluorescence, indicating that EcN gfp Completely encapsulated in microspheres ( Figure 2 B, C). LGG@CT@CA has good tolerance to the gastrointestinal environment. The microspheres have no obvious morphological changes in artificial gastric juice within 0.5 hours, and no obvious morphological changes in artificial small intestinal juice within 1 hour. After 1 hour, they swell slightly and release some bacteria. In artificial colonic juice, they swell quickly within 1 hour and release the encapsulated bacteria ( Figure 3 The particle size of the microspheres measured by the wet particle size analyzer showed a normal distribution, with an average particle size of 219.2 μm ( Figure 4). For the microspheres EcN containing fluorescent bacteria gfp @CT@CA performs confocal 3D scanning, showing bacteria in EcN gfp @CA and EcN gfp @CA@CT is evenly distributed in the microspheres, and the coating modification makes the bacterial agglomerates evenly dispersed in the spheres, enhancing the adhesion and aggregation of bacteria in the microspheres ( Figure 5 ).

[0052] Experimental Example 2. Probiotic microsphere preparation prolongs the retention of probiotics in the intestine and enhances colonization effect

[0053] Materials: C57BL / 6J mice, male, SPF grade, weight 20±2g; EcN prepared according to Example 1 gfp @CT@CA and LGG@CT@CA, fecal total DNA extraction kit, real-time fluorescence quantitative PCR instrument, and confocal microscope.

[0054] Methods: C57BL / 6J male mice were randomly divided into 3 groups, 12 mice in each group, and divided into EcN gfp Group, EcN gfp @CT Group, EcN gfp @CT@CA group, mice in each group were gavaged with EcN gfp (10 8 CFU / 0.2mL / each), EcN gfp @CT(10 8 CFU / 0.2mL / each) and EcN gfp @CT@CA(10 8 CFU / 0.2mL / mouse). Three mice were randomly selected from each group at 1h, 4h, 24h and 72h after intragastric administration to collect the contents of the cecum and colon. Total DNA from the intestinal contents was extracted and EcN gfp The expression of the specific gene sfGFP was quantified by qPCR, and then the EcN in the intestinal contents was detected. gfp Quantitative.

[0055] The retention of oral probiotics in the mouse intestine was evaluated by fluorescence in situ hybridization (FISH). C57BL / 6J male mice were randomly divided into 3 groups, 4 mice in each group, namely LGG, LGG@CT, and LGG@CT@CA groups. All mice were fed with 2 mg / mL streptomycin for 3 days to keep the intestinal bacteria level consistent. After the feeding, each group of mice was given LGG (10 9 CFU / 0.2mL / each), LGG@CT(10 9 CFU / 0.2mL / each), LGG@CT@CA(10 9CFU / 0.2mL / mouse) and gavage for 3 consecutive days. One week after the last gavage, the abdomen of the mouse was gently massaged to promote the emptying of feces, and the mouse colon was collected, fixed, embedded, and sliced. The lra primer modified with the fluorescent probe FAM was used for in situ hybridization with LGG in the colon tissue section to observe the distribution and colonization of LGG in the mouse colon.

[0056] Result: EcN gfp The group had the shortest intestinal transit time and was enriched in the cecum and colon at 1h and 4h after oral administration. gfp The number is significantly higher than EcN gfp @CT and EcN gfp @CT@CA group. 24h after oral administration, EcN gfp Only a small number of strains were present in the cecum and colon, while EcN gfp The bacterial counts in the EcNgfb@CT and EcNgfb@CT@CA groups were significantly higher than those in the EcN gfp group, indicating that coating modification can improve bacterial adhesion to the intestine ( Figure 6 A, B). 72h after intragastric administration, EcN gfp There were almost no bacteria in the intestines of the EcN group. gfp Only a small amount of bacteria were detected in the CT group, while in the EcN group gfp In the @CT@CA group, a certain number of bacteria were still detected in the colon, and this number was close to the distribution in the colon at 24 hours. This shows that the combination of coating modification and microsphere encapsulation can prolong the retention of strains in the colon.

[0057] The results of in situ fluorescence hybridization showed that after three consecutive intragastric administrations of LGG solution, only a small amount of LGG was distributed in the outer mucus layer of the colon ( Figure 7 ). The distribution of strains in the LGG@CT group was close to the deep mucus layer of the colon. The strains in the LGG@CT@CA group were concentrated in the inner layer of the colon mucus, close to the intestinal epithelial barrier. This phenomenon is consistent with the characteristic of LGG tending to colonize close to the intestinal epithelial barrier, indicating that the combination of coating modification and microsphere encapsulation can promote bacterial colonization in the colon.

[0058] Experimental Example 3. Oral administration of LGG@CT@CA to prevent and treat radiation-induced intestinal injury in mice

[0059] Materials: C57BL / 6J mice, male, SPF grade, weight 23±2 g; LGG@CT and LGG@CT@CA prepared according to Example 1, 60 Co γ-radiation device, real-time fluorescence quantitative PCR instrument, inhibition and generation of superoxide anion free radical assay kit.

[0060] Methods: C57BL / 6J male mice were randomly divided into 5 groups, 10 mice in each group, namely Healthy, Model, Amifostine, LGG and LGG@CT@CA groups. Except for the Healthy group, all mice were anesthetized and fixed, and the abdomen was exposed to receive 13Gy 60 Coγ-ray irradiation. Model, LGG, and LGG@CT@CA groups were gavaged with normal saline (0.2 mL / mouse), LGG (5×10 8 CFU / 0.2mL / piece), LGG@CT@CA(5×10 8 CFU / 0.2mL / mouse), and the drug was administered for a total of 6 days. The Amifostine group was intraperitoneally injected with the drug (150mg / kg, 0.2mL / mouse) 0.5h before irradiation. The body weight of mice was monitored daily. On the 3.5th day after irradiation, 5 mice in each group were euthanized. A 1cm intestinal segment was taken from the small intestine 5cm below the stomach of the mice for fixation and pathological embedding, and the remaining small intestinal tissue was temporarily stored at -80℃. The remaining 5 mice in each group continued to be observed, and the death of mice was recorded until the end of the experiment on the 7th day after irradiation.

[0061] H&E staining and Ki67 immunohistochemical staining were performed on the mouse small intestinal tissue pathological sections. The mouse small intestinal tissue was taken, 10% tissue homogenate was prepared, and the supernatant was collected by centrifugation to detect the small intestinal tissue resistance to superoxide anion (O2 ·- ) ability. 10 mg of small intestinal tissue was taken, total RNA was extracted, and reverse transcribed into cDNA. The expression levels of IL-10, SLC39A14 and GPX4 genes in small intestinal tissue were detected by qPCR.

[0062] Results: Whole abdominal irradiation caused a decrease in mouse body weight ( Figure 8 A), Model and LGG groups had death on the 3rd day after irradiation, and all died on the 4th day after irradiation, while LGG@CT@CA group had one death on the 4th day and one death on the 6th day after irradiation ( Figure 8 B). Compared with the Model group, LGG@CT@CA prolonged the survival of mice.

[0063] Compared with the Model and LGG groups, LGG@CT@CA protected the crypt morphology of the small intestinal tissue, maintained a certain number of Ki67-positive cells, and protected the stemness and repair ability of the small intestinal tissue ( Fig. 9 LGG@CT@CA group small intestinal tissue O2 ·- It has a certain clearance ability (**P<0.01), and the effect is equivalent to that of the positive drug Amifostine group ( Fig.10 ). Compared with the model group, LGG@CT@CA significantly increased the gene expression level of anti-inflammatory factor IL-10 in small intestinal tissue (*P<0.05)( Fig.11 A), reduced the gene expression level of SLC39A14 in small intestinal tissue (****P<0.0001), increased the gene expression level of GPX4 (***P<0.001), and inhibited ferroptosis in small intestinal tissue ( Fig.11 B, C).

[0064] Experimental Example 4. Oral administration of LR@CT@CA prevents whole-body irradiation-induced hematopoietic system damage in mice

[0065] Materials: C57BL / 6J mice, male, SPF grade, weight 20±2g, 32 mice in total, LR@CT@CA prepared according to Example 1, 60 Coγ radiation device, blood cell analyzer.

[0066] Methods: C57BL / 6J male mice were randomly divided into 4 groups, 8 mice in each group, namely Healthy, Model, LR and LR@CT@CA groups. Except for the Healthy group, all mice received 6.5 Gy whole body 60 Coγ-ray irradiation. Model, LR, and LR@CT@CA groups were gavaged with normal saline (0.2 mL / mouse), LR (5×10 8 CFU / 0.2mL / piece), LR@CT@CA(5×10 8 CFU / 0.2mL / mouse), and the drug was administered for 9 days in total. The weight of mice was monitored daily, and 20 μL of peripheral blood was collected from the tail vein 2 days before irradiation and on the 4th, 7th, 10th, 13th, 18th, and 22nd days after irradiation to measure the changes of white blood cells (WBC), red blood cells (RBC), and platelets (PLT). On the 7th day after irradiation, 3 mice in each group were euthanized, and the spleen and femur of the mice were collected for fixation and embedding, and H&E staining and Ki67 immunohistochemical staining were performed.

[0067] Results: After whole-body irradiation, the body weight of mice showed a slow growth trend ( Fig.12 ). Compared with the Model group, LR@CT@CA promoted the recovery of RBC and PLT in peripheral blood ( Fig.13 ). The nucleated cells in the bone marrow tissue of the Model group were significantly reduced, and there were a large number of vacuoles, indicating that the bone marrow of the irradiated mice was severely damaged; compared with the Model group, LR@CT@CA significantly increased the number of nucleated cells in the bone marrow tissue, and there were fewer vacuoles. Ki67 is an antigen related to proliferating cells, which mainly marks cells in the proliferation cycle and indirectly reflects the proliferation ability of nucleated cells in the bone marrow of irradiated mice. Compared with the Model group, the number of Ki67-positive cells in LR@CT@CA increased significantly ( Fig.14). The spleen is an immune organ of the body and is highly sensitive to radiation. In the Model group, the boundary between the red and white pulp of the spleen was not obvious, there was no obvious germinal center, and the area of ​​the white and red pulp was significantly reduced. In the LR@CT@CA group, the boundary between the red and white pulp was more obvious, the area of ​​the white pulp was enlarged, obvious germinal centers were visible, and the proportion of Ki67 positive cells was significantly increased ( Fig.15 ).

Claims

1. A probiotic gel microsphere comprising probiotics and gel microsphere material.

2. The probiotic gel microspheres as claimed in claim 1 have an obvious protective effect against ionizing radiation damage when taken orally.

3. The probiotic gel microspheres as claimed in claim 1, wherein the probiotics are selected from a single type of probiotic microorganism or a mixed microorganism composed of multiple types of probiotics.

4. The probiotic gel microspheres as claimed in claim 1, wherein the probiotics are coated probiotics.

5. The probiotic gel microspheres as claimed in claim 1, wherein the probiotics are coated probiotics, and the coating material is selected from sodium alginate, chitosan, tannic acid, polylysine, polydopamine, and whey protein.

6. The probiotic gel microspheres as claimed in claim 1, wherein the probiotics are selected from Lactobacillus rhamnosus, Lactobacillus reuteri, Akkermansia muciniphila, Clostridium butyricum, and Bifidobacterium.

7. The probiotic gel microspheres as claimed in claim 1, wherein the gel microsphere material is selected from sodium alginate, konjac glucomannan and chitosan.

8. The probiotic gel microspheres according to claim 1, wherein the preparation method comprises the following steps: (1) mixing the probiotics with a chitosan solution to coat the probiotics with chitosan, then adding a tannic acid solution to electrostatically adsorb the chitosan to form a second coating, and adding sodium tripolyphosphate to cross-link the chitosan to promote the stable attachment of the double-layer coating to the surface of the strain; (2) mixing the coated probiotics and the sodium alginate solution evenly; (3) The sodium alginate solution containing the coated probiotics is sprayed into fine droplets under the action of an electrostatic field and dropped into a CaCl2 solution to solidify the surface to obtain probiotic gel microspheres.

9. The probiotic gel microspheres according to claim 1, wherein the particle size is selected from 1 micron to 1000 microns.

10. The probiotic gel microspheres according to claim 1, wherein the preparation method comprises the following steps: Chitosan was added to physiological saline containing 2% acetic acid to swell, and the pH was adjusted to 6 with NaOH solution to obtain a 2 mg / mL chitosan solution; Lactobacillus rhamnosus LGG was cultured anaerobically, and the bacteria in the logarithmic growth phase were collected, washed with physiological saline and collected by centrifugation; LGG was dispersed in the above chitosan solution, stirred at room temperature for 30 minutes, centrifuged and washed with physiological saline to obtain LGG@CS; LGG@CS was dispersed in physiological saline, and 2 mg / mL tannic acid solution was added dropwise under stirring, and stirring was continued for 10 minutes after the addition was completed, and 5 mg / mL sodium tripolyphosphate solution was continued to be added dropwise, and stirring was continued for 10 minutes after the addition was completed; The product was collected by centrifugation and washed with physiological saline to obtain LGG@CT; sodium alginate was added to physiological saline for swelling and dissolution to obtain a 3% sodium alginate solution; LGG@CT was re-dispersed with physiological saline, an equal volume of 3% sodium alginate solution was added, mixed, and bubbles were removed; the dispersion was loaded into a syringe, the syringe was installed in an electrospinning instrument, and the liquid was pushed at a rate of 0.1 mL / h. Under an 8 kV electrostatic field, the dispersion sprayed out micron-sized spherical droplets, which entered a 3 mg / mL calcium chloride solution, and the outer layer solidified to form gel microspheres; the gel microspheres were collected, passed through a 200-mesh sieve, and washed with physiological saline to obtain probiotic gel microspheres LGG@CA@CT.

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