CN116617199A - Use of gamma-aminobutyric acid for preventing and/or improving related cell change caused by thermal stimulation and method for realizing same - Google Patents
Use of gamma-aminobutyric acid for preventing and/or improving related cell change caused by thermal stimulation and method for realizing same Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/40—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing nitrogen
- A61K8/44—Aminocarboxylic acids or derivatives thereof, e.g. aminocarboxylic acids containing sulfur; Salts; Esters or N-acylated derivatives thereof
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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
- A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The present application discloses the use of gamma-aminobutyric acid for preventing and/or ameliorating reduced expression of TJ related proteins in cells caused by thermal stimulation and/or enhanced cell permeability caused by thermal stimulation and a method for preventing and/or ameliorating reduced expression of TJ related proteins in cells caused by thermal stimulation and/or enhanced cell permeability caused by thermal stimulation comprising administering gamma-aminobutyric acid to a site subjected to thermal stimulation.
Description
Technical Field
The application relates to the technical field of personal care products, in particular to application of gamma-aminobutyric acid in preventing and/or improving related cell change caused by thermal stimulation and a method for realizing the application.
Background
Epidemiological evidence of very hot beverages and human cancers has been strengthened over time, with positive associations and trends in studies that consider qualitative grading of temperature. In addition, new laboratory animal studies have shown that hot water above 65 ℃ can be used as a tumor promoter. People in China have eating habits of eating hot drinks and hot foods, and researches show that human periodontal ligament cells (HPDL) have cytotoxicity after being treated for 2 hours at 45 ℃; heat stress at 54 ℃ promotes the occurrence of esophageal squamous cell carcinoma. Therefore, the study of protection against thermal stimulation in the oral cavity is particularly important.
The oral cavity is often affected by temperature changes such as drinking hot drinks or dental treatments, and the temperature in the oral cavity can rise, resulting in persistent thermal pain sensation, producing an inflammatory response. When the temperature is too high, heat stress can destroy the oral epithelium and increase oral permeability, allowing pathogens and endotoxins to enter the circulation causing organ dysfunction.
Disclosure of Invention
In view of the adverse effect of thermal stimulation on cells, the present application has been found through experimental studies that gamma-aminobutyric acid can effectively prevent and/or ameliorate this adverse effect, so that it can be applied to personal care products to alleviate the sites susceptible to thermal stimulation, and has completed the present application.
The specific technical scheme of the application is as follows:
1. use of gamma-aminobutyric acid for preventing and/or improving reduced expression of TJ related proteins in cells caused by thermal stimulation and/or increased cell permeability caused by thermal stimulation.
2. The use of item 1, wherein the TJ-related protein comprises one or more of a ZO-1 protein, an occludin protein, a claudin-1 protein, a claudin-2 protein, and a claudin-4 protein.
3. The use of item 1 or 2, the cell comprising: one or more than two of human oral epithelial cells, human periodontal ligament cells, esophageal squamous cells and human colon epithelial cells.
4. The use of any one of claims 1-3, wherein the thermal stimulus comprises a thermal stimulus having a temperature of 40 ℃ or higher.
5. A method of preventing and/or ameliorating reduced expression of TJ-related proteins in cells caused by thermal stimulation, and/or increased cell permeability caused by thermal stimulation, the method comprising administering gamma-aminobutyric acid to a site subjected to thermal stimulation.
6. The method of item 5, wherein the TJ-related protein comprises one or more of a ZO-1 protein, an occludin protein, a claudin-1 protein, a claudin-2 protein, and a claudin-4 protein.
7. The method of item 5 or 6, wherein the cell comprises: one or more than two of human oral epithelial cells, human periodontal ligament cells, esophageal squamous cells and human colon epithelial cells.
8. The method of any one of claims 5-7, the thermal stimulus comprising a thermal stimulus having a temperature of 40 ℃ or greater.
Effects of the application
The application applies gamma-aminobutyric acid (GABA) or a product containing GABA to a position subjected to thermal stimulation, can effectively slow down the change of the thermal stimulation to cells, and widens the application range of the gamma-aminobutyric acid.
Further, by the relieving or improving effect of gamma-aminobutyric acid on thermal stimulation, it is expected that it can also prevent adverse effects when thermal stimulation is applied to a site susceptible to thermal stimulation.
Drawings
The drawings are included to provide a better understanding of the application and are not to be construed as unduly limiting the application. Wherein:
FIG. 1 shows the relative fluorescence intensity of FD4 in bottom chamber media after treatment of different groups in example 1;
FIG. 2 is a fluorescence photograph of ZO-1 protein, occludin protein, claudin-1 protein after treatment of different groups in example 2;
FIG. 3 shows the ZO-1 protein relative fluorescence intensity after semi-quantitative analysis of various groups of treatments by Image J software in example 2.
Detailed Description
Exemplary embodiments of the application are described below, including various details of embodiments of the application to facilitate understanding, which should be considered as merely exemplary. Accordingly, those of ordinary skill in the art will recognize that various changes and modifications of the embodiments described herein can be made without departing from the scope and spirit of the application. Also, descriptions of well-known functions and constructions are omitted in the following description for clarity and conciseness.
The application provides a novel application of gamma-aminobutyric acid and a method for realizing the application.
In one aspect, the application provides the use of gamma-aminobutyric acid for preventing and/or ameliorating reduced expression of TJ related proteins in cells caused by thermal stimulation, and/or increased cell permeability caused by thermal stimulation.
The term gamma-aminobutyric acid (GABA) refers to gamma-aminobutyric acid, which is a widely occurring amino acid in vertebrates, plants and microorganisms and is also an important inhibitory neurotransmitter.
The TJ is called light Junction in the whole and is called Tight connection in the Chinese, which is an important connection mode among cells, and a primary barrier is formed in a cell side space to play an important regulation role. TJ structure not only has the functions of regulating intercellular substance transport and maintaining epithelial polarity, but also the related protein molecules are involved in information transmission and regulation of processes such as cell proliferation differentiation, tumor inhibition, gene transcription and the like. TJ related protein molecules have been found to be more than 40. TJ-related proteins of the application are within the conventional definition in the art and may be any type of TJ-related protein, may be one type of TJ-related protein or a collection of types of TJ-related proteins, including but not limited to ZO-1 protein, ZO-2 protein, occludin protein, claudin-1 protein, claudin-2 protein, claudin-3 protein or claudin-4, and the like.
The expression is referred to in the art as a process by which a cell converts genetic information stored in a DNA sequence into a protein molecule having biological activity through transcription and translation during its life. In the present application, the expression is in accordance with the general definition in the art, specifically refers to the expression of TJ-related protein, and the reduced expression refers to the reduced or no expression of TJ-related protein, i.e., the expression of TJ-related protein lower than the normal TJ-related protein in the animal body may be referred to as reduced expression of TJ-related protein.
Cell permeability is susceptible to chemical and physical factors such as surfactants, lipids, sugars, ultrasound, etc., and is not stable; as the life cycle of the cell itself continues, such as aging, the cell permeability also changes. In certain pathological conditions (e.g., allergies, wounds, burns, hypoxia, etc.), the permeability of the semipermeable membrane is also enhanced by disrupting its normal structure and function.
The cell permeability enhancement can influence the balance of normal intracellular and extracellular substances, change the content of the intracellular and extracellular substances, accelerate the removal of the intracellular substances, easily cause the imbalance of intracellular environment, cause the imbalance of intracellular osmotic pressure, and influence the normal vital movement of cells; some substances in the cells such as proteins, lipids and the like leak out, cause immune reaction and the like.
The cell permeability enhancement, namely the life activity change, specifically the cell permeability enhancement caused by heat stimulation, can obviously reduce the cell permeability enhancement caused by heat stimulation.
The application discovers that the gamma-aminobutyric acid can effectively prevent and/or improve the reduction of the TJ related protein expression in cells caused by the thermal stimulation.
Further, the present application provides the use of gamma-aminobutyric acid in the preparation of a personal care product for preventing and/or improving the decrease of TJ-related protein expression in cells caused by thermal stimulation and/or the increase of cell permeability caused by thermal stimulation, wherein the personal care product can be an oral preparation or an external preparation, and the specific preparation type is not limited, and the person skilled in the art can select the preparation according to the use requirement in the prior art, for example, the oral preparation can be powder, granule, capsule, liquid preparation, suspension, etc., and the external preparation can be an application, spray, cream, liquid coating preparation, etc.
In some embodiments, the personal care product comprises a mouthwash, toothpaste, powder, lozenge, or oral patch. The present application is not limited in any way with respect to the preparation method of mouthwash, toothpaste, powder, lozenge or oral patch, and it can be prepared by the preparation method conventional in the art.
In some embodiments, the personal care product further comprises one or more of an antibacterial agent, an anticaries agent, an antiallergic agent, an anticalculus agent, an anti-inflammatory agent, a whitening agent, and a humectant.
Further, the present application provides the use of gamma-aminobutyric acid for the non-therapeutic purpose of preventing and/or ameliorating reduced expression of TJ related proteins in cells caused by thermal stimulation and/or enhanced cell permeability caused by thermal stimulation.
The non-therapeutic purpose, i.e., not for diagnosis or treatment of a disease, may be for prophylactic purposes, for improvement purposes, for alleviation, etc.
Further, the present application provides a method for preventing and/or ameliorating reduced expression of TJ-related proteins in cells caused by thermal stimulation, and/or enhanced cell permeability caused by thermal stimulation, the method comprising administering gamma-aminobutyric acid to a site subjected to thermal stimulation. The mode of administration may be any of those in the art including, but not limited to, painting, buccal, nebulization, oral administration, injection, and the like.
The site may be any site in the body of the animal, preferably a site susceptible to thermal stimulation, such as the oral cavity, palate, throat, stomach, intestinal tract, skin, etc. of the animal. The animals include, but are not limited to, humans, cats, dogs, pigs, sheep, cows, fish, etc., preferably humans. The method is a method of non-therapeutic interest.
In a preferred embodiment of the application, any one of the above uses or methods, wherein said TJ-related protein comprises one or two or three or four or more of a ZO-1 protein, an occludin protein, a claudin-1 protein, a claudin-2 protein or a claudin-4 protein.
The thermal stimulus as described above may be any thermal stimulus as long as it is capable of causing the change in expression of the TJ-related protein or causing the enhancement of cell permeability, and in a preferred embodiment, the thermal stimulus includes thermal stimuli having a temperature of 40 ℃ or higher, for example, 41 ℃ or higher, 42 ℃ or higher, 45 ℃ or higher, 50 ℃ or higher, 55 ℃ or higher, 60 ℃ or higher, 70 ℃ or higher, 80 ℃ or higher, 90 ℃ or higher, 100 ℃ or higher, 110 ℃ or higher, preferably 40 to 100 ℃.
The heat stimulus may be, but not limited to, liquid heat stimulus such as hot water, gas heat stimulus such as steam, solid heat stimulus such as hot food, radiation such as infrared or irradiation, and the like.
The application of the gamma-aminobutyric acid in preventing and/or improving the reduction of TJ related protein expression in cells caused by thermal stimulation or the method for preventing and/or improving the reduction of TJ related protein expression in cells caused by thermal stimulation can obviously enhance the expression of TJ related proteins, such as ZO-1 protein, occludin protein and claudin-1 protein, and the analysis of the ZO-1 protein expression quantity semi-quantitatively by Image J software can find that the ZO-1 protein expression quantity in cells treated by gamma-aminobutyric acid has extremely obvious statistical difference with the ZO-1 protein expression quantity in cells not treated by gamma-aminobutyric acid, so that the gamma-aminobutyric acid has stronger effect of resisting thermal stimulation and simultaneously prevents and/or improves adverse effects when being subjected to thermal stimulation.
The application of the gamma-aminobutyric acid in reducing the cell permeability enhancement caused by the heat stimulus or the method for reducing the cell permeability enhancement caused by the heat stimulus can prove that the cell experiment shows that the cell permeability after the gamma-aminobutyric acid treatment and the cell permeability after the non-gamma-aminobutyric acid treatment have obvious statistical differences, so that the gamma-aminobutyric acid can effectively reduce the cell permeability caused by the heat stimulus and simultaneously prevent and/or improve the adverse effect when the gamma-aminobutyric acid is subjected to the heat stimulus.
Examples
The materials used in the test and the test methods are described generally and/or specifically in the examples which follow,% represents wt%, i.e. weight percent, unless otherwise specified. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.
Example 1 Effect of GABA on cell permeability under thermal stimulation
Gamma-aminobutyric acid (hereinafter abbreviated as GABA, yu Huaxi organisms) was weighed and dissolved in Ca9-22 cell complete medium (containing 10% FBS, 1% penicillin) to prepare a GABA solution of 100 μg/mL.
The testing method comprises the following steps: ca9-22 cells (human oral epithelial cancer cells) were inoculated into a Transwell upper chamber at a density of 30000 cells/well at 37℃with 5% CO 2 Culturing under the condition for standby. After 24h cell attachment, the experimental group was added with 100. Mu.g/mL GABA and 100. Mu.L/well, and a control group and a blank group were simultaneously set. After 72h incubation, the 24-well plates were placed in a 41.5℃incubator for 3h. mu.L of fluorescein isothiocyanate-dextran (FD 4) 1mg/ml was added to the medium in the upper chamber of the Transwell. After incubation for 2 hours, medium was collected from the bottom chamber and added to a 96-well plate, and the fluorescence intensity (excitation 492nm; emission 520 nm) of FITC (fluorescein isothiocyanate) -Dextran 4 (FD 4) was detected with a fluorescence microplate reader,
blank group: GABA is not added, the incubator treatment at 41.5 ℃ is not carried out,
control group: GABA was not added, and the treatment was performed in an incubator at 41.5 ℃,
experimental group: GABA (100. Mu.g/mL) was added thereto, and the incubator treatment at 41.5℃was performed.
Analysis was performed using Graphpad Prism statistical software. The comparisons were performed using the t-test, P <0.05 indicated a statistical difference, P <0.01 indicated a significant statistical difference, and P <0.001 indicated a very significant statistical difference.
The experimental results are shown in fig. 1, and as can be seen from the graph, the comparison of the blank group and the control group can show that the thermal stimulation can significantly enhance the cell permeability and improve the FD4 fluorescence intensity of the bottom chamber culture medium. When 100 μg/mL of GABA was added to the experimental group, the fluorescence intensity in the basal chamber medium was significantly reduced, and GABA was effective in reducing the increase in cell permeability caused by thermal stimulation as compared to the control group.
Example 2: influence of GABA on the production of the light Junction related protein under thermal stimulation
Gamma-aminobutyric acid (hereinafter abbreviated as GABA, yu Huaxi organisms) was weighed and dissolved in Ca9-22 cell complete medium (containing 10% FBS, 1% penicillin) to prepare a GABA solution of 100 μg/mL.
Culturing Ca9-22 cells in DMEM medium in T75 flask until cell density is about 80%, inoculating into 24-well plate with cover glass, and culturing at 37deg.C and 5% CO 2 Culturing under the condition. After 24h the supernatant was aspirated off, and the experimental group was incubated with 100. Mu.g/mLGABA while the control and blank groups were set. Placing at 37deg.C and 5% CO 2 Incubator incubation for 72h, experimental and control groups were incubated for 3h at 41.5℃in incubator. After the incubation, the supernatant was aspirated, the cells were washed twice, and the cells were fixed with ice methanol at-20 ℃, followed by addition of TJ related protein, respectively: ZO-1 protein, occludin protein and claudin-1 protein primary antibody are incubated at 4 ℃ for overnight, the next day is washed three times by PBS, the corresponding secondary antibody is added, the secondary antibody is incubated at the dark room temperature for 1.5 hours, the nuclear sealing sheet is stained with DAPI, then the photographs are observed under a fluorescence microscope, finally the fluorescence data of the photographs are semi-quantitatively analyzed by Image J software, wherein,
blank group: GABA is not added, the incubator treatment at 41.5 ℃ is not carried out,
control group: GABA was not added, and the treatment was performed in an incubator at 41.5 ℃,
experimental group: GABA (100. Mu.g/mL) was added thereto, and the incubator treatment at 41.5℃was performed.
The experimental results are shown in fig. 2, and according to the analysis of fig. 2, the TJ related proteins of the control group after heat stimulation are compared with those of the blank group: the fluorescence of ZO-1 protein, occludin protein, claudin-1 protein (fluorescent region of FIG. 2) was significantly reduced in brightness, confirming that heat treatment affected TJ-related protein expression. Meanwhile, 100 mug/mL GABA is added in the experimental group, so that the expression quantity of three proteins can be obviously enhanced, and the brightness of a fluorescent region is enhanced. Therefore, the GABA effectively inhibits the reduction of TJ related protein expression caused by thermal stimulation, and has stronger effect of resisting the thermal stimulation.
FIG. 3 shows half-quantitative analysis of ZO-1 protein expression by Image J software, and analysis of Image J analysis data by Graphpad Prism statistical software. The comparisons were performed using the t-test, P <0.05 indicated a statistical difference, P <0.01 indicated a significant statistical difference, and P <0.001 indicated a very significant statistical difference.
As can be seen from fig. 3, compared with the control group, the ZO-1 expression level before and after thermal stimulation has a significant difference, the protein expression level after thermal stimulation in the control group is reduced, the relative fluorescence intensity is reduced, and the ZO-1 protein expression level caused by thermal stimulation can be significantly inhibited from being reduced by adding GABA in the experimental group, and the relative fluorescence intensity is increased, which indicates that the GABA effectively inhibits the reduction of the TJ-related protein expression caused by thermal stimulation, and has a stronger effect of resisting thermal stimulation.
Although the embodiments of the present application have been described above in connection with the above, the present application is not limited to the above-described specific embodiments and fields of application, which are merely illustrative, instructive, and not restrictive. Those skilled in the art, having the benefit of this disclosure, may effect numerous forms of the application without departing from the scope of the application as claimed.
Claims (8)
1. Use of gamma-aminobutyric acid for preventing and/or improving reduced expression of TJ related proteins in cells caused by thermal stimulation and/or increased cell permeability caused by thermal stimulation.
2. The use of claim 1, wherein the TJ-related protein comprises one or more of a ZO-1 protein, an occludin protein, a claudin-1 protein, a claudin-2 protein, and a claudin-4 protein.
3. The use according to claim 1 or 2, the cell comprising: one or more than two of human oral epithelial cells, human periodontal ligament cells, esophageal squamous cells and human colon epithelial cells.
4. Use according to any one of claims 1-3, said thermal stimulus comprising a thermal stimulus having a temperature above 40 ℃.
5. A method of preventing and/or ameliorating reduced expression of TJ-related proteins in cells caused by thermal stimulation, and/or increased cell permeability caused by thermal stimulation, the method comprising administering gamma-aminobutyric acid to a site subjected to thermal stimulation.
6. The method of claim 5, wherein the TJ-related protein comprises one or more of a ZO-1 protein, an occludin protein, a claudin-1 protein, a claudin-2 protein, and a claudin-4 protein.
7. The method of claim 5 or 6, the cell comprising: one or more than two of human oral epithelial cells, human periodontal ligament cells, esophageal squamous cells and human colon epithelial cells.
8. The method of any one of claims 5-7, the thermal stimulus comprising a thermal stimulus having a temperature above 40 ℃.
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