CN117982544A - Application of Lactobacillus reuteri Glory LR15 in regulation of obese asthma products - Google Patents
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Abstract
The invention discloses an application of lactobacillus reuteri Glory LR15 in the regulation of obesity-type asthma products, wherein the lactobacillus reuteri Glory LR15 is preserved in China general microbiological culture Collection center (CGMCC) No.26972, the preservation time is 2023 and 31 days, and the preservation address is North Star Xiu No. 1,3 of the Chao Yangjinggao area of Beijing, and the institute of microbiology of China academy of sciences. The lactobacillus reuteri Glory LR15 disclosed by the invention can effectively reduce the central airway resistance of mice in vivo experiments, reduce neutrophils in alveolar lavage fluid, obviously reduce the specific IgE level in mouse serum, obviously reduce Th2 cells in spleen and obviously increase Treg cells in spleen, and has the potential of being applied to regulation of obesity asthma. The invention can provide new raw materials for preparing and developing products related to preventing or regulating obesity and asthma.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to application of lactobacillus reuteri Glory LR15 in regulation of obesity-type asthma products.
Background
Bronchial asthma (asthma for short) is a major public health problem affecting millions of people throughout the world. Epidemiological studies have shown that obesity and overweight lead to an increased risk of asthma and exacerbate asthma symptoms. Currently, obese asthma is considered a refractory asthma phenotype. Compared to simple asthma, the respiratory symptoms of obesity type asthma are more severe and more frequent. Obesity can lead to a range of metabolic changes, such as insulin resistance leading to systemic and tissue-specific metabolic defects such as inflammation, oxidative stress, endoplasmic reticulum stress, lipotoxicity, and mitochondrial dysfunction. Meanwhile, metabolic changes in obesity (such as diabetes and insulin resistance) are associated with the pathogenesis of asthma. Obesity-type asthma patients often have abnormal glycolipid metabolism and insulin resistance and are characterized by metabolic syndrome, but the specific mechanism between obesity and asthma is still unknown. There is growing evidence that alterations in mitochondrial number and function play an important role in the pathogenesis of obese asthma, while mitochondrial targeting antioxidants have a protective effect on leptin-induced increases in human bronchial epithelial reactive oxygen species (reactive oxygen species, ROS).
The improvement of modern eating habits and living standards has caused obesity to become popular worldwide, leading to an increase in the incidence of a range of complications. There is a large population of data to find that obesity is an important risk factor for asthma, and that the prevalence of asthma in overweight and obese children and adults is higher than that in normal individuals. The obese asthmatic population identified by BMI had elevated neutrophils in sputum, whereas eosinophils were not. In addition, obesity may exacerbate asthma conditions by ways other than allergic asthma, such as by promoting polarization of macrophages from M2 to M1.
It is currently believed that the pathogenesis of obesity-type asthma involves mainly non-Th 2-type inflammation. non-Th 2 type inflammation is characterized by inflammatory infiltration of Th1, th17 and neutrophils with their corresponding cytokines such as TNF-alpha, IL-17, etc. Studies show that IL-17 of the obese asthma patient is higher than that of the common asthma patient, and obvious TH17 inflammation exists in the obese asthma patient and has a certain correlation with obesity. IL-17A expression in the lung was reduced in obese asthmatic mice by weight loss. Thus, inhibition of TH17 overexpression may be one of the therapeutic targets for obesity-type asthma. Studies have shown that obese asthmatics present with systemic chronic inflammatory conditions, elevated levels of TNF- α, IL-6 and leptin in serum, and reduced levels of adiponectin, which is positively correlated with the severity of clinical symptoms of asthma, and that increased systemic inflammatory mediators associated with obesity may exacerbate pulmonary inflammation, thereby exacerbating asthma. In addition, activated lymphocytes in the intestinal mucosal immune system can also function by blood reaching multiple mucosal-related lymphoid tissues including the respiratory tract. Intestinal mucosa innate immune cells such as Th17 cells, ILC2s and ILC3s can migrate to respiratory tract directly via peripheral circulation, and directly participate in respiratory tract immune cell activation and immune homeostasis maintenance. Thus, the onset of obesity-type asthma is associated with an immune imbalance in the body, and inflammation of the airways may be caused by infiltration of Th17 cells, ILC2s, ILC3s and cytokines thereof, resulting in onset or exacerbation of obesity-type asthma.
Most of the research on probiotics is limited to lactobacillus and bifidobacterium, and the treatment with bifidobacterium can improve airway hyperresponsiveness of asthmatic patients and reduce the concentration of TH 2-mediated inflammation markers. Clinical treatment finds that the treatment mode or the medicine of normal asthma is not completely effective for obese asthma people, so that the search for the treatment mode and the medicine for preventing or relieving the obese asthma is urgent.
Disclosure of Invention
In view of the shortcomings of the prior art, the invention aims to provide application of lactobacillus reuteri Glory LR15 in regulation of obese asthma products, and the lactobacillus reuteri Glory LR15 is found to be beneficial to relieving obese asthma through an obese asthma mouse model.
In order to achieve the above purpose, the present invention provides the following technical solutions:
an application of Lactobacillus reuteri GLORY LR15 in the preparation of obesity-related asthma products, wherein the Lactobacillus reuteri GLORY LR15 is preserved in China general microbiological culture collection center (CGMCC) No.26972, the preservation time is 2023, 3 and 31 days, and the preservation address is North Star XILU 1, 3, the institute of microbiology, national academy of sciences.
Preferably, the product is a food and/or pharmaceutical product.
Preferably, in said product, lactobacillus reuteri Glory LR15 is a live strain.
Preferably, in the product, the viable count of Lactobacillus reuteri Glory LR15 is not less than 1.0X10 11 CFU/mL or 1.0X10 11 CFU/g.
Preferably, the lactobacillus reuteri Glory LR15 is capable of modulating the efficacy of obese asthma in reducing central airway resistance and reducing neutrophil content in alveolar lavage fluid in obese mice.
Preferably, the lactobacillus reuteri Glory LR15 is capable of reducing inflammatory cell infiltration and mucogenesis or subepithelial collagen deposition.
Preferably, the lactobacillus reuteri Glory LR15 is capable of modulating the efficacy of obese asthma is a significant reduction in serum total IgE, allergen-specific IgE, specific IgG1 and specific IgG2a levels, and a significant reduction in Th2 cells in the spleen.
Compared with the prior art, the invention has the following beneficial effects:
Experimental results show that the lactobacillus reuteri Glory LR15 provided by the invention can reduce the central airway resistance of mice, remarkably reduce the levels of total IgE, OVA specific IgE, specific IgG1 and specific IgG2a in serum, remarkably reduce the content of neutrophils in alveolar lavage fluid and remarkably reduce Th2 cells in spleen, is beneficial to relieving obesity asthma, and can provide a new raw material for preparing and developing products related to preventing or regulating obesity asthma.
Drawings
FIG. 1 is a phylogenetic tree constructed using the method of adjacency as described in example 1. Wherein DSM 20016T, CECT8605, RC-14 and ZLR003 are Lactobacillus reuteri.
FIG. 2 is a thermal diagram of ANI values as described in example 1;
FIG. 3 is a diagram of the iprobiotics platform prediction results described in example 1;
FIG. 4 is a graph showing the effect of Lactobacillus reuteri Glory LR15 on central airway resistance in obese asthmatic mice as described in example 2;
FIG. 5 is a graph showing the effect of Lactobacillus reuteri Glory LR15 on immune cell typing in alveolar lavage fluid of obese asthmatic mice as described in example 2;
FIG. 6 shows the lung HE staining of the mice described in example 2 at 30X magnification;
FIG. 7 is a graph of MASSON staining of the lungs of the mice described in example 2, at 30X magnification;
FIG. 8 is a graph showing the effect of Lactobacillus reuteri Glory LR15 on cytokines in serum of obese asthmatic mice described in example 2, wherein A is total IgE level in serum, B is OVA-specific IgE level in serum, C is OVA-specific IgG1 in serum, D is OVA-specific IgG2a level in serum;
FIG. 9 shows the effect of Lactobacillus reuteri Glory LR15 on immune cells in the spleen of obese asthmatic mice described in example 2, wherein A is the proportion of Th1 cells in CD4 + T cells in the spleen of each group of mice, B is the proportion of Th2 cells in CD4 + T cells in the spleen of each group of mice, and C is the proportion of Treg cells in CD4 + T cells in the spleen of each group of mice.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The data of the test results of the following examples are all expressed as mean.+ -. Standard error of mean (means.+ -. SEM). Statistical analysis used One-way analysis of variance (One-WAY ANALYSIS of variance), with Tukey multiple comparison assay (Tukey's Multiple Comparison Test) as post-test comparison group differences. When p < 0.05 (/ x) or < 0.01 (/ x) or < 0.001 (/ x) or < 0.0001 (/ x), it is considered that the differences between the control group and the other groups are statistically significant.
Ovalbumin (OVA), methyl choline, and ELISA kits were all available from sigma company in the united states; control feeds (rat and mouse maintenance feed, cat No. 1010088, SPF grade irradiation sterilization) and 60% high fat feeds (cat No. XTHF, SPF grade irradiation sterilization) were purchased from Jiangsu co-biology limited; antibodies L/D-FSV700, CD3-APC-CY7, CD4-FITC, T-bet-PC5.5, GATA3-BV421, and streaming buffers used in flow cytometry were purchased from BD company.
The invention provides a lactobacillus reuteri, which is classified and named as lactobacillus reuteri (Limosilactobacillus reuteri) Glory LR15 and is preserved in China general microbiological culture collection center (CGMCC), address: the collection date is 2023, 3 and 31 days of the national academy of sciences of China, the Qingyang area North Star, the West way No. 1, the No. 3 and the collection number is CGMCC No.26972.
Example 1
Separation, purification and genome identification of lactobacillus reuteri Glory LR15
1ML of yogurt samples were vortexed in 9mL of sterile 0.85% physiological saline solution (NaCl), 10 -1 dilutions, and so on, serially diluted to a gradient of 10 -8, then 1mL of dilutions were each aspirated into a petri dish, and poured into MRS agar medium at about 45℃for mixing, anaerobic incubation at 37℃for 24h, colony diameters of about 1mm when grown on MRS (ManRogosa Sharpe Broth) agar medium, were shiny, dome-shaped opaque, and displayed tackiness when contacted with an inoculating loop. Suspected colonies were picked with an inoculating needle according to the above biological characteristics and streaked in MRS plates, cultured at 37℃for 24 hours, and then analyzed for their whole genome.
As shown in Table 1, limosilactobacillus reuteriGlory LR chromosome genome length is 2.24Mb, and there are 2,330 CDSs (coding genes) in total; the plasmid genome was 1.31kb in length. ANI heat maps and phylogenetic tree maps constructed with 1201 core genes were drawn with Limosilactobacillus reuteriCECT8605, limosilactobacillus reuteri RC-14 and Limosilactobacillus reuteri ZLR003 using Limosilactobacillus reuteriDSM 20016T as model strain. Combining phylogenetic tree (figure 1) and ANI results (figure 2) shows that ANI between Gly LR15 and Limosilactobacillus reuteriDSM and 20016T is 95.39%, and ANI value between each strain in the strain is in the range of 94.87% -98.74%, which shows that Limosilactobacillus reuteri kernel nucleotides have lower consistency. The Gly LR15 and RC-14 are gathered on the same branch, and have common ancestor, but are far apart. As shown in FIG. 3, the probability of Gly LR15 being a probiotic was 99.96% and slightly higher than RC-14 (98.97%) as determined by the iProbiotics platform Model1: probiotic Predictor analysis.
TABLE 1 Table of essential information on genome of strains
Example 2
Effect of Lactobacillus reuteri Gly LR15 on obese asthmatic mice
(1) 28 SFP-class female BALB/c mice of 5-6 weeks of age were randomly divided into four groups of 7, control group, control asthma group, high-fat asthma group, lactobacillus reuteri Glory LR15 group, respectively. After one week of adaptive feeding, the control group and the control asthma group were fed with the control feed for 12 weeks, and the high-fat asthma group and the lactobacillus reuteri Glory LR15 group were fed with the high-fat feed for 12 weeks. At the same time, the lactobacillus reuteri Glory LR15 group of mice were given 3×10 9 CFU/lactobacillus reuteri Glory LR15 (mixed with sterile PBS shaking) daily and the remaining group of mice were given an equal volume of sterile PBS daily by gavage. Subsequently, 200. Mu.L of ovalbumin solution (containing 50. Mu.g ovalbumin and 2mg aluminum hydroxide gel) was injected intraperitoneally into mice of the control asthma group, the high-fat asthma group, and the Lactobacillus reuteri Glory LR15 group, respectively, on days 1, 7, and 14, and finally, nasal drip excitation was performed using 200. Mu.L of ovalbumin solution (containing 200. Mu.g ovalbumin) on days 22-24.
(2) 24 Hours after the last challenge, the mice were intubated with an anesthetic injected into the abdomen and the central airway resistance was measured using a pulmonary function machine with a stimulus of methyl choline 0, 3.125, 6.25, 12.5, 25, 50 mg/mL. As shown in fig. 4, lactobacillus reuteri Glory LR15 group was able to reduce central airway resistance in obese asthma, reducing airway responsiveness. This suggests that lactobacillus reuteri can alleviate asthma in obese mice.
(3) The mice were sacrificed by cervical removal and the lungs were rinsed with PBS to obtain alveolar lavage fluid for immunocytotyping. As shown in FIG. 5, lactobacillus reuteri Glory LR15 was able to reduce neutrophil content. Obesity exacerbates mouse asthma by recruiting more neutrophils, and thus the results suggest that lactobacillus reuteri can alleviate obesity asthma.
(4) A portion of the lung tissue was removed and fixed in 4% paraformaldehyde at 24 h, dehydrated using a dehydrator, and then transparently treated with xylene. Finally, paraffin-embedded sections were stained with Hematoxylin and Eosin (HE) and scanned. The results are shown in figure 6, where lactobacillus reuteri bronchial wall thickness is thin and inflammatory cell infiltration around bronchioles is reduced compared to the high-fat asthma group.
(5) And (5) taking the lung tissue wax block in the step (4) for dyeing treatment. Nuclei were first stained with Regaud's hematoxylin stain or Weigert hematoxylin and thoroughly washed. The tissue samples were then placed in a 1% aniline blue dye until the collagen fibers appeared blue. The tissue samples were further stained by immersing in acid fuchsin dye. Alcohol is used for dehydration and dibenzoate or similar solvents for cleaning. Finally, the slide is blocked with a blocking agent and scanned. The results are shown in fig. 7, where lactobacillus reuteri significantly reduced collagen deposition in the airways of high-fat asthma, demonstrating that lactobacillus reuteri was able to attenuate airway remodeling.
(6) After the measurement is completed, the eyeball is picked up and blood is taken. Total IgE, specific IgG1 and specific IgG2a levels in mouse serum were determined using ELISA kit. As shown in FIG. 8, lactobacillus reuteri Glory LR15 can reduce serum total IgE level of obese asthma mice, and can also significantly reduce OVA-specific IgE, OVA-specific IgG1 and OVA-specific IgG2a levels in serum of obese asthma mice. This suggests that lactobacillus reuteri Glory LR15 significantly reduces OVA allergic asthma in obese mice.
(7) Spleens of mice were taken for flow cytometry. As shown in fig. 9, lactobacillus reuteri Glory LR15 significantly increased Th1 cell and Treg cell content in the spleen of obese asthmatic mice, significantly reduced Th2 cell content. Lactobacillus reuteri Glory LR15 can reduce symptoms of obesity asthma by decreasing Th2 cells involved in allergic asthma and increasing Treg cells that regulate allergic reactions.
In conclusion, the lactobacillus reuteri disclosed by the invention can obviously reduce the OVA specific IgE antibodies of obese asthma mice and regulate immune cells such as Th1, th2 and Treg cells in organisms, so that inflammatory cell infiltration in lungs and airway collagen deposition are reduced, and the obese asthma reaction is obviously relieved. Thus, the lactobacillus reuteri can be used for relieving obesity asthma.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (7)
1. The application of lactobacillus reuteri Gly LR15 in the regulation of obesity-type asthma products is characterized in that the lactobacillus reuteri Gly LR15 is preserved in China general microbiological culture Collection center (CGMCC) No.26972, the preservation time is 2023 and 31 days, and the preservation address is North Star Xiu No. 1, 3 of the Chao Yangjing area of Beijing, and the institute of microbiology of China academy of sciences.
2. Use according to claim 1, wherein the product is a food and/or pharmaceutical product.
3. Use according to claim 1, characterized in that in the product lactobacillus reuteri Glory LR15 is a live strain.
4. Use according to claim 1, characterized in that in the product the viable count of lactobacillus reuteri Glory LR15 is not lower than 1.0 x 10 11 CFU/mL or 1.0 x 10 11 CFU/g.
5. The use according to claim 1, wherein the lactobacillus reuteri Glory LR15 is capable of modulating the efficacy of obesity asthma in reducing central airway resistance and reducing neutrophil content in alveolar lavage fluid in obese mice.
6. The use according to claim 1, wherein the lactobacillus reuteri Glory LR15 is capable of reducing inflammatory cell infiltration and airway myxogenesis or subepithelial collagen deposition.
7. The use according to claim 1, wherein the lactobacillus reuteri Glory LR15 is capable of modulating the efficacy of obesity asthma is a significant reduction in serum total IgE, allergen specific IgE, specific IgG1 and specific IgG2a levels, and a significant reduction in Th2 cells in spleen.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106794207A (en) * | 2014-10-29 | 2017-05-31 | 雀巢产品技术援助有限公司 | Using lactobacillus reuteri in life early recovery micropopulation ecological disturbance |
| CN114717127A (en) * | 2021-03-31 | 2022-07-08 | 青岛蔚蓝生物股份有限公司 | Lactobacillus reuteri capable of preventing and relieving allergic asthma symptoms and application thereof |
| WO2022164784A1 (en) * | 2021-01-26 | 2022-08-04 | Seed Health Inc. | Methods of probiotic treatment to improve human health |
| CN117042779A (en) * | 2021-01-26 | 2023-11-10 | 种子保健有限公司 | Probiotic treatment method for improving human health |
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| CN106794207A (en) * | 2014-10-29 | 2017-05-31 | 雀巢产品技术援助有限公司 | Using lactobacillus reuteri in life early recovery micropopulation ecological disturbance |
| WO2022164784A1 (en) * | 2021-01-26 | 2022-08-04 | Seed Health Inc. | Methods of probiotic treatment to improve human health |
| CN117042779A (en) * | 2021-01-26 | 2023-11-10 | 种子保健有限公司 | Probiotic treatment method for improving human health |
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