CN114452306B - Use of heat-inactivated clostridium butyricum as and/or in the preparation of a product for the prophylaxis and/or treatment of asthma - Google Patents
Use of heat-inactivated clostridium butyricum as and/or in the preparation of a product for the prophylaxis and/or treatment of asthma Download PDFInfo
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- 241000193171 Clostridium butyricum Species 0.000 title claims abstract description 83
- 208000006673 asthma Diseases 0.000 title claims abstract description 42
- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- 238000011282 treatment Methods 0.000 title abstract description 15
- 238000011321 prophylaxis Methods 0.000 title description 2
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- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention discloses an application of heat-inactivated clostridium butyricum in serving as and/or preparing products such as medicines for preventing and treating asthma, and belongs to the technical field of medicines. The heat-inactivated clostridium butyricum can be used as a single preparation, can also be compatible with other medicines, is added with a proper amount of pharmaceutically acceptable auxiliary materials, and is prepared into a pharmaceutically acceptable dosage form by adopting the prior art. The invention is proved by experiments: the aerosol inhalation heat-inactivated clostridium butyricum and the oral heat-inactivated clostridium butyricum can obviously reduce airway inflammation and airway mucus secretion of asthmatic mice, the aerosol inhalation heat-inactivated clostridium butyricum can play an anti-asthmatic role by adjusting immune cells and cytokines, and the aerosol inhalation heat-inactivated clostridium butyricum and the oral heat-inactivated clostridium butyricum can be applied to the prevention or treatment of asthma.
Description
Technical Field
The invention relates to an application of heat-inactivated clostridium butyricum in serving as and/or preparing a product for preventing and treating asthma, belonging to the technical field of medicines.
Background
The current treatment with drugs such as inhaled glucocorticoids, beta adrenergic receptor agonists, leukotriene receptor antagonists and theophyllines can control the symptoms of asthma, but the asthma cannot be radically cured, and the drugs have long treatment time, certain side effects, no tolerance for a large part of patients and poor compliance. Even with standardized treatment according to the global asthma control program, about 10% of asthmatics are not effectively controlled. The finding of an effective control scheme is of great significance.
Numerous epidemiological studies have shown that exposure to beneficial microorganisms can reduce the development of asthma and can be used for the prevention and treatment of asthma. The beneficial microorganism live bacteria or dead bacteria regulate the immune function of the human body mainly through the respiratory tract and the digestive tract, and inhibit the occurrence and development of asthma. Probiotics are a class of microorganisms that are ingested in suitable amounts to bring benefits to the health of the host. A number of studies have found that: the probiotics have immunoregulatory and anti-infective activities, and can be used for preventing and treating asthma.
Clostridium butyricum (latin academy: clostridium butyricum), also known as: clostridium butyricum, clostridium butyricum and clostridium butyricum are clinically commonly used probiotics, and researches report that clostridium butyricum has an anti-asthma effect and can be used for preventing or treating asthma. Both clostridium butyricum and heat-inactivated clostridium butyricum have an immunoregulatory function, but whether the heat-inactivated clostridium butyricum has an anti-asthma effect is unclear, and related researches on the heat-inactivated clostridium butyricum in the aspect of asthma prevention and treatment are not reported at present.
Disclosure of Invention
The invention aims to provide the application of heat-inactivated clostridium butyricum in serving as and/or preparing a product for preventing and treating asthma, and provides a new scheme for preventing or treating asthma.
In order to achieve the above purpose, the present invention provides the following technical solutions:
the present invention discloses the use of heat-inactivated clostridium butyricum as and/or in the manufacture of a product for the prevention and/or treatment of asthma, said product comprising a medicament.
Furthermore, the invention discloses application of the heat-inactivated clostridium butyricum in serving as and/or preparing a medicament for preventing and treating asthma. In practical application, the heat-inactivated clostridium butyricum can be used as a single preparation, can also be matched with other medicines, is added with a proper amount of pharmaceutically acceptable auxiliary materials, and is prepared into a pharmaceutically acceptable dosage form by adopting the prior art.
The dosage forms of the medicine comprise aerosol, inhalant, granule, capsule, tablet, pill, oral liquid or injection.
The heat-inactivated clostridium butyricum is applied to the preparation of medicines for preventing and treating asthma, and in practical application, the medicines can be prepared into microcapsules, microspheres, nanoparticles and liposomes.
The use of the heat-inactivated clostridium butyricum of the invention as and/or in the manufacture of a medicament for the prevention and treatment of asthma, in which the mode of administration comprises respiratory tract administration and digestive tract administration.
The research experiment shows that the invention has the following advantages:
1. the atomized inhalation heat-inactivated clostridium butyricum, the oral heat-inactivated clostridium butyricum and the oral clostridium butyricum viable bacteria can obviously reduce lung inflammation of asthmatic mice, wherein the atomized inhalation heat-inactivated clostridium butyricum has the best effect.
2. The aerosol inhalation heat-inactivated clostridium butyricum, the oral heat-inactivated clostridium butyricum and the oral clostridium butyricum viable bacteria can obviously reduce airway mucus secretion of asthmatic mice, wherein the aerosol inhalation heat-inactivated clostridium butyricum has the best effect.
3. Aerosol inhalation of heat-inactivated clostridium butyricum significantly reduces the content of IL-4 in alveolar lavage fluid (BALF) of asthmatic mice, and increases the content of IFN-gamma.
4. Aerosol inhalation of heat-inactivated clostridium butyricum significantly increases the proportion of spleen Treg cells in asthmatic mice, and reduces the proportion of Th17 cells and the ratio of Th17/Treg cells.
The beneficial effects of the invention are as follows:
1. the invention is proved by experiments: the aerosol inhalation heat-inactivated clostridium butyricum, the oral heat-inactivated clostridium butyricum and the oral clostridium butyricum viable bacteria can obviously reduce airway inflammation and airway mucus secretion of asthmatic mice, wherein the aerosol inhalation heat-inactivated clostridium butyricum has the best effect; aerosol inhalation of heat-inactivated clostridium butyricum can exert an anti-asthmatic effect by regulating immune cells and cytokines; both aerosol inhalation 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 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 present inventors have studied to find that: both aerosol inhalation and oral heat-inactivated clostridium butyricum have the function of resisting asthma, can be applied to the prevention or treatment of asthma, is favorable for preventing and controlling asthma, is worthy of further clinical popularization and application, and has better market prospect.
3. The invention discloses application of heat-inactivated clostridium butyricum in serving as and/or preparing a medicament for preventing and treating asthma, and provides a new path and scheme for safe and effective clinical prevention and treatment of asthma. And compared with the live clostridium butyricum, the heat-inactivated clostridium butyricum has more outstanding effect of treating asthma, and has remarkable 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; the stability is good, standardized production can be realized, the storage is convenient, the use is convenient, the biological potency is high, and the like.
Drawings
FIG. 1 alveolar lavage fluid total inflammatory cells and eosinophil ratio;
FIG. 2 lung tissue HE staining;
FIG. 3 PAS staining of lung tissue;
FIG. 4 concentration levels of alveolar lavage fluid IFN-gamma and IL-4.
Detailed Description
1. Materials and methods
1.1 materials: clostridium butyricum CGMCC NO:0313-1 is supplied by Shandong XingBio-pharmaceutical Co., ltd;
1.2 Experimental methods
1.2.1 heat-inactivating clostridium butyricum: firstly, measuring the bacterial content of clostridium butyricum powder, then weighing a certain weight of clostridium butyricum powder, and placing the clostridium butyricum powder in 110kpa and 121 ℃ for high-temperature high-pressure heat inactivation for 20 minutes to obtain heat-inactivated clostridium butyricum. Naturally cooling to room temperature, and storing at-20deg.C.
1.2.2 preparation of heat-inactivated clostridium butyricum bacterial liquid: before use, heat-inactivated clostridium butyricum is added into Phosphate Buffer (PBS) to prepare the clostridium butyricum with bacterial content (bacterial content before inactivation) of 5X10 8 CFU/ml suspension. The ultrasonic cytoclasis instrument is broken 4 times with 40% power to promote dissolution, each time the ultrasonic duration is 15s, and the interval is 20s. PBS was then added to prepare the desired concentration for use.
1.2.3 experimental animal groups: the 64 BALB/C male mice were randomly divided into 8 groups, 8 in each group, which were respectively a normal control group (A group), an asthma control group (B group), an aerosol inhalation low-dose heat-inactivated clostridium butyricum intervention group (C group), an aerosol inhalation high-dose heat-inactivated clostridium butyricum intervention group (D group), an oral low-dose heat-inactivated clostridium butyricum intervention group (E group), an oral high-dose heat-inactivated clostridium butyricum intervention group (F group), an oral low-dose clostridium butyricum live bacterium intervention group (G) and an oral high-dose clostridium butyricum live bacterium intervention group (H group).
1.2.4 establishment of asthma model: mice were intraperitoneally sensitized on days 0, 7, and 14 of the experiment with 0.2ml of sensitization solution (25. Mu.g OVA+1mg aluminum hydroxide+200. Mu.l PBS) using Ovalbumin (OVA) sensitization. On day 21, mice were placed in 30cm X20cm X30cm nebulization chambers, nebulized for inhalation of 2% ova challenge, 30min each time, 1 time per day, for 7 consecutive days, and asthma models were established.
1.2.5 intervention in mice of each group: B. c, D, E, F, G and H groups were modeled as described above. Group a was intraperitoneally injected and nebulized with PBS instead of OVA. C. D, E, F, G and H groups were subjected to drug intervention on days 0-13 of the experiment, 1 time per day, for 14 consecutive days.
The drug intervention treatment is as follows: group C contains 1X10 7 20ml of CFU heat-inactivated clostridium butyricum liquid is atomized and inhaled, and each time is atomized for 30 minutes; group D contains 1X10 8 CFU heat-inactivated clostridium butyricum20ml of liquid is atomized and inhaled, and each time is atomized for 30 minutes; each mouse of group E contained 1X10 per day 7 CFU heat-inactivated clostridium butyricum liquid 0.2ml is irrigated; group F mice each contained 1X10 per day 8 CFU heat-inactivated clostridium butyricum liquid 0.2ml is irrigated; each mouse of group G contained 1X10 per day 7 CFU clostridium butyricum viable bacteria liquid 0.2ml is irrigated; h groups of mice each contain 1X10 daily 8 CFU clostridium butyricum live bacteria liquid 0.2ml is irrigated with stomach.
1.2.6BALF inflammatory cells and differential counts: mice were sacrificed within 24 hours of the last challenge, total BALF line inflammatory cells and differential counts.
1.2.7 staining and scoring of lung tissue pathology: left lung tissue was HE stained and PAS stained. Airway inflammation scoring was performed according to the degree of peribronchial inflammatory cell infiltration: no inflammatory cell infiltration, 0 min; a small amount of inflammatory cells infiltrate, 1 minute; 1 layer of inflammatory cells infiltrate, 2 minutes; 2-4 layers of inflammatory cells infiltrate, 3 minutes; more than 4 layers of inflammatory cells infiltrate, 4 minutes; PAS scoring was performed according to the percentage of goblet cells to bronchiolar epithelial cells: bronchioles have no goblet cells, 0 minutes; 0< goblet cell fraction <25%,1 min; the goblet cell proportion is more than or equal to 25 percent and less than or equal to 50 percent, and the goblet cell proportion is 2 minutes; 50% < goblet cell ratio less than or equal to 75%,3 min; 75% < goblet cell fraction, 4 min.
1.2.8ELISA method detects IFN-gamma and IL-4 concentration levels in BALF.
1.2.9 flow cytometry examined spleen Th17 and Treg cell ratios.
2. Statistical methods: metering data conforming to normal distribution and uniform varianceThe two-by-two comparisons within the group are shown using the LSD test; the non-conformity to the normal distribution is expressed in median (quarter bit spacing) and analyzed using rank sum test. P (P)<0.05 is significant. Comparison with group A, P<0.05; compared 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 other 5 mice in the intervention group have no obvious adverse reaction; asthma attack-like symptoms appear in the excitation process of the group B mice, and the asthma attack-like symptoms of the group 6 drug intervention group are all relieved.
3.2 total BALF inflammatory cells and cell sorter counts: the total inflammatory cell count and eosinophil proportion were significantly increased in group B mice compared to group a (P < 0.05); the total inflammatory cells and eosinophil fraction were significantly reduced (P < 0.05) in the 6 drug intervention group compared to group B, with the nebulized inhalation high dose heat-inactivated clostridium butyricum intervention group (group D) having the best effect, see figure 1.
3.3 pathological observation of lung tissue: compared with group A, the lung tissue of group B mice sees massive inflammatory cell infiltration around the airway and increased airway mucus secretion, and airway inflammation score and PAS score are significantly increased (P < 0.05); compared with group B, the 6 drug intervention groups had reduced airway inflammatory cell infiltration and airway mucus secretion, and the airway inflammatory and PAS scores were significantly reduced (P < 0.05), with the high dose heat inactivated clostridium butyricum intervention group (group D) with the best effect of nebulization inhalation, as shown in figures 2, 3 and Table 1.
TABLE 1 airway inflammation score and PAS score M (P25, P75)
3.4 IFN-gamma and IL-4 concentration levels in BALF: compared with group A, the IFN-gamma concentration level of the group B mice is obviously reduced (P < 0.05), and the IL-4 concentration level is obviously increased (P < 0.05); compared to group B, the level of IFN- γ concentration in the aerosolized inhalation high dose heat-inactivated clostridium butyricum intervention group (group D) was significantly increased (P < 0.05), the level of IL-4 concentration was significantly decreased (P < 0.05), see fig. 4.
3.5 ratio of spleen Th17 and Treg cells: the Th17 cell fraction and Th17/Treg ratio were significantly increased in group B mice compared to group a (P < 0.05); compared to group B, the Treg cells of the aerosolized inhalation high dose heat-inactivated clostridium butyricum intervention group (group D) were significantly increased (P < 0.05), the Th17 cell ratio and Th17/Treg ratio were significantly decreased (P < 0.05), see table 2.
TABLE 2 spleen Th17 and Treg cells M (P25, P75)
Conclusion: the aerosol inhalation heat-inactivated clostridium butyricum, the oral heat-inactivated clostridium butyricum and the oral clostridium butyricum viable bacteria can obviously reduce airway inflammation and airway mucus secretion of asthmatic mice, wherein the aerosol inhalation heat-inactivated clostridium butyricum has the best effect; aerosol inhalation of heat-inactivated clostridium butyricum can exert an anti-asthmatic effect by regulating immune cells and cytokines; both aerosol inhalation and oral heat-inactivated clostridium butyricum can be used for the prevention or treatment of asthma.
Claims (4)
1. Use of heat-inactivated clostridium butyricum in the preparation of a medicament for treating asthma, wherein the clostridium butyricum is clostridium butyricum CGMCC NO:0313-1.
2. The use according to claim 1, characterized in that: the heat-inactivated clostridium butyricum is used as a single preparation or is compatible with other medicines, a proper amount of pharmaceutically acceptable auxiliary materials are added, and the heat-inactivated clostridium butyricum is prepared into a pharmaceutically acceptable dosage form by adopting the prior art.
3. The use according to claim 2, characterized in that: the dosage forms of the medicine comprise aerosol, inhalant, granule, capsule, tablet, pill, oral liquid or injection.
4. The use according to claim 2, characterized in that: the medicine is prepared into microcapsule, microsphere, nanoparticle or liposome.
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