New uses of semaglutide

Abstract

The present application discloses the use of semaglutide in the preparation of drugs for the prevention and / or treatment of skin fibrosis. The present application is the first to show that semaglutide has a significant effect in treating skin fibrosis, and experiments have confirmed that semaglutide can improve the skin fibrosis state of the classic skin fibrosis model BLM mice; and semaglutide can inhibit the expression of collagen-1 in primary skin fibroblasts induced by TGF-β, as well as the migration of primary skin fibroblasts.

Description

Technical Field

[0001] The present application relates to the technical field of new drug uses, and in particular to the use of semaglutide in the preparation of drugs for preventing and / or treating skin fibrosis. Background Art

[0002] Fibrosis is the excessive accumulation of pathological extracellular matrix (ECM). Continuous progression can lead to organ structural destruction and functional impairment, and even failure, seriously threatening human health and life.

[0003] Fibrosis can affect multiple organs, such as the lungs, liver, kidneys, peritoneum, blood vessels, pancreas, skin, etc., leading to progressive tissue scarring and organ dysfunction. Fibroblasts are the central cell type in the process of skin fibrosis, which can lead to ECM accumulation and inflammation. Fibrotic skin diseases involve the accumulation of ECM components in the dermis, including scleroderma, hypertrophic scars, keloids, etc.; in mild cases, sweat gland secretion is reduced, thereby affecting body temperature regulation, and in severe cases, joint contracture leads to dysfunction.

[0004] So far, the drugs currently used in clinical treatment of skin fibrosis have not achieved good results, so it is necessary to find and develop new anti-skin fibrosis drugs. Summary of the invention

[0005] In order to solve the above technical problems, the purpose of the present invention is to provide the use of semaglutide in the preparation of a drug for preventing and / or treating skin fibrosis; the present application experimentally confirms that semaglutide can improve the skin fibrosis state of the classic skin fibrosis model BLM mice. And semaglutide can inhibit the expression of collagen-1 in primary skin fibroblasts induced by TGF-β, as well as the migration of primary skin fibroblasts.

[0006] The technical solution provided by the present invention is as follows:

[0007] Use of semaglutide in preparing a drug for preventing and / or treating skin fibrosis.

[0008] Preferably, semaglutide has the effects of reducing skin thickness and reducing collagen deposition.

[0009] Preferably, semaglutide has the effect of reducing the expression levels of COL1 and α-SMA.

[0010] Preferably, semaglutide has the effect of reducing the expression levels of TGF-β and IL-6.

[0011] Preferably, the skin fibrosis is induced by bleomycin.

[0012] Preferably, semaglutide has the effect of attenuating the increase in COL1 expression caused by TGF-β1 stimulation.

[0013] Preferably, semaglutide has the efficacy of inhibiting TGF-β-induced migration of human primary skin fibroblasts.

[0014] Preferably, semaglutide is administered by subcutaneous injection.

[0015] Preferably, the medicament comprises an effective dose of semaglutide.

[0016] Preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0017] Semaglutide is a long-acting glucagon-like peptide-1 (GLP-1) analog that can stimulate insulin secretion in a glucose concentration-dependent manner, thereby improving blood sugar control in patients with type 2 diabetes. It is currently widely used to treat type 2 diabetes.

[0018] This application is the first study to show that semaglutide has a significant effect in treating skin fibrosis, providing an efficient and safe drug source for the treatment of skin fibrosis diseases.

[0019] The present application uses semaglutide to treat mice injected subcutaneously with bleomycin (BLM). The results of HE and masson staining of the mouse skin show that after BLM mice were treated with semaglutide, skin thickening and collagen deposition were significantly reduced compared with the BLM control group. Western blot results show that the expression of collagen-1 (COL1) and smooth muscle α-actin (SMA) in BLM mice treated with semaglutide was significantly downregulated compared with the BLM control group. The results of the present invention prove that semaglutide can improve the skin fibrosis state of BLM mice, a classic skin fibrosis model.

[0020] In addition, experiments showed that semaglutide could inhibit the expression of collagen-1 and the migration of primary skin fibroblasts induced by TGF-β.

[0021] Therefore, the present application provides the use of semaglutide for preparing a drug for preventing and / or treating skin fibrosis, which can be used to prepare a drug for treating skin fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present application 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 recorded in the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 HE and Masson staining photos of mouse skin; showing that semaglutide reduces the degree of fibrosis in mice with bleomycin-induced skin fibrosis model. The skin thickness and collagen deposition of the BLM model group were significantly higher than those of the PBS group. The skin thickness and collagen deposition of the semaglutide treatment group were significantly decreased compared with the BLM model group. In the figure, *** represents P < 0.001, **** represents P < 0.0001; 1 is the blank control group, 2 is the BLM model group, 3 is the semaglutide treatment group; 4 is the semaglutide group alone;

[0024] Figure 2 The results of Western blot of mouse skin; compared with the blank control group, the expression levels of COL1 and α-SMA in the BLM model group were significantly increased; after semaglutide treatment, the expression levels of COL1 and α-SMA in the semaglutide treatment group were significantly decreased compared with the BLM model group; 1 is the blank control group, 2 is the BLM model group, 3 is the semaglutide treatment group; 4 is the semaglutide group alone;

[0025] Figure 3 The results of mouse serum ELISA show that the expression of TGF-β and IL-6 in the serum of mice in the BLM model group was significantly higher than that in the blank control group, and the semaglutide treatment group was significantly decreased compared with the BLM model group. In the figure, * represents <0.05; ** represents <0.01; 1 is the blank control group, 2 is the BLM model group, and 3 is the semaglutide treatment group;

[0026] Figure 4 This is the western blot result of healthy adult primary fibroblasts. After 24 hours of TGFβ1 stimulation, the protein expression of COL1 in the positive control group was significantly increased compared with the blank control group, while 2nM and 20nM semaglutide treatment could attenuate the increase in COL1 expression caused by TGF-β1 stimulation, and there was no difference between 2nM and 20nM.

[0027] Figure 5 This is a graph showing the migration results of primary fibroblasts from healthy adults. The 24h and 48h results both show that semaglutide significantly inhibited the TGF-β-induced migration of primary human skin fibroblasts. In the graph, ns represents >0.05, * represents <0.05; ** represents <0.01, and *** represents <0.001. DETAILED DESCRIPTION

[0028] In order to enable those skilled in the art to better understand the technical solutions in this application, the technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0029] It should be noted that when an element is referred to as being "fixed on" or "set on" another element, it can be directly on the other element or indirectly set on the other element; when an element is referred to as being "connected to" another element, it can be directly connected to the other element or indirectly connected to the other element.

[0030] It should be understood that the terms "length", "width", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", etc., indicating the orientation or position relationship, are based on the orientation or position relationship shown in the drawings, and are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present application.

[0031] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of this application, "multiple" and "several" mean two or more, unless otherwise clearly and specifically defined.

[0032] It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings of this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the conditions under which this application can be implemented. Therefore, they have no substantive technical significance. Any structural modification, change in proportional relationship or adjustment of size should still fall within the scope of the technical content disclosed in this application without affecting the effects and purposes that can be achieved by this application.

[0033] 1. Mouse Experiment

[0034] The following experiment will study the effect of semaglutide on the degree of skin fibrosis induced by bleomycin (BLM) in mice to prove that semaglutide can be used as a drug for the treatment of skin fibrosis.

[0035] The skin fibrosis model was established by continuous subcutaneous injection of bleomycin for 28 days. After the fibrosis model was established, semaglutide was given subcutaneously for 21 consecutive days (once a day) to observe the effect of semaglutide on skin fibrosis mice.

[0036] 1. Animal model construction

[0037] The mice used in the experiment were BALB / c mice (8 weeks old, 18-22 g), all purchased from Hunan Slake Jingda Experimental Animal Co., Ltd. All experiments were approved by the Animal Ethics Review Committee of Central South University. The experimental mice were fed in plastic cages with a standard diet and normal drinking water, and the room temperature was controlled at 20-25°C, the relative humidity was 50-60%, and the light cycle was 12 hours.

[0038] BALB / c mice were placed in the barrier environment at 6 weeks of age and allowed to adapt for 2 weeks. Experimental treatment began at 8 weeks of age. Twelve mice were randomly assigned to PBS plus saline (blank control group) group (n=3), BLM plus saline group (BLM model group) (n=3), BLM followed by semaglutide treatment group (semaglutide treatment group) (n=3), and PBS plus semaglutide group (semaglutide simple group) (n=3).

[0039] The BLM model group, semaglutide treatment group, and simple semaglutide group were first injected subcutaneously with PBS / BLM to establish the model: after shaving the back of the mouse, the skin of the shaved area of ​​the mouse and the injection needle were first disinfected with 75% alcohol cotton balls. One person fixed the head and tail of the mouse, and the other person gently pinched the skin of the shaved area with one hand, and used a 1mL syringe with the other hand to absorb the prepared working solution, and inserted the needle in the center of the shaved area, so that the needle broke through the skin and entered the subcutaneous area, and then slowly injected 100μL. The BLM injection dose for each mouse was 40ug / d. After the injection, the needle was slowly withdrawn, and the mouse skin and needle were disinfected with alcohol cotton balls again. The injection was continued for 28 days to establish a skin fibrosis model, which is the most classic and most commonly used skin fibrosis model.

[0040] The blank control group was injected with 100 μL of normal saline every day for 28 consecutive days.

[0041] 2 Drug intervention and outcome management

[0042] 2.1 After successful modeling, the semaglutide treatment group was given drug intervention for 21 days. The mice in the semaglutide treatment group and the simple semaglutide group were subcutaneously injected with semaglutide (0.12ug / kg / d), and the mice in the blank control group and the BLM model group were subcutaneously injected with an equal volume of normal saline.

[0043] 2.2 Drug intervention On the 21st day, mice in each group were killed, and skin tissue and whole blood were collected. The skin tissue was divided into two parts, one of which was fixed in 4% neutral formaldehyde and then HE and Masson staining were performed for each group of samples. The other part was placed at -80℃ and Western blot (to detect COL1 and α-SMA) was performed later. The results are shown in the attached Figure 1-3 .

[0044] in, Figure 1 HE and Masson staining showed that semaglutide reduced the degree of fibrosis in bleomycin-induced skin fibrosis model mice. The skin thickness and collagen deposition of the BLM model group were significantly higher than those of the PBS group. The skin thickness and collagen deposition of the semaglutide treatment group were significantly decreased compared with the BLM model group. In the figure, *** represents P < 0.001, **** represents P < 0.0001.

[0045] Figure 2 : Western blot analysis showed that the expression levels of COL1 and α-SMA in the BLM model group were significantly increased compared with the blank control group; the expression levels of COL1 and α-SMA in the semaglutide treatment group mice were significantly decreased after semaglutide treatment compared with the BLM model group.

[0046] Figure 3 : ELISA results showed that the expression of TGF-β and IL-6 in the serum of mice in the BLM model group was significantly higher than that in the blank control group, and the expression of TGF-β and IL-6 in the semaglutide treatment group was significantly decreased compared with the BLM model group. In the figure, * represents <0.05; ** represents <0.01.

[0047] 2. Cell Experiment

[0048] 1Primary skin fibroblasts were successfully extracted from a normal adult skin tissue (male foreskin tissue, 25-year-old male) and cultured. They were placed in DMEM culture medium containing 10% fetal bovine serum, 100 units / ml penicillin and 100ug / ml streptomycin, and incubated in a 37°C, 5% CCh incubator. Cells were digested with 0.25% trypsin for cell passage. The medium was changed every other day until the cells adhered to the wall and grew to a sub-confluent state (reaching 75% to 80% of the bottom area), then passaged, divided into groups, and balanced with serum-free medium for 12 hours. After passage to the third generation, they were used for experiments; then the groups were stimulated separately,

[0049] 2 Stimulation: The original culture medium was removed, and the cells were washed three times with PBS. The blank control group was cultured with DMEM culture medium containing 0.02% fetal bovine serum, 100 units / ml penicillin and 100ug / ml streptomycin. The TGF-β1 (10ng / ml) positive control group was cultured with the same culture medium as the blank control group, with the addition of 10ng / ml TGF-β1. The TGF-β1 (10ng / ml) + semaglutide (2nM) group was cultured with the same culture medium as the positive control group, with the addition of 2nM semaglutide. The TGF-β1 (10ng / ml) + semaglutide (20nM) group was cultured with the same culture medium as the positive control group, with the addition of 20nM semaglutide. After incubation in a 37°C, 5% CCh incubator for 24 hours, the cells were digested with 0.25% trypsin, the cell proteins were extracted, and Western blot detection of COL1 was performed. Stimulation of fibroblasts with TGF-β can promote the high expression of COL1, which is closely related to fibrosis, and make the fibroblasts secrete extracellular matrix COL1 function active.

[0050] Western blot grouping types: ① blank control group; ② TGF-β1 (10 ng / ml) positive control group; ③ TGF-β1 (10 ng / ml) + semaglutide (2 nM); ④ TGF-β1 (10 ng / ml) + semaglutide (20 nM).

[0051] The results are as follows Figure 4 shown.

[0052] Figure 4 The results showed that after 24 hours of TGFβ1 stimulation, the protein expression of COL1 in the positive control group was significantly increased compared with the blank control group, while semaglutide treatment could attenuate the increase in COL1 expression caused by TGF-β1 stimulation, and there was no difference between 2nM and 20nM; the high expression of TGF-β in fibroblasts caused fibroblasts to secrete excessive extracellular matrix, which is the reason for promoting SSc fibrosis.

[0053] 3. Cell migration scratch test: After the well-grown 4th generation human skin primary fibroblasts were digested, they were inoculated into 6-well plates. The experiment was divided into: ① blank control group, ② TGF-β1 (10ng / ml) group, ③ TGF-β1 (10ng / ml) + semaglutide (20nM) group. The experiment was carried out when the cells grew to 80-90%. A 200ul gun tip was used to make scratches, and then culture medium without fetal bovine serum (FBS) was added according to the grouping. The medium was changed every 24h, and photos were taken every 24h. The changes in the scratch distance of each group were observed under a 200x microscope for 48h. ImageJ software was used to analyze the migration distance of the cells.

[0054] The results are as follows Figure 5 shown.

[0055] Figure 5 The results showed that both the 24h and 48h results showed that semaglutide significantly inhibited the migration of human primary skin fibroblasts induced by TGF-β. The high expression of TGF-β in fibroblasts caused the rapid migration of fibroblasts, which promoted SSc fibrosis. In the figure, ns represents > 0.05, * represents < 0.05; ** represents < 0.01, and *** represents < 0.001.

[0056] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Use of semaglutide in the preparation of a drug for preventing and / or treating skin fibrosis; The skin fibrosis was induced by bleomycin.

2. The use according to claim 1, characterized in that Semaglutide has the effect of reducing skin thickness and collagen deposition.

3. The use according to claim 1, characterized in that Semaglutide has the effect of reducing the expression of COL1 and α-SMA.

4. The use according to claim 1, characterized in that Semaglutide has the effect of reducing the expression of TGF-β and IL-6.

5. The use according to claim 1, characterized in that: Semaglutide has the effect of attenuating the increase in COL1 expression caused by TGF-β1 stimulation.

6. The use according to claim 1, characterized in that: Semaglutide has the efficacy of inhibiting TGF-β-induced migration of human primary skin fibroblasts.

7. The use according to any one of claims 1 to 6, characterized in that Semaglutide is administered by subcutaneous injection.

8. The use according to any one of claims 1 to 6, characterized in that The drug comprises an effective dose of semaglutide.

9. The use according to claim 8, characterized in that The drug also includes pharmaceutically acceptable excipients.

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