CN115381928B - Application of fungus extract in preparation of medicine for treating ulcerative colitis - Google Patents
Application of fungus extract in preparation of medicine for treating ulcerative colitis Download PDFInfo
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- CN115381928B CN115381928B CN202211321879.0A CN202211321879A CN115381928B CN 115381928 B CN115381928 B CN 115381928B CN 202211321879 A CN202211321879 A CN 202211321879A CN 115381928 B CN115381928 B CN 115381928B
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Abstract
The invention provides application of a fungus extract in preparation of a medicine for treating ulcerative colitis, and belongs to the technical field of ulcerative colitis. The fungus extract provided by the invention is Agrocybin polypeptide, and the experimental result shows that the Agrocybin polypeptide can effectively inhibit macrophages from generating inflammatory factors TNF-a and IL-6, inhibit NF-KB signal channels and effectively reduce the permeability of intestinal epithelial cells. The Agrocybin polypeptide can therefore be used for the development of new therapeutic drugs for ulcerative colitis.
Description
Technical Field
The invention belongs to the technical field of ulcerative colitis, and particularly relates to application of a fungus extract in preparation of a medicine for treating ulcerative colitis.
Background
Ulcerative colitis is a chronic nonspecific inflammatory bowel disease, the clinical symptoms of patients mainly include abdominal pain, diarrhea, mucopurulent bloody stool and other intestinal symptoms, and severe patients are often accompanied by high fever and toxic symptoms. In recent years, the incidence of ulcerative colitis has been on the rise in both our country and worldwide.
In the development process of ulcerative colitis, the inflammatory environment of the intestinal tract can cause damage to the barrier structure of the intestinal mucosa, cause abnormal oxidative stress reaction, cause the permeability of the intestinal epithelial mucosa to be increased, enable antigens in the intestinal cavity to enter the mucosa and submucosa, induce immune disorder and aggravate intestinal inflammation. Therefore, it would be helpful to treat ulcerative colitis by inhibiting intestinal inflammation and reducing intestinal damage.
At present, the clinically used therapeutic drugs for ulcerative colitis are mainly aminosalicylic acid preparations, steroid hormone preparations and immunosuppressants, and although the drugs are effective, the drugs are easy to cause serious side effects. Therefore, development of a new and safe therapeutic agent for ulcerative colitis is essential.
The Agrocybin polypeptide is a polypeptide extracted from Agrocybin, the NCBI number of the polypeptide is 91208278, and existing researches show that the Agrocybin polypeptide can have antibacterial, fungal and viral effects, however, the effect of the Agrocybin polypeptide in ulcerative colitis is not related to research.
Disclosure of Invention
The invention aims to provide a new application of Agrocybin polypeptide in preparing medicines for treating ulcerative colitis.
In order to achieve the purpose, the invention provides the following technical scheme:
in a first aspect, the invention provides an application of a fungus extract in preparing a medicine for treating ulcerative colitis, wherein the fungus extract is Agrocybin polypeptide.
Preferably, the Agrocybin polypeptide reduces macrophage secretion of inflammatory factors, such as TNF-a and IL-6.
Preferably, the Agrocybin polypeptide reduces intestinal epithelial permeability, and the Agrocybin polypeptide increases protein expression of the intestinal epithelial cell connexins ZO-1 and Occludin.
Preferably, the Agrocybin polypeptide inhibits the NF-KB signaling pathway.
In a second aspect, the invention provides the use of an Agrocybin polypeptide in the preparation of a medicament for inhibiting secretion of an inflammatory factor by a macrophage.
Preferably, the inflammatory factors are TNF-a and IL-6.
In a third aspect, the invention provides an application of Agrocybin polypeptide in preparing a medicament for reducing intestinal epithelial cell permeability.
Preferably, the medicament reduces intestinal epithelial cell permeability by increasing protein expression of the cellular connexins ZO-1 and Occludin.
The invention has the beneficial effects that:
the invention provides a new application of Agrocybin polypeptide in inhibiting macrophage from secreting inflammatory factor and reducing permeability of intestinal epithelial cells, provides possibility for further developing Agrocybin polypeptide, and provides a new safe and effective medicament for treating ulcerative colitis.
Drawings
FIG. 1 shows the results of the cytotoxicity of Agrocybin polypeptide against RAW264.7 cells;
FIG. 2 shows the results of the cytotoxicity of Agrocybin polypeptide against Caco-2 cells;
FIG. 3 is a graph showing the effect of Agrocybin polypeptide on TNF-a secretion from RAW264.7 cells;
FIG. 4 is a graph of the effect of Agrocybin polypeptide on IL-6 secretion from RAW264.7 cells;
FIG. 5 is a graph of the effect of Agrocybin polypeptide on the NF-KB pathway of RAW264.7 cells;
FIG. 6 is a graph of the effect of Agrocybin polypeptide on electrical resistance of Caco-2 cells;
FIG. 7 is a graph of the effect of Agrocybin polypeptides on protein expression of connexins ZO-1 and Occludin by Caco-2 cells.
Detailed Description
The examples are given for the purpose of better illustration of the invention, but the invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.
Example 1
(1) RAW264.7 and Caco-2 were mixed in a 2.5X 10 ratio 4 The seed/ml is inoculated in a 96-well plate, and each well is inoculated with 100 mu l;
(2) Placing a 96-well plate in a cell culture box, after overnight culture, adding the Agrocybin polypeptide containing 25,50,100,200,400 micromole/L into experimental groups 1-5, replacing a new culture medium for a control group, arranging 3 repeated holes in each group, after 24 hours of treatment, adding 10 mu L cck-8, incubating for 3 hours, detecting an OD value of 450nm, and calculating the survival rate of cells.
The results of the assay are shown in FIG. 1 (RAW 264.7 cells) and FIG. 2 (Caco-2 cells), from which we can conclude that the Agrocybin polypeptide is not toxic to RAW264.7 and Caco-2 cells.
Example 2
(1) According to2×10 5 Inoculating RAW264.7 cells into a 24-pore plate per hole, slightly shaking in a cross way to uniformly distribute the cells in a culture medium, and putting the 24-pore plate into a cell culture box for culture;
(2) After overnight culture, the experimental groups 1 and 2 are respectively added with Agrocybin polypeptide containing 200,400 μmol/L for pretreatment for 2h, the culture medium is replaced and the culture medium containing 500ng/ml LPS is added for treatment for 10h, 1 control group is replaced with the culture medium without adding drugs, 2 control groups are added with LPS, and each group of treatment is provided with 3 times of repetition;
(3) After the treatment, the content of TNF-a and IL-6 in the supernatant was measured by ELISA method, and the results of the measurement are shown in FIGS. 3 and 4.
From FIGS. 3 and 4, we can conclude that pre-treatment with Agrocybin polypeptide is effective in reducing LPS-induced TNF-a and IL-6 upregulation, i.e., agrocybin polypeptide is effective in inhibiting LPS-induced inflammation.
Example 3
(1) According to 5X 10 5 Inoculating RAW264.7 cells into a 6-hole plate per hole, slightly shaking in a cross way to uniformly distribute the cells in a culture medium, and putting the 6-hole plate into a cell culture box for culture;
(2) After overnight culture, the experimental groups 1 and 2 are respectively added with Agrocybin polypeptide containing 200,400 μmol/L for pretreatment for 2h, the culture medium is replaced and the culture medium containing 500ng/ml LPS is added for treatment for 10h, 1 control group is replaced with the culture medium without adding drugs, 2 control groups are added with LPS, and each group of treatment is provided with 3 times of repetition;
(3) After the treatment is finished, the culture medium is sucked and PBS is added to clean the cells, after 2 times of cleaning, 100 mu l of cell protein lysate is added, after the cells are scraped, the cells are collected in a centrifuge tube, and after 30min of ice cracking, the supernatant is centrifugally collected;
(4) Measuring the protein concentration of each group by using a BCA method, adding a loading buffer, uniformly mixing, placing the centrifuge tube in a water bath, and heating at 95 ℃ for 10min;
(5) Preparing 10% separation glue, flattening by using isopropanol, removing the isopropanol, washing by using double distilled water, adding the prepared 5% concentrated glue, and inserting into a comb for a subsequent experiment;
(6) Adding protein samples of different experimental groups into sample holes, adding a protein Marker, and adding an electrophoresis solution for electrophoresis;
(7) The voltage of the concentrated gel is 90V, the voltage of the separation gel is 100V, and electrophoresis is stopped when bromophenol blue reaches the bottom of the gel;
(8) Assembling an electric transfer clamp according to the sequence of red surface, sponge, three layers of filter paper, PVDF film, polyacrylamide gel, three layers of filter paper, sponge and black surface, adding electric transfer liquid after the assembly is finished, and carrying out constant current 250mA film transfer for 90min;
(9) After the electrotransformation is finished, transferring the PVDF into a sealing solution, and sealing for 1h at room temperature;
(10) Configuring primary antibodies of NF-KB, P-NF-KB and GAPDH according to the instruction of the antibodies, incubating the PVDF membrane with the primary antibodies, and incubating overnight at 4 ℃;
(11) After the incubation is finished, washing the membrane for three times by using PBS (phosphate buffer solution), wherein each time is 10min;
(12) After washing the membrane, the secondary antibody was incubated, and after incubation at room temperature for 2h, the secondary antibody was removed, washed 3 times with PBS, and developed and photographed.
The experimental results are shown in FIG. 5, and it can be shown that the protein expression of P-NF-KB can be obviously improved after LPS stimulation, and the protein expression of P-NF-KB can be reduced by the Agrocybin polypeptide, which indicates that the Agrocybin polypeptide can play an anti-inflammatory role through an NF-KB channel.
Example 4
(1) Preparation of Caco-2 to 15X 10 4 Adding 1ml of cell suspension into an upper microporous membrane of a 12-hole Transwell plate, adding 2ml of complete cell culture solution into a lower microporous membrane, and continuously culturing for 21 days to obtain a monolayer of cells;
(2) The experimental groups 1 and 2 are respectively added with Agrocybin polypeptide containing 200,400 micromol/L for pretreatment for 2h, the culture medium is replaced and the culture medium containing 500ng/ml LPS is added for treatment for 10h, 1 control group is replaced with the culture medium without adding medicines, 2 control groups are added with LPS, and each group of treatment is provided with 3 times of repetition;
(3) After the treatment was completed, the resistance value of each group was measured using a cell resistance meter.
The experimental result is shown in fig. 6, LPS can significantly reduce the magnitude of the TER value, and after the Agrocybin polypeptide is used for pretreatment, the TER value can be significantly increased, so that the Agrocybin polypeptide treatment can effectively reduce the cell barrier permeability of intestinal epithelial cells.
Example 5
(1) Preparation of Caco-2 to 15X 10 4 Adding 1ml of cell suspension into an upper microporous membrane of a 12-hole Transwell plate, adding 2ml of complete cell culture solution into a lower microporous membrane, and continuously culturing for 21 days to obtain a monolayer of cells;
(2) The experimental groups 1 and 2 are respectively added with Agrocybin polypeptide containing 200,400 micromol/L for pretreatment for 2h, the culture medium is replaced and the culture medium containing 500ng/ml LPS is added for treatment for 10h, 1 control group is replaced with the culture medium without adding medicines, 2 control groups are added with LPS, and each group of treatment is provided with 3 times of repetition;
(3) After the treatment is finished, removing the culture medium by suction, adding PBS to clean the cells, cleaning for 2 times, adding 100 mu l of cell protein lysate, scraping the cells, collecting the cells in a centrifuge tube, cracking the cells on ice for 30min, and centrifugally collecting the supernatant;
(4) Measuring the protein concentration of each group by using a BCA method, adding a loading buffer, uniformly mixing, placing the centrifuge tube in a water bath, and heating at 95 ℃ for 10min;
(5) Preparing 10% separation glue, flattening by using isopropanol, removing the isopropanol, washing by using double distilled water, adding the prepared 5% concentrated glue, and inserting into a comb for a subsequent experiment;
(6) Adding protein samples of different experimental groups into sample holes, adding a protein Marker, and adding an electrophoresis solution for electrophoresis;
(7) The voltage of the concentrated gel is 90V, the voltage of the separation gel is 100V, and electrophoresis is stopped when bromophenol blue reaches the bottom of the gel;
(8) Assembling an electric transfer clamp according to the sequence of red surface, sponge, three layers of filter paper, PVDF film, polyacrylamide gel, three layers of filter paper, sponge and black surface, adding electric transfer liquid after the assembly is finished, and carrying out constant current 250mA film transfer for 90min;
(9) After the electrotransformation is finished, transferring the PVDF into a sealing solution, and sealing for 1h at room temperature;
(10) Configuring primary antibodies of ZO-1, occludin and GAPDH according to the antibody specification, incubating the primary antibodies with a PVDF membrane, and incubating overnight at 4 ℃;
(11) After the incubation is finished, washing the membrane for three times by using PBS (phosphate buffer solution), wherein each time is 10min;
(12) After washing the membrane, the secondary antibody was incubated, and after incubation at room temperature for 2h, the secondary antibody was removed, washed 3 times with PBS, and developed and photographed.
The experimental result is shown in figure 7, LPS can effectively reduce the protein expression level of ZO-1 and Occludin of intestinal epithelial cells, and the protein expression level of ZO-1 and Occludin can be effectively improved by the treatment of the Agrocybin polypeptide, so that the Agrocybin polypeptide can reduce the cell barrier permeability of the intestinal epithelial cells by improving the protein expression of ZO-1 and Occludin.
In view of the above examples, the present inventors concluded that Agrocybin polypeptides can be used to treat ulcerative colitis by lowering the production of inflammatory factors TNF-a and IL-6 by macrophages through the condition P-NF-KB, while lowering the permeability of intestinal epithelial cells by providing protein expression of ZO-1 and Occludin.
Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.
Claims (4)
1. The application of a fungus extract in preparing a medicine for treating ulcerative colitis is characterized in that the fungus extract is Agrocybin polypeptide.
2. The use of claim 1, wherein the Agrocybin polypeptide reduces macrophage secretion of inflammatory factors, wherein the inflammatory factors are TNF-a and IL-6.
3. The use of claim 1, wherein the Agrocybin polypeptide decreases intestinal epithelial permeability and increases protein expression of the intestinal epithelial cell connexins ZO-1 and Occludin.
4. The use of claim 1, wherein said Agrocybin polypeptide inhibits the NF-KB signaling pathway.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102604843A (en) * | 2012-03-31 | 2012-07-25 | 福建师范大学 | Preparation method of fungus fermentation product and application thereof in prevention and treatment of rice diseases |
| CN104031883A (en) * | 2014-05-16 | 2014-09-10 | 浙江大学 | Model for protecting mouse ulcerative colitis by antibacterial peptide C-BF |
| CN104428036A (en) * | 2012-03-14 | 2015-03-18 | 诺瓦生命科学有限公司 | Polypeptides and their use |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104428036A (en) * | 2012-03-14 | 2015-03-18 | 诺瓦生命科学有限公司 | Polypeptides and their use |
| CN102604843A (en) * | 2012-03-31 | 2012-07-25 | 福建师范大学 | Preparation method of fungus fermentation product and application thereof in prevention and treatment of rice diseases |
| CN104031883A (en) * | 2014-05-16 | 2014-09-10 | 浙江大学 | Model for protecting mouse ulcerative colitis by antibacterial peptide C-BF |
Non-Patent Citations (2)
| Title |
|---|
| 微生物来源的新抗菌肽;恽冬杰等;《抗感染药学》;20061225(第04期);第149-153页 * |
| 田头菇属真菌研究进展;周会明等;《中国食用菌》;20091115(第06期);第3-8页 * |
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