CN112870157A - Inflammation-inhibiting gamma aminobutyric acid enema and preparation method and application thereof - Google Patents
Inflammation-inhibiting gamma aminobutyric acid enema and preparation method and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
- A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0031—Rectum, anus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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Abstract
The invention belongs to the technical field of medicines, and particularly relates to a novel gamma aminobutyric acid enema for inhibiting inflammation, and a preparation method and application thereof. The compounds have good anti-inflammatory immunosuppressive activity and higher safety, and the enema can be used for preparing medicines for treating or assisting in treating inflammatory bowel diseases and has good application prospect.
Description
Technical Field
The invention belongs to the technical field of medicines, relates to a novel inflammation-inhibiting Gamma-aminobutyric acid enema, and a preparation method and application thereof, and particularly relates to application of Gamma-aminobutyric acid (GABA) in preparation of a medicine for treating or assisting in treating inflammatory bowel diseases.
Background
The prior art discloses that Inflammatory Bowel Disease (IBD) is characterized by disturbance of the intestinal immune system, can affect Inflammatory diseases of different parts of the digestive tract, has the characteristic of recurrent attacks, and the mechanism of IBD is not completely clear; inflammatory bowel disease is largely divided into two distinct types, including Ulcerative Colitis (UC) and Crohn's Disease (CD). In recent years, practice has shown that the incidence of the disease is remarkably increased and the disease tends to be gradually younger. The current main clinical treatment modes are mainly nutritional therapy, drug therapy, biological therapy related to monoclonal antibodies and immunosuppressant therapy, and the treatment practice shows that the therapies cannot completely and effectively control the development of inflammation, wherein part of patients often develop to a stage requiring surgical intervention, and the life quality of the patients is seriously influenced.
Studies have analyzed a significant increase in macrophage numbers in local intestinal and mesenteric lymph nodes in inflammatory bowel disease; macrophages are mainly classified into M1 type macrophages and M2 type macrophages: m1 type macrophage highly expresses Nitric oxide synthase (NOS 2), can synthesize Nitric Oxide (NO) and active oxygen (ROS) in a large amount, and generate a large amount of proinflammatory factors such as IFN gamma, IL-1, IL-6, IL-12, IL-23 and TNF alpha, and further promote the progress of inflammation; m2 type macrophages, high CD206 molecules, mainly secrete IL-10, TGF beta and other anti-inflammatory factors to inhibit the progression of inflammation. Research shows that in inflammatory bowel diseases, M1 type macrophages are increased remarkably in local intestinal tracts and mesenteric lymph nodes to generate proinflammatory functions, and under the pathological conditions of the inflammatory bowel diseases, the intestinal mucosa of an inflammatory part highly expresses a chemokine CCL2, so that proinflammatory M1 type macrophages can be recruited from blood to infiltrate into the local intestinal tracts in large quantity, and meanwhile, the macrophage group secretes proinflammatory cytokines such as TNF alpha, IL-1, IL-12, IL-6, IL-23, IFN gamma and the like in large quantity to further aggravate local inflammatory responses.
Gamma-aminobutyric acid (GABA) is an important inhibitory neurotransmitter in the central nervous system, mainly acts in inhibitory nerve synapses and plays a role in reducing excitability of nerve cells, and dysfunction of GABA is closely related to occurrence of various nervous system diseases. Research shows that in vivo, GABA is mainly synthesized by Glutamate catalyzed by Glutamate decarboxylase (GAD), and research finds that GABA receptor is expressed on the surface of immune cells, so GABA is probably an important transmitter for neuro-immune regulation; the GABA receptors are largely divided into two types, including: a type receptor (GABAAR) and B type receptor (GABABR), wherein the A type receptor is mainly expressed on the surface of the T cell, the GABA can inhibit the proliferation of the T cell after stimulation, and the GABA can inhibit the production of IL-6 and IL-12 induced by LPS in macrophages in mouse abdominal cavity. The current research shows that GABA has a therapeutic effect in animal models of various immune diseases, but reports on GABA in treating inflammatory bowel diseases are not found yet.
Based on the current situation of the prior art, the inventors of the present application intend to provide a novel inflammation-inhibiting Gamma-aminobutyric acid enema, and a preparation method and an application thereof, and particularly relate to an application of Gamma-aminobutyric acid (GABA) in preparation of a medicament for treating or adjunctively treating inflammatory bowel disease.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a novel inflammation-inhibiting Gamma-aminobutyric acid enema and a preparation method thereof, and particularly relates to application of Gamma-aminobutyric acid (GABA) in preparation of a medicine for treating or assisting in treating inflammatory bowel diseases.
The invention provides an inflammation-inhibiting Gamma-aminobutyric acid enema, which is prepared by using Gamma-aminobutyric acid (GABA) as an active ingredient, preparing an enema solution with the concentration of 40mg/mL by using double distilled water, and filtering, removing impurities and sterilizing by using a 0.22 mu m filter to obtain the GABA enema; the enema can be prepared on site; gamma-aminobutyric acid (GABA) was purchased from Sigma company, usa;
the results of in vitro and in vivo experiments show that the GABA enema can effectively improve the bed symptoms of colitis such as weight loss, diarrhea, hematochezia and the like of experimental animals, relieve the pathological damage of colon tissues, inhibit the infiltration of inflammatory cells of experimental mice to the colon tissues and improve the local immune pattern; experimental results prove that the GABA enema has good anti-inflammatory immunosuppressive activity and higher safety, and can be further used for preparing medicines for clinical treatment and adjuvant treatment of inflammatory bowel diseases.
Specifically, in order to achieve the above object, the technical scheme adopted by the invention is as follows:
1. dextran Sodium Sulfate (DSS) molding and animal grouping
C57BL/6 female mice were modeled after two weeks of acclimatization (six weeks of age); setting a control group: ACh intervention group: performing GABA enema intervention on the 2 nd, 4 th and 6 th days of the experiment;
DSS was purchased from MP Biomedical, usa and formulated as a 3% DSS solution with double distilled water; filtering with 0.22 μm filter to remove impurities and bacteria, and mixing;
the general conditions of each group of mice, anus blood and diarrhea conditions of the mice are observed and recorded in the experimental process;
2. medicine preparation and enema method
Gamma-aminobutyric acid (GABA) is purchased from Sigma company in America, and is used for preparing a solution for enema by double distilled water, and is used for filtration, impurity removal and sterilization and is used as the preparation;
performing conventional clysis operation;
3. intestinal sampling
Treating the experimental mouse conventionally, and taking a full colorectal from an anus to the tail end of a cecum; observing the general morphology of the colon of each group of mice, measuring the colorectal length (the confluent end of the small intestine and the cecum to the anus); observing the condition of the inner wall of the intestinal tract and taking a picture; taking the lesion tissues of the intestinal part, and carrying out H.E. staining pathological examination;
4. carrying out intestinal tissue lymphocyte separation by adopting a conventional method;
5. adopting conventional methods for cell surface and nuclear staining and flow cytometry analysis;
6. statistical analysis was performed using GraphPad Prism 7.0 software; the measured data are expressed by Means of mean numbers (Means) ± Standard Error (SEM), two-by-two comparison of the measured data is performed by independent sample t test (paired t test and unpaired t test), and the difference is that P is less than 0.05, which has statistical significance.
The experimental results show that:
the GABA enema is given to the mice with DSS modeling, the enteritis of the mice can be remarkably relieved: after administration of GABA enema, local M1 type macrophages and Th2 cells in intestinal tracts of DSS modeling mice are obviously reduced, and NOS 2-negative non-M1 type macrophages are obviously increased:
proved by in vivo and in vitro experiments, the prepared GABA enema can effectively improve the bed symptoms of colitis such as weight loss, diarrhea, hematochezia and the like of experimental animals, relieve the pathological damage of colon tissues, inhibit the infiltration of inflammatory cells of experimental mice to the colon tissues and improve the local immune pattern; furthermore, the compound can be used for preparing medicines for clinical treatment and adjuvant treatment of inflammatory bowel diseases.
Drawings
Figure 1a. mouse weight change; b & C, length of intestinal tract of mouse; D. HE staining of intestinal tissue sections of mice; p < 0.05, p < 0.001;
FIG. 2 intestinal local macrophages after GABA enema (CD45+ CD11b + F4/80+), dendritic cells (CD45+ CD11b + CD11c +), M1 type macrophages (CD45+ CD11b + F4/80+ NOS2+), NOS 2-non-M1 type macrophages (CD45+ CD11b + F4/80+ NOS 2-); p is < 0.05;
FIG. 3 intestinal local T cell changes following GABA enema, Th1(CD4+ Tbet +), Th2(CD4+ GATA3+), Th17(CD4+ ROR γ +), Treg (CD4+ Foxp3 +); p is < 0.05.
Detailed Description
Example 1
Dextran Sodium Sulfate (DSS) molding and animal grouping:
c57BL/6 female mice were modeled after two weeks of acclimatization (six weeks of age); control group: drinking water normally, and killing after 7 days; model group: 3% DSS drinking water, lasting for 7 days, and replacing mouse DSS drinking water every 2-3 days in the water feeding stage; ACh intervention group: 3% DSS drinking water, lasting for 7 days, replacing mouse DSS drinking water every 2-3 days in the water feeding stage, and performing GABA enema intervention on the 2 nd, 4 th and 6 th days of the experiment;
DSS was purchased from MP Biomedical, usa and formulated as a 3% DSS solution with double distilled water; filtering with 0.22 μm filter to remove impurities and bacteria, and mixing;
the general conditions of each group of mice, anus blood and diarrhea conditions of the mice are observed and recorded in the experimental process;
the preparation and enema method of the medicine comprises the following steps:
GABA is purchased from Sigma company in America, and is prepared into enema solution with the concentration of 40mg/mL by double distilled water, and the solution is filtered by a 0.22 Hongm filter to remove impurities and bacteria, and is used at present;
the sausage filling operation method comprises the following steps: exposing the anus of the mouse after the mouse is normally held, lightly touching the anus with a cotton swab to stimulate the defecation of the mouse, connecting a stomach tube with the diameter of 2mm to a 1mL syringe, sucking an enema solution and lubricating the stomach tube with glycerol; slowly inserting the stomach tube into the anus of a mouse for about 4cm, slowly withdrawing the stomach tube, injecting about 100uL of solution while injecting, and then completely withdrawing the stomach tube, wherein the anus is free from liquid outflow;
intestinal sampling:
killing the mouse by dislocation of cervical vertebra, longitudinally cutting open the abdominal cavity along the abdominal midline, dissociating colon and distal ileum, and taking the whole colorectal from the anus to the tail end of the cecum; observe the general morphology of the colon of each group of mice, measure the colorectal length (small intestine merging with cecum to anus); and (3) dissecting the colon and rectum in longitudinal rows along the colonic zone, washing the colon and rectum with precooled sterile normal saline, observing the condition of the inner wall of the intestinal tract and taking a picture. Cutting the intestinal tract along the long axis, soaking part of lesion tissues in 4% paraformaldehyde at 4 deg.C overnight, embedding in paraffin, and slicing for H.E. staining pathological examination;
and (3) intestinal tissue lymphocyte separation:
the method comprises the following steps of (1) quickly disinfecting a mouse after the mouse is killed, opening the abdominal cavity layer by layer, taking down the colon as soon as possible, placing the colon in precooled PBS + 2% FBS liquid, excising mesentery tissues, cutting open an intestinal canal along a longitudinal axis, carefully removing residual excrement and rinsing;
transferring the intestinal tissue into a new centrifuge tube, cutting into pieces with sterile scissors, adding 2-3mL of digestive juice, and horizontally shaking at 230rpm/37 ℃ for 30 min;
centrifuging at 400g/4 ℃ for 5min, discarding the supernatant, adding a proper amount of precooled PBS and 2% FBS for resuspending tissues, and filtering through a 70-micron sterile cell filter to remove impurities;
centrifuging at 400g/4 ℃ for 5min, resuspending the tissue with 2.4mL of RMPI 1640 medium + 2% FBS, mixing with 1.6mL of 100% Percoll to prepare 40% Percoll, slowly adding the 40% Percoll to the upper layer of the 70% Percoll separating medium, and then slowly adding 30% Percoll to the surface of the 40% Percoll to form a 30%/40%/70% Percoll separating system, taking care not to damage the liquid plane;
centrifuging at 2000 rpm/room temperature for 20min, and setting the speed increasing and decreasing gradient as 1;
collecting 40%/70% Percoll interlayer cells, washing twice by using a large amount of PBS, and then suspending in 1mL of PBS + 2% FBS and placing at 4 ℃ for later use;
cell surface, nuclear staining and flow cytometry analysis:
1) counting intestinal immune cells of the mice and calculating the cell activity, wherein the cell activity is not lower than 90%;
2) transferring the cells into a flow tube, centrifuging at 400g/4 ℃ for 5min, and resuspending in 500 μ L PBS;
3) adding appropriate amount of surface staining antibody into each tube according to the instruction, and incubating at 4 deg.C in dark for 30 min;
4) adding 2mL PBS to wash the cells, and centrifuging for 5min at 400g/4 ℃; after the repetition of this step, the procedure is repeated,
5) adding 1mL of fixing/membrane-breaking liquid, and fixing for 30min at room temperature;
6) adding 2mL of 1 Xmembrane-breaking solution to wash cells, and centrifuging at 400g/4 ℃ for 5 min; after the repetition of this step, the procedure is repeated,
7) adding appropriate amount of nuclear staining antibody into each tube according to the instruction requirement, and incubating for 30-40min at room temperature in the dark;
8) adding 2mL of 1 Xmembrane-breaking solution to wash cells, and centrifuging at 400g/4 ℃ for 5 min;
9) add 300. mu.L PBS to resuspend, prepare the flow cytometry to detect;
10) data analysis was performed using Flowjo X.
The experiment was statistically analyzed using GraphPad Prism 7.0 software, the measurements were expressed as Means (Means) ± Standard Error (SEM), the measurements were compared pairwise using independent sample t-tests (paired t-test and unpaired t-test), and P < 0.05 was statistically significant for differences.
The experimental results show that:
1. the GABA enema is given to obviously relieve the enteritis of DSS model-making mice
Compared with the DSS modeling and water enema group (DSS + H20 group), the DSS modeling and GABA (GABA) enema intervention group (DSS + GABA group) significantly relieved the weight loss (fig. 1A), and the length of the intestinal tract was significantly increased (fig. 1B, C); HE staining of intestinal tissue section shows that the disruption of villi and glandular tube structures of the intestinal mucosa epithelial layer of the DSS + H20 group is obvious, the intestinal mucosa epithelial layer of the DSS + GABA group is relatively complete, and the villi and glandular tube structures are slightly disrupted (FIG. 1D).
2. After GABA enema is given, local M1 type macrophages and Th2 cells in intestinal tracts of DSS modeling mice are obviously reduced, and NOS 2-negative non-M1 type macrophages are obviously increased
Analyzing the change of local immune pattern of intestinal tracts of DSS model mice after the enema of GABA solution by flow cytometry, and finding that DSS + GABA group contrasts DSS + H2Group O, macrophages were significantly reduced (fig. 2B), while DC cells were not significantly different (fig. 2D); a significant reduction in macrophages of type M1 (fig. 2C), while non M1-negative macrophages of type NOS 2-were significantly increased (fig. 2E); th2 cells were significantly reduced (fig. 3D), while none of CD8+ T cells, CD4+ T cells, Th1, Th17, Treg cells were significantly altered (fig. 3A, B, C, E, F).
In conclusion, the enema prepared by locally administering GABA in the intestinal tract can obviously improve the intestinal inflammation state of mice with DSS-induced enteritis and regulate the disordered intestinal immunity pattern, so the enema prepared by locally administering GABA for the adjuvant treatment of inflammatory bowel diseases has good development and application prospects.
Claims (5)
1. An anti-inflammation Gamma-aminobutyric acid enema is characterized in that Gamma-aminobutyric acid (GABA) is used as an active ingredient, double distilled water is used for preparing an enema solution, and the enema solution is filtered, purified and sterilized to obtain the GABA enema.
2. The anti-inflammatory enema of gamma aminobutyric acid according to claim 1, wherein the enema solution is prepared to have a concentration of 40 mg/mL.
3. The inflammation-suppressing gamma aminobutyric acid enema according to claim 1, wherein the prepared solution for enema is filtered by a 0.22 μm filter to remove impurities and bacteria to obtain GABA enema.
4. The anti-inflammatory GABA enema according to claim 1, wherein said GABA enema is formulated at the time of use.
5. The anti-inflammatory gaba enema according to claim 1, wherein the use of gaba in the manufacture of a medicament for the treatment or co-treatment of inflammatory bowel disease.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6127418A (en) * | 1997-08-20 | 2000-10-03 | Warner-Lambert Company | GABA analogs to prevent and treat gastrointestinal damage |
| WO2003045360A2 (en) * | 2001-11-29 | 2003-06-05 | Laves Arzneimittel Gmbh | Use of gaba for the treatment of intestinal inflammations |
| WO2017058074A1 (en) * | 2015-08-30 | 2017-04-06 | Diamyd Medical Ab | Combination therapy using gliadin and gamma aminobutyric acid |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6127418A (en) * | 1997-08-20 | 2000-10-03 | Warner-Lambert Company | GABA analogs to prevent and treat gastrointestinal damage |
| WO2003045360A2 (en) * | 2001-11-29 | 2003-06-05 | Laves Arzneimittel Gmbh | Use of gaba for the treatment of intestinal inflammations |
| WO2017058074A1 (en) * | 2015-08-30 | 2017-04-06 | Diamyd Medical Ab | Combination therapy using gliadin and gamma aminobutyric acid |
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