Lactic acid bacteria composition for regulating immune cell differentiation and / or reducing inflammatory hormone secretion and use thereof
By using the composition of Lactobacillus reuteri and Lactobacillus rhamnosus F-1 strain, the differentiation of immune cells and the secretion of CXCL10 was adjusted, and the polarization of macrophages into M1 type in allergic rhinitis was solved. The problem of inflammatory response has achieved the effect of alleviating inflammatory response and improving symptoms.
Patent Information
- Application Number
- CN202410020814.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2023-08-02
- Filing Date
- 2024-01-05
- Publication Date
- 2025-02-14
AI Technical Summary
In patients with allergic rhinitis, macrophages tend to polarize into the M1 type, resulting in a strong inflammatory response. The prior art is difficult to effectively regulate immune cell differentiation and reduce inflammatory hormone secretion.
The combination of the Limosilactobacillus reuteri GL-104 strain and the Lacticaseibacillus rhamnosus F-1 strain was used to regulate immune cell differentiation and reduce CXCL10 secretion by mixing different proportions.
This composition can reduce the inflamed hormone CXCL10 secreted by macrophages, promote the polarization of macrophages into the M2 type, thereby reducing the inflammation response and improving the symptoms of allergic rhinitis.
Smart Images
Figure CN119424484A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field related to probiotics, and particularly relates to a lactic acid bacteria composition for regulating immune cell differentiation and / or reducing inflammatory hormone secretion, and uses of the composition, such as treating or preventing allergic rhinitis. Background Art
[0002] Allergic rhinitis is a symptom of rhinitis caused by the immune system being affected by allergens in the air. The main symptoms include: excessive nasal discharge, nasal congestion, frequent sneezing, itchy eyes, and watery eyes. Common allergens include: pollen, dust mites, and pet dander. Although allergic rhinitis does not pose a threat to life, it may have a serious negative impact on the quality of daily life, such as: lack of concentration, insomnia, dizziness, fatigue, etc.
[0003] The occurrence of allergic rhinitis is related to the differentiation of immune cells. Monocytes can differentiate into macrophages, which can be divided into three different types: M0, M1, and M2. M0 macrophages can polarize into M1 or M2 macrophages according to environmental conditions. The receptor CD86 is highly expressed on the surface of M1 macrophages, and M1 macrophages induce inflammatory responses, which helps to resist infection, enhance T cell activity, and resist or clear diseased tissues. The receptor CD206 is highly expressed on the surface of M2 macrophages, and M2 macrophages are involved in eliminating inflammatory responses and tissue repair. In patients with allergic rhinitis, macrophages tend to polarize into the M1 type, resulting in an excessively strong inflammatory response, causing symptoms such as runny nose, nasal congestion, sneezing, and itchy eyes. When the number of M2 macrophages decreases, the ability to suppress allergies will decrease, causing allergic symptoms to worsen.
[0004] CXCL10 (CXC motif chemokine 10) is also known as "10kD IFN-γ inducible protein (interferon-γinducible protein 10kD, IP-10)", which can recruit T cells and other white blood cells, such as natural killer cells or dendritic cells. Studies have shown that the expression of CXCL10 in the nasal mucosa of patients with allergic rhinitis is increased, and the degree of expression is proportional to the severity of symptoms. In animal models of allergic rhinitis, it was found that inhibiting the expression of CXCL10 can relieve rhinitis symptoms.
[0005] Allergic reactions are closely related to intestinal health; that is, the composition of intestinal flora is directly related to the occurrence of allergic diseases. Generally speaking, the higher the diversity of intestinal flora, the better the health. Studies have shown that probiotics can regulate the response of the immune system, maintain the tightness between intestinal cells, inhibit harmful microorganisms, produce short-chain fatty acids, and reduce allergic reactions caused by intestinal leakage. In vitro experiments show that probiotics can promote the polarization of macrophages to the M2 form, which has the effects of reducing inflammation and improving the symptoms of allergic rhinitis. This can provide a new perspective for the development of relief of related symptoms.
[0006] Therefore, it is necessary to develop a safe and long-term probiotic formula that can regulate the immune system. As we all know, probiotics are food or health products, over-the-counter drugs, and usually do not have adverse effects on the human body. Therefore, proposing a probiotic formula that can improve immune-related diseases is indeed one of the topics that people in this technical field are actively pursuing. Summary of the invention
[0007] The present invention is based on the discovery that a combination of specific lactic acid bacteria strains can reduce the inflammatory hormone CXCL10 produced by immune cells and increase the CD206 / CD86 value on the cell surface.
[0008] Accordingly, one object of the present invention is to provide a lactic acid bacteria composition for regulating immune cell differentiation and / or reducing inflammatory hormone secretion, the composition comprising: (a) Lactobacillus reuteri GL-104 strain; and (b) Lactobacillus rhamnosus F-1 strain; wherein the GL-104 strain is deposited in the China Center for Type Culture Collection with the deposit number CCTCC M209138, and the F-1 strain is deposited in the China Center for Type Culture Collection with the deposit number CCTCC M2011124.
[0009] For example, based on the total strain amount of GL-104 strain and F-1 strain, GL-104 strain accounts for 10% to 90%, and F-1 strain accounts for 10% to 90%.
[0010] For example, based on the total strain amount of GL-104 strain and F-1 strain, GL-104 strain accounts for 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, and F-1 strain accounts for 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.
[0011] For example, based on the total strain amount of GL-104 strain and F-1 strain, GL-104 strain accounts for 30% and F-1 strain accounts for 70%.
[0012] For example, the GL-104 strain is an active strain or an inactivated strain, and the F-1 strain is an active strain or an inactivated strain.
[0013] For example, the lactic acid bacteria composition further includes: (c) an excipient, a diluent, or a carrier.
[0014] Illustratively, the excipient, diluent, or carrier is a food-acceptable excipient, diluent, or carrier, or a pharmaceutically acceptable excipient, diluent, or carrier.
[0015] Another object of the present invention is to provide a use of the lactic acid bacteria composition, which is used to prepare a composition for treating or preventing allergic rhinitis.
[0016] For example, the prepared composition achieves the treatment or prevention of allergic rhinitis by reducing the production of inflammatory hormone CXCL10 by immune cells.
[0017] For example, the prepared composition achieves the treatment or prevention of allergic rhinitis by increasing the cell surface CD206 / CD86 value of immune cells. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] To make the above and / or other objects, features, advantages, and embodiments of the present invention more clearly understood, detailed descriptions of the accompanying drawings are as follows:
[0019] Figure 1 A histogram comparing the effects of mixing GL-104 strain and F-1 strain in different ratios on the expression of inflammatory hormone CXCL10 by M1 macrophages, where the symbol * indicates P value < 0.05, the symbol ** indicates P value < 0.01, the symbol *** indicates P value < 0.001, and the symbol **** indicates P value < 0.0001;
[0020] Figure 2 A histogram comparing the effects of GL-104, F-1, and other strains on the expression of inflammatory hormone CXCL10 by M1 macrophages, where the symbol * indicates P value < 0.05, and the symbol **** indicates P value < 0.0001;
[0021] Figure 3 A histogram comparing the effects of GL-104 strain, F-1 strain, and any combination of other strains on the expression of inflammatory hormone CXCL10 by M1 macrophages, where the symbol * indicates P value < 0.05, and the symbol *** indicates P value < 0.001;
[0022] Figure 4is a histogram comparing the effects of mixing GL-104 strain and F-1 strain in different ratios on the polarization of M1 macrophages into M2 macrophages, where the symbol *** indicates P value < 0.001, and the symbol **** indicates P value < 0.0001;
[0023] Figure 5 is a histogram comparing the effects of GL-104 strain, F-1 strain, and other strains on the polarization of M1 macrophages into M2 macrophages, where the symbol * indicates P value < 0.05, and the symbol *** indicates P value < 0.001;
[0024] Figure 6 The figure is a histogram comparing the effects of GL-104 strain, F-1 strain and any combination of two of the other strains on the polarization of M1 macrophages into M2 macrophages, wherein the symbol * indicates P value < 0.05, and the symbol ** indicates P value < 0.01. DETAILED DESCRIPTION
[0025] In order to make the above and / or other purposes, effects, and features of the present invention more clearly understood, preferred embodiments are specifically described below in detail as follows:
[0026] The lactic acid bacteria strains mentioned in this article are deposited in the form of freeze-dried cultures at the China Center for Type Culture Collection, Wuhan University, Wuhan, China. The deposit details are listed in Table 1:
[0027] Table 1. Strain deposit information
[0028]
[0029] In this article, it was found that the Lactobacillus reuteri GL-104 strain and the Lactobacillus rhamnosus F-1 strain listed in Table 1 can work together to reduce the secretion of CXCL10 by macrophages and promote the polarization of macrophages to the M2 morphology, so the obtained lactic acid bacteria combination formula can be used to treat or prevent allergic rhinitis.
[0030] According to one embodiment of the present invention, a lactic acid bacteria composition for regulating immune cell differentiation and / or reducing inflammatory hormone secretion is provided. The composition comprises: a Lactobacillus reuteri GL-104 strain deposited in the China Center for Type Culture Collection under the deposit number CCTCC M209138; and a Lactobacillus rhamnosus F-1 strain deposited in the China Center for Type Culture Collection under the deposit number CCTCCM2011124.
[0031] The composition may further include: excipients, diluents, or carriers to make the composition present in different forms. The excipients, diluents, or carriers may be, but are not limited to, excipients, diluents, or carriers acceptable to food, or excipients, diluents, or carriers acceptable to medicine.
[0032] The composition is a food composition when a food-acceptable excipient, diluent, or carrier is used. Examples of food-acceptable excipients, diluents, or carriers are food, but are not limited thereto. Food may be fluid dairy products (e.g., milk or condensed milk), fermented dairy products (e.g., yogurt), milk powder, ice cream, cheese, cottage cheese, soy milk, fermented soy milk, vegetable and fruit juices, fruit juices, sports drinks, jellies, biscuits, energy bars, health foods, animal feeds, or dietary supplements, but are not limited thereto.
[0033] The composition is a pharmaceutical composition when a pharmaceutically acceptable excipient, diluent, or carrier is used. Examples of pharmaceutically acceptable excipients, diluents, or carriers include solvents, buffers, emulsifiers, suspending agents, decomposers, disintegrants, dispersants, binders, stabilizers, chelating agents, gelling agents, wetting agents, lubricants, absorption delaying agents, or liposomes, but are not limited thereto.
[0034] In addition, based on the total strain amount of GL-104 strain and F-1 strain, the GL-104 strain can account for 10% to 90%, and the F-1 strain can account for 10% to 90%; preferably, the GL-104 strain accounts for 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, and the F-1 strain accounts for 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%; more preferably, the GL-104 strain accounts for 30% and the F-1 strain accounts for 70%.
[0035] As mentioned above, the composition can reduce the secretion of CXCL10 by macrophages and promote the polarization of macrophages to the M2 type, and can therefore be used to treat or prevent diseases or symptoms caused by excessive secretion of CXCL10 by macrophages or excessive polarization of macrophages to the M1 type, such as allergic rhinitis.
[0036] According to another embodiment of the present invention, a use of the above-mentioned lactic acid bacteria composition is provided, which is used to prepare a composition for treating or preventing allergic rhinitis. Specifically, the prepared composition can reduce the production of inflammatory hormone CXCL10 by immune cells (such as macrophages) or increase the cell surface CD206 / CD86 value of immune cells (such as macrophages) to achieve the purpose of treating or preventing allergic rhinitis.
[0037] More specifically, the prepared composition can be administered to an individual who has a need for treatment or prevention of allergic rhinitis to achieve treatment or prevention of allergic rhinitis. The so-called "administration" can be administered by, but is not limited to, oral, sublingual, rectal, nasal, vaginal, abdominal, transdermal, epidermal, intra-articular, intraocular, or ocular surface administration. The so-called "individual" can be, but is not limited to, mammals; examples of mammals are primates, cats, dogs, mice, rats, rabbits, cows, horses, goats, sheep, or pigs, but are not limited thereto; examples of primates are chimpanzees, humans, gorillas, bonobos, orangutans, or monkeys, but are not limited thereto.
[0038] When the subject is a human, the dosage may be 10 6 Up to 10 10 CFU of GL-104 strain and F-1 strain as a whole, but not limited thereto; preferably, 10 per kg body weight per day 7 Up to 10 9 CFU of GL-104 strain and F-1 strain as a whole; preferably, 10 per kg body weight per day 8 CFU of GL-104 strain and F-1 strain as a whole.
[0039] The present invention is illustrated by the following embodiments:
[0040] <Example 1: Morphology and general properties of strains>
[0041] The taxonomic characteristics of the strains were confirmed based on the results of 16S rDNA sequence analysis and API bacterial identification system analysis. The morphological and general characteristics of the strains are listed in Table 2:
[0042] Table 2. Morphological and general characteristics of the strains
[0043]
[0044] <Example 2: Preparation of bacterial solution>
[0045] The strain was stored at -80°C with 20% glycerol. After being taken out of the -80°C environment, the strain was activated with MRS broth containing 0.05% cysteine at 37°C for 24 hours. Afterwards, an appropriate amount of PBS was taken to disperse the suspended strain and adjust the strain concentration of the obtained bacterial solution to 10 9 CFU / mL.
[0046] Please refer to Table 3 for the composition and strain amount of the probiotic samples obtained by mixing bacterial solutions of different strains.
[0047] Table 3. Composition and strain quantity of probiotic samples
[0048]
[0049] The carbon source of the strain culture solution used in this embodiment can be glucose, fructose, lactose, sucrose, maltose, galactose, mannose, trehalose, starch, molasses, potato starch, corn starch, malt extract, maltodextrin, or any combination thereof. For example, the strain culture solution used contains a mixture of 2wt% to 5wt% of glucose and maltodextrin; preferably, the liquid culture solution contains a mixture of 3wt% of glucose and maltodextrin.
[0050] The nitrogen source of the strain culture solution used in this embodiment can be (NH4)2SO4, (NH4)2PO4, NH4NO3, NH4Cl, casamino acid, urea, peptone, polypeptone, tryptone, meat extract, yeast extract, yeast powder, milk, soybean flour, whey, or any combination thereof. For example, the strain culture solution used contains at least one of 5wt% to 30wt% of milk and 1wt% to 10wt% of soybean flour.
[0051] <Example 3: Expression test of inflammatory hormone CXCL10>
[0052] Here, THP-1 cells were selected to evaluate the ability of probiotics to regulate immunity. THP-1 cells can differentiate into three different types of macrophages: M0, M1, and M2.
[0053] With a density of 1x10 6 THP-1 cells were seeded in a 10 cm dish at 10 cells / mL and treated with 200 ng / mL PMA for 24 hours to differentiate the cells into M0 macrophages. Then, the cells were washed with PBS and treated with 5 mL trypsin. The cells were centrifuged at 1,500 rpm for 15 minutes to obtain cell pellets. 5The cells were seeded in a 12-well culture dish at 10 cells / mL and rested for 24 hours. After the cells were differentiated into M1 macrophages with 40ng / mL IFN-γ and 200ng / ml LPS, the culture was continued for 48 hours. The probiotic sample was dissolved in antibiotic-free cell culture medium and co-cultured with M1 macrophages for 48 hours (the ratio of macrophage number to strain number was 1:100). After culture, the culture medium was centrifuged at 2,000rpm for 5 minutes, the bacteria were removed and the supernatant was obtained. Finally, the CXCL10 content in the supernatant was analyzed by ELISA, and the results of different groups were compared by Ordinary one-way ANOVA method.
[0054] Please refer to Figure 1 , GL-104 strain and F-1 strain mixed in any ratio can inhibit the activity of M1 macrophages to express the inflammatory hormone CXCL10 better than the single GL-104 strain and the single F-1 strain. In particular, the inhibitory effect obtained by mixing 70% F-1 strain and 30% GL-104 strain ("70% F-1 + 30% GL-104" group) is the most significant.
[0055] Please refer to Figure 2 , with the same total strain amount, GL-104 and RC-14 belong to the same group, Lactobacillus reuteri, and the former has a better inhibitory activity on the expression of inflammatory hormone CXCL10 in M1 macrophages than the latter; F-1 and LGG belong to the same group, Lactobacillus rhamnosus, and the former has a better inhibitory activity on the expression of inflammatory hormone CXCL10 in M1 macrophages than the latter. In addition, the inhibitory activity of the combination of GL-104 and F-1 is better than that of the single GL-104 and single F-1 strains.
[0056] Please refer to Figure 3 When the strain amounts of all single strains were the same, the combination of GL-104 strain and F-1 strain had a better activity in inhibiting the expression of inflammatory hormone CXCL10 by M1 macrophages than other combinations.
[0057] It should be noted that Figures 1 to 3 The "relative amount of CXCL10" indicated on the Y-axis is calculated based on the expression of the inflammatory hormone CXCL10 in untreated M1 macrophages.
[0058] In summary, it was confirmed that the GL-104 strain and the F-1 strain can act synergistically on M1 macrophages to inhibit their expression of the inflammatory hormone CXCL10.
[0059] <Example 4: Test of polarization toward M2 macrophages>
[0060] With a density of 1x10 6THP-1 cells were seeded in a 10 cm dish at 10 cells / mL and treated with 200 ng / mL PMA for 24 hours to differentiate the cells into M0 macrophages. Then, the cells were washed with PBS and treated with 5 mL trypsin. The cells were centrifuged at 1,500 rpm for 15 minutes to obtain cell pellets. 5 Cells were seeded in 12-well culture plates at 10 cells / mL and rested for 24 hours. Cells were differentiated into M1 macrophages with 40ng / mL IFN-γ and 200ng / ml LPS, or into M2 macrophages with 40ng / mL IL-4, and then cultured for 48 hours. Probiotic samples were dissolved in antibiotic-free cell culture medium and co-cultured with M1 macrophages for 48 hours (the ratio of macrophage number to strain number was 1:100). Finally, after washing the cells, anti-CD206 antibody and anti-CD86 antibody were used as primary antibodies, and the corresponding antibodies were used as secondary antibodies for fluorescent staining to analyze the CD206 / CD86 value. In addition, the results of different groups were compared by the Ordinary one-way ANOVA method.
[0061] Please refer to Figure 4 , GL-104 strain and F-1 strain mixed in any ratio can promote the activity of polarization of M1 macrophages into M2 macrophages better than single GL-104 strain and single F-1 strain. In particular, the most significant promotion effect was obtained by mixing 70% F-1 strain and 30% GL-104 strain ("70% F-1 + 30% GL-104" group).
[0062] Please refer to Figure 5 , under the same total strain amount, GL-104 strain and RC-14 strain belong to Lactobacillus reuteri, and the former has better activity in promoting polarization of M1 macrophages into M2 macrophages than the latter; F-1 strain and LGG strain belong to Lactobacillus rhamnosus, and the former has better activity in promoting polarization of M1 macrophages into M2 macrophages than the latter. In addition, the promotion activity of the combination of GL-104 strain and F-1 strain is better than that of GL-104 strain alone and F-1 strain alone. Moreover, the CD206 / CD86 value obtained by the combination of GL-104 strain and F-1 strain acting on M1 macrophages is close to that of untreated M2 macrophages.
[0063] Please refer to Figure 6 , when the strain amount of all single strains is the same, the combination of GL-104 strain and F-1 strain has a better activity than other combinations in promoting the polarization of M1 macrophages into M2 macrophages. Moreover, the CD206 / CD86 value obtained by the combination of GL-104 strain and F-1 strain acting on M1 macrophages is close to that of untreated M2 macrophages.
[0064] In summary, it was confirmed that the GL-104 strain and the F-1 strain can act synergistically on M1 macrophages to promote the polarization of M1 macrophages into M2 macrophages.
[0065] However, what is described above is only a preferred embodiment of the present invention, but it cannot be used to limit the scope of implementation of the present invention; therefore, all simple equivalent changes and modifications made according to the scope of patent protection of the present invention and the content of the invention specification are still within the scope of patent protection of the present invention.
[0066]
Biological material storage
[0067] 1. CN China, China Center for Type Culture Collection, 2009 / 08 / 07, CCTCC M209138
[0068] 2. CN China, China Center for Type Culture Collection, 2011 / 04 / 10, CCTCC M2011124
Claims
1. A lactic acid bacteria composition for regulating immune cell differentiation and / or reducing inflammatory hormone secretion, characterized in that: include: (a) Lactobacillus reuteri GL-104 strain; and (b) Lactobacillus rhamnosus F-1 strain; Among them, the GL-104 strain was deposited in the China Center for Type Culture Collection with the deposit number CCTCC M209138, and the F-1 strain was deposited in the China Center for Type Culture Collection with the deposit number CCTCC M2011124.
2. The lactic acid bacteria composition according to claim 1, characterized in that Based on the total strain amount of the GL-104 strain and the F-1 strain, the GL-104 strain accounts for 10% to 90%, and the F-1 strain accounts for 10% to 90%.
3. The lactic acid bacteria composition according to claim 1, characterized in that Based on the total strain amount of the GL-104 strain and the F-1 strain, the GL-104 strain accounts for 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, and the F-1 strain accounts for 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.
4. The lactic acid bacteria composition according to claim 1, characterized in that Based on the total strain amount of the GL-104 strain and the F-1 strain, the GL-104 strain accounts for 30% and the F-1 strain accounts for 70%.
5. The lactic acid bacteria composition according to claim 1, characterized in that The GL-104 strain is an active strain or a deactivated strain, and the F-1 strain is an active strain or a deactivated strain.
6. The lactic acid bacteria composition according to claim 1, characterized in that Also includes: (c) Excipient, diluent, or carrier.
7. The lactic acid bacteria composition according to claim 6, characterized in that The excipient, diluent, or carrier is a food-acceptable excipient, diluent, or carrier, or a pharmaceutically acceptable excipient, diluent, or carrier.
8. A use of the lactic acid bacteria composition according to claim 1, characterized in that: Used for preparing a composition for treating or preventing allergic rhinitis.
9. The use according to claim 8, characterized in that The prepared composition achieves the treatment or prevention of allergic rhinitis by reducing the production of inflammatory hormone CXCL10 by immune cells.
10. The use according to claim 8, characterized in that The prepared composition achieves the purpose of treating or preventing allergic rhinitis by increasing the CD206 / CD86 value on the cell surface of immune cells.