Application of calycosin-7-glucoside in preparation of medicine for treating skin fibrosis diseases

Abstract

The invention discloses an application of calycosin-7-glucoside in preparation of a medicine for treating skin fibrosis diseases. In-vitro and in-vivo experiments show that calycosin glucose can effectively inhibit proliferation of primary scleroderma cells, promote apoptosis of the primary scleroderma cells and effectively inhibit development of mouse skin fibrosis. Therefore, the calycosin-7-glucoside can be used for developing the medicine for treating the skin fibrosis diseases, and an effective way is provided for treating the skin fibrosis diseases.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and particularly relates to application of calycosin isoflavone glucoside in preparing medicines for treating skin fibrosis diseases. Background Art

[0002] Skin fibrosis refers to the abnormal proliferation of collagen fibers in the skin tissue, which causes the skin to become hard and lose elasticity. Skin fibrosis diseases include systemic sclerosis (SSc), etc. Systemic sclerosis is a chronic autoimmune connective tissue disease, which is mainly characterized by vascular lesions, immune imbalance, and progressive fibrosis of the skin and multiple organs. According to the extent of the affected skin, it can be mainly divided into localized cutaneous systemic sclerosis (lcSSc) and diffuse cutaneous systemic sclerosis (dcSSc). Due to the frequent occurrence of various complications such as interstitial lung disease, pulmonary hypertension, and fingertip ulcers, the quality of life of patients is poor. In addition, the pathogenesis of systemic sclerosis is complex and has not yet been fully understood, and its treatment options are very limited. Traditional treatment is still mainly hormone combined with immunosuppressants (mycophenolate mofetil, cyclophosphamide, methotrexate, etc.) and other vasodilators, supplemented by symptomatic treatment. Although these therapies have improved patients' symptoms such as inflammation and fibrosis to a certain extent, there is still a lack of drugs to reverse SSc fibrosis.

[0003] Astragali Radix, also known as Huangqi, Mianqi, etc., is the dried root of the leguminous plant Mongolian Astragalus, which can replenish qi and blood, promote diuresis and reduce swelling. Astragali contains a variety of active ingredients, such as polysaccharides, saponins, flavonoids, etc., which have antioxidant, antiviral, hypoglycemic, immune-enhancing, and cardiovascular protective effects. Among them, Calycosin-7-glucoside (CG) is one of the active ingredients in Astragali (Li Liangliang, Huang Jinzhi. Research progress on the pharmacological effects of Calycosin-7-glucoside [J]. Journal of Hainan Medical College, 2020, 26 (2): 156-160.), with a structural formula as shown in Formula I, which has antioxidant and myocardial function-improving effects.

[0004] Formula I.

[0005] Related reports show that CG can promote the healing of diabetic skin wounds (Chen J, Ma H, Meng Y, LiuQ, Wang Y, Lin Y, Yang D, Yao W, Wang Y, He X, Li P. Analysis of the mechanism underlying diabetic wound healing accelerationby Calycosin-7-glycoside using network pharmacology and molecular docking. Phytomedicine. 2023 Jun; 114:154773.), and in the study of liver cancer, CG was also found to have 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 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, TanYH, Wang LS. Combination of paeoniflorin and calycosin-7-glucosidealleviatesischaemic stroke injury via the PI3K / AKT signalling pathway. Pharm Biol. 2022Dec;60(1):1469-1477.). However, at present, no research has shown that calycosin glucoside can be used to treat skin fibrosis. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide an application of calycosin isoflavone glucoside in the preparation of a drug for treating skin fibrosis diseases.

[0007] The purpose of the present invention is achieved through the following technical solutions: Application of calycosin isoflavone glucoside in the preparation of medicines for treating skin fibrosis diseases.

[0008] The chemical structural formula of the calycosin isoflavone glucoside is shown in Formula I: Formula I.

[0009] The fibrotic skin diseases include systemic scleroderma (SSc).

[0010] The systemic sclerosis (SSc) includes localized cutaneous systemic sclerosis (lcSSc) and diffuse cutaneous systemic sclerosis (dcSSc).

[0011] Application of calycosin isoflavone glucoside in preparing a medicine for inhibiting proliferation of primary scleroderma cells and / or promoting apoptosis of primary scleroderma cells.

[0012] The concentration of the calycosin glucoside is 0.01 μmol / L or more; more preferably 0.01 to 1 μmol / L; and more preferably 1 μmol / L.

[0013] Compared with the prior art, the present invention has the following advantages and effects: The present invention combines in vitro and in vivo experiments and finds that calycosin glucoside can inhibit the proliferation of primary scleroderma cells and promote apoptosis, and inhibit the development of mouse skin fibrosis. Therefore, calycosin glucoside can provide a new drug source for the treatment or adjuvant treatment of skin fibrosis (including systemic scleroderma). BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a graph showing the effect of calycosin glucoside on the activity of primary scleroderma cells.

[0015] Figure 2 This is a graph showing the effect of calycosin glucoside on the proliferation of primary scleroderma cells.

[0016] Figure 3 This is a graph showing the effect of calycosin glucoside on collagen expression in primary scleroderma cells.

[0017] Figure 4 This is a graph showing the effect of calycosin glucoside on mouse skin thickness. DETAILED DESCRIPTION

[0018] The present invention is further described in detail below in conjunction with examples, but the embodiments 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 art. The test methods for which specific experimental conditions are not specified in the following examples are usually based on conventional experimental conditions. Unless otherwise specified, the reagents and materials used in the present invention can be obtained commercially.

[0019] The calycosin glucoside (CG) (CAS No.: 20633-67-4) involved in the embodiments of the present invention was purchased from Chengdu Mansite Biotechnology Co., Ltd.

[0020] Example 1: Calycosin Glucose Can Inhibit the Development of Systemic Scleroderma 1. Calycosin glucoside inhibits proliferation and promotes apoptosis of primary scleroderma cells In vitro experiments: Primary cells derived from scleroderma were treated with different drug concentrations, and then cell activity was detected by CCK-8 and clone formation experiments to study the effects of different drug concentration gradients on cell proliferation. The specific steps of the cell activity detection experiment are as follows: 1) Take skin tissue from patients with scleroderma (from volunteers, determined by collagen indicators (reference: ZixuanCheng, 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 / FLI1axis, Bioengineered, 12:1, 1212-1225, DOI: 10.1080 / 21655979.2021.1907128), that is, colla1 of scleroderma fibroblasts (type I collagen α1, see Figure 3) levels were significantly increased, which can be diagnosed as systemic sclerosis (SSc) patients) (Note: Skin tissues of 10 scleroderma patients were collected in this experiment, but because the experimental results were basically the same, only the results of primary scleroderma cells from one of the patients 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 filtered through a filter to remove undigested tissue fragments; the collected cells were resuspended in DMEM cell culture medium (purchased from Life Technologies, Gaithersburg, MD, USA) to obtain a large number of primary scleroderma skin fibroblasts (SSCF); 2) SSCF were plated into 96-well plates, with 3,000 cells per well; 3) Dissolve calycosin glucoside (CG, purchased from Chengdu Mansite Biotechnology Co., Ltd.) in DMSO (dimethyl sulfoxide) to prepare a stock solution with a concentration of 224 mM, and then dilute the stock solution with DMEM cell culture medium (purchased from LifeTechnologies, Gaithersburg, MD, USA) to prepare working solutions with final concentrations of 0 mM, 0.01 mM, 0.1 mM, and 1 mM; 4) After the cells in step 2) adhered to the wall, the experiment was divided into an experimental group and a control group: ① Add different concentrations of the working solution prepared in step 3 at a volume of 100 μL per well at the same time; ② After treatment for 24, 48, and 72 hours, the cell activity was detected using the CCK-8 cell proliferation and cytotoxicity detection kit (purchased from Biotech Co., Ltd.); the above experimental steps were repeated three times.

[0021] The results are as follows Figures 1 to 3 Shown: Activity of primary scleroderma cells ( Figure 1 ), cell proliferation ability ( Figure 2 ) (CG concentration 1mM) and collagen expression ( Figure 3 ) (CG concentration 1 mM) was affected by the stamen isoflavone glucoside.

[0022] 2. Calycosin glucoside can inhibit the development of skin fibrosis in mice Forty six-week-old female mice (balb / c) (purchased from Guangdong Experimental Animal Center) were selected, including 8 mice in the control group and 32 mice in the experimental group, to construct a scleroderma mouse model: (1) The hair on the back of the mice was shaved and marked with a marker pen. Two marked points were placed on the upper and lower sides of each mouse. Bleomycin was subcutaneously injected every day, with 0.1 ml injected at each point. The concentration of bleomycin was 200 ug / ml. The control group was subcutaneously injected with normal saline, with 0.1 ml injected at each point. (2) Nude mice were anesthetized before the experiment, and the degree of anesthesia was assessed by painless and painful stimulation to ensure that the mice were in an anesthetized state; (3) Drug treatment: On the day of subcutaneous injection, drugs were administered by gavage. Three drug combinations were set up 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 drugs were administered once every two days. The drug (CG) was first dissolved in edible oil (Golden Dragon Fish blending oil), and 100 μL was administered to each mouse each time; the control group was given edible oil, and 100 μL was administered to each mouse each time, and the weight of the mice was measured each time. After 28 days, the mice were euthanized and skin tissues were collected.

[0023] The experimental results of mouse skin thickness are as follows Figure 4 As shown, the thickness measurement of mouse skin showed that calycosin glucoside could significantly inhibit the development of mouse skin fibrosis, and the high-dose CG group had the best effect.

[0024] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Application of calycosin isoflavone glucoside in the preparation of drugs for treating skin fibrosis diseases.

2. The use according to claim 1, characterized in that: The skin fibrosis diseases include systemic scleroderma.

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

4. Use of calycosin isoflavone glucoside in the preparation of a drug for inhibiting the proliferation of primary scleroderma cells and / or promoting the apoptosis of primary scleroderma cells.

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

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

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