Application of GHK-Cu and analogue thereof in preparation of medicine for treating ulcerative colitis

Abstract

The invention relates to the technical field of biological medicine, in particular to application of GHK-Cu and an analogue thereof in preparation of a medicine for treating ulcerative colitis, and the GHK-Cu is glycine-L-histidine-L-lysine-Cu < 2 + >. The invention finds that GHK-Cu and the analogue thereof show a remarkable curative effect in an animal ulcerative colitis model, and can effectively relieve the symptoms of ulcerative colitis.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the application of GHK-Cu and its analogs in the preparation of drugs for treating ulcerative colitis. Background Art

[0002] Ulcerative colitis, referred to as UC, is a chronic nonspecific inflammatory bowel disease (IBD) that often occurs in the colorectal mucosa and submucosa. Its symptoms include abdominal pain, diarrhea, and mucus, pus, and blood in the stool. UC is prone to recurrence, so the course of the disease is long. It can occur in all age groups, but is more common in people aged 20 to 40, and there is no gender difference in the incidence rate. In recent years, with the changes in people's living conditions and eating habits, the incidence of UC has been increasing year by year, seriously affecting the quality of life of patients.

[0003] The pathological process of UC can be summarized as: the intestinal inflammatory response is stimulated, continued and expanded by unknown reasons. As the inflammatory response area expands and the severity increases, a large number of inflammatory cells such as lymphocytes, neutrophils and macrophages gather in the damaged area, thereby producing a large number of inflammatory mediators, leading to intestinal damage and physiological dysfunction. At present, conventional treatment drugs for UC mainly include aminosalicylic acid preparations, such as 5-aminosalicylic acid and 5-ASA, but these drugs have low clinical efficacy and many adverse reactions. There is an urgent need to develop a new drug for the treatment of ulcerative colitis. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a use of GHK-Cu and its analogs in the preparation of a drug for treating ulcerative colitis, so as to improve the therapeutic effect of ulcerative colitis.

[0005] The present invention is achieved through the following technical solutions:

[0006] Application of GHK-Cu and its analogs in the preparation of drugs for treating ulcerative colitis, wherein the GHK-Cu is glycine-L-histidine-L-lysine-Cu 2+ .

[0007] Preferably, the analogs of GHK-Cu are palmitoyl hexapeptide-25 and alanine / histidine / lysine polypeptide copper HCl.

[0008] Preferably, GHK-Cu or its analogs are the only active ingredient of the drug for treating ulcerative colitis.

[0009] Preferably, the content of GHK-Cu or its analogues in the preparation of the drug for treating ulcerative colitis is ≥20 mg / kg.

[0010] Preferably, the content of GHK-Cu or its analogues in the preparation of the drug for treating ulcerative colitis is 20 mg / kg.

[0011] Preferably, the drug includes the GHK-Cu or its analogs and also includes pharmaceutically acceptable excipients.

[0012] Preferably, the pharmaceutically acceptable excipient is one or more of a diluent, a disintegrant, a precipitation inhibitor, a glidant, a binder, a dispersant, a suspending agent, an isotonic agent, a thickener, an emulsifier, a preservative and a stabilizer.

[0013] Preferably, the dosage form of the drug includes enema, tablet, capsule, granule, oral suspension, powder or paste.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention experiments GHK-Cu on animal models to show the effect of relieving ulcerative colitis. In animal experiments, GHK-Cu shows significant therapeutic effects and can effectively alleviate the symptoms of ulcerative colitis, such as weight loss, diarrhea, blood in the stool, etc. Compared with existing therapeutic drugs, such as 5-aminosalicylic acid and 5-ASA, it has a higher therapeutic effect. In addition, preliminary animal experiments show that while GHK-Cu relieves ulcerative colitis, no obvious toxic reactions or side effects are observed. This provides safety for its future clinical application. The discovery of the therapeutic effect of GHK-Cu on ulcerative colitis is expected to provide new treatment options for a large number of patients. In addition, its potential mechanism of action may also be applicable to the treatment of other inflammatory bowel diseases or autoimmune diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 The figures are the weight change results and DAI score figures of each group of mice in the present invention.

[0018] Figure 1 In the figure, A is the weight change results of mice in each group; B is the DAI score diagram; compared with the control group, #P<0.05; ## P < 0.01; ### P<0.001; compared with the model group, *P<0.05; **P<0.01; ***P<0.001.

[0019] Figure 2This is a graph showing the results of GHK-Cu of the present invention improving DSS-induced colon injury and inflammatory cell infiltration in mice.

[0020] Figure 2 In the figure, A is H&E staining of colon sections, 100×, 200×; red arrows indicate areas of high inflammatory cell infiltration; black arrows indicate areas of erosion and ulceration; green arrows point to structural damage in the colon crypts; yellow arrows indicate the presence of edema and fibrosis; B is the colon pathological score of mice in each group; compared with the control group, ### P<0.001; compared with the model group, *P<0.05; ***P<0.001.

[0021] Figure 3 The figure shows the result that GHK-Cu of the present invention inhibits the decrease of the number of goblet cells and the reduction of mucus secretion in the intestinal tract of mice induced by DSS.

[0022] Figure 3 In the figure, A is AB-PAS staining of colon sections, 40×, 200×; B is the count of intestinal goblet cells in each group of mice; compared with the control group, ## P<0.01; **P<0.01 compared with the model group.

[0023] Figure 4 This is the WB band diagram of inflammatory factors in the colon tissue of each group of mice in the present invention.

[0024] Figure 5 This is a graph showing the promoting effect of GHK-Cu on MCECs migration.

[0025] Figure 5 In the figure, A is a schematic diagram of the co-culture model of MCECs and MPMs; B is a diagram of the scratch test results; C is a diagram of the migration rate results of each group; compared with the control group, ### P<0.001; ***P<0.001 compared with the model group.

[0026] Figure 6 This is a graph showing the results that GHK-Cu of the present invention can increase the expression levels of tight junction proteins ZO-1 and occludin in MCECS.

[0027] Figure 6 Middle is the WB band image of tight junction protein ZO-1 and occludin.

[0028] Figure 7 This is a graph showing the effect of GHK-Cu on the SIRT1 / STAT3 signaling pathway in the in vivo model of the present invention; Figure 7 Western blot images of SIRT1, p-STAT3, STAT3 and RORγT.

[0029] Figure 8 This is a graph showing the effect of GHK-Cu on the SIRT1 / STAT3 signaling pathway in an in vitro model of the present invention; Figure 8 Western blot images of SIRT1, p-STAT3, STAT3 and RORγT. DETAILED DESCRIPTION

[0030] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively below, and preferred embodiments of the present invention are provided. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0032] The inventive concept of the present invention is as follows:

[0033] At present, the clinical efficacy of conventional therapeutic drugs for UC is not ideal and there are many adverse reactions, and it is urgent to develop a new therapeutic drug for ulcerative colitis. To solve the above problems, the present invention provides the use of GHK-Cu in the preparation of a drug for treating ulcerative colitis to improve the therapeutic effect of ulcerative colitis.

[0034] The present invention first uses 3% sodium dextran sulfate DSS to construct an animal model of UC, and studies the inhibitory effect of GHK-Cu on DSS-induced UC at the whole animal level. The results show that GHK-Cu shows the effect of relieving ulcerative colitis in animal models, and GHK-Cu can inhibit the expression of inflammatory factors in colon tissue. On this basis, DSS-induced mice were used to extract peritoneal macrophages MPMs and mouse colon epithelial cells MCECs to construct a co-culture system through a transwell chamber to simulate the inflammatory environment in colitis mice, and study the effect of GHK-Cu on promoting DSS-induced macrophages to inhibit the healing of colon epithelial cells. The results show that GHK-Cu can promote the expression of tight junction proteins ZO-1 and occludin proteins. In summary, GHK-Cu may exert its therapeutic effect by inhibiting the release of inflammatory mediators and promoting intestinal mucosal repair. In addition, while GHK-Cu relieves ulcerative colitis, no obvious toxic reactions or side effects were observed, which provides safety for its future clinical application. Therefore, the present invention demonstrates the effectiveness and safety of GHK-Cu in treating ulcerative colitis, and is expected to provide a new treatment option and innovative approach for a large number of ulcerative colitis patients. In addition, its potential mechanism of action may also be applicable to the treatment of other inflammatory bowel diseases or autoimmune diseases.

[0035] The specific implementation steps of the present invention are as follows:

[0036] 1. Study of Glycine-L-Histidine-L-Lysine-Cu by in vivo model 2+ , that is, the therapeutic effect of GHK-Cu on ulcerative colitis UC.

[0037] The animal model of UC was established using 3% dextran sulfate sodium DSS, and the inhibitory effect of GHK-Cu on DSS-induced UC was studied at the whole animal level.

[0038] a. The mice were randomly divided into a normal group, a DSS enteritis model group, a positive control group, and a GHK-Cu group. The survival, mental state, hair condition, body weight, stool characteristics, blood in stool, and the presence or absence of anal fistula of the animals in the blank group, model group, and drug-treated group were compared, and the disease activity index DAI was calculated.

[0039] b. Hematoxylin-eosin staining was used to investigate the therapeutic effect of GHK-Cu on inflammatory damage and the inhibitory effect on inflammatory cell infiltration, and pathological scoring was performed on the colon lesions of mice.

[0040] c. AB-PAS staining was used to evaluate the changes in the morphology and number of intestinal goblet cells.

[0041] d. Western blot experiment was used to detect the effect of GHK-Cu on the expression levels of IL-6, TNF-α, and IL-1β proteins.

[0042] 2. Study the effect of GHK-Cu by in vitro experiments

[0043] Peritoneal macrophages (MPMs) extracted from DSS-induced mice were used to construct a co-culture system with mouse colon epithelial cells (MCECs) using a transwell chamber to simulate the inflammatory environment in colitis mice, and to study the effect of GHK-Cu on promoting the healing of DSS-induced macrophages and inhibiting colon epithelial cells.

[0044] a. The cell scratch repair experiment was used to investigate the promoting effect of GHK-Cu on MCECs migration.

[0045] b. Western blot experiment was used to detect the effect of GHK-Cu on the expression levels of tight junction proteins ZO-1 and occludin.

[0046] 3. Through in vivo and in vitro experiments, the target of GHK-Cu in the treatment of UC was preliminarily verified.

[0047] a. Effects of GHK-Cu on the expression of RORγT, SIRT1 and p-STAT3 in colon tissue.

[0048] Using the in vivo animal model as described above, Western blots were performed to detect the expression levels of RORγT, SIRT1, STAT3 and p-STAT3 in colon tissues.

[0049] b. Effect of GHK-Cu on the expression of RORγT, SIRT1 and STAT3 in the co-culture system.

[0050] A co-culture system was constructed using DSS-induced MPMs and MCECs, and Western Blots were used to detect the effect of GHK-Cu on the expression levels of RORγT, SIRT1, STAT3 and p-STAT3 proteins in MCECs.

[0051] The technical solution of the present invention will be further described below in conjunction with specific embodiments.

[0052] Example 1

[0053] The therapeutic effect of GHK-Cu on ulcerative colitis (UC) was studied using an in vivo model.

[0054] The animal model of UC was established using 3% dextran sulfate sodium (DSS), and the inhibitory effect of GHK-Cu on DSS-induced UC was studied at the whole animal level.

[0055] Specific operation: Mice were randomly divided into a normal group, a DSS enteritis model group, a positive control group, and a GHK-Cu group, n = 8. Except for the control group, 3% DSS solution was added to the drinking water of the other three groups for 12 days, followed by normal drinking water for 2 days. The mice in the control group and the model group were gavaged with normal saline every day for 14 consecutive days, and the mice in the drug-treated group were gavaged with 5-ASA 40 mg / kg or GHK-Cu 20 mg / kg. All mice were euthanized on the 15th day.

[0056] a. The mice were randomly divided into a normal group, a DSS enteritis model group, a positive control group, and a GHK-Cu group. The survival, mental state, hair condition, body weight, stool characteristics, blood in stool, and the presence or absence of anal fistula of the animals in the blank group, model group, and drug-treated group were compared, and the disease activity index DAI was calculated. The results are shown in Figure 1 As shown. The results showed that during the modeling process, mice in the DSS model group showed reduced motor activity, reduced food and water consumption, dull hair, and depression-like behaviors such as hunchback. In addition, the model mice showed swelling and hematuria, accompanied by diarrhea and bloody stools, and GHK-Cu improved these symptoms. The body weight of mice in the DSS group decreased significantly, and GHK-Cu reversed this trend on the 12th day of administration. The DAI score in the DSS group increased sharply from the 4th day, and GHK-Cu treatment significantly reduced the DAI score. The colon length of DSS model mice was significantly shorter than that of control mice, and GHK-Cu significantly alleviated the reduction in colon length. GHK significantly reduced the increase in the colon mucosal injury index CMDI score caused by DSS.

[0057] b. The H&E staining experiment was used to detect the colon tissue of mice to investigate the therapeutic effect of GHK-Cu on inflammatory damage and the inhibitory effect on inflammatory cell infiltration, and the pathological scoring of the colon lesions of mice was performed; AB-PAS staining was used to evaluate the changes in the morphology and number of intestinal goblet cells, such as Figure 2 and Figure 3 As shown. The results showed that pathological changes occurred in the colonic epithelium of DSS model mice, characterized by erosion and ulceration, and the colonic crypts were significantly deformed and distorted, accompanied by irregular shortening and even ablation of these structures. The mucosa and submucosa were extensively infiltrated by inflammatory cells, mainly composed of lymphocytes and neutrophils, indicating a severe inflammatory response. In addition, the submucosa became widened, which is a hallmark feature suggesting edema. Notably, the number of goblet cells decreased significantly, emphasizing the further destruction of the colonic structure. However, these pathological lesions were significantly alleviated after GHK-Cu intervention.

[0058] c. Western blot was used to detect the effect of GHK-Cu on the expression levels of IL-6, TNF-α, and IL-1β proteins. The results are as follows: Figure 4 The results showed that the expression of IL-1β, IL-6 and TNF-α in the colon tissue of the model mice was significantly increased, and after GHK-Cu treatment, the proportion of inflammatory cytokines IL-1β, IL-6 and TNF-α in the colon tissue was significantly reduced.

[0059] Example 2

[0060] The effects of GHK-Cu were studied by in vitro experiments.

[0061] Peritoneal macrophages (MPMs) extracted from DSS-induced mice were used to construct a co-culture system with mouse colon epithelial cells (MCECs) using transwell chambers. MCECs were inoculated into 24-well plates and given DSS solution to drink for 5 days. Peritoneal macrophages (PMs) were collected and introduced into transwell chambers with a pore size of 0.4 μm. They were co-cultured with 24-well culture plates to simulate the inflammatory environment in mice with colitis. After treatment with 62.5 μM GHK-Cu for 24 hours, the effect of GHK-Cu on promoting the healing of DSS-induced macrophages and inhibiting colon epithelial cells was studied.

[0062] a. Use cell scratch repair experiment to observe the scratch width and take pictures to calculate the coverage. Figure 5 As shown, peritoneal macrophages of DSS model mice inhibited the migration ability of MCECs, while GHK-Cu promoted the migration of MCECs.

[0063] b. Western blot was used to detect the effect of GHK-Cu on the expression levels of tight junction proteins ZO-1 and occludin. The results are as follows Figure 6 As shown, GHK-Cu promoted the expression levels of ZO-1 and occludin proteins.

[0064] Example 3

[0065] Through in vivo and in vitro experiments, the target of GHK-Cu in treating UC was preliminarily verified.

[0066] a. Effects of GHK-Cu on the expression of RORγT, SIRT1 and p-STAT3 in colon tissue.

[0067] Using the in vivo animal model described above, the expression levels of RORγT, SIRT1, STAT3, and p-STAT3 in colon tissue were detected by Western blots. Figure 7 As shown, GHK-Cu inhibited the expression of RORγT, SIRT1 and p-STAT3 in colon tissues.

[0068] b. Effects of GHK-Cu on the expression of RORγT, SIRT1 and STAT3 in co-culture system

[0069] The co-culture system was constructed by using DSS-induced MPMs and MCECs, and the effect of GHK-Cu on the expression levels of RORγT, SIRT1, STAT3 and p-STAT3 proteins in MCECs was detected by Western Blots. Figure 8 As shown, GHK-Cu inhibited the expression of RORγT, SIRT1, and p-STAT3 in MCECs in the co-culture system.

[0070] In addition, the present invention uses palmitoyl hexapeptide-25, an analog of GHK-Cu, and alanine / histidine / lysine polypeptide copper HCl to carry out the above experiment. The results show that after treatment with palmitoyl hexapeptide-25, an analog of GHK-Cu, or alanine / histidine / lysine polypeptide copper HCl, the proportion of inflammatory cytokines IL-1β, IL-6, and TNF-α in colon tissue is significantly reduced; and it can promote DSS-induced macrophages to inhibit the healing of colon epithelial cells, the migration of MCECs, and promote the expression levels of ZO-1 and occludin proteins; at the same time, it inhibits the expression of RORγT, SIRT1, and p-STAT3 in colon tissue, and inhibits the expression of RORγT, SIRT1, and p-STAT3 in MCECs in the co-culture system.

[0071] In summary, it is shown that GHK-Cu and its analogs can effectively treat ulcerative colitis.

[0072] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0073] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. The use of GHK-Cu and its analogs in the preparation of drugs for treating ulcerative colitis, characterized in that: The GHK-Cu is glycine-L-histidine-L-lysine-Cu 2+ .

2. The use according to claim 1, characterized in that: The analogs of GHK-Cu are palmitoyl hexapeptide-25 and alanine / histidine / lysine polypeptide copper HCl.

3. The use according to claim 1, characterized in that: GHK-Cu or its analogues are used as the only active ingredient of the drug for treating ulcerative colitis.

4. The use according to claim 1, characterized in that: The content of the GHK-Cu or its analogues in the preparation of the drug for treating ulcerative colitis is ≥20 mg / kg.

5. The use according to claim 1, characterized in that: The content of the GHK-Cu or its analogues in the preparation of the drug for treating ulcerative colitis is 20 mg / kg.

6. The use according to claim 1, characterized in that: The drug comprises the GHK-Cu or its analogs and also comprises pharmaceutically acceptable excipients.

7. The use according to claim 6, characterized in that: The pharmaceutically acceptable drug excipient is one or more of a diluent, a disintegrant, a precipitation inhibitor, a glidant, a binder, a dispersant, a suspending agent, an isotonic agent, a thickener, an emulsifier, a preservative and a stabilizer.

8. The use according to claim 7, characterized in that: The dosage form of the drug includes enema, tablet, capsule, granule, oral suspension, powder or ointment.

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