Application of Calycosin-7-O-β-D-glucoside in the preparation of drugs for treating skin fibrosis diseases

Abstract

The present invention discloses the application of calycosin-7-O-β-D-glucoside in the preparation of a medicament for treating skin fibrosis diseases. Through in vitro and in vivo experiments, the present invention finds that calycosin-7-O-β-D-glucoside can effectively inhibit the proliferation of primary scleroderma cells, promote the apoptosis of primary scleroderma cells, and effectively inhibit the development of skin fibrosis in mice. Therefore, calycosin-7-O-β-D-glucoside can be used to develop a medicament for treating skin fibrosis diseases, providing an effective way for the treatment of skin fibrosis diseases.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technologies, and particularly relates to the use of calycosin-7-glucoside in the preparation of a medicament for treating skin fibrosis diseases. Background Art

[0002] Skin fibrosis refers to the abnormal proliferation of collagen fibers in skin tissues, resulting in hardening and loss of elasticity of the skin. Skin fibrosis diseases include systemic sclerosis (SSc), etc. Systemic sclerosis is a chronic autoimmune connective tissue disease, whose main characteristics are vascular lesions, immune imbalance, and progressive fibrosis of the skin and multiple organs. According to the scope of the affected skin, it can be mainly divided into limited cutaneous systemic sclerosis (lcSSc) and diffuse cutaneous systemic sclerosis (dcSSc). Due to various complications such as interstitial lung disease, pulmonary hypertension, digital ulcers, etc., the quality of life of patients is poor. Moreover, the pathogenesis of systemic sclerosis is complex and has not been fully understood yet, and its treatment options are very limited. Traditional treatments mainly still include hormones combined with immunosuppressants (mycophenolate mofetil, cyclophosphamide, methotrexate, etc.) and other vasodilator drugs, supplemented with symptomatic treatment. Although these therapies have improved the symptoms such as inflammation and fibrosis of patients to a certain extent, there is still a lack of drugs for reversing SSc fibrosis at present.

[0003] Astragali Radix, also known as Huangqi, Mianqi, etc., is the dried root of the leguminous plant Astragalus membranaceus var. mongholicus, which can replenish qi and generate blood, promote diuresis and alleviate edema. Astragali Radix contains various active ingredients such as polysaccharides, saponins, flavonoids, etc., and has effects such as antioxidation, antivirus, hypoglycemic, improving the body's immunity, protecting the cardiovascular system, etc. Among them, calycosin-7-glucoside (CG) is one of the active ingredients in Astragali Radix (Li Liangliang, Huang Jinzhi. Research progress on the pharmacological effects of calycosin-7-glucoside [J]. Journal of Hainan Medical University, 2020, 26(2): 156-160.), and its structural formula is shown in Formula I, and it has effects such as antioxidation and improving myocardial function.

[0004] Formula I.

[0005] Related reports show that CG can promote the healing of diabetic skin wounds (Chen J, Ma H, Meng Y, Liu Q, Wang Y, Lin Y, Yang D, Yao W, Wang Y, He X, Li P. Analysis of the mechanism underlying diabetic wound healing acceleration by Calycosin-7-glycoside using network pharmacology and molecular docking. Phytomedicine. 2023 Jun; 114:154773.), and it has also been found in the study of liver cancer that CG has a good inhibitory effect on the proliferation of liver cancer cells (Wei X, Zeng Y, Meng F, Wang T, Wang H, Yuan Y, Li D, Zhao Y. Calycosin-7-glucoside promotes mitochondria-mediated apoptosis in hepatocellular carcinoma by targeting thioredoxin 1 to regulate oxidative stress. Chem Biol Interact. 2023 Apr 1; 374:110411.). In addition, there are also literature reports that CG has a certain therapeutic effect on ischemic stroke injury (Wang PC, Wang SX, Yan XL, He YY, Wang MC, Zheng HZ, Shi XG, Tan YH, Wang LS. Combination of paeoniflorin and calycosin-7-glucoside alleviates ischemic stroke injury via the PI3K / AKT signalling pathway. Pharm Biol. 2022 Dec;60(1):1469-1477.). However, at present, there is no study showing that calycosin-7-glucoside can be used to treat skin fibrosis diseases. Summary of the Invention

[0006] The object of the present invention is to overcome the disadvantages and deficiencies of the prior art and provide the use of calycosin-7-glucoside in the preparation of a drug for treating skin fibrosis diseases.

[0007] The object of the present invention is achieved by the following technical solutions:

[0008] Application of calycosin-7-O-β-D-glucoside in preparing a drug for treating skin fibrosis diseases.

[0009] The chemical structural formula of the calycosin-7-O-β-D-glucoside is shown as Formula I:

[0010] Formula I.

[0011] The skin fibrosis diseases include systemic sclerosis (SSc).

[0012] The systemic sclerosis (SSc) includes limited cutaneous systemic sclerosis (lcSSc) and diffuse cutaneous systemic sclerosis (dcSSc).

[0013] Application of calycosin-7-O-β-D-glucoside in preparing a drug for inhibiting the proliferation of primary scleroderma cells and / or promoting the apoptosis of primary scleroderma cells.

[0014] The using concentration of the calycosin-7-O-β-D-glucoside is above 0.01 μmol / L; further preferably 0.01 - 1 μmol / L; more preferably 1 μmol / L.

[0015] The present invention has the following advantages and effects compared with the prior art:

[0016] By combining in vitro and in vivo experiments, the present invention finds that calycosin-7-O-β-D-glucoside can inhibit the proliferation of primary scleroderma cells and promote apoptosis, and inhibit the development of skin fibrosis in mice. Therefore, calycosin-7-O-β-D-glucoside can provide a new drug source for the treatment or adjuvant treatment of skin fibrosis (including systemic sclerosis). Description of the Drawings

[0017] Figure 1 It is a diagram showing the effect of calycosin-7-O-β-D-glucoside on the activity of primary scleroderma cells.

[0018] Figure 2 It is a diagram showing the effect of calycosin-7-O-β-D-glucoside on the proliferation of primary scleroderma cells.

[0019] Figure 3 It is a diagram showing the effect of calycosin-7-O-β-D-glucoside on the collagen expression of primary scleroderma cells.

[0020] Figure 4 It is a diagram showing the effect of calycosin-7-O-β-D-glucoside on the skin thickness of mice. Detailed Embodiments

[0021] The present invention will be further described in detail below in conjunction with embodiments, but the implementation manners of the present invention are not limited thereto. Unless otherwise specified, the reagents and methods used in the present invention are conventional reagents and methods in the technical field. The test methods without specific experimental conditions specified in the following embodiments are usually carried out under conventional experimental conditions. Unless otherwise specified, the reagents and materials used in the present invention can be obtained commercially.

[0022] Calycosin-7-O-β-D-glucoside (CG) (CAS No.: 20633-67-4) involved in the embodiments of the present invention was purchased from Chengdu Mansite Biotechnology Co., Ltd.

[0023] Example 1: Calycosin-7-O-β-D-glucoside can inhibit the development of systemic sclerosis

[0024] 1. Calycosin-7-O-β-D-glucoside inhibits the proliferation of primary scleroderma cells and promotes apoptosis

[0025] In vitro experiment: Primary cells derived from scleroderma were treated with different drug concentrations, and then the cell viability was detected by CCK-8 and colony formation assays to study the effect of different drug concentration gradients on cell proliferation. The specific steps of the cell viability detection experiment are as follows:

[0026] 1) Take the skin tissue of scleroderma patients (from volunteers, determined according to the collagen index (Reference: Zixuan Cheng, Jialin Zhang, Wanying Deng, Shaojian Lin, Donghai Li, Ke Zhu & Qing Qi (2021) Bushen Yijing Decoction (BSYJ) exerts an anti-systemic sclerosis effect via regulating MicroRNA-26a / FLI1 axis, Bioengineered, 12:1, 1212-1225, DOI: 10.1080 / 21655979.2021.1907128)), that is, colla1 of scleroderma fibroblasts (type I collagen α1, see Figure 3The level is significantly elevated, and it can be diagnosed as a patient with systemic sclerosis (SSc). (Note: In this experiment, skin tissues from 10 patients with scleroderma were collected. However, since the experimental results were basically the same, only the results of primary scleroderma cells from one patient source are shown here.) The tissue samples were washed with PBS to remove blood and other impurities, and then the tissues were cut into small pieces. The tissues were digested with 0.25% trypsin-EDTA (Gibco, USA) and the undigested tissue fragments were removed by filtration. The collected cells were resuspended in DMEM cell culture medium (purchased from Life Technologies, Gaithersburg, MD, USA) and cultured to obtain a large number of primary scleroderma skin fibroblasts (SSCF).

[0027] 2) SSCF were seeded into 96-well plates, 3000 cells per well.

[0028] 3) Calycosin-7-O-β-D-glucoside (CG, purchased from Chengdu Mansite Biotechnology Co., Ltd.) was dissolved in DMSO (dimethyl sulfoxide) to prepare a stock solution with a concentration of 224 mM, and then the stock solution was diluted with DMEM cell culture medium (purchased from Life Technologies, Gaithersburg, MD, USA) to prepare working solutions with final concentrations of 0 mM, 0.01 mM, 0.1 mM, and 1 mM.

[0029] 4) After the cells in step 2) adhered to the wall, the experiment was divided into an experimental group and a control group: ① Different concentrations of the working solutions prepared in step 3 were added simultaneously at a volume of 100 μL per well; ② After treatment for 24, 48, and 72 hours respectively, the cell viability was detected using a CCK-8 cell proliferation and cytotoxicity detection kit (purchased from Beyotime Biotechnology Co., Ltd.). The above experimental steps were repeated 3 times for biological replicates.

[0030] The results are as Figures 1 to 3 shown: The viability of primary scleroderma cells ( Figure 1 ), the cell proliferation ability ( Figure 2 )(CG concentration 1 mM), and the collagen expression ( Figure 3 )(CG concentration 1 mM) were affected by calycosin-7-O-β-D-glucoside.

[0031] 2. Calycosin-7-O-β-D-glucoside can inhibit the development of skin fibrosis in mice

[0032] Forty 6-week-old female mice (balb / c) (purchased from Guangdong Laboratory Animal Center) were selected, with 8 in the control group and 32 in the experimental group, to establish a mouse model of scleroderma:

[0033] (1) Shave the hair on the back of the mice, mark them with a marker pen, with two marking points on each mouse, and inject bleomycin subcutaneously every day, injecting 0.1 ml at each point, and the concentration of bleomycin is 200 μg / ml; the control group is injected with normal saline subcutaneously, injecting 0.1 mL at each point;

[0034] (2) Anesthetize the nude mice before the experiment, and evaluate the anesthetic level through painless and painful stimuli to determine that the mice are in an anesthetized state;

[0035] (3) Drug treatment: On the day of subcutaneous injection, start administering the drug by gavage. A total of 3 drug combinations are set in this experiment, namely CG 50 mg / kg (low-dose CG group), CG 100 mg / kg (medium-dose CG group) and CG 200 mg / kg (high-dose CG group), with 8 mice in each group, and the drug is administered once every two days. First, dissolve the drug (CG) in edible oil (Jinlongyu blended oil), and administer 100 μL of the drug to each mouse each time; the control group is given edible oil, and 100 μL of the drug is administered to each mouse each time, and the body weight of the mice is measured each time the drug is administered. After 28 days, euthanize the mice and collect the skin tissues.

[0036] The experimental results of the skin thickness of the mice are as Figure 4 shown. The measurement of the skin thickness of the mice shows that calycosin-7-O-β-D-glucoside can significantly inhibit the development of skin fibrosis in mice, and the high-dose CG group has the best effect.

[0037] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Use of calycosin isoflavone glucoside as the sole active ingredient in the preparation of a drug for treating skin fibrosis, characterized in that: The skin fibrosis disease is systemic scleroderma.

2. The use according to claim 1, characterized in that: The systemic scleroderma includes localized cutaneous systemic scleroderma and diffuse cutaneous systemic scleroderma.

3. The use according to claim 1, characterized in that: The calycosin isoflavone glucoside has the effect of inhibiting the proliferation of primary scleroderma cells and / or promoting the apoptosis of primary scleroderma cells.

4. The use according to claim 3, characterized in that: The usage concentration of the calycosin isoflavone glucoside is above 0.01 μmol / L.

5. The use according to claim 4, characterized in that: The usage concentration of the calycosin isoflavone glucoside is 0.01-1 μmol / L.

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