CN114452306A - Application of heat-inactivated clostridium butyricum in preparation of products for preventing and treating asthma - Google Patents
Application of heat-inactivated clostridium butyricum in preparation of products for preventing and treating asthma Download PDFInfo
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Abstract
The invention discloses application of heat-inactivated clostridium butyricum in preparation of products such as medicines for preventing and treating asthma and the like, and belongs to the technical field of medicines. The heat inactivated clostridium butyricum can be used as a single preparation and can also be compatible with other medicines, and a proper amount of pharmaceutically acceptable auxiliary materials are added to prepare a medicinal preparation by adopting the prior art. The invention is proved by tests that: the aerosol inhalation heat-inactivated clostridium butyricum and the oral administration heat-inactivated clostridium butyricum can both obviously reduce airway inflammation and airway mucus secretion of asthmatic mice, the aerosol inhalation heat-inactivated clostridium butyricum can play an anti-asthma role by regulating immune cells and cytokines, and the aerosol inhalation heat-inactivated clostridium butyricum and the oral administration heat-inactivated clostridium butyricum can both be applied to prevention or treatment of asthma.
Description
Technical Field
The invention relates to application of heat-inactivated clostridium butyricum in preparation of products for preventing and treating asthma, belonging to the technical field of medicines.
Background
At present, the symptoms of asthma can be controlled by the treatment of medicaments such as inhaled glucocorticoids, beta-adrenoceptor agonists, leukotriene receptor antagonists, theophylline and the like, but the asthma cannot be radically treated, the treatment time of the medicaments is long, certain side effects are generated, a large number of patients cannot tolerate the medicaments, and the compliance is poor. Even with standardized treatment according to the protocol of "initiative for global asthma control", about 10% of asthmatics cannot be effectively controlled. The search for an effective prevention and treatment scheme is of great significance.
A large number of epidemiological studies show that beneficial microorganisms can be contacted with the extract to reduce the occurrence and development of asthma, and the extract can be used for preventing and treating asthma. The beneficial live or dead microorganisms mainly regulate the immune function of the human body through respiratory tracts and digestive tracts and inhibit the development of asthma. Probiotics are a class of microorganisms that provide benefits to the health of the host when ingested in appropriate amounts. A large number of researches find that: the probiotic has immunoregulatory and anti-infectious activities, and can be used for preventing and treating asthma.
Clostridium butyricum (latin scientific name:Clostridium butyricum) Also called as: the clostridium butyricum, the butyric acid bacteria, the clostridium butyricum, the butyric acid bacteria and the probiotics commonly used in clinic are reported to have the anti-asthma effect, and can be used for preventing or treating asthma. Both clostridium butyricum and heat-inactivated clostridium butyricum have an immunoregulation function, but whether the heat-inactivated clostridium butyricum has an anti-asthma effect is not clear, and no relevant research report on the aspect of preventing and treating asthma of the heat-inactivated clostridium butyricum exists at present.
Disclosure of Invention
The invention aims to provide a new scheme for preventing or treating asthma by providing application of heat-inactivated clostridium butyricum as and/or in preparation of products for preventing and treating asthma.
In order to achieve the purpose, the invention provides the following technical scheme:
the invention discloses application of heat-inactivated clostridium butyricum in preparing products for preventing and treating asthma, wherein the products comprise but are not limited to medicines, health products, enteral nutrition preparations and dietary supplements.
Furthermore, the invention discloses application of the heat-inactivated clostridium butyricum in preparing and/or preventing asthma medicines. In practical application, the heat-inactivated clostridium butyricum can be used as a single preparation or can be compatible with other medicines, and a proper amount of pharmaceutically acceptable auxiliary materials are added to prepare a medicinal preparation by adopting the prior art.
The dosage form of the medicine comprises aerosol, inhalant, granules, capsules, tablets, pills, oral liquid or injection.
The heat-inactivated clostridium butyricum is applied to and/or prepared into a medicament for preventing and treating asthma, and in practical application, the medicament can be prepared into microcapsules, microspheres, nanoparticles and liposomes.
The heat-inactivated clostridium butyricum is applied to and/or preparing a medicament for preventing and treating asthma, and in practical application, the administration modes comprise respiratory administration and digestive tract administration.
Research experiments show that:
1. the atomizing inhalation heat-inactivated clostridium butyricum, the oral administration heat-inactivated clostridium butyricum and the oral administration live clostridium butyricum bacteria can obviously reduce the lung inflammation of asthma mice, wherein the effect of atomizing inhalation heat-inactivated clostridium butyricum is the best.
2. The aerosol inhalation heat-inactivated clostridium butyricum, the oral administration heat-inactivated clostridium butyricum and the oral administration live clostridium butyricum bacteria can obviously reduce the airway mucus secretion of asthma mice, wherein the effect of the aerosol inhalation heat-inactivated clostridium butyricum is the best.
3. The atomized inhalation heat-inactivated clostridium butyricum obviously reduces the content of IL-4 in alveolar lavage fluid (BALF) of asthmatic mice and increases the content of IFN-gamma.
4. The atomization inhalation heat-inactivated clostridium butyricum obviously increases the proportion of spleen Treg cells of asthma mice, and reduces the proportion of Th17 cells and the ratio of Th17/Treg cells.
The invention has the beneficial effects that:
1. the invention is proved by tests that: the atomizing inhalation heat-inactivated clostridium butyricum, the oral administration heat-inactivated clostridium butyricum and the oral administration live clostridium butyricum bacteria can obviously reduce airway inflammation and airway mucus secretion of asthmatic mice, wherein the effect of atomizing inhalation heat-inactivated clostridium butyricum is the best; the atomized inhaled heat-inactivated clostridium butyricum can play an anti-asthma role by regulating immune cells and cytokines; both aerosolized inhaled and oral heat-inactivated clostridium butyricum can be used for the prevention or treatment of asthma.
2. The respiratory tract immunity has the advantages of small required dosage, strong immunoregulation, high safety, good tolerance and the like, and the digestive tract administration has the advantages of simplicity, safety, economy and the like. The inventor researches and discovers that: the aerosol inhalation and oral administration of the heat-inactivated clostridium butyricum have an anti-asthma effect, can be applied to prevention or treatment of asthma, is beneficial to prevention and control of asthma, is worthy of further clinical popularization and application, and has a good market prospect.
3. The invention discloses application of heat-inactivated clostridium butyricum in preparing products such as medicines and health products for preventing and treating asthma, and provides a new way and scheme for safe and effective clinical prevention and treatment of asthma. Compared with the live clostridium butyricum, the heat-inactivated clostridium butyricum has more prominent effect of treating asthma and has significant progress. In addition, compared with the live clostridium butyricum, the heat-inactivated clostridium butyricum also has the advantages of safety, reliability and no infection risk; good stability, standardized production, convenient storage, convenient use, high biological value and the like.
Drawings
FIG. 1: total number of alveolar lavage fluid inflammatory cells and proportion of eosinophils;
FIG. 2: lung tissue HE staining;
FIG. 3: PAS staining of lung tissues;
FIG. 4: concentration levels of IFN-. gamma.and IL-4 in alveolar lavage fluid.
Detailed Description
1. Materials and methods
1.1 materials: clostridium butyricum CGMCC NO: 0313-1 is provided by Shandong Kexing biopharmaceutical corporation;
1.2 Experimental methods
1.2.1 Heat-inactivated Clostridium butyricum: the method comprises the steps of firstly measuring the bacterium content of the clostridium butyricum powder, then weighing a certain weight of the clostridium butyricum powder, and placing the clostridium butyricum powder in a high-temperature high-pressure heat inactivation environment at 110kpa and 121 ℃ for 20 minutes to obtain the heat-inactivated clostridium butyricum. Naturally cooling to room temperature and storing at-20 ℃.
1.2.2 preparation of heat-inactivated Clostridium butyricum bacterial liquid: prior to use, heat-inactivated Clostridium butyricum was formulated with Phosphate Buffered Saline (PBS) to give a bacteria content (the bacteria content before inactivation) of 5X108CFU/ml suspension. Lysis was facilitated by 4 power disruptions of the sonicator at 40% for a duration of 15s with 20s intervals per sonication. PBS was then added to make up the desired concentration for use.
1.2.3 groups of experimental animals: the 64 BALB/C male mice are randomly divided into 8 groups, and each group comprises 8 mice, namely a normal control group (A group), an asthma control group (B group), an atomization inhalation low-dose heat-inactivation clostridium butyricum intervention group (C group), an atomization inhalation high-dose heat-inactivation clostridium butyricum intervention group (D group), an oral low-dose heat-inactivation clostridium butyricum intervention group (E group), an oral high-dose heat-inactivation clostridium butyricum intervention group (F group), an oral low-dose clostridium butyricum viable bacteria intervention group (G) and an oral high-dose clostridium butyricum viable bacteria intervention group (H group).
1.2.4 establishment of asthma model: by adopting an Ovalbumin (OVA) sensitization method, 0.2ml of sensitization solution (25 mu g of OVA, 1mg of aluminum hydroxide and 200 mu l of PBS solution) is injected into the abdominal cavity of the mouse on the 0 th day, the 7 th day and the 14 th day of the experiment. From day 21, the mice were placed in a 30cm X20 cm X30 cm nebulization chamber, and 2% OVA was inhaled by nebulization for 30min each time, 1 time per day, for 7 consecutive days, to establish an asthma model.
1.2.5 intervention in groups of mice: B. c, D, E, F, G and group H an asthma model was established as described above. Group a was intraperitoneally injected and nebulized with PBS instead of OVA. C. D, E, F, G and group H were medicated on days 0-13 of the experiment, 1 time per day for 14 consecutive days.
The drug intervention treatment was as follows: group C contains 1X107Atomizing 20ml of CFU heat-inactivated clostridium butyricum liquid for inhalation for 30 minutes each time; group D contains 1X108Atomizing 20ml of CFU heat-inactivated clostridium butyricum liquid for inhalation for 30 minutes each time; e groups of mice each were given 1X10 daily7Performing intragastric lavage by using 0.2ml of CFU heat-inactivated clostridium butyricum liquid; group F mice were given 1X10 daily8Performing intragastric lavage by using 0.2ml of CFU heat-inactivated clostridium butyricum liquid; g groups of mice each administered 1X10 daily70.2ml of CFU clostridium butyricum viable bacteria liquid is perfused into the stomach; h groups each mouse was given 1X10 daily8And (3) performing intragastric lavage on 0.2ml of CFU clostridium butyricum viable bacteria liquid.
1.2.6 BALF inflammatory cells and differential counts: mice were sacrificed within 24 hours of the last challenge, BALF rows for total inflammatory cells and differential counts.
1.2.7 pathological staining and scoring of lung tissue: left lung tissue was stained with HE and PAS. Airway inflammation scoring according to the degree of infiltration of inflammatory cells around bronchioles: no inflammatory cell infiltration, 0 point; a small amount of inflammatory cells infiltrate, 1 point; there were 1 layer of inflammatory cell infiltration, 2 points; 2-4 layers of inflammatory cells are infiltrated, and 3 minutes are spent; more than 4 layers of inflammatory cell infiltration, 4 points; PAS scoring was performed as a percentage of goblet cells to bronchiolar epithelial cells: bronchioles without goblet cells, 0 min; 0 < goblet cell ratio < 25%, 1 point; the proportion of goblet cells is more than or equal to 25 percent and less than or equal to 50 percent, 2 minutes; 50% < the proportion of goblet cells is less than or equal to 75%, 3 minutes; 75% < ratio of goblet cells, 4 points.
1.2.8 ELISA method for detecting IFN-gamma and IL-4 concentration levels in BALF.
1.2.9 flow cytometry splenic Th17 and Treg cell ratios were examined.
2. The statistical method comprises the following steps: the measurement data conforming to normal distribution and having uniform variance
Indicating that LSD test is used for pairwise comparison in the group; non-fit to normal distributions are expressed in median (interquartile range) and analyzed using rank sum test. P<A difference of 0.05 was significant. Comparison with group A. P<0.05; comparison with group B, # P<0.05。
3. Results
3.1 behavioral symptom changes: diarrhea appears in the intervention process of 2 mice in the H group, and the mice in the other 5 groups of intervention groups have no obvious adverse reaction; asthma attack-like symptoms appear in the B group mice in the excitation process, and asthma attack-like symptoms are relieved in the 6 groups of medicine intervention groups.
3.2 BALF Total inflammatory cell count and cell differential count: the total number of inflammatory cells and the proportion of eosinophils were significantly increased in group B mice compared to group a (P < 0.05); the total number of inflammatory cells and the proportion of eosinophils were significantly reduced in the 6 drug intervention groups compared to group B (P < 0.05), with the best results in the aerosolized inhalation high dose heat-inactivated clostridium butyricum intervention group (group D), see figure 1.
3.3 pathological observation of lung tissues: compared with the group A, the lung tissues of the mice in the group B show a large amount of inflammatory cell infiltration around the airway and increased airway mucus secretion, and the airway inflammation score and PAS score are obviously increased (P is less than 0.05); compared with group B, the 6 groups of drug intervention groups have reduced airway inflammatory cell infiltration and airway mucus secretion, and significantly reduced airway inflammation score and PAS score (P < 0.05), wherein the inhalation of atomized high-dose heat-inactivated clostridium butyricum intervention group (group D) has the best effect, and the attached figures 2 and 3 and the table 1 show that the medicine intervention group is a drug intervention group.
3.4 IFN-. gamma.and IL-4 concentration levels in BALF: compared with the group A, the IFN-gamma concentration level of the mice in the group B is remarkably reduced (P < 0.05), and the IL-4 concentration level is remarkably increased (P < 0.05); the IFN- γ concentration levels were significantly increased (P < 0.05) and IL-4 concentration levels were significantly decreased (P < 0.05) in the aerosolized inhalation high dose heat-inactivated clostridium butyricum intervention group (group D) compared to group B, see figure 4.
3.5 ratio of splenic Th17 and Treg cells: compared with the group A, the proportion of Th17 cells and the ratio of Th17/Treg of the mice in the group B are obviously increased (P < 0.05); compared with group B, the Treg cells in the aerosolized inhalation high-dose heat-inactivated clostridium butyricum intervention group (group D) were significantly increased (P < 0.05), the Th17 cell ratio and the Th17/Treg ratio were significantly decreased (P < 0.05), see table 2.
And (4) conclusion: the atomizing inhalation heat-inactivated clostridium butyricum, the oral administration heat-inactivated clostridium butyricum and the oral administration live clostridium butyricum bacteria can obviously reduce airway inflammation and airway mucus secretion of asthmatic mice, wherein the effect of atomizing inhalation heat-inactivated clostridium butyricum is the best; the atomized inhaled heat-inactivated clostridium butyricum can play an anti-asthma role by regulating immune cells and cytokines; both aerosolized inhaled and oral heat-inactivated clostridium butyricum can be used for the prevention or treatment of asthma.
Claims (5)
1. Application of heat-inactivated clostridium butyricum in preparing products for preventing and treating asthma.
2. The use according to claim 1, wherein the product comprises a pharmaceutical, nutraceutical, enteral nutritional, dietary supplement.
3. Use according to claim 2, characterized in that: the heat-inactivated clostridium butyricum can be used as a single preparation and can also be compatible with other medicines, and a proper amount of pharmaceutically acceptable auxiliary materials are added to prepare a medicinal preparation by adopting the prior art.
4. Use according to claim 3, characterized in that: the dosage form of the medicine comprises aerosol, inhalant, granules, capsules, tablets, pills, oral liquid or injection.
5. Use according to claim 3, characterized in that: the medicine can be prepared into microcapsules, microspheres, nanoparticles and liposome.
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