Application of pseudoprevotella griseus in preparation of medicine for preventing radiation intestinal injury

Abstract

The invention relates to an application of pseudoprevotella griseus in preparation of a medicine for preventing intestinal injury caused by ionizing radiation. The medicine disclosed by the invention can be used for preventing radiation intestinal injury, has the effects of relieving rectum villus shedding and reduction of the number of crypts and goblet cells after radiation injury, can be used for remarkably improving the survival rate, and has the advantages of small side effect and good curative effect; under the condition of lack of schemes for preventing intestinal injury caused by ionizing radiation clinically at present, the application prospect is good.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and particularly relates to application of Pseudomonas griseus in preparing medicine for preventing radiation intestinal damage. Background Art

[0002] With the widespread application of nuclear energy and nuclear technology in the fields of industry, health care, agriculture, etc., radiation damage caused by ionizing radiation has received more and more attention. In recent years, the Fukushima nuclear power plant accident in Japan and the subsequent discharge of nuclear contaminated water have once made the public "talk about nuclear fear". The human intestine is a radiation-sensitive organ. When exposed to a one-time whole-body ionizing radiation of more than 10Gy, the intestinal tissue will suffer irreversible damage, causing acute intestinal radiation sickness, and there have been no surviving cases so far. In addition, for patients with abdominal and pelvic malignancies who require high-dose ionizing radiation radiotherapy in clinical practice, severe intestinal side effects are also the main factor restricting the efficacy. At present, there is an extreme shortage of drugs to prevent radiation-induced intestinal damage worldwide, and effective intervention strategies are urgently needed to be developed.

[0003] The main pathological manifestations of severe intestinal damage caused by ionizing radiation include: loss or shortening of intestinal villi, a sudden decrease in the number of small intestinal crypts, and loss of goblet cells. In addition to the direct damage of radiation to intestinal epithelial cells, a large number of studies have confirmed that intestinal flora disorders play an important role in radiation intestinal damage (Moraitis, I. et al. Trends Endocrinol Metab, 2023, 34 (8): 489-501). In a healthy intestinal environment, more than 100 trillion microorganisms are colonized in the human intestinal cavity; these microorganisms coexist harmoniously with us, help digest food and maintain the function of the intestinal immune system. After ionizing radiation acts on the human intestine, it can cause flora disorders, mainly manifested as abnormal ratio of Firmicutes / Bacteroidetes, reduced abundance of probiotics, and increased abundance of pathogenic bacteria (Y. Jian, et al. Front Cell Infect Microbiol, 2021, 11, 586392). Compared with normal mice, germ-free mice are more tolerant to radiation damage, suggesting that microbiota homeostasis is a new target for preventing radiation-induced intestinal damage (PACrawford, et al. Proc Natl Acad Sci USA, 2005, 102, 13254-13259). Based on the above strategies, exogenous supplementation of probiotics has been shown to effectively reduce radiation-induced intestinal damage (Guo, H. et al. Science, 2020, 370, 6516). In recent studies, we found that human intestinal defensin 5-derived peptides have the effect of regulating intestinal microbiota homeostasis and reducing radiation-induced intestinal damage; microbiota diversity analysis results suggest that a probiotic that can produce short-chain fatty acids, Pseudomonas prevotella, may play an important role in this (Zhao G, et al. J Radiat Res, 2024, 65(2): 194-204).

[0004] Alloprevotellarava (A. rava) is a common species of the genus Alloprevotellarava, a Gram-negative anaerobic bacillus that is mostly colonized in the human oral cavity and intestines. Recent studies have revealed that the reduced abundance of A. rava in the intestinal lumen is associated with the occurrence of human obesity (LEITE, G. et al. Am J Gastroenterol, 2024, 119: 1141–1153), the latter of which often has manifestations such as intestinal flora disorders and abnormal mucosal barrier function. This conclusion suggests that exogenous supplementation of A. rava is a potential means of maintaining intestinal mucosal barrier function and may have a protective effect against intestinal radiation damage. In addition, studies have found that an increase in the abundance of Alloprevotellarava bacteria can increase the content of short-chain fatty acids in the intestinal lumen (Zhu H. et al. Cell Metab, 2021, 33: 1926–42). Since short-chain fatty acids can promote the repair of intestinal epithelial damage, A. rava may exert a radioprotective effect by increasing short-chain fatty acids.

[0005] At present, there is no definite research data reported at home and abroad on the effect of A. rava supplementation on preventing intestinal radiation damage. Summary of the invention

[0006] The purpose of the present invention is to provide a use of Pseudomonas griseus in the preparation of a drug for preventing radiation-induced intestinal damage. The drug of the present invention can reduce intestinal damage caused by high-dose ionizing radiation, has the effect of reducing the shedding of ileal villi, the reduction in the number of crypts and goblet cells after radiation damage, and has a significant effect of improving the survival rate of radiation damage, and has the advantages of small side effects and high efficacy.

[0007] The technical solution of the present invention is:

[0008] Application of Pseudomonas griseus in the preparation of drugs for preventing radiation-induced intestinal damage.

[0009] The injury is caused by whole abdomen irradiation.

[0010] The damage is caused by intestinal epithelial shedding.

[0011] The damage is caused by a decrease in the number of intestinal mucosal crypts.

[0012] The damage is caused by the reduction of intestinal epithelial goblet cells.

[0013] The drug is administered orally.

[0014] The oral Pseudomonas griseus was 1×10 9 CFU / day.

[0015] The application of the drug of the present invention can prevent radiation intestinal damage, reduce intestinal damage caused by large doses of ionizing radiation, reduce the loss of ileal villi and the reduction in the number of crypts and goblet cells after radiation damage, and significantly improve the survival rate. It also has the advantages of few side effects and good efficacy, and has good application prospects.

[0016] (1) Pharmacological activity of the present invention: Animal experiments by the applicant have shown that supplementation with Pseudomonas griseus can improve the survival rate of mice with whole-abdomen radiation injury; intestinal tissue pathological analysis found that supplementation with Pseudomonas griseus can significantly reduce the shedding of intestinal mucosa caused by ionizing radiation, and has the effect of resisting intestinal radiation injury. (2) Pseudomonas griseus is a probiotic in the human oral cavity and intestinal cavity, with low toxicity and side effects, and has the advantages of good therapeutic effect.

[0017] The applicant's experiments have shown that the survival rate of Pseudomonas griseus significantly increased to 40% in the case of whole-abdomen radiation damage, and the damage caused by intestinal epithelial shedding due to ionizing radiation was significantly alleviated. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Curve showing that Pseudomonas griseus improves the survival of mice with whole abdominal radiation injury.

[0019] Figure 2 Pathological section showing that Prevotella griseus alleviates intestinal epithelial exfoliation in radiation-damaged mice.

[0020] Figure 3 Statistical results of the effect of Pseudomonas griseus on increasing the height of intestinal villi in mice with radiation-induced intestinal damage.

[0021] Figure 4 Statistical results of Prevotella griseus increasing the number of small intestinal crypts in mice with radiation-induced intestinal damage.

[0022] Figure 5 Pathological sections and statistical results showing that Prevotella griseus alleviates the reduction of goblet cells in radiation-damaged intestinal tract of mice. DETAILED DESCRIPTION

[0023] The substantial content of the present invention is further illustrated by the following embodiments, but the content of the present invention is not limited thereto.

[0024] Main experimental materials

[0025] 1. Male C57BL / 6J mice aged 6-8 weeks and weighing 20-22 g (Wei Tong Li Hua Laboratory Animal Technology Co., Ltd., Beijing)

[0026] 2. Carnoy's fixative (G1120-500mL, Seville, Hubei)

[0027] 3. Ethanol (Sinopharm, Chongqing)

[0028] 4. Xylene (X112051, Aladdin, Shanghai)

[0029] 5. Hematoxylin and eosin (HE) staining kit (C0105S, Bio-Tech, Shanghai)

[0030] 6. Neutral gum (ZLI-9555, Zhongshan Jinqiao, Beijing)

[0031] 7. Scanning microscope (M8, PreciPoint, France)

[0032] 8. PBS phosphate buffer (pH 7.2-7.4) (ZLI-9062, Zhongshan Jinqiao, Beijing)

[0033] 9. Automatic biological tissue pathology dehydration machine (ZT-12M2, Yaguang Pathology, Hubei)

[0034] 10. Biological tissue paraffin embedding machine (YB-7LF, Yaguang Pathology, Hubei)

[0035] 11. Paraffin slicer (Leica RM2245, Leica, Germany)

[0036] 12. Alcian blue-nuclear fast red staining kit (C0155M, Bio-Tech, Shanghai)

[0037] 13. Pseudomonas griseus (BNCC358370, Beina Biotech, Beijing)

[0038] Other reagents were commercially available.

[0039] Example 1: Effect of Pseudomonas griseus on the survival rate of mice with whole-abdomen radiation injury

[0040] 1. Male 6-8 week old C57BL / 6J mice (weight 20-22 g) were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. and cared for and treated in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal experiments were approved by the Animal Experiment Ethics Committee of the Army Medical University.

[0041] 2. Experimental groups:

[0042] Male C57BL / 6J mice were randomly divided into four groups (10 mice in each group):

[0043] (1) Control group (Sham); (2) P. griseus group; (3) Total abdominal irradiation (TAI) group; (4) P. griseus + TAI group.

[0044] 3. Radiation treatment:

[0045] use 60 The mice were irradiated with a single whole abdomen irradiation using a Coγ-ray source, with a dose of 14 Gy and a dose rate of 0.48 Gy / min.

[0046] 4. Administration and dosage:

[0047] The drug was administered by intragastric gavage.

[0048] The mice in the control group and the whole abdominal irradiation (TAI) group were gavaged with 200 μl of normal saline every other day for 14 consecutive days, and the mice in the P. griseus group were gavaged with 1×10 9 CFU / day of P. griseus for 14 consecutive days. After drug treatment, the mice in the whole abdomen irradiation group (TAI) and P. griseus + TAI group were irradiated with 14 Gy, and the survival of mice in each group was observed and recorded every day after irradiation.

[0049] 5. Experimental observation indicators:

[0050] After radiation injury, the mortality rate of mice in each group was observed for 30 days.

[0051] 6. It has been tested that oral administration of Pseudomonas griseus before irradiation can significantly improve the survival rate of mice with whole-abdomen radiation injury (see Figure 1 ). After radiation injury, the mortality rate of experimental mice reached 90% within 30 days; after oral administration of Pseudomonas griseus, the survival rate of radiation-injured mice increased significantly to 40%.

[0052] Example 2: Effect of Pseudomonas griseus on intestinal epithelial shedding induced by ionizing radiation

[0053] 1. Animal model construction and drug administration:

[0054] Referring to steps 1-4 in Example 1, a whole-abdomen radiation injury mouse model was established and the mice were administered with Pseudomonas griseus.

[0055] 2. Identification of intestinal tissue damage:

[0056] Three days after the whole abdomen of the mice was irradiated, the mice were killed by cervical dislocation, and the ileum segment was removed and rinsed with pre-cooled PBS. The small intestine was then rolled up with a small wooden stick, with the villi facing inward, and fixed with a pin. After being fixed overnight with Carnoy's fixative, the tissue was dehydrated with gradient alcohol concentrations and the alcohol in the tissue was replaced with xylene. The transparent tissue block was placed in the dissolved paraffin and kept warm in a wax dissolving box. After the embedded wax block was fixed on a microtome, it was cut into 4μm slices and stained with hematoxylin and eosin (HE) for observation.

[0057] 3. HE staining:

[0058] Use xylene to remove paraffin from mouse small intestine tissue sections, then transfer to distilled water through high-concentration to low-concentration alcohol. Take out the sections, put them in hematoxylin for 2 minutes, tap water for 1 minute, 0.6% hydrochloric acid alcohol for 15 seconds, rinse with tap water for 15 minutes, and place the sections in distilled water for a while. Move to 1% alcohol solution eosin for 30 seconds, dehydrate the sections with alcohol, make them transparent with xylene, drop neutral gum on them, and cover them with coverslips for sealing.

[0059] 4. The pathological changes of small intestinal tissues were observed by Precipoint M8 scanning microscope. After single 14 Gy whole abdominal γ-ray irradiation, the small intestinal mucosal barrier of mice was severely damaged, intestinal epithelial cells were significantly shed, and small intestinal villi were atrophied. Before irradiation, the administration of Pseudomonas griseus significantly alleviated the intestinal epithelial shedding caused by radiation, and the small intestinal villi could maintain a good morphology (see Figure 2 ). The scale bar indicates 200 μm.

[0060] Example 3: Effect of Pseudomonas griseus on the height of small intestinal villi in radiation-induced intestinal damage

[0061] According to steps 3-4 in "Example 2", the mouse small intestinal tissue was HE stained and observed. The height of the small intestinal villi was measured using ViewPoint software (version setup_70). Six well-oriented full-length crypt villus units were measured in each field of view. After testing, the average height of the ileal villi of normal mice and mice treated with Pseudomonas griseus alone were 309 and 310 μm, respectively; after a single 14Gy whole-abdomen γ-ray irradiation, the intestinal villi were reduced to 165 μm. Administration of Pseudomonas griseus can maintain the intestinal villus height at 278 μm, which is significantly different from that of irradiated mice, see Figure 3 ,in **** , P<0.0001, with statistically significant difference. It can be seen that supplementation of Pseudomonas griseus can alleviate the intestinal villus damage caused by ionizing radiation.

[0062] Example 4: Effect of Pseudomonas griseus on the number of small intestinal crypts in radiation-induced intestinal damage

[0063] According to steps 3-4 in "Example 2", the mouse small intestine tissue was stained with HE and observed. The number of crypts in the small intestine tissue was detected by Precipoint M8 scanning microscope. The number of crypts in 6 different fields of view was detected for each slide. After testing, the number of crypts in normal mice was 14 per millimeter; after a single 14Gy whole-abdomen γ-ray irradiation, the number of crypts decreased to 2 per millimeter. Administration of Pseudomonas glycyrrhizus maintained the number of crypts at 5 per millimeter, which was significantly different from that of irradiated mice, see Figure 4 ,in ***, P<0.001, with statistically significant difference. Therefore, supplementation of P. griseus can reduce the loss of small intestinal crypts caused by ionizing radiation.

[0064] Example 5: Effect of Pseudomonas griseus on the number of goblet cells in the small intestine after radiation-induced intestinal damage

[0065] According to steps 3-4 in "Example 2", the small intestinal tissue of mice was stained with Alcian blue-nuclear fast red and observed. The number of goblet cells in the small intestinal tissue was detected by Precipoint M8 scanning microscope. The number of goblet cells in 6 different fields of view was detected for each slide. After testing, the number of goblet cells in normal mice was 97 per millimeter; after a single 14Gy whole-abdomen γ-ray irradiation, the number of goblet cells decreased to 34 per millimeter. Administration of Pseudomonas glycyrrhizus can maintain the number of goblet cells at 62 per millimeter, which is significantly different from that of irradiated mice, see Figure 5 ,in *** , P<0.001, with statistically significant difference. The results suggest that supplementation of P. griseus can reduce the loss of intestinal epithelial goblet cells caused by ionizing radiation.

Claims

1. Application of Pseudomonas griseus in the preparation of drugs for preventing radiation-induced intestinal damage.

2. The use according to claim 1, characterized in that: The injury is caused by whole abdomen irradiation.

3. The use according to claim 1, characterized in that: The damage is caused by intestinal epithelial shedding.

4. The use according to claim 1, characterized in that: The damage is caused by a decrease in the number of intestinal mucosal crypts.

5. The use according to claim 1, characterized in that: The damage is caused by the reduction of intestinal epithelial goblet cells.

6. The use according to claim 1, characterized in that: The drug is administered orally.

7. The use according to claim 1, characterized in that: The oral Pseudomonas griseus was 1×10 9 CFU / day.

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