Application of CST-14 in the preparation of drugs for treating diabetic skin ulcers and damage

Abstract

The present invention discloses the use of the oxidative stress antagonist molecule CST-14 in the preparation of a drug for treating diabetic skin ulcers. CST-14 can antagonize the production of reactive oxygen species (ROS), reduce oxidative stress, and play a protective role in diabetic skin ulcers. Cell experiments have shown that CST-14 reduces the level of oxidative stress in vascular endothelial cells. Furthermore, animal models have shown that topical application of CST-14 can promote the repair of diabetic skin ulcers and improve the condition. Long-term application has no significant toxic side effects. CST-14 can be used in the fields of diabetic foot and diabetic skin ulcer repair, and has broad application value and market prospects.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to application of CST-14 in preparing a medicine for treating diabetic skin ulcers and damages. Background Art

[0002] Diabetic skin ulcers, particularly diabetic foot ulcers, are common clinically. Medical treatments are generally ineffective, and surgical debridement is often the final option for these patients. However, surgical risks and complications lead to a continuous increase in surgical failures. Current conservative treatments primarily focus on maintaining blood sugar stability, physical therapy such as thermal light irradiation, and oral neurotrophic medications, but their effectiveness remains limited. Recent studies have identified topical antioxidant therapy as a potential treatment for diabetic skin ulcers. CST-14 is a neurotrophic peptide with potent anti-inflammatory and antioxidant properties. Our preliminary studies have demonstrated that CST-14 improves the progression of diabetic skin ulcers, potentially offering a potential approach to alleviate diabetic skin ulcer symptoms with long-term use. Summary of the Invention

[0003] The present invention aims to provide a method for preparing a drug for treating diabetic skin ulcers using an oxidative stress antagonist, CST-14. As a ROS antagonist, CST-14 can alleviate oxidative stress and improve the condition of diabetic skin ulcers, such as diabetic foot. Furthermore, in vitro and in vivo experiments have shown no cytotoxicity or tissue toxicity, making long-term drug use safer.

[0004] The technical solution of the present invention is:

[0005] Cortistatin 14 (abbreviated as CST-14), CAS: 186901-48-4, has a molecular weight of 1.721 kD.

[0006] CST-14 has an amino acid sequence of Pro-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Ser-Ser-Cys-Lys (disulfide bridge: Cys2-Cys13). Its simplicity of preparation is a key advantage over other oxidative stress antagonists. Furthermore, its synthetic purity has reached over 99.5%, meeting pharmaceutical production requirements.

[0007] The present invention provides use of an oxidative stress antagonist CST-14 in preparing a drug for treating diabetic skin ulcers. In particular, the oxidative stress antagonist CST-14 is used as an oxidative stress antagonist to antagonize ROS.

[0008] Specifically, it is especially used in the preparation of drugs for treating diabetic foot.

[0009] It also includes applications in the preparation of other medicines for diabetic skin ulcers and damages, such as diabetic dermatitis and diabetic gangrene, which are all related to skin tissue damage caused by diabetes. It is foreseeable that CST will also produce corresponding therapeutic effects.

[0010] The medicine of the present invention further includes a pharmaceutically acceptable carrier, adjuvant or diluent.

[0011] The specific application modes of the present invention are microneedle administration, nanoparticle administration, and drug microsphere administration.

[0012] The form of the drug is selected from one of the following: spray, lotion, gel, ointment, paste, emulsion, etc.

[0013] Preferably, the drug is in the form of a cream or ointment.

[0014] If economic cost is not a concern, the drug can be administered orally or by injection.

[0015] In vitro binding experiments, in vivo animal model studies, and molecular interaction studies have demonstrated that cortistatin 14 (CST-14) can directly antagonize reactive oxygen species (ROS) and exert a protective effect against diabetic skin ulcers, including diabetic foot ulcers. CST-14 is a novel oxidative stress antagonist that can be directly synthesized using existing technologies, thus reducing costs. Furthermore, long-term use of CST-14 in animal models has shown no significant toxic side effects, demonstrating its safe use in the treatment of diabetic foot ulcers and other diabetic skin ulcers. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a visual illustration of the therapeutic effect of CST-14 on diabetic skin defects in rats.

[0017] Figure 2 This is a statistical graph showing the percentage of skin defect repair achieved with CST-14 in diabetic rat skin defects.

[0018] Figure 3 This is a histochemical staining image of CST-14 in the treatment of diabetic skin defects in rats.

[0019] Figure 4 It is the protective antagonistic effect of CST-14 in hydrogen peroxide-induced oxidative stress in vascular endothelial cells. DETAILED DESCRIPTION

[0020] The experimental animals, reagents, culture media, and buffers used in the following examples were obtained from: Cortistatin 14 (purity 98.4%, CAS: 186901-48-4, amino acid sequence: Pro-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Ser-Ser-Cys-Lys (Disulfide bridge: Cys2-Cys13)) (Gill Biochemical Co., Ltd.)

[0021] Streptozotocin (STZ), (McLean)

[0022] SD male rats (Animal Center of Shandong University)

[0023] PBS buffer (Biyuntian Bioreagent Company)

[0024] RIPA cell protein extraction and lysis buffer (Thermo Fisher, Pierce)

[0025] Protease inhibitors (Beijing Solebow Technology Co., Ltd.)

[0026] BCA protein quantification kit (Shanghai Yanxi Biotechnology Co., Ltd.)

[0027] Complete EDTA-Free (Roche Biopharmaceuticals)

[0028] Xylene (Sinopharm Chemical Reagent Co., Ltd.)

[0029] Neutral rubber (Shanghai Titan Technology Co., Ltd.)

[0030] Concentrated hydrochloric acid (Sinopharm Chemical Reagent Co., Ltd.)

[0031] Yi Hong (Shanghai Titan Technology Co., Ltd.)

[0032] Hematoxylin (Shanghai Titan Technology Co., Ltd.)

[0033] Methanol (Sinopharm Chemical Reagent Co., Ltd.)

[0034] Citrate buffer (0.01 M, pH = 6.0) (Shanghai Sangon Biotech Co., Ltd.)

[0035] 10% NGS (Shanghai Sangon Biotech Co., Ltd.)

[0036] Hydrogen peroxide (H2O2) (Sinopharm Chemical Reagent Co., Ltd.)

[0037] BSA (Shanghai Sangon Biotech Co., Ltd.)

[0038] Anhydrous ethanol (Sinopharm Chemical Reagent Co., Ltd.)

[0039] The cell counter was purchased from Thermo Fisher, USA

[0040] Microscope purchased from Shanghai Caikang Optical Instrument Co., Ltd.

[0041] Centrifuge purchased from Jinan Olaibo Medical Equipment Co., Ltd.

[0042] Electronic balance purchased from Jinan Oulaibo Medical Equipment Co., Ltd.

[0043] Enzyme-linked immunosorbent assay (ELISA) was purchased from Beijing Meihuayi Technology Co., Ltd.

[0044] Flow cytometer was purchased from BD

[0045] Ice maker purchased from Jinan Olaibo Medical Equipment Co., Ltd.

[0046] The ultrapure water system was purchased from Jinan Olaibo Medical Equipment Co., Ltd.

[0047] Vortex mixer purchased from Jinan Olabo Medical Instrument Co., Ltd.

[0048] 1. Construction of Mouse Model of Skin Inflammation

[0049] A streptozotocin (STZ)-induced diabetic rat model was established in 14-week-old wild-type SD male rats (14 rats in total). First, the dorsal skin of all rats was shaved with a razor and depilatory cream. STZ 65 mg / kg body weight was injected intraperitoneally. Three days later, the tail was clipped to test blood glucose. Rats with random blood glucose greater than 16.7 mmol / L were included in the experiment. After shaving and disinfection, a 1 cm diameter punch was used to create a full-thickness skin defect ulcer model. 25 μl PBS was added to the wound surface as a control. In the treatment group, 25 μl CST-14 (500 μg / ml) was applied topically to the wound surface every two days, and the wound surface was covered with sterile cloth. The dorsal skin was photographed on the day of surgery, 7 days, and 14 days after surgery, and the healing rate of the ulcer wound was statistically analyzed. After 14 days, all rats in all groups were euthanized, and skin specimens from the dorsal lesion area were collected for subsequent experiments.

[0050] Figure 1 、 Figure 2 This is the result of this part of the experiment. Through intuitive photos and skin defect regeneration ratio, it is shown that the diabetic skin ulcers in rats are greatly alleviated under CST-14 treatment.

[0051] 2. Tissue Slice Preparation

[0052] Skin from the lesional area of ​​the dorsal region of rats from all groups was fixed in 10% formalin for at least 72 hours at room temperature. Tissue was then dehydrated sequentially with 50% ethanol (60 minutes), 70% ethanol (60 minutes), 85% ethanol (60 minutes), 95% ethanol (60 minutes), 100% ethanol (30 minutes), and 100% ethanol (30 minutes). Tissue was then treated sequentially with ethanol and xylene (60 minutes), followed by xylene (60 minutes). Tissue was then cleared with xylene and paraffin (60 minutes), followed by paraffin (80 minutes). Tissue was placed in a cassette, filled with paraffin, and then placed on the cold stage of a paraffin embedding machine. The embedded paraffin block was placed on a microtome and sectioned to a thickness of approximately 4 μm. The paraffin slide containing the tissue was gently applied to 42°C water. After complete flattening, the sections were gently lifted with a clean glass slide. The sections were placed on slides, numbered, and baked in a 68°C oven for at least 6 hours.

[0053] 3. Hematoxylin / eosin staining

[0054] The sections were dewaxed with conventional fat-soluble solvents until they were hydrated (xylene twice, 15 minutes each time; 100% alcohol for 5 minutes; 95% alcohol for 5 minutes; 75% alcohol for 5 minutes; 50% alcohol for 5 minutes), then stained with hematoxylin staining solution for 5 minutes. After rinsing with clean water, the sections were stained with eosin staining solution for 5 minutes. After rinsing with clean water, the sections were dehydrated (50% alcohol for 5 minutes; 75% alcohol for 5 minutes; 95% alcohol for 5 minutes; 100% alcohol for 5 minutes; xylene twice, 15 minutes each time). After the sections were dried, they were sealed with neutral gum and observed and analyzed under an optical microscope.

[0055] Figure 3 This is the result of the experiment in this part. Histochemical HE staining shows that local CST-14 treatment has excellent function in reducing skin inflammatory cell infiltration and improving soft tissue regeneration and repair process.

[0056] 4. Cultivation and stimulation of human umbilical vein endothelial cells (HUVEC)

[0057] HUVECs were revived and cultured synchronously with nucleus pulposus cells in DMEM / F-12 medium (Hyclone, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 1% 100 u / ml penicillin, and 100 mg / ml streptomycin (Hyclone, USA) at pH 7.2 (95% air, 5% CO₂, 37°C). The medium was changed every 3 days, and cells were passaged when they reached 80–90% confluence. Second- or third-passage cells were used for the indicated experiments. HUVECs were stimulated with 600 μM hydrogen peroxide for 24 hours to activate the oxidative stress response and then treated with either PBS (control group) or CST-14 peptide (experimental group).

[0058] 4. Western Blot

[0059] After in vitro culture and stimulation, HUVEC cells in each group were placed on ice, treated, and washed with ice water. Proteins were extracted using lysis buffer (p0013c, Beyotime Biotechnology) after harvesting. The cultured HUVECs were lysed in RIPA lysis buffer (p0013c, Beyotime Biotechnology) and total protein was collected from each sample. The proteins in loading buffer were heated at 100°C for 10 minutes (ThermoFisher). Protein electrophoresis was performed using 10% SDS-PAGE gels (beyondtime Biotechnology) (30 g per lane) and transferred to a nitrocellulose membrane. The membranes were blocked with Tween 20 (10 mM Tris-HCl, pH 8.0; 150 mM NaCl; 0.5% Tween 20) in 5% nonfat dry milk for 2 h. The membranes were incubated with specific primary antibodies (Caspase-3, Bax, Bcl-2, GAPDH) for 1 h at 37°C, washed three times with PBS, and incubated overnight at 4°C. A secondary antibody (dilution 1:2000) containing horseradish peroxidase was added and incubated for 1 h at room temperature. The membranes were removed with blunt forceps and rinsed at least three times with PBS. 1 ml of working buffer (p0018s, Biotime Biotechnology) was added to each membrane and detected (Amersham Life Sciences, Ellington, IL, USA). Statistical analysis of grayscale values ​​was performed using Image J software.

[0060] Figure 4 This is the experimental result. By using Western blot, we observed the abnormal levels of apoptosis markers caused by hydrogen peroxide stimulation, and CST-14 treatment alleviated this abnormal phenomenon in HUVEC cells under oxidative stress stimulation.

[0061] 5. Statistical Analysis

[0062] All data are presented as the mean ± standard deviation of at least three independent experiments. Statistical analysis of two groups of data was performed using paired t-test, and statistical analysis of more than two groups of data was performed using one-way analysis of variance.

[0063] These experiments confirmed that CST-14 can directly antagonize ROS production and oxidative stress-induced tissue cell damage, demonstrates therapeutic efficacy in animal models of diabetic skin ulcer wounds, and effectively mitigates endothelial cell apoptosis in response to oxidative stress. Furthermore, CST-14 is a polypeptide molecule with a molecular weight of 1.7 kDa, which can reduce single-use dosages and provide more flexible administration methods.

[0064] More importantly, CST-14 is simple to prepare, the preparation method is mature, and it can be directly synthesized, which reduces costs and thus reduces the financial burden on patients.

[0065] The above is a detailed description of an embodiment of the present invention. However, the content described is only a preferred embodiment of the present invention and should not be considered to limit the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. Use of CST-14 in the preparation of a drug for treating diabetic skin ulcers and lesions. CST-14 reduces the level of oxidative stress in vascular endothelial cells by antagonizing the production of reactive oxygen species (ROS). In an in vitro hydrogen peroxide-induced oxidative stress model of vascular endothelial cells, CST-14 treatment reduces the expression levels of apoptosis markers Caspase-3 and Bax, and exerts a protective effect against diabetic skin ulcers.

2. Application of CST-14 in the preparation of drugs for the treatment of diabetic foot.

3. The use according to any one of claims 1-2, characterized in that: The drug further includes a pharmaceutically acceptable carrier.

4. The use according to any one of claims 1-2, characterized in that: The form of the drug is selected from one of the following: spray, lotion, gel, ointment, paste, emulsion.

5. The use according to claim 4, characterized in that: The drug is in the form of a cream or ointment.

6. The use according to any one of claims 1-2, characterized in that: The specific application method is microneedle drug delivery.

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