CN116036072A - Application of isoginkgetin in preparation of medicines for preventing and/or treating ulcerative colitis - Google Patents
Application of isoginkgetin in preparation of medicines for preventing and/or treating ulcerative colitis Download PDFInfo
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- CN116036072A CN116036072A CN202310104337.6A CN202310104337A CN116036072A CN 116036072 A CN116036072 A CN 116036072A CN 202310104337 A CN202310104337 A CN 202310104337A CN 116036072 A CN116036072 A CN 116036072A
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- inflammatory
- intestinal mucosa
- isoginkgetin
- intestinal
- dss
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Abstract
The invention discloses application of isoginkgetin in preparation of a medicament for preventing and/or treating ulcerative colitis, and relates to the technical field of biological medicines. Based on a DSS-induced mouse colonitis model, the invention discovers that the isoginkgetin has the functions of relieving the inflammation of intestinal mucosa and maintaining the barrier of the intestinal mucosa for the first time. Specifically, administration of the DSS-induced isoborneolin treatment in a mouse colitis model can significantly slow down weight loss, reduce disease activity index, reduce colon tissue damage, reduce inflammatory cell infiltration, reduce inflammatory immune cell infiltration in intestinal mucosa, down regulate Th1/Th17 cell production in intestinal mucosa of a DSS non-human mammal model, weaken intestinal mucosa inflammatory reaction, repair impaired intestinal barrier function of the non-human mammal model, and reduce secretion of serum inflammatory cytokines. Thus, the isoginkgetin is suggested to be used for clinically preventing and/or treating ulcerative colitis patients in the future.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to application of isoginkgetin in preparation of a medicament for preventing and/or treating ulcerative colitis.
Background
Ulcerative colitis (Ulcerative colitis, UC) is an inflammatory bowel disease, which is mainly inflammatory and ulcerative lesions of the mucous membrane and submucosa of the rectum, sigmoid colon, and is mainly manifested by recurrent intense abdominal pain, diarrhea, rectal bleeding and hematochezia, and in severe cases can invade the whole colon and terminal ileum, even with risk of canceration.
Many researches consider that the onset of UC is mostly related to host susceptibility genes, individual immune dysfunction, impaired intestinal barrier, intestinal flora imbalance, unhealthy eating habits and the like, but the specific etiology is still unknown, and the characteristics of long course, easy recurrence and many complications are added, so far, no cure strategy exists, and the UC is already listed as a modern refractory disease by the world health organization.
The most commonly used intervention drugs in clinic at present mainly comprise three types of 5-amino salicylate, hormone and immunosuppressant. Although the short-term curative effect is better, the long-term curative effect is still poor, the recurrence rate after stopping the medicine is high, the adverse reaction is large, and the like. Therefore, searching for natural anti-colitis drugs and adjuvant therapies that are efficient, safe, and free of toxic and side effects has become an urgent task to be solved in modern medical treatment.
In recent years, traditional Chinese medicine has unique advantages in the aspects of regulating immune function, repairing intestinal mucosa, relieving intestinal inflammation and the like, and has the advantages of lasting effect, low price, high safety, easy acceptance by patients and the like, and gradually becomes a research hotspot for developing anti-colitis medicines and novel immunosuppressants. Clinical practice proves that for the early-stage and early-stage light patients of the colitis, the intestinal inflammation and the mucous membrane damage degree can be effectively improved by adding the intervention of the traditional Chinese medicine, and the danger of the intestinal inflammation and the mucous membrane damage degree to the clinical colitis and colon cancer is reduced.
Isoginkgetin (IGK) is a natural biflavanoid, and was first discovered in the traditional Chinese medicine gingko by researchers such as Briencon-Scheid in 1983. Studies have shown that IGK can inhibit the production of proinflammatory cytokines such as TNF-a, IL-6 and the like in RAW 264.7 macrophages activated by LPS. In addition, IGK has good protection effect on heart diseases caused by obesity by enhancing Nrf2/ARE signal paths. However, it is not yet reported whether IGK has therapeutic effects on ulcerative colitis.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a novel application of isoginkgetin in preparing a medicament for preventing and/or treating ulcerative colitis, thereby providing medicament support for patients suffering from ulcerative colitis in clinical prevention and/or treatment.
The invention is realized in the following way:
in a first aspect, the invention provides the use of isoginkgetin in the manufacture of a medicament for the prevention and/or treatment of ulcerative colitis.
The inventor finds that Isoginkgetin (IGK) has the functions of relieving intestinal mucosa inflammation and maintaining intestinal mucosa barrier for the first time based on a DSS-induced mouse colitis model, and shows good treatment effect in the DSS-induced mouse colitis model, thereby prompting that the Isoginkgetin (IGK) can be used for clinically preventing and/or treating ulcerative colitis patients in the future.
It should be noted that the isoginkgetin provided by the invention can be used for preventing and/or treating mild, moderate and severe ulcerative colitis. After the isoginkgetin is used, the UC symptoms of the patients are relieved, the effect of reducing colonitis is obvious, and the like, which belong to the category of treatment. Including but not limited to rectal bleeding, mucosal crypt abscess (mucosal crypt abscess), inflammatory pseudopolyps, abdominal pain, diarrhea, and ulcers of the colon and rectum.
In a preferred embodiment of the present invention, the medicament further comprises pharmaceutically acceptable excipients.
In certain embodiments, pharmaceutically acceptable excipients, including one or more of fillers, stabilizers, solvents, dispersants, coatings, absorption enhancers, controlled release agents, and one or more inert excipients; inert excipients include starches, polyols, granulating agents, microcrystalline cellulose (e.g., celpher), diluents, lubricants, binders, disintegrating agents and the like.
Pharmaceutically acceptable excipients, including, but not limited to, starch, dextrin, collagen scaffold, matrigel, skin repair film, aminated gelatin, chitosan, silk fibroin, cellulose polylactic acid, tropoelastin, hyaluronic acid, and the like.
In a preferred embodiment of the present invention, the dosage form of the above-mentioned drug is selected from the group consisting of oral liquid, capsule, tablet, effervescent tablet, powder injection, injection solution, injection, aerosol, microemulsion, gel or nano-preparation.
In an alternative embodiment, the dosage form of the above-mentioned medicament is selected from injection (including injection, freeze-dried powder), through intraperitoneal injection; oral preparation.
In certain embodiments, the pharmaceutical compositions described above comprise a pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solution (e.g., balanced salt solution or physiological saline), dispersion, suspension, or emulsion. For general principles regarding the formulation of the pharmaceutical composition reference is made to cell therapy, which is written by g.morstyn and w.shelidan: stem Cell transplantation, gene Therapy and cellular immunotherapy (Cell Therapy: stem Cell Transplantation, gene Therapy, and Cellular Immunotherapy), cambridge university press, 1996; and hematopoietic stem cell therapy (Hematopoietic Stem Cell Therapy), E.D.Ball, J.Lister & P.Law, churchillLivingstone,2000.
In a second aspect, the invention also provides an application of the isoginkgetin in preparing a medicament for jointly treating ulcerative colitis.
In a preferred embodiment of the present invention, the agent used in the combination therapy comprises isoginkgetin and at least one of the following agents:
aminosalicylic acid formulations, glucocorticoids, immunosuppressants, equisetol, antibiotics, monoclonal antibodies, microecologics and topical therapeutic agents;
the aminosalicylic acid preparation is one or more selected from 5-aminosalicylic acid (5-ASA), mesalamine, sulfasalazine, and oxalazine He Baliu nitrogen;
the glucocorticoid is selected from prednisone (prednisone), budesonide (budesonide), beclomethasone (beclomethasone), beclomethasone dipropionate and hydrocortisone;
immunosuppressants are selected from mercaptopurine drugs, methotrexate (MTX), cyclosporin A (CsA), tacrolimus (FK 506) and mycophenol (MMF); in an alternative embodiment, the mercaptopurine drug is selected from azathioprine or 6-mercaptopurine;
the antibiotic is selected from penicillin, tobramycin, novel cephalosporin, metronidazole or cephalosporin;
the monoclonal antibody is selected from IgG4 monoclonal antibody of anti-TNF-alpha monoclonal antibody and anti-alpha 24 Integrin (Integrin);
the microecological preparation is selected from probiotics, prebiotics and synbiotics.
Probiotics are preparations containing a specific sufficient number of living bacteria; the prebiotics are functional oligosaccharides which are not easy to be digested and absorbed by human bodies, but can be absorbed and utilized by beneficial bifidobacteria in intestinal tracts to play a role in promoting bifidobacteria; the synbiotics are mixed preparations of probiotics and prebiotics, or vitamins, microelements and the like are added.
In an alternative embodiment, the monoclonal antibody is selected from Infliximab (IFX), adalimumab (Adalimumab), natalizumab, or cetuximab (Certolizumab Pegol).
In a preferred embodiment of the present invention, the topical therapeutic agent is selected from at least one of enema, foam and suppository. Local administration greatly reduces systemic side effects.
In a third aspect, the invention also provides the use of an isoginkgetin in the manufacture of a medicament for the prevention and/or treatment of a non-human mammalian model of ulcerative colitis, the non-human mammal having at least one of the following characteristics after administration of the isoginkgetin treatment:
(1) A reduced weight loss in a DSS non-human mammalian model;
(2) Decreasing disease activity index;
(3) Reducing colon tissue damage;
(4) Reducing inflammatory cell infiltration;
(5) Reducing inflammatory immune cell infiltration in intestinal mucosa;
(6) Down-regulating intestinal intrathecal Th1/Th17 cell production in a DSS non-human mammalian model;
(7) The inflammatory response of the intestinal mucosa is weakened;
(8) Repairing an impaired intestinal barrier function in a non-human mammalian model;
(9) Reducing secretion of serum inflammatory cytokines;
the non-human mammal is selected from a mouse, a rat, a monkey, a sheep or a cow.
In a preferred embodiment of the application of the present invention, in the above feature (7), the weakening of inflammatory response of intestinal mucosa comprises: reduce the secretion of inflammatory cytokines in the intestinal mucosa.
In a preferred embodiment of the present invention, the inflammatory cytokine in the intestinal mucosa is at least one cytokine selected from the group consisting of:
IL-6, TNF-a and IL-1b.
In a preferred embodiment of the use of the present invention, in the above feature (9), the serum inflammatory cytokine is at least one selected from the group consisting of:
IL-6, TNF-a and IL-1b.
The invention has the following beneficial effects:
the invention is based on a DSS-induced mouse colonitis model, and discovers that the isoginkgetin has the functions of relieving intestinal mucosal inflammation and maintaining intestinal mucosal barrier for the first time, and has good treatment effect in the DSS-induced mouse colonitis model. Specifically, administration of the DSS-induced isoborneolin treatment in a mouse colitis model can significantly slow down weight loss, reduce disease activity index, reduce colon tissue damage, reduce inflammatory cell infiltration, reduce inflammatory immune cell infiltration in intestinal mucosa, down regulate Th1/Th17 cell production in intestinal mucosa of a DSS non-human mammal model, weaken intestinal mucosa inflammatory reaction, repair impaired intestinal barrier function of the non-human mammal model, and reduce secretion of serum inflammatory cytokines.
Thus, the isoginkgetin is suggested to be used for clinically preventing and/or treating ulcerative colitis patients in the future.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the effect of IGK treatment on clinical symptoms in mice with colon inflammation; (a) a mouse weight change profile; (B) A curve of the activity index (disease activity index, DAI) of colitis in mice. The sample size of each group of mice n=10.
Fig. 2 is a graph of colon HE staining and score statistics for reduction of pathological lesions of intestinal mucosa in colon inflammatory mice after IGK treatment. (a) a colon HE staining picture of mice; and (B) pathology score statistics. * P <0.01, the sample size n=10 per group of mice.
FIG. 3 is a graph showing the cell statistics of reduced intestinal mucosa innate immune cell infiltration in colitis mice following IGK treatment. (A) Analyzing a loop-gate strategy map by using a mouse colon cell flow cytometry; colon cell count statistics in mice: (B) Total myeloid lineage cells, (C) dendritic cells, (D) neutrophils, (E) total macrophages, (F) pro-inflammatory macrophages, (G) anti-inflammatory macrophages. * p <0.05, p <0.01, ns, no statistical difference, per group of mice sample size n=10.
FIG. 4 is a graph showing the cytostatistical results of Th1/Th17 cytopenia in the intestinal mucosa of mice after IGK treatment. (A) Analyzing a loop-gate strategy map by using a mouse colon cell flow cytometry; colon cell count statistics in mice: (B) Th1 cells, (C) Th17 cells. * p <0.05, the sample size n=10 per group of mice.
FIG. 5 is a graph of statistical results of the effect of IGK on inflammatory factor levels in the intestinal mucosa of mice with colon inflammation. ELISA detects IL-6, TNF-a, IL-1b levels in the intestinal mucosa of mice (left to right), respectively. The sample size of each group of mice n=10, p <0.05, p <0.01.
FIG. 6 is a graph showing the effect of IGK on inflammatory factor levels in serum of mice with colon inflammation. ELISA measures IL-6, TNF-a, IL-1b levels in mouse serum (left to right), respectively. The sample size of each group of mice n=10, p <0.05, p <0.001.
Fig. 7 is a graph of experimental results of IGK alleviating damage to the intestinal mucosal barrier of colitis mice. ELISA detects levels of (A) FITC-dextran, (B) LPS, and (C) Zoneulin in mouse serum, respectively. The sample size of each group of mice n=10, p <0.0001.
FIG. 8 is a graph showing the comparison of the therapeutic effects of IGK and mesalamine (mesalazine) on a murine model of colitis. Evaluating the difference in efficacy of two different groups of drugs to improve clinical symptoms in colitis mice using DAI scores; sample size n=15 for each group of mice.
Fig. 9 is a schematic diagram of the experimental scheme.
Detailed Description
Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.
Unless otherwise indicated, practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the ability of a person skilled in the art. This technique is well explained in the literature, as is the case for molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989); oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait et al, 1984); animal cell culture (Animal Cell Culture) (r.i. freshney, 1987); methods of enzymology (Methods in Enzymology) (Academic Press, inc.), experimental immunology handbook (Handbook of Experimental Immunology) (D.M.Weir and C.C.Blackwell, inc.), gene transfer vectors for mammalian cells (Gene Transfer Vectors for Mammalian Cells) (J.M.Miller and M.P.calos, inc., 1987), methods of contemporary molecular biology (Current Protocols in Molecular Biology) (F.M.Ausubel et al, inc., 1987), PCR: polymerase chain reaction (PCR: the Polymerase Chain Reaction, inc., 1994), and methods of contemporary immunology (Current Protocols in Immunology) (J.E.Coligan et al, 1991), each of which is expressly incorporated herein by reference.
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
The following inventors first constructed a mouse colitis model. Specifically, mice were fed with DSS solution (Dextran sulfate sodium salt (DSS)) for 7 days DSS solution+3 days drinking water, and a model of mouse colitis was constructed. Male mice with the background of C57BL/B6J are selected, and the mice are 8-10 weeks old and have normal morphology, activity and development. DSS model mice were divided into 2 groups, each group of mice was not less than 8 mice, one group received IGK treatment, and the other group was a non-treatment control group. IGK (treatment group, 4 mg/kg/day) or PBS (control group) was administered daily by intraperitoneal injection, respectively, from day 0 to day 10 of the molding. The experimental protocol is shown with reference to fig. 9.
Two groups of mice were then sacrificed on day 10, colon tissues were collected separately and the pathological damage to colitis was assessed by H-E staining. And detecting the influence of the IGK on the inflammatory reaction of the mouse DSS model intestinal mucosa by enzyme-linked immunosorbent assay on inflammatory cytokines in serum and the intestinal mucosa. The effect of IGK on inflammatory immune cell infiltration of intestinal mucosa was analyzed by flow cytometry. The effect of IGK on intestinal wall permeability in a mouse model of colitis was assessed by detecting the levels of LPS, FITC-dextran, zonulin in the serum of the mice.
The colitis model construction and related efficacy evaluation test are specifically as follows:
example 1
Construction of a mouse model of DSS colitis.
(1) Male mice of 20-25g, 8-10 weeks old, were selected from healthy, normal-rearing laboratory mice (C57 BL/6J) and randomly divided into experimental and control groups.
(2) Preparing a DSS solution: 4g of DSS powder was dissolved in distilled water, and the volume was then set to 200ml with distilled water to prepare a 2% DSS solution.
(3) The 2% DSS aqueous solution was changed once for 3 days, dosing was stopped after seven days, and normal drinking water was changed to feed for three days, which was one cycle.
(4) The mental state, hair condition, weight, stool characteristics, presence or absence of hematochezia, etc. of the mice of the experimental group and the control group were recorded once daily in one period, so as to evaluate the disease development and modeling progress of the mice. Disease activity index (disease activity index, DAI) scoring system is as follows:
based on the constructed DSS-induced mouse colitis model, the curative effect of IGK on colitis is firstly explored. As shown in fig. 1A, the weight change of mice was recorded daily during the modeling period, and the weight loss of IGK-treated DSS mice was significantly lower than that of non-treated mice, and the therapeutic efficacy of IGK on clinical symptoms of enteritis mice was evaluated according to the disease activity index (disease activity index, DAI) scoring system by observing the stool characteristics of mice, whether or not there were clinical symptoms such as diarrhea, mucopurulent stool, and the like. Higher DAI scores indicate more severe disease.
As shown in B in fig. 1, the day of mice DAI was recorded, and it was found that the clinical symptoms of the colitis in the IGK-treated group were significantly reduced as compared with the control group (PBS-treated group), and it was primarily suggested that the IGK had a therapeutic effect on colitis.
Example 2
The present example evaluates the severity of intestinal lesions in mice by means of sections of the colon paraffin of the mice. The results of the paraffin sections of the colon are mainly used for comprehensively evaluating the severity of intestinal lesions from the damage degree of colon tissues and the infiltration degree of inflammatory cells.
1. Colon paraffin section of mice
(1) Fixing: the colon of the dissected mouse is taken to be fixed in 4% PFA fixing solution by a tissue block with the length of about 0.5cm, and the colon is placed in a refrigerator at the temperature of 4 ℃ for more than 48 hours.
(2) Dehydrating: the fixed intestinal tissue pieces were removed and dehydrated using an automatic dehydrator using ethanol graded at different concentrations of 70%, 80%, 90%, 95%, 100%.
(3) And (3) transparency: the dehydrated tissue is sequentially put into a mixed solution prepared by 100 percent of ethanol and dimethylbenzene according to the proportion of 1:1, and soaked in the dimethylbenzene solution for 30min.
(4) Wax dipping: after the soaking, adding mixed solution of xylene and paraffin at 60 ℃ according to the proportion of 1:1, and washing away redundant xylene in paraffin at 60 ℃ for 1h.
(5) Embedding: the samples are placed into a paraffin embedding machine for embedding, and the direction and the plane of the tissue pre-section are considered in the embedding process.
(6) Slicing: slicing by paraffin slicer, oven baking at 60deg.C for 2 hr to obtain slices with thickness of 3-6 μm.
2. HE staining of mouse colo-paraffin sections
(1) And (5) putting the baked paraffin sections into a xylene solution for 10-20min to remove the paraffin.
(2) Take out the sections and place them in 1:1 and xylene for 10min, then sequentially adding 100%, 95%, 90%, 80% and 70% ethanol with different concentrations for 3min, and finally adding distilled water for 3min.
(3) Taking out paraffin section, soaking in hematoxylin solution for 10min for staining, and optionally shrinking
Short or prolonged dyeing times.
(4) After dyeing, flowing water for 10s, washing with 1% ethanol hydrochloride for 3s, and flowing water for 10s.
(5) The sections were soaked in eosin dye for about 5min.
(6) And sequentially placing the slices into 70%, 80%, 90%, 95% and 100% ethanol with different concentration grades for 30s, mixing 100% ethanol and xylene according to the mixed solution for 3min (1:1 concentration), and respectively dehydrating and transparentizing the xylene solution for 3min.
(7) The surface of the slice is slightly dry, and the slice is sealed by neutral resin.
(8) The pathological scoring criteria for the colitis lesions of the mice are shown in the following table:
after comparing the two paraffin sections, the mucous membrane layer of the non-treatment control group is seriously diseased, the goblet cells and the crypts are seriously damaged, the diseased range exceeds the mucous membrane layer, and the mucous membrane layer and the submucosa are accompanied by infiltration of a large amount of lymphocytes. In contrast, the crypt and goblet cells of the treated group were kept relatively intact without massive lymphocyte infiltration, as shown in fig. 2A (the right side of fig. 2A is the modeling treated group and the left side is the modeling non-treated control group). Combining inflammatory cell infiltration score and tissue injury score. After quantitative statistics of the scores, the treatment group is found to have significantly lower inflammatory injury degree of the colonic mucosa than the control group, and the results are statistically significant, as shown in B in FIG. 2. Thus, the above results indicate that IGK is effective in alleviating DSS-induced experimental colitis in mice.
Example 3
Because of the massive inflammatory immune cell infiltration within the intestinal mucosa, an imbalance in pro-inflammatory/anti-inflammatory immune responses is an important pathophysiological mechanism of ulcerative colitis intestinal mucosal inflammatory lesions. Thus, this example further analyzes the effect of IGK treatment on inflammatory immune cell infiltration in the intestinal mucosa.
As shown in fig. 3, after IGK treatment, the infiltration of innate immune cells in the colon mucosa of DSS mice was significantly reduced compared to the non-treated control group. Wherein the pro-inflammatory subtype in macrophages is also affected by IGK and reduced in number.
In addition, the Th1/Th17 cellular immune response is also an important pathogenic element in colitis. Through flow cytometry analysis, IGK treatment was found to significantly down-regulate Th1/Th17 cell production in intestinal mucosa of mice with colitis (see fig. 4).
Example 4
This example further explores the therapeutic effect of IGK on intestinal mucosal inflammation in mice with colon inflammation. The levels of inflammatory factors in the mouse intestinal mucosa and serum were determined by ELISA, respectively.
Enzyme-linked immunosorbent assay (ELISA)
(1) Antigen coating: the antigen was diluted to 10mg/ml with carbonate coating solution, 100ml was added to each microplate well, and the mixture was placed in a refrigerator at 4℃overnight. The antigen liquid was aspirated and each well was washed with 100ml of PBST and once every 5min.
(2) Closing: 100ml of blocking solution (5% BSA) was added to each of the microplate wells, incubated at room temperature for 60min, the blocking solution was removed by pipetting, and washed with PBST in a volume of 100ml for 5min.
(3) Incubation resistance: the washing solution was removed, and a primary antibody diluted 500 to 1000 times with 5% BSA was added to each of the wells, and the wells were incubated at room temperature for 1 hour in a volume of 100 ml.
(4) Washing: the liquid in the wells of the enzyme-labeled wells was aspirated, and each well was washed three times with PBST in a volume of 100ml per well for 5min.
(5) Adding enzyme-labeled antibody: the wash was blotted clean and 100ml of secondary antibody diluted 1:2000 with 5% BSA was added to each well and incubated at room temperature for 1h.
(6) Washing: the liquid in the wells was removed and washed three times with PBST for 5min each with 100ml per well volume, and then 100ml PBS was added to each well for a further 5min.
(7) Color development: 100ml of TMB substrate solution was added to each well, and the reaction was carried out at room temperature for 40min while keeping out light during the addition.
(8) Terminating the reaction: the reaction was stopped by adding 100ml of 2M sulfuric acid to each microplate.
(9) Reading a plate: the microplate was placed on the microplate reader for reading.
Through ELISA measurement, the levels of inflammatory cytokines IL-6, TNF-a and IL-1b in the intestinal mucosa (shown in figure 5) and serum (shown in figure 6) of the colonitis mice treated by the IGK are obviously lower than those of non-treated groups, so that the IGK is indicated to have good treatment effect on the inflammatory reaction of the intestinal mucosa of a colonitis model of the mice.
Example 5
Impaired intestinal barrier function, i.e. increased intestinal permeability, is also one of the main pathophysiological manifestations of colitis. Thus, this example performed an intestinal permeability test.
First, after the mice were fasted overnight, the mice were given FITC-dextran lavage treatment. After 4 hours, it was found that the permeation rate of FITC-dextran in serum of colitis mice treated with IGK was significantly reduced compared to PBS mice, indicating that IGK could repair the impaired intestinal barrier function of colitis mice (as shown in fig. 7 a). In addition, the levels of LPS and Zoneulin in serum are common indicators reflecting the permeability of the intestinal wall. The levels of LPS and Zonulin in the serum of mice, respectively, were found to be significantly reduced in both serum of the colitis mice given IGK treatment compared to the untreated group using ELISA (as shown by B, C in fig. 7). Surface IGK can alleviate colonitis mice intestinal mucosal barrier damage.
Example 6
To further evaluate the clinical promise of IGK for treatment of ulcerative colitis, we compared the therapeutic effects of mesalazine (purchased from Sigma-Aldrich,0.52 g/kg/day) with IGK (4 mg/kg/day) based on a DSS-induced colitis mouse model. Mesalazine is currently the most commonly used drug clinically for the treatment of ulcerative colitis.
The result of the treatment effect is shown in figure 8, and the result of recording the DAI in the whole modeling process shows that the treatment effect of the IGK on the clinical symptoms of mice with the colon inflammation is superior to that of mesalamine.
In summary, the inventor discovers that IGK can demonstrate good treatment effect in a mouse colonitis model induced by DSS through relieving intestinal mucosal inflammation and maintaining intestinal mucosal barrier function for the first time, and prompts that the IGK can be used for clinically treating ulcerative colonitis patients in the future.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. Application of isoginkgetin in preparing medicine for preventing and/or treating ulcerative colitis is provided.
2. The use according to claim 1, wherein the medicament further comprises pharmaceutically acceptable excipients.
3. The use according to claim 2, wherein the pharmaceutical dosage form is selected from the group consisting of oral liquid, capsule, tablet, effervescent tablet, powder injection, water injection, nebuliser, microemulsion, gel or nano-preparation.
4. The application of isoginkgetin in preparing medicine for treating ulcerative colitis is provided.
5. The use according to claim 4, wherein the agent used in the combination therapy comprises isoginkgetin and at least one of the following agents:
aminosalicylic acid formulations, glucocorticoids, immunosuppressants, equisetol, antibiotics, monoclonal antibodies, microecologics and topical therapeutic agents;
the aminosalicylic acid preparation is one or more selected from 5-aminosalicylic acid (5-ASA), mesalamine, sulfasalazine and olsalazine He Baliu nitrogen;
the glucocorticoid is selected from prednisone (prednisone), budesonide (budesonide), beclomethasone (beclomethasone), beclomethasone dipropionate and hydrocortisone;
the immunosuppressant is selected from mercaptopurine drugs, methotrexate (MTX), cyclosporin A (CsA), tacrolimus (FK 506) and mycophenol (MMF); preferably, the mercaptopurine drug is selected from azathioprine or 6-mercaptopurine;
the antibiotic is selected from penicillin, tobramycin, novel cephalosporin, metronidazole or cephalosporin;
the monoclonal antibody is selected from the group consisting of anti-TNF- ɑ IgG4 mab against α24 Integrin (interserin);
the microecological preparation is selected from probiotics, prebiotics and synbiotics;
preferably, the monoclonal antibody is selected from Infliximab (IFX), adalimumab (Adalimumab), natizumab or cetuximab (certolizumab pegol).
6. The use according to claim 5, wherein the topical therapeutic agent is selected from at least one of an enema, a foam and a suppository.
7. Use of isoginkgin the manufacture of a medicament for preventing and/or treating a non-human mammalian model of ulcerative colitis, characterized in that said non-human mammal has at least one of the following characteristics after administration of isoginkgin treatment:
(1) A reduced weight loss in a DSS non-human mammalian model;
(2) Decreasing disease activity index;
(3) Reducing colon tissue damage;
(4) Reducing inflammatory cell infiltration;
(5) Reducing inflammatory immune cell infiltration in intestinal mucosa;
(6) Down-regulating intestinal intrathecal Th1/Th17 cell production in a DSS non-human mammalian model;
(7) The inflammatory response of the intestinal mucosa is weakened;
(8) Repairing an impaired intestinal barrier function in a non-human mammalian model;
(9) Reducing secretion of serum inflammatory cytokines;
the non-human mammal is selected from a mouse, a rat, a monkey, a sheep, or a cow.
8. The use according to claim 7, wherein in feature (7) the reduction of the inflammatory response of the intestinal mucosa comprises: reduce the secretion of inflammatory cytokines in the intestinal mucosa.
9. The use according to claim 8, wherein the inflammatory cytokine in the intestinal mucosa is selected from at least one of the following cytokines:
IL-6, TNF-a and IL-1b.
10. The use according to claim 7, characterized in that in feature (9) the serum inflammatory cytokine is selected from at least one of the following cytokines:
IL-6, TNF-a and IL-1b.
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Non-Patent Citations (2)
| Title |
|---|
| HAMSA N S等: "PHARMACOPHORE ELUCIDATION AND DOCKING STUDIES ON ANTIINFLAMMATORY COMPOUNDS OF MEDICINAL PLANTS FOR ULCERATIVE COLITIS", ASIAN J PHARM CLIN RES, vol. 6, no. 3, 31 July 2013 (2013-07-31) * |
| 印晓星等主编: "《临床药理学》", vol. 2016, 31 January 2016, 中国医药科技出版社, pages: 16 * |
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