CN114470004A - Application of probiotic spore shadow in preparation of medicine for preventing or treating radioactive intestinal injury - Google Patents

Application of probiotic spore shadow in preparation of medicine for preventing or treating radioactive intestinal injury Download PDF

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CN114470004A
CN114470004A CN202210065271.XA CN202210065271A CN114470004A CN 114470004 A CN114470004 A CN 114470004A CN 202210065271 A CN202210065271 A CN 202210065271A CN 114470004 A CN114470004 A CN 114470004A
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shadow
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王蕾
郑翠霞
孔月月
陈丹丹
牛梦亚
巩爽
王芳芳
冯倩华
宋庆龄
赵洪娟
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Zhengzhou University
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Abstract

The invention provides an application of probiotic spore shadow in preparation of a medicine for preventing or treating radioactive intestinal injury, belongs to the technical field of biomedicine, and aims to solve the problem of prevention or treatment of acute radioactive intestinal injury. The probiotic spore shadow is used as an effective component of the medicament for preventing or treating the radioactive intestinal injury, so that the intestinal injury of animals generated after the intestinal tracts are subjected to radioactive irradiation can be effectively reduced, and a new technical support is provided for preventing and treating the radioactive intestinal injury.

Description

Application of probiotic spore shadow in preparation of medicine for preventing or treating radioactive intestinal injury
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of probiotic spore shadows in preparation of a medicine for preventing or treating radioactive intestinal injury.
Background
Radiotherapy (RT) is an important cornerstone of modern cancer therapy, with no alternatives in its position in clinical cancer therapy. RT can not only directly control the progress of local tumors, but also activate the immune system of the organism, and generate a synergistic amplification effect with immunotherapy, thereby not only inhibiting the development of in-situ tumors, but also effectively improving the invasion and metastasis of the tumors and improving the prognosis. Although during radiotherapy the radiation is mainly concentrated at the focal site, the surrounding normal tissues are not protected from accidental irradiation. The intestinal tract serves as one of the target organs for radiotherapy side effects, and radiotherapy intestinal injury is the most common side effect, particularly the radiotherapy of the abdominal cavity or the pelvic cavity. Radiation-induced intestinal injury is mainly manifested by ectopic intestinal flora, intestinal epithelial cell death, intestinal villus shedding, mucosal barrier disruption and mucosal inflammation. The intestinal flora has a very close mutualistic symbiosis relationship with the gastrointestinal health of a host, and influences the stability of the internal environment of the host and the health of the host. To date, with the increasing survival rate of cancer patients, research into strategies for preventing or treating acute radiation-induced intestinal injury has become an urgent issue.
Disclosure of Invention
In order to solve the problem of prevention or treatment of acute radioactive intestinal injury, the invention provides application of the probiotic spore ghost in preparation of a medicament for preventing or treating the radioactive intestinal injury.
The invention is realized by the following technical scheme:
the invention provides application of probiotic spore shadow in preparation of a medicine for preventing or treating radioactive intestinal injury.
Further, the dosage form of the medicament for preventing or treating the radioactive intestinal injury comprises an oral preparation.
Further, the dosage form of the medicament for preventing or treating the radioactive intestinal injury comprises any one of tablets, capsules, granules, suspensions and pills.
Further, in the preventive or therapeutic agent for radiation damage of intestine, the site of the intestine damage to be prevented or treated includes at least one of small intestine, colon and rectum, and the radiation source includes x-ray and/or gamma-ray.
Furthermore, the probiotic spore ghost is prepared by taking at least one of bacillus subtilis, bacillus coagulans and bacillus licheniformis as a raw material.
Further, the probiotic spore shadow is prepared by the following method:
culturing the probiotics to enable the probiotics to produce spores;
separating the spore inner core from the capsid by physical means or chemical means to obtain a complete spore capsid, namely the probiotic spore shadow.
Further, the probiotic spore shadow is prepared by the following method:
extracting spores: inoculating 1-5% of probiotics in logarithmic phase into a spore production culture medium, performing shake culture at 37 ℃ for 48-72 h, collecting bacterial liquid, heating at 80 ℃ for 25-40 min, cooling, centrifuging, precipitating, washing, and freeze-drying the obtained spores for later use;
preparing a probiotic spore shadow: separating the inner core from the capsid by a spore through a physical means or a chemical means, washing and centrifuging the separated capsid to obtain the probiotic spore shadow;
wherein the physical means is to extrude spores through a high-pressure filter membrane with the aperture of 0.8 mu m to obtain the spore shadow of the probiotics;
the chemical means is to culture spores by using a probiotic culture medium containing at least one of a strong oxidant, a surfactant and an antibacterial compound to obtain the probiotic spore shadow. The principle is as follows: in the stage of spore germination initiation, under the condition that the permeability of the shell of the spore is increased, the genetic material of the spore can be destroyed by the strong oxidant, the surfactant and the antibacterial compound, and the spore shadow is obtained.
In the present invention, the surfactant may be higher fatty alcohol sulfates (e.g., sodium dodecylsulfonate, sodium hexadecyl sulfate, etc.), sulfuric acid compounds (e.g., sulfoaryl sulfonate, sulfonaphthyl sulfonate); the strong oxidant can adopt hydrogen peroxide; the antibacterial compound can be antibiotic antibacterial agent, antibacterial peptide, inorganic antibacterial agent (such as metal ions of silver, copper, zinc, etc.), organic antibacterial agent (such as acylaniline, imidazole, thiazole, phenol, biguanide, etc.).
Optionally, the probiotic spore shadow is prepared by the following method:
b, extraction of bacillus subtilis spores: inoculating bacillus subtilis in a logarithmic growth phase into a spore production culture medium in a proportion of 3%, wherein the spore production culture medium comprises the following raw material components in percentage by mass: 0.3% of beef extract, 0.5% of peptone, 0.5% of NaCl and MnSO4·H20.005% of O, shaking and culturing at 37 ℃ for 72h, then collecting bacterial liquid, heating at 80 ℃ for 35min, cooling, centrifuging, precipitating and washing to obtain spore freeze-dried for later use;
preparing a bacillus subtilis spore shadow: separating the inner core and the capsid by the bacillus subtilis spore through a physical means or a chemical means, washing and centrifuging the separated capsid, and obtaining the probiotic spore shadow.
Optionally, the probiotic spore shadow is prepared by the following method:
extraction of bacillus coagulans spores: the bacillus coagulans in the logarithmic growth phase is inoculated in a spore production culture medium in a proportion of 1%, and the spore production culture medium comprises the following raw material components in percentage by mass: beef extract 0.3%, peptone 1%, NaCl 0.5%, MnSO4·H20.005% of O, shaking and culturing at 37 ℃ for 72h, then collecting bacterial liquid, heating at 80 ℃ for 30min, cooling, centrifuging, precipitating and washing to obtain spore freeze-dried for later use;
preparation of a spore shadow of bacillus coagulans: separating the inner core and the capsid by bacillus coagulans spores through a physical means or a chemical means, washing and centrifuging the separated capsid, and obtaining the probiotic spore shadow.
Optionally, the probiotic spore shadow is prepared by the following method:
b, extraction of bacillus licheniformis spores: inoculating bacillus licheniformis in a logarithmic growth phase into a spore production culture medium in a proportion of 5%, wherein the spore production culture medium comprises the following raw material components in percentage by mass: 0.45% of beef extract, 1.5% of peptone, 0.75% of NaCl0, and MnSO4·H20.005% of O, shaking and culturing at 37 ℃ for 48h, then collecting the bacterial liquid, heating at 80 ℃ for 35min, cooling, centrifuging, precipitating and washing to obtain spore freeze-dried for later use;
preparing a bacillus licheniformis spore shadow: separating the inner core and the capsid by bacillus licheniformis spore through physical means or chemical means, washing and centrifuging the separated capsid to obtain the probiotic spore shadow.
One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:
the probiotic spore shadow is high in tolerance to extreme environments and good in biocompatibility, firstly, the probiotic spore shadow can protect the stability of normal dominant flora in gastrointestinal tracts, improve the growth environment of anaerobic probiotics such as lactobacillus, bifidobacterium, streptococcus digestus and the like, increase the richness and diversity of intestinal flora, and prevent the ectopic intestinal flora, the death of intestinal epithelial cells and the falling of intestinal villi caused by ray irradiation; secondly, the spore shadow of the probiotics can remove active oxygen generated in the radiotherapy process, and the characteristics lay a foundation for the spore shadow of the probiotics to be used for preventing radioactive intestinal injury; thirdly, the spore shadow of the probiotics can relieve the inflammation of mucous membrane by inhibiting proinflammatory factors such as IL-1 beta, TNF-alpha, IL-6 and the like, has an anti-inflammatory effect, and can be used for treating the radioactive intestinal injury; finally, calcium ions and the like contained in the spore shadows of the probiotics can promote the repair of the damaged epithelial barrier and promote the rapid reconstruction of the intestinal barrier, thereby achieving the treatment effect on the acute intestinal injury induced by radiotherapy; the probiotic spore shadow disclosed by the invention is simple in preparation method, low in production cost and stable in property, provides a new technical support for preventing and treating radioactive intestinal injury, is an innovation on an oral micro-ecological preparation, and has great economic and social benefits.
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In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.
FIG. 1 is a TEM image of Bacillus subtilis spores of the present invention.
FIG. 2 is a TEM image of spores of Bacillus coagulans of the present invention.
FIG. 3 is a TEM image of spores of Bacillus licheniformis of the present invention.
FIG. 4 is a TEM image of a spore image of Bacillus subtilis according to the present invention.
FIG. 5 is a TEM image of a spore image of Bacillus coagulans of the present invention.
FIG. 6 is a TEM image of the spore shadow of Bacillus licheniformis of the present invention.
FIG. 7 is a graph showing the change in body weight of mice after X-ray abdominal irradiation in examples 4-6 of the present invention.
FIG. 8 is an MPO expression profile of mouse intestinal tissue after abdominal X-ray irradiation in examples 4-6 of the present invention.
Detailed Description
The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are illustrative of the invention and are not to be construed as limiting the invention.
Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.
In order to solve the technical problems, the embodiment of the invention provides the following general ideas:
the bacillus probiotics belong to gram-positive bacteria and are easy to survive, colonize and propagate. The spore of the probiotic bacteria can be lower than the severe external environment, and benefits from the special structure. Wherein the core of the spore is positioned at the innermost part of the spore, contains DNA, RNA and most enzymes of the spore and is a storage area of probiotic genetic material. The exterior of the spore coat is compact, and the spore coat has the characteristics of strong acid-base environment tolerance, toxic chemical components, ionizing radiation, protease, lysozyme and the like, thereby having a protective effect on spore genetic materials. The core of the spore is removed by physical or chemical means to form a spore vacant shell without genetic material, the vacant shell is similar to a bacterial ghost, and the complete shell structure which is the same as that of the natural spore is kept, namely the spore ghost. Repeated experiments show that the spore shadow not only can eliminate active oxygen and has biological activity such as anti-inflammation, but also can prevent and treat radioactive intestinal injury.
Based on the above, the invention provides the application of the spore shadow of the probiotic in the preparation of the medicine for preventing or treating the radioactive intestinal injury, and no public report is found so far.
The application of the spore shadow of the probiotic bacteria in the preparation of the medicament for preventing or treating the radioactive intestinal injury is described in detail in the following by combining the examples and experimental data.
Example 1
A preparation method of spore shadow of bacillus subtilis comprises the following steps:
(1) b, extraction of bacillus subtilis: inoculating 3% Bacillus subtilis in logarithmic growth phase into spore production medium (beef extract 0.3%, peptone 0.5%, NaCl)0.5％，0.005％MnSO4·H2O), culturing in a constant-temperature shaking incubator at 37 ℃ for 72h, collecting bacterial liquid, heating in an environment at 80 ℃ for 35min, standing to room temperature, centrifuging for 10min under the condition of 10000r/min, washing the obtained precipitate for 3 times by using 0.85% sodium chloride solution, dispersing the obtained spores by using ultrapure water, and freeze-drying for later use, wherein a TEM image of the spores prepared in the embodiment is shown in figure 1.
(2) B, preparation of bacillus subtilis ghost: separating the core from the shell of the spore obtained in the step (1) by a 0.8-micron high-pressure filter membrane extrusion system, washing the shell obtained by separation for 2-4 times by using ultrapure water, and centrifuging at 10000r/min to obtain a spore shadow.
The yield of the obtained spore image is 10% -20%, and the TEM image of the spore image is shown in FIG. 4.
Example 2
A preparation method of spore shadow of bacillus coagulans comprises the following steps:
(1) extraction of bacillus coagulans spores: bacillus coagulans in logarithmic growth phase is inoculated into spore production medium (beef extract 0.3%, peptone 1%, NaCl 0.5%, 0.005% MnSO) at a ratio of 1%4·H2O), culturing in a constant-temperature shaking incubator at 37 ℃ for 72h, collecting bacterial liquid, heating in an environment at 80 ℃ for 30min, and standing to room temperature. Centrifuging at 12000r/min for 10min, washing the obtained precipitate with 0.85% sodium chloride solution for 3 times, dispersing the obtained spore with ultrapure water, and freeze-drying for use, wherein the TEM image of the spore prepared in this example is shown in FIG. 2.
(2) Preparation of bacillus coagulans spore shadow: and (2) separating the core from the shell of the spore obtained in the step (1) by a 0.8-micron high-pressure filter membrane extrusion system, washing the shell obtained by separation for 2-4 times by using ultrapure water, and centrifuging at 8000r/min to obtain a spore shadow.
The yield of the obtained spore image is 20% -30%, and the TEM image of the spore image is shown in FIG. 5.
Example 3
A preparation method of spore shadow of Bacillus licheniformis comprises the following steps:
(1) b, extraction of bacillus licheniformis: inoculating Bacillus subtilis in logarithmic growth phase at a ratio of 5%Inoculating to spore-forming culture medium (beef extract 0.45%, peptone 1.5%, NaCl 0.75%, and MnSO 0.005%)4·H2O), culturing in a constant-temperature shaking incubator at 37 ℃ for 48h, collecting bacterial liquid, heating in an environment at 80 ℃ for 35min, and standing to room temperature. Centrifuging at 10000r/min for 10min, washing the obtained precipitate with 0.85% sodium chloride solution for 3 times, dispersing the obtained spores with ultrapure water, and freeze-drying for later use, wherein TEM image of the spores prepared in the example is shown in FIG. 3.
(2) B, preparation of bacillus licheniformis ghost: separating the core from the shell of the spore obtained in the step (1) by a 0.8-micron high-pressure filter membrane extrusion system, washing the shell obtained by separation for 2-4 times by using ultrapure water, and centrifuging at 10000r/min to obtain a spore shadow.
The yield of the obtained spore shadows is 30% -45%, and the TEM image of the spore shadows is shown in FIG. 6.
Example 4
An application of spore shadow of bacillus subtilis in preventing and treating radioactive intestinal injury comprises the following steps:
experiment 1: morphology characterization and determination of bacillus subtilis spore shadow
The bacillus subtilis spore shadow is taken and dripped on a copper mesh ultrathin film, after drying, the appearance of the bacillus subtilis spore shadow is observed by using a transmission electron microscope, the spore shadow is a hollow shell shape and is complete, and the size of the spore shadow is not obviously different from that of the bacillus subtilis.
Experiment 2: yield determination of bacillus subtilis ghost
Weighing spore lyophilized powder, and recording its weight as M0. Dispersing the spore ghost in ultrapure water, extruding, washing and centrifuging to obtain a spore ghost solution, freeze-drying the solution to obtain freeze-dried powder of the spore ghost, weighing and recording as M1. The spore shadow yield calculation formula is M1/M0The calculated yield of bacillus subtilis ghost was 14.73%.
Experiment 3: biosafety investigation of bacillus subtilis ghost
Healthy Balb/c female mice (Beijing Beffy Bio Inc.) were used for this experiment with Bacillus subtilis ghost (0.5mg/mL) orally administered. Thereafter, the MOP of the blood routine, liver function, kidney function, small intestine and colon tissues were performed on days 1, 4 and 7, respectively.
According to experimental results, after the bacillus subtilis ghost is orally taken, the levels of red blood cells, white blood cells and platelets of a mouse are not obviously different from those of a healthy mouse; there was also no significant difference between glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase and normal mice; the same creatinine and urea nitrogen are also at normal levels. The results show that the bacillus subtilis spore shadow has good biological safety. The business of the mouse small intestine and colon tissue MOP is obviously different from that of a healthy mouse, and the bacillus subtilis spore shadow is proved not to influence the change of the mouse intestinal tract.
Experiment 4: protective effect of bacillus subtilis spore shadow on radioactive intestinal injury
The experiment takes healthy Balb/c female mice (Beijing sbeful biology, Ltd.) as an animal model, and researches the prevention and treatment effect of bacillus subtilis spore shadow on radioactive intestinal injury. After bacillus subtilis spore is administrated by gastric lavage (0.5mg/mL) for 48h, the abdomen of the mouse is irradiated by X-ray with the irradiation dose of 10 Gy. Following irradiation, bacillus subtilis spore lavage dosing was continued for 3 days during which time mouse body weight was monitored. After the experiment was completed, mouse small intestine and colon tissues were removed, H & E staining was performed, and pathological analysis was performed thereon. In addition, the expression of inflammatory factors and MOP in small intestine and colon tissues were examined.
The results showed that the mice in the spore shadow group had a weight loss compared to the X-Ray + saline group, but entered the weight recovery phase earlier than in the radiotherapy alone group. The pathological analysis result shows that the pathological changes of intestinal tissues of each group are not obvious and no obvious damage condition exists on the first day after irradiation. On the seventh day after irradiation, the small intestine was severely damaged and villi were absent in the X-Ray + normal saline group, and only a small number of crypt structures were visible. The remaining lamina propria was seen to be more infiltrated with inflammatory cells. In the X-Ray + bacillus subtilis ghost group, intestinal villus is relatively slightly damaged, and the number and height of crypts are larger than those in the X-Ray + physiological saline group. Under high power microscope, the inflammation of the lamina propria is relatively complete, and the infiltration of inflammatory cells is obviously less than that of the X-Ray + normal saline group. Meanwhile, the levels of proinflammatory cytokines (IL-1 beta and TNF-alpha) and MOP of the X-Ray + bacillus subtilis ghost group are obviously lower than those of the X-Ray + physiological saline group. The results show that the spore shadow has obvious prevention and treatment effect on the radioactive intestinal injury.
Example 5
An application of spore shadow of bacillus coagulans in preventing and treating radioactive intestinal injury comprises the following steps:
experiment 1: morphology characterization and determination of bacillus coagulans spore shadow
The bacillus coagulans spore shadow is taken and dripped on a copper mesh ultrathin film, after drying, the morphology of the bacillus coagulans spore shadow is observed by using a transmission electron microscope, the spore shadow is a hollow shell shape and is complete, the long diameter and the short diameter of the bacillus coagulans spore shadow are uniform, and the source spores are relatively close.
Experiment 2: productivity determination of Bacillus coagulans ghost
Weighing spore lyophilized powder, and recording its weight as M0. Dispersing the spore ghost in ultrapure water, extruding, washing and centrifuging to obtain a spore ghost solution, freeze-drying the solution to obtain freeze-dried powder of the spore ghost, weighing and recording as M1. The spore shadow yield calculation formula is M1/M0The calculated yield was 27.44%.
Experiment 3: biosafety investigation of bacillus coagulans ghost
Healthy Balb/c female mice (Beijing Beford Biotech, Inc.) were used for this experiment to orally administer Bacillus coagulans spore shadow (0.5 mg/mL). Thereafter, the MOP of the blood routine, liver function, kidney function, small intestine and colon tissues were performed on days 1, 4 and 7, respectively.
According to the experimental result, after the bacillus coagulans ghost is orally taken, the red blood cell, white blood cell and platelet levels of the mouse are not obviously different from those of a healthy mouse; there was also no significant difference between glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase and normal mice; the same creatinine and urea nitrogen are also at normal levels. The results show that the spore shadow has good biological safety. The business of the mouse small intestine and colon tissue MOP is obviously different from that of a healthy mouse, and the spore shadow is proved not to influence the intestinal tract change of the mouse.
Experiment 4: protective effect of bacillus coagulans spore shadow on radioactive intestinal injury
The experiment takes healthy Balb/c female mice (Beijing Befort biology, Ltd.) as an animal model, and researches the prevention and treatment effect of bacillus coagulans spore shadow on radioactive intestinal injury. After bacillus coagulans spore administration (0.5mg/mL) for 48 hours, X-ray irradiation is carried out on the abdomen of the mouse, and the irradiation dose is 10 Gy. After irradiation, the bacillus coagulans gavage was continued for 3 days during which the mice were monitored for body weight. After the experiment is finished, the tissues of the small intestine and the colon of the mouse are removed, H & E staining is carried out, pathological analysis is carried out on the tissues, the cecal resolution is removed, and the intestinal tract 16S RNA analysis is carried out on the intestinal tract flora state. In addition, the expression of inflammatory factors and MOP in small intestine and colon tissues were examined.
The results show that the body weight of mice treated with bacillus coagulans ghost rapidly entered the convalescent phase. And the villi and crypts of the small intestine of the bacillus coagulans spore group are relatively complete, and only slight abnormality occurs on the fourth day after X-ray irradiation. In the X-ray + normal saline group, the villi, crypt, serosal layer and lamina propria of the small intestine are seriously damaged in the seventh day after irradiation. Although at the time of the fourteenth day, part of the intestinal barrier of the mouse also entered the repair phase, intact structures were not visible. The 16SRNA results also indicate that the bacillus coagulans ghost can obviously increase the richness and diversity of the intestinal flora. The proinflammatory cytokines (IL-1 beta, TNF-alpha) and MOP levels of the X-Ray + Bacillus coagulans ghost group are significantly lower than those of the X-Ray + saline group. The results show that the spore shadow has obvious prevention and treatment effect on radioactive intestinal injury.
Example 6
The application of spore shadow of Bacillus licheniformis in preventing and treating radioactive intestinal injury comprises the following steps:
experiment 1: shape characterization and determination of bacillus licheniformis spore shadow
The bacillus licheniformis spore shadow is taken and dripped on a copper mesh ultrathin film, after drying, the shape of the bacillus licheniformis spore shadow is observed by using a transmission electron microscope, the spore shadow is a hollow shell shape and is complete, the length is about 0.75 mu m, and the size of the spore shadow is not obviously different from that of the spore.
Experiment 2: yield determination of bacillus licheniformis ghost
Weighing spore jellyDry powder, its weight is denoted as M0. Dispersing the spore ghost in ultrapure water, extruding, washing and centrifuging to obtain a spore ghost solution, freeze-drying the solution to obtain freeze-dried powder of the spore ghost, weighing and recording as M1. The spore shadow yield calculation formula is M1/M0The calculated yield was 33.45%.
Experiment 3: biological safety investigation of Bacillus licheniformis ghost
Healthy Balb/c female mice (Beijing Beeford Biometrics, Inc.) were used for this experiment to orally administer Bacillus licheniformis ghost (0.5 mg/mL). Thereafter, the MOP of the blood routine, liver function, kidney function, small intestine and colon tissues were performed on days 1, 4 and 7, respectively.
According to the experimental result, after the bacillus licheniformis ghost is orally taken, the red blood cell, white blood cell and platelet levels of the mouse are not obviously different from those of a healthy mouse; there was also no significant difference between glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase and normal mice; the same creatinine and urea nitrogen are also at normal levels. The results show that the spore shadow has good biological safety. The business of the mouse small intestine and colon tissue MOP is obviously different from that of a healthy mouse, and the spore shadow is proved not to influence the intestinal tract change of the mouse.
Experiment 4: protective effect of bacillus licheniformis ghost on radioactive intestinal injury
The experiment takes healthy Balb/c female mice (Beijing Beeford biology, Inc.) as an animal model to explore the prevention and treatment effect of bacillus licheniformis spore shadow on radioactive intestinal injury. After bacillus licheniformis ghost is administrated by gastric gavage (0.5mg/mL) for 48 hours, the abdomen of the mouse is irradiated by X-ray, and the irradiation dose is 10 Gy. Following irradiation, the administration of bacillus licheniformis spores by gavage was continued for 3 days during which time the weight of the mice was monitored. After the experiment was completed, mouse small intestine and colon tissues were removed, H & E staining was performed, and pathological analysis was performed thereon. In addition, the expression of inflammatory factors and MOP in small intestine and colon tissues were examined.
The results show that the sporulated mice lost only slightly and entered the phase of weight recovery earlier than the X-Ray + saline group. The pathological analysis result shows that most intestinal villi on the small intestinal canal wall of the X-Ray + normal saline group are lost, only a small amount of intestinal villi and crypt structures exist, the muscular layer is thin, part of the inner muscular layer is broken, and the gap between the inner muscular layer and the outer muscular layer is obvious. The residual lamina propria can show more lymphocytes and neutrophils, the number of the cup-shaped cells and Pan cells in the residual crypt is reduced, and the part of mesothelial cells in the serosal layer can be seen to drop. In the X-Ray + Bacillus licheniformis ghost group, the intestinal villus part is separated from the epithelium, and the goblet cells are relatively few. Under high power microscope, there are few lymphocytes in mucosa epithelium, no obvious abnormality in lamina propria cells, few lymphocytes in intestinal crypt, and no obvious abnormality in muscular layer and serosal layer cells. Meanwhile, the levels of proinflammatory cytokines (IL-1 beta, TNF-alpha and IL-6) and MOP of the X-Ray + bacillus licheniformis ghost group are obviously lower than those of the X-Ray + normal saline group. The results show that the spore shadow has obvious prevention and treatment effect on radioactive intestinal injury.
The invention successfully constructs the bacillus ghost, explains the application of the bacillus ghost in preventing and treating the radioactive intestinal injury, is an innovation of radioactive intestinal injury prevention and treatment medicines, and has great social and economic benefits.
Detailed description of figures 7 and 8:
the results in FIG. 7 show that the weight of each group of mice decreased after X-ray irradiation. Compared with the X-ray + normal saline group, the three spore shadows can relieve the weight loss of mice caused by X-ray irradiation, wherein the bacillus licheniformis spore shadow relieving effect is the most obvious. In addition, mice in the three spore shadow groups all entered the weight recovery period earlier than in the X-ray + normal saline group, and the weight recovery speed of each group was faster than that in the X-ray + normal saline group.
The results in FIG. 8 show that the Myeloperoxidase (MPO) content of the intestinal tissue of mice is significantly increased after X-ray irradiation, indicating increased neutrophil infiltration of the intestinal tissue. Compared with the X-ray + normal saline group, the three spore ghosts can obviously reduce the expression of MPO, and the spore ghosts can reduce inflammatory cell infiltration of intestinal tissues after irradiation.
Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims (10)

1. Application of probiotic spore shadow in preparation of medicine for preventing or treating radioactive intestinal injury.
2. The use of probiotic spore ghosts in the preparation of a medicament for the prevention or treatment of radiation-induced bowel injury according to claim 1, wherein the dosage form of the medicament for the prevention or treatment of radiation-induced bowel injury comprises an oral preparation.
3. The use of probiotic spore shadow in the preparation of a medicament for preventing or treating radiation-induced intestinal injury according to claim 1, wherein the dosage form of the medicament for preventing or treating radiation-induced intestinal injury comprises any one of tablets, capsules, granules, suspensions and pills.
4. The use of probiotic spore shadows according to claim 1 in the preparation of a medicament for preventing or treating radiation-induced intestinal injury, wherein the site of intestinal injury to be prevented or treated comprises at least one of the small intestine, colon and rectum, and the radiation source comprises x-rays and/or gamma-rays.
5. The use of a probiotic spore shadow according to claim 1 in the preparation of a medicament for the prevention or treatment of radiation-induced bowel injury, wherein the probiotic spore shadow is prepared from at least one of bacillus subtilis, bacillus coagulans and bacillus licheniformis.
6. Use of a probiotic spore shadow according to claim 1 in the preparation of a medicament for the prevention or treatment of radiation-induced bowel injury, wherein the probiotic spore shadow is prepared by the following method:
culturing the probiotics to enable the probiotics to produce spores;
separating the spore inner core from the capsid by physical means or chemical means to obtain a complete spore capsid, namely the probiotic spore shadow.
7. Use of a probiotic spore shadow according to claim 1 in the preparation of a medicament for the prevention or treatment of radiation-induced bowel injury, wherein the probiotic spore shadow is prepared by the following method:
extracting spores: inoculating 1-5% of probiotics in logarithmic phase into a spore production culture medium, performing shake culture at 37 ℃ for 48-72 h, collecting bacterial liquid, heating at 80 ℃ for 25-40 min, cooling, centrifuging, precipitating, washing, and freeze-drying the obtained spores for later use;
preparing a probiotic spore shadow: separating the inner core from the capsid by a spore through a physical means or a chemical means, washing and centrifuging the separated capsid to obtain the probiotic spore shadow;
wherein the physical means is to extrude spores through a high-pressure filter membrane with the aperture of 0.8 mu m to obtain the spore shadow of the probiotics;
the chemical means is to culture spores by using a probiotic culture medium containing at least one of a strong oxidant, a surfactant and an antibacterial compound to obtain the probiotic spore shadow.
8. Use of probiotic spore shadow according to claim 7 for the preparation of a medicament for the prevention or treatment of radioactive bowel injury, wherein the probiotic spore shadow is prepared by the following method:
b, extraction of bacillus subtilis spores: inoculating bacillus subtilis in a logarithmic growth phase into a spore production culture medium in a proportion of 3%, wherein the spore production culture medium comprises the following raw material components in percentage by mass: beef extract 0.3%, peptone 0.5%, NaCl 0.5%, MnSO4·H20.005% of O, shaking and culturing at 37 ℃ for 72h, then collecting bacterial liquid, heating at 80 ℃ for 35min, cooling, centrifuging, precipitating and washing to obtain spore freeze-dried for later use;
preparing a bacillus subtilis spore shadow: separating the inner core and the capsid by the bacillus subtilis spore through a physical means or a chemical means, washing and centrifuging the separated capsid, and obtaining the probiotic spore shadow.
9. Use of probiotic spore shadow according to claim 7 for the preparation of a medicament for the prevention or treatment of radioactive bowel injury, wherein the probiotic spore shadow is prepared by the following method:
extraction of bacillus coagulans spores: the bacillus coagulans in the logarithmic growth phase is inoculated in a spore production culture medium in a proportion of 1%, and the spore production culture medium comprises the following raw material components in percentage by mass: beef extract 0.3%, peptone 1%, NaCl 0.5%, MnSO4·H20.005% of O, shaking and culturing at 37 ℃ for 72h, then collecting bacterial liquid, heating at 80 ℃ for 30min, cooling, centrifuging, precipitating and washing to obtain spore freeze-dried for later use;
preparation of a spore shadow of bacillus coagulans: separating the inner core and the capsid by bacillus coagulans spores through a physical means or a chemical means, washing and centrifuging the separated capsid, and obtaining the probiotic spore shadow.
10. Use of probiotic spore shadow according to claim 7 for the preparation of a medicament for the prevention or treatment of radioactive bowel injury, wherein the probiotic spore shadow is prepared by the following method:
b, extraction of bacillus licheniformis spores: inoculating bacillus licheniformis in a logarithmic growth phase into a spore production culture medium in a proportion of 5%, wherein the spore production culture medium comprises the following raw material components in percentage by mass: 0.45% of beef extract, 1.5% of peptone, 0.75% of NaCl0, and MnSO4·H20.005% of O, shaking and culturing at 37 ℃ for 48h, then collecting the bacterial liquid, heating at 80 ℃ for 35min, cooling, centrifuging, precipitating and washing to obtain spore freeze-dried for later use;
preparing a bacillus licheniformis spore shadow: separating the inner core and the capsid by bacillus licheniformis spore through physical means or chemical means, washing and centrifuging the separated capsid to obtain the probiotic spore shadow.
CN202210065271.XA 2022-01-18 2022-01-18 Application of probiotics spore shadow in preparation of medicine for preventing or treating radioactive intestinal injury Active CN114470004B (en)

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