CN116509904A - Application of bifidobacterium bifidum NX-7 in preparation of medicines for preventing or treating inflammatory diseases - Google Patents
Application of bifidobacterium bifidum NX-7 in preparation of medicines for preventing or treating inflammatory diseases Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
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- A61K35/74—Bacteria
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- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
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- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
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Abstract
The invention discloses an application of bifidobacterium bifidum NX-7 in preparing a medicament for preventing or treating inflammatory diseases, and belongs to the technical field of microorganisms. The application of the bifidobacterium bifidum NX-7 in preparing the medicine for preventing or treating the inflammatory diseases can inhibit the aggregation of neutrophils and macrophages to the inflammation part of the zebra fish tail fin in an in-vivo inflammation model; bifidobacterium bifidum NX-7 can inhibit the increase of inflammatory factors IL-1 beta, IL-6 and TNF-alpha induced by LPS; the method provides theoretical reference and guiding basis for developing medicines for preventing or treating inflammatory diseases by using bifidobacterium bifidum NX-7.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to application of bifidobacterium bifidum NX-7 in preparation of medicines for preventing or treating inflammatory diseases.
Background
Inflammation (inflammation) is what is commonly called "inflammation" and is a basic pathological process of biological tissues, which is mainly based on defense reaction, and is caused by the stimulation of the biological tissues by a certain stimulation factor such as trauma, infection and the like. The local manifestations of inflammation are redness, swelling, heat, pain and dysfunction, and systemic reactions such as fever and changes in peripheral blood leukocyte count are also accompanied. Inflammation is often beneficial and is an automatic defensive response in humans. However, inflammation is sometimes also detrimental, such as attack on the body's own tissues, inflammation occurring in transparent tissues, and the like. Current traditional modes of treatment include the use of antibiotics and anti-inflammatory agents: in the case of infectious inflammation caused by infection, it is possible to use what is known as "anti-inflammatory" or "anti-inflammatory needles" in general, and it is actually an antibiotic, which kills or inhibits the proliferation of bacteria, and thus reduces inflammation caused by infection. If not inflammation caused by infection, non-steroidal anti-inflammatory agents or steroids are used in medicine to suppress the inflammatory response, often referred to as "anti-inflammatory agents". However, the continued use of the above agents brings significant drawbacks and side effects; for example, associated with sustained steroid use are significant side effects including gastric ulcers and bleeding. Probiotics are used as a type of microorganism beneficial to human health, and when a certain number of living probiotics are planted in the intestinal tracts of a host, beneficial health effects can be exerted by changing the intestinal microecology and other ways.
Therefore, providing the use of bifidobacterium bifidum NX-7 in the preparation of a medicament for preventing or treating inflammatory diseases is a problem to be solved by the person skilled in the art.
Disclosure of Invention
In view of this, the present invention provides the use of bifidobacterium bifidum NX-7 in the manufacture of a medicament for the prevention or treatment of inflammatory diseases.
Inflammation is a reaction of the immune system to tissue injury and infection and is mainly characterized by the accumulation of leukocytes (granulocytes, macrophages) around the infected tissue. The immune system of zebra fish is very similar to mammals. When trauma occurs, neutrophils, macrophages respond to traumatic inflammation almost simultaneously, and macrophages and neutrophils recruit to the site of injury.
Cutting off the tail fin of the zebra fish by a scalpel, inducing acute injury and promoting the zebra fish neutrophil to generate immune response. The transgenic neutrophil fluorescent fish (green) is adopted, and the number of the zebra fish wound neutrophil truncated by the tail fin can be obviously increased compared with that of a normal zebra fish under a fluorescent microscope.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the application of bifidobacterium bifidum NX-7 in preparing a medicament for preventing or treating inflammatory diseases is characterized in that the preservation number of the bifidobacterium bifidum (Bifidobacterium bifidum) NX-7 is CGMCC No.20115 (see patent number 202011356472.2).
Further, the bifidobacterium bifidum NX-7 is applied to the preparation of medicines for inhibiting neutrophils and macrophages from gathering at the inflammation position of the zebra fish tail fin.
Further, the bifidobacterium bifidum NX-7 is applied to the preparation of medicines for inhibiting inflammatory factors IL-1 beta, IL-6 and TNF-alpha.
Further, the bifidobacterium bifidum NX-7 is a bacterial suspension.
At the same concentration, bifidobacterium bifidum NX-7 has stronger inhibition effect on the aggregation of granulocytes and macrophages to the inflammation position of the zebra fish tail fin in an in-vivo inflammation model than bifidobacterium bifidum 29521, and has good effect of preventing or treating inflammatory diseases. At the same concentration, the inhibition effect of the bifidobacterium bifidum NX-7 on the LPS to improve the contents of inflammatory factors IL-1 beta, IL-6 and TNF-alpha in the zebra fish body is stronger than that of the bifidobacterium bifidum 29521, and the preparation method has the potential of preventing or treating inflammatory diseases.
Compared with the prior art, the application of the bifidobacterium bifidum NX-7 in preparing the medicines for preventing or treating the inflammatory diseases is disclosed, and the bifidobacterium bifidum NX-7 can inhibit the aggregation of neutrophils and macrophages to the inflammation part of the zebra fish tail fin in an in-vivo inflammation model; bifidobacterium bifidum NX-7 can inhibit the increase of inflammatory factors IL-1 beta, IL-6 and TNF-alpha induced by LPS; the method provides theoretical reference and guiding basis for developing medicines for preventing or treating inflammatory diseases by using bifidobacterium bifidum NX-7.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a visual graph showing the effect of bifidobacterium bifidum NX-7 on neutrophil and macrophage aggregation to inflammation of zebra fish tail fin;
wherein A: normal group; b: a model group; c: a positive control group; d: bifidobacterium bifidum 29521; e: bifidobacterium bifidum NX-7;
FIG. 2 is a graph showing the effect of bifidobacterium bifidum NX-7 on neutrophil and macrophage aggregation to inflammation of zebra fish tail fin;
FIG. 3 is a graph showing the effect of bifidobacterium bifidum NX-7 of the present invention on LPS to increase the IL-1β content of inflammatory factor in zebra fish;
FIG. 4 is a graph showing the effect of bifidobacterium bifidum NX-7 of the present invention on LPS to increase the IL-6 content of inflammatory factors in zebra fish;
FIG. 5 is a graph showing the effect of bifidobacterium bifidum NX-7 of the present invention on LPS to increase the amount of inflammatory factor TNF-alpha in zebra fish.
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.
Zebra fish are AB-line and Tg (corola: eGFP) -line zebra fish.
Interleukin 6 (IL-6) enzyme-linked immunosorbent assay kit, tumor necrosis factor alpha (TNF-alpha) enzyme-linked immunosorbent assay kit and interleukin 1 beta (IL-1 beta) enzyme-linked immunosorbent assay kit are all purchased from Wuhan Huamei bioengineering Co., ltd; lipopolysaccharide (LPS) was purchased from Sigma company; tricaine was purchased from Beijing Wash chemical Co., ltd; bifidobacterium bifidum 29521 (ATCC 29521) was purchased from bio-technology limited of beijing Bai-o-pegbo.
EXAMPLE 1 preparation of Bifidobacterium bifidum NX-7 suspension (thallus)
Inoculating bifidobacterium bifidum NX-7 after activation culture in a BS liquid culture medium, culturing for 24 hours at 37 ℃, and centrifuging for 10 minutes at 4 ℃ and 6000r/min to obtain bacterial precipitate; after the bacterial cell precipitate is washed twice by PBS, the bacterial cell is resuspended by PBS, and the cell concentration is regulated to be 1 multiplied by 10 6 CFU/mL gave a bacterial suspension (cell).
EXAMPLE 2 preparation of Bifidobacterium bifidum 29521 bacterial suspension (thallus)
Inoculating bifidobacterium bifidum 29521 into a BS liquid culture medium after activating and culturing for 24 hours at 37 ℃, and centrifuging for 10 minutes at 4 ℃ and 6000r/min to obtain a bacterial precipitate; after the bacterial cell precipitate is washed twice by PBS, the bacterial cell is resuspended by PBS, and the cell concentration is regulated to be 1 multiplied by 10 6 CFU/mL gave a bacterial suspension (cell).
Example 3 Effect of Bifidobacterium bifidum NX-7 on the accumulation of neutrophils and macrophages at inflammation of the zebra fish tail fin
Healthy zebra fish Tg (corola: eGFP) developed to 3dpf (days post fertilization) was selected and placed in 6-well cell culture plates, 25 strips/well, and PBS was added to both the normal (untapped) and model groups; positive control (indomethacin) was added with 0.4. Mu.g/mL indomethacin; bifidobacterium bifidum 29521 intervention group (1×10 6 CFU/mL) was added 1X 10 6 CFU/mL bifidobacterium bifidum 29521; bifidobacterium bifidum NX-7 intervention group (1×10) 6 CFU/mL) was added 1X 10 6 CFU/mL bifidobacterium bifidum NX-7; 5mL of each hole is placed in a biochemical incubator for incubation at 28 ℃ and new solution is replaced every 24 hours; after 72h incubation, zebra fish tail fins are cut off by a surgical knife under a stereoscopic microscope, then placed in a 6-hole cell culture plate, 20 strips/hole are added, PBS is added to both the normal group (without tail cutting) and the model group, and 0.4 mug/mL indomethacin is added to the positive control group (indomethacin); bifidobacterium bifidum 29521 intervention group(1×10 6 CFU/mL) was added 1X 10 6 CFU/mL bifidobacterium bifidum 29521; bifidobacterium bifidum NX-7 intervention group (1×10) 6 CFU/mL) was added 1X 10 6 CFU/mL bifidobacterium bifidum NX-7, 5mL per well, placing in a biochemical incubator at 28 ℃ for incubation for 6 hours, anaesthetizing zebra fish with cocaine, observing the aggregation of neutrophils and macrophages at the wound of the tail fin under a fluorescence microscope, and photographing and recording. The numbers of neutrophils and macrophages were counted using the area within 150 μm from the incision as the count range. SPSS 19.0 software is adopted for statistical data processing, and experimental data are all adoptedData represent, analyzed by T-test, compared to normal group: ### P<0.005, compared to model group: *** P<0.005。
as can be seen from fig. 1 and 2, there was almost no neutrophil and macrophage aggregation (1.05±0.21) at the zebra fish tail fins of the normal group. When 6 hours are cut off, a large amount of neutrophils and macrophages are gathered at the wound of the zebra fish tail fin of the model group; meanwhile, the number of neutrophils and macrophages at the positions of the zebra fish tail fin wound of the model group is 21.65+/-1.46, and compared with the normal group (1.05+/-0.21), the model group has obvious dissimilarity (P < 0.005), which indicates that the zebra fish inflammation model is successfully established.
As can be seen from fig. 1 and 2, there was only a small amount of neutrophil and macrophage aggregation at the wound of the zebra fish tail fin of the positive control group (indomethacin). Meanwhile, the number of neutrophils and macrophages at the positions of the zebra fish tail fin wound of the positive control group is 10.40+/-0.68, and the difference is obvious (P) compared with the model group (21.65+/-1.46)<0.005). Therefore, indomethacin has the effect of relieving inflammation, and is consistent with clinical results, which shows that the test for evaluating the anti-inflammatory efficacy is effective. Bifidobacterium bifidum 29521 intervention group (1×10 6 CFU/mL) of zebra fish tail fin wound with a large number of neutrophil and macrophage aggregates; simultaneous bifidobacterium bifidum 29521 intervention group (1×10 6 The number of neutrophils and macrophages at the wound site of the zebra fish tail fin is 18.90+/-1.35, compared with a model group (21.65+/-1.46)No significant difference (P)>0.05). In addition, bifidobacterium bifidum NX-7 intervention group (1X 10) 6 CFU/mL) were also only slightly aggregated with neutrophils and macrophages at the zebra fish tail fin wound, similar to the positive control (indomethacin); bifidobacterium bifidum NX-7 intervention group (1×10) 6 The number of neutrophils and macrophages at the wound of the zebra fish tail fin is 13.55+/-0.79, and the variability is obvious (P) compared with the model group (21.65+/-1.46)<0.005). Therefore, the results show that at the same concentration, bifidobacterium bifidum NX-7 has stronger inhibition effect on the aggregation of granulocytes and macrophages to the inflammation site of the zebra fish tail fin in an in-vivo inflammation model than bifidobacterium bifidum 29521, and has good effect of preventing or treating inflammatory diseases.
Example 4 Effect of Bifidobacterium bifidum NX-7 on LPS to increase the content of inflammatory factors IL-6, TNF-alpha and IL-1β in zebra fish
Healthy AB-series zebra fish which develop to 3dpf (days post fertilization) are selected and placed in 6-hole cell culture plates, 40 zebra fish/hole are arranged, and 6 compound holes are arranged in each group; adding PBS into the normal group; the model group was added with 5. Mu.g/mL LPS solution; adding indomethacin and 5 mug/mL LPS solution with final concentration of 0.4 mug/mL into the positive control group; bifidobacterium bifidum 29521 intervention group (1×10 6 CFU/mL) was added at a final concentration of 1X 10 6 CFU/mL bifidobacterium bifidum 29521 and 5 μg/mL LPS solution; bifidobacterium bifidum NX-7 intervention group (1×10) 6 CFU/mL) was added at a final concentration of 1X 10 6 CFU/mL bifidobacterium bifidum NX-7 and 5 mug/mL LPS solution, 5mL per well, placing in a biochemical incubator for incubation at 28 ℃ and replacing new solution every 24 hours; after 72h incubation, wash each well of zebra fish 3 times with PBS, and then collect each well of zebra fish to 1.5mL centrifuge tube, 40 zebra fish per tube, 6 tubes per experimental group; after the water in the centrifuge tube is sucked dry, 100 mu L of PBS is added, the zebra fish homogenate is crushed by an S-18KS handheld micro-electric tissue homogenate device until no obvious tissue fragments exist, the supernatant is collected by centrifugation at 12000 Xg and 4 ℃ for 10min, and the content of the supernatant IL-6, TNF-alpha and IL-1 beta is detected by an ELISA kit. SPSS 19.0 software is adopted for statistical data processing, and experimental data are all adoptedData represent, analyzed by T-test, compared to normal group: ### P<0.005, compared to model group: * P<0.05， ** P<0.01， *** P<0.005。
as can be seen from FIG. 3, the amount of inflammatory factor IL-1β (124.73 + -5.60 pg/mL) in zebra fish in the model group was significantly increased compared to the normal group (70.17 + -1.98 pg/mL) (P)<0.005 Indicating that the LPS induced inflammation model is successfully established. As can be seen from FIG. 3, the content of inflammatory factor IL-1 beta in zebra fish of the positive control group (indomethacin) is 82.41 + -2.72 pg/mL, and the difference is significant (P) compared with the model group (124.73 + -5.60 pg/mL)<0.005). Therefore, indomethacin has the effect of relieving inflammation, and is consistent with clinical results, which shows that the test for evaluating the anti-inflammatory efficacy is effective. Bifidobacterium bifidum 29521 intervention group (1×10 6 CFU/mL) of the zebra fish, the IL-1 beta content of the zebra fish is 116.52+/-5.57 pg/mL, and no significant difference (P) is generated compared with a model group (124.73 +/-5.60 pg/mL)>0.05). In addition, bifidobacterium bifidum NX-7 intervention group (1X 10) 6 CFU/mL) the content of inflammatory factor IL-1 beta in zebra fish body is 98.81+ -4.07 pg/mL, and the difference is obvious compared with the model group (124.73 + -5.60 pg/mL) (P<0.01)。
As can be seen from FIG. 4, the amount of inflammatory factor IL-6 (17.91.+ -. 1.11 pg/mL) in zebra fish in the model group was significantly increased compared to that in the normal group (9.55.+ -. 0.81 pg/mL) (P)<0.005 Indicating that the LPS induced inflammation model is successfully established. As can be seen from FIG. 4, the content of inflammatory factor IL-6 in zebra fish of the positive control group (indomethacin) was 11.18.+ -. 0.88pg/mL, and the difference was significant (P) compared with the model group (17.91.+ -. 1.11 pg/mL)<0.005). Therefore, indomethacin has the effect of relieving inflammation, and is consistent with clinical results, which shows that the test for evaluating the anti-inflammatory efficacy is effective. Bifidobacterium bifidum 29521 intervention group (1×10 6 CFU/mL) was 16.21+ -0.49 pg/mL, and there was no significant difference (P) from the model group (17.91+ -1.11 pg/mL)>0.05). In addition, bifidobacterium bifidum NX-7 intervention group (1X 10) 6 CFU/mL) was 13.45+ -1.14 pg/mL, and was different from the model group (17.91+ -1.11 pg/mL)Significance (P)<0.05)。
As can be seen from FIG. 5, the amount of inflammatory factor TNF-. Alpha.in the zebra fish (36.79.+ -. 1.90 pg/mL) was significantly increased in the model group compared to the normal group (17.58.+ -. 1.01 pg/mL) (P)<0.005 Indicating that the LPS induced inflammation model is successfully established. As can be seen from FIG. 5, the inflammatory factor TNF-. Alpha.content in the zebra fish of the positive control group (indomethacin) was 23.43.+ -. 1.96pg/mL, and the difference was significant (P) compared with the model group (36.79.+ -. 1.90 pg/mL)<0.005). Therefore, indomethacin has the effect of relieving inflammation, and is consistent with clinical results, which shows that the test for evaluating the anti-inflammatory efficacy is effective. Bifidobacterium bifidum 29521 intervention group (1×10 6 CFU/mL) was 32.19+ -1.65 pg/mL, and there was no significant difference (P) from the model group (36.79+ -1.90 pg/mL)>0.05). In addition, bifidobacterium bifidum NX-7 intervention group (1X 10) 6 CFU/mL) the inflammatory factor TNF-alpha content in zebra fish is 25.59+ -2.15 pg/mL, and the difference is significant (P) compared with the model group (36.79+ -1.90 pg/mL)<0.01)。
Therefore, the results show that the inhibition effect of the bifidobacterium bifidum NX-7 on the LPS to improve the content of inflammatory factors IL-1 beta, IL-6 and TNF-alpha in the zebra fish body is stronger than that of the bifidobacterium bifidum 29521 at the same concentration, and the preparation method has the potential of preventing or treating inflammatory diseases.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (6)
1. The application of bifidobacterium bifidum NX-7 in preparing a medicament for preventing or treating inflammatory diseases is characterized in that the preservation number of the bifidobacterium bifidum NX-7 is CGMCC No.20115.
2. Use of bifidobacterium bifidum NX-7 according to claim 1, in the manufacture of a medicament for the prevention or treatment of inflammatory diseases, wherein said bifidobacterium bifidum NX-7 is a bacterial suspension.
3. Use of bifidobacterium bifidum NX-7 as defined in claim 1 for the preparation of a medicament for inhibiting the aggregation of neutrophils and macrophages to inflammation of the zebra fish tail fin.
4. Use of bifidobacterium bifidum NX-7 according to claim 3, in the preparation of a medicament for inhibiting the aggregation of neutrophils and macrophages to the inflammation of the zebra fish tail fin, wherein said bifidobacterium bifidum NX-7 is a bacterial suspension.
5. Use of bifidobacterium bifidum NX-7 as claimed in claim 1 in the manufacture of a medicament for inhibiting inflammatory factors IL-1 beta, IL-6, TNF-alpha.
6. The use of bifidobacterium bifidum NX-7 according to claim 5, in the preparation of a medicament for inhibiting inflammatory factors IL-1 β, IL-6, TNF- α, wherein said bifidobacterium bifidum NX-7 is a bacterial suspension.
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