CN114949174A - Application of CST-14 in preparation of medicine for treating diabetic skin ulcer and diabetic skin breakage - Google Patents

Abstract

The invention discloses an application of an oxidative stress antagonistic molecule CST-14 in preparing a medicine for treating diabetic skin ulcer. CST-14 can antagonize reactive oxygen species ROS production, relieve oxidative stress, and exert a protective effect in diabetic skin ulcers. Cell experiments show that CST-14 reduces the level of oxidative stress of vascular endothelial cells. Meanwhile, animal models show that the local application of CST-14 can promote the repair of diabetic skin ulcer and improve the disease condition, has no obvious toxic or side effect after long-term application, can be used in the fields of diabetic foot, diabetic skin ulcer repair and the like, and has wide application value and market prospect.

Description

Application of CST-14 in preparation of medicine for treating diabetic skin ulcer and diabetic skin breakage

Technical Field

The invention relates to the field of medicine, in particular to application of CST-14 in preparing a medicine for treating diabetic skin ulcer and damage.

Background

At present, diabetic skin ulcers, especially diabetic feet, are common in clinic, the curative effect of general medicines of patients is poor, and operation debridement is often the final choice of patients, however, operation-related risks and operation failure cases caused by complications are continuously appeared. At present, conservative treatment methods mainly comprise physiotherapy such as maintaining stable blood sugar, baking lamp irradiation and the like, and oral administration of nerve nutrition medicines, but still have limited curative effect. In recent years, researches find that local antioxidant stress is a potential treatment method for diabetic skin ulcer, and CST-14 is a neurotrophic polypeptide and has stronger anti-inflammatory and antioxidant effects. Application research on CST-14 through earlier experiments shows that the composition has an improvement function on the diabetic skin ulcer process, and can provide a potential method for alleviating the diabetic skin ulcer condition after long-term use.

Disclosure of Invention

The invention aims to provide application of an oxidative stress antagonist CST-14 in preparation of a medicine for treating diabetic skin ulcer, wherein the CST-14 is used as a ROS antagonistic molecule and has the effects of relieving oxidative stress reaction and improving the conditions of diabetic skin ulcer diseases such as diabetic foot and the like, in-vivo and in-vitro experiments show that the CST-14 has cell and tissue toxicity, and the medicine is safer to use for a long time.

The technical scheme of the invention is as follows:

cortistatin14 (Cortistatin14, abbreviated as CST-14), CAS: 186901-48-4. Its molecular weight is 1.721 kD.

The amino acid sequence of CST-14 is: Pro-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Ser-Ser-Cys-Lys (Disulfide bridge: Cys2-Cys13), and is simple to prepare, and the advantages of the compound compared with other oxidative stress antagonistic medicaments are also achieved. Meanwhile, the synthesis purity of the compound reaches over 99.5 percent at present, and the purity meets the requirement of medicine preparation.

The invention provides application of an oxidative stress antagonist CST-14 in preparation of a medicine for treating diabetic skin ulcer, in particular to the application of the oxidative stress antagonist CST-14 serving as an oxidative stress antagonist for antagonizing ROS function.

In particular to the application in preparing the medicine for treating diabetic foot.

The application of the CST also comprises the application of preparing other medicines for treating diabetic skin ulcer and damage, such as diabetic dermatitis, diabetic gangrene and the like, which are related to the skin tissue damage caused by diabetes, and the CST can also generate corresponding treatment effect in expectation.

The medicine also comprises a pharmaceutically acceptable carrier, an auxiliary agent or a diluent.

The application of the invention is specifically microneedle administration, nanoparticle administration and drug microsphere administration.

The form of the medicament is selected from one of the following forms: sprays, lotions, gels, ointments, pastes, creams and the like.

Preferably, the form of the medicament is selected from the following creams or ointments.

The administration can also be carried out by oral or parenteral pharmaceutical means, without consideration of economic cost.

The results of intermolecular interaction experiments through in vitro combination experiments and in vivo animal model experiments show that the cortistatin14 (CST-14) can directly antagonize the function of Reactive Oxygen Species (ROS) and can play a protective role in diabetic skin ulcers such as diabetic foot and the like. CST-14 belongs to a novel oxidative stress antagonist drug which can be directly synthesized by the prior art, thereby reducing the cost. In addition, in the animal model, the long-term application of the CST-14 has no obvious toxic or side effect, and can be safely used in the fields of treatment of diabetic foot, diabetic skin ulcer and the like.

Drawings

FIG. 1 is a graph of the effect of CST-14 on the treatment of diabetic skin defects in rats.

FIG. 2 is a statistical plot of the percent skin defect repair for CST-14 in diabetic skin defects in rats.

FIG. 3 is a graph of histochemical staining of CST-14 in the treatment of diabetic skin defects in rats.

FIG. 4 is a protective antagonism of CST-14 in hydrogen peroxide-induced oxidative stress of vascular endothelial cells.

Detailed Description

Sources of experimental animals, reagents, media and buffers referred to in the following examples: cortisan 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 (Disulide bridge: Cys2-Cys13)) (Gill Biochemical Co., Ltd.)

Streptozotocin (STZ), (Meclin Co.)

SD male rat (Shandong university animal center)

PBS buffer (Biyuntian biological reagent company)

RIPA cell protein extraction lysate (Thermo Fisher, Pierce)

Protease inhibitor (Beijing Solaibao Tech Co., Ltd.)

BCA protein quantification kit (Shanghai Yanxi Biotech Co., Ltd.)

Complete EDTA-Free (Roche biomedicine)

Xylene (national drug group chemical reagent Co., Ltd.)

Neutral gum (Shanghai Tantake skill Co., Ltd.)

Concentrated hydrochloric acid (national drug group chemical reagent Co., Ltd.)

Eosin (Shanghai Tantake skill Co., Ltd.)

Hematoxylin (Shanghai Tai Tankou Tech Co., Ltd.)

Methanol (national medicine group chemical reagent Co., Ltd.)

Citrate buffer (0.01M, pH 6.0) (Biotechnology engineering, Shanghai Co., Ltd.)

10% NGS (biological engineering Shanghai share Co., Ltd.)

Hydrogen peroxide (H2O2) (national drug group chemical Co., Ltd.)

BSA (Biotechnology Shanghai GmbH)

Absolute ethyl alcohol (national medicine group chemical reagent Co., Ltd.)

Cell counter was purchased from Thermo Fisher, USA

Microscope from Shanghai Caikang optical instruments Ltd

Centrifuge from Jinan Olaibo medical instruments Ltd

Electronic balance from medical instruments ltd, denna olabo

Enzyme-linked immunosorbent assay (ELISA) instrument purchased from Beijing Meihua apparatus science and technology Limited

Flow cytometer from BD company

Ice machine from Jinan Ou Laibo medical instruments Ltd

Ultra pure water system is commercially available from Jinan Oolabo medical instruments Ltd

Vortex mixer from Jinan Olaibo medical instruments Ltd

1. Construction of mouse model for skin inflammation

Streptozotocin (STZ) -induced diabetic rat models (14 total) were established in 14-week-old wild-type SD male rats. First, all rats were shaved of their back skin with a razor and depilatory cream. The STZ65mg/kg body weight is injected into the abdominal cavity, the blood sugar is detected by cutting the tail after three days, and the random blood sugar is more than 16.7mmol/L and is included in the experiment. After shaving and disinfection, a punch with a diameter of 1cm was used to create a full-thickness skin defect ulcer model, 25 μ l PBS was added to the wound surface as a control, 25 μ l CST-14(500 μ g/ml) was administered topically to the wound surface of the treatment group once every two days, and the wound surface was aseptically covered. The dorsal skin was photographed on the day after the operation, 7 days and 14 days, respectively, and statistical analysis of the ulcer wound healing ratio was performed. After 14 days, all groups of rats were euthanized and skin specimens were collected from the area of the back skin lesions for subsequent testing.

FIG. 1 and FIG. 2 are graphs showing the results of this part of the experiment, showing that the diabetic skin ulcer of rats was greatly relieved under the CST-14 treatment by means of visual photographs and the regeneration ratio of skin defects.

2. Tissue slice preparation

The skin of the dorsal lesions of rats from all groups were fixed in 10% formalin for at least 72 hours at room temperature. Sequentially dehydrating the tissue with 50% ethanol (60 min), 70% ethanol (60 min), 85% ethanol (60 min), 95% ethanol (60 min), 100% ethanol (30 min); sequentially treating the mixture with ethanol, xylene (60 minutes) and xylene (60 minutes); then clear with xylene and paraffin (60 min), paraffin (80 min); the tissue was placed in a cassette, filled with paraffin, and then placed on the cold plate of a paraffin embedding machine. Placing the embedded tissue paraffin block on a microtome and sectioning the tissue to a thickness of about 4 μm; the organized paraffin pieces were lightly smeared in water at 42 ℃. After the glass is completely flattened, a clean glass sheet is used for gently pulling up the slices; the sections were placed on glass slides, numbered, and baked in an oven at 68 ℃ for at least 6 hours.

3. Hematoxylin/eosin staining

The slices were dewaxed with a conventional fat-soluble solvent to water (xylene twice, 15 min/each time; 100% alcohol 5 min; 95% alcohol 5 min; 75% alcohol 5 min; 50% alcohol 5 min), then stained with hematoxylin staining solution for 5 min, rinsed with clear water, then stained with eosin staining solution for 5 min, rinsed with clear water, dehydrated (50% alcohol 5 min; 75% alcohol 5 min; 95% alcohol 5 min; 100% alcohol 5 min; xylene twice, 15 min/each time), after which the slices were air-dried, they were sealed with neutral gum, and observed and analyzed under an optical microscope.

FIG. 3 is a graph of the results of this section of the experiment showing that topical CST-14 treatment has excellent function for reducing skin inflammatory cell infiltration and improving soft tissue regenerative repair process by histochemical HE staining.

4. HUVEC culture and stimulation of human umbilical vein endothelial cells

HUVEC cells were recovered by adding 10% bovine serum (FBS, gibco, USA), 1% 100u/ml penicillin and 100mg/ml streptomycin (Hyclone, USA) to DMEM/F-12 medium (Hyclone, Thermo CO), and nucleus pulposus cells were cultured simultaneously at pH7.2 (95% air, 5% CO2,37 ℃). The medium was changed every 3 days and passaged when the cells reached 80-90% confluence. Second or third generation cells were used for the indicated experiments. Oxidative stress activation was achieved by stimulating HUVEC24 hours with 600. mu.M hydrogen peroxide, treated with PBS (control) or CST-14 polypeptide (experimental).

Western Blot protein immunoblotting experiment

The HUVEC cells of each group after in vitro culture and stimulation were placed on ice, treated and washed with ice water. After collection, proteins were extracted by adding lysis buffer (p0013c, best biotechnology), and cultured HUVECs were added to RIPA lysis buffer (p0013c, best biotechnology), and total proteins were collected from each sample. The protein in the loading buffer was heated at 100 ℃ for 10min (thermolfisher). Protein electrophoresis (30 g per lane) was performed using a 10% SDS-PAGE gel (beyontiamebic technology), and after electrophoresis, the proteins were electrophoretically transferred onto nitrocellulose membranes. Tween 20(10mm tris-hcl, ph 8.0; 150mm nacl; 0.5% tween 20) was used for 2h barrier in 5% skim dried milk, incubated with specific primary antibodies (Caspase-3, Bax, Bcl-2, GAPDH) for 1h at 37 ℃, washed 3 times with PBS, overnight at 4 ℃, horseradish peroxidase secondary antibody (dilution 1:2000) was added, and incubated for 1h at room temperature. The membrane was removed with blunt forceps and rinsed at least three times with PBS. 1ml of working solution (p0018s, beiime Biotechnology) was added per membrane and tested (Amersham life sciences, illinton, illinois, usa). Statistical analysis of band grey values was performed using Image J software.

FIG. 4 is a graph showing the results of this experiment, in which abnormal levels of apoptosis markers due to hydrogen peroxide stimulation were observed using Western blot, whereas CST-14 treatment reduced this abnormality in HUVEC cells under oxidative stress.

5. Statistical analysis

All data are expressed as mean standard deviations of at least three independent experiments. Statistical analysis for two sets of data statistical analysis using paired t-tests more than two sets of data statistical analysis using one-way analysis of variance.

The experiment proves that CST-14 can directly antagonize ROS generation and histiocyte damage caused by oxidative stress, has exact curative effect on a diabetic skin ulcer wound animal model, and effectively lightens the apoptosis process of vascular endothelial cells in oxidative stress reaction. Meanwhile, CST-14 is a polypeptide molecule with a molecular weight of 1.7kD, can reduce single-use dosage and has more flexible administration method.

More importantly, the CST-14 is simple to prepare, the preparation method is pure and mature, direct synthesis can be realized, and the cost is reduced, so that the economic burden of patients is reduced.

While one embodiment of the present invention has been described in detail, the description is only a preferred embodiment of the present invention and should not be taken as limiting the scope of the invention. All equivalent changes and modifications made within the scope of the present invention shall fall within the scope of the present invention.

Claims (7)

1. Application of CST-14 in preparing medicine for treating diabetic skin ulcer and diabetic skin breakage is disclosed.
2. Application of CST-14 in preparing a medicament for treating diabetic foot.
3. Application of CST-14 in preparing medicine for treating diabetic dermatitis and diabetic gangrene is disclosed.
4. 4. Use according to any one of claims 1 to 3, characterized in that: the medicament also comprises a pharmaceutically acceptable carrier, auxiliary agent or diluent.
5. 5. Use according to any one of claims 1 to 3, characterized in that: the form of the medicament is selected from one of the following forms: spray, lotion, gel, ointment, paste, emulsion.
6. 6. Use according to claim 5, characterized in that: the medicament is in the form of a cream or ointment.
7. 7. Use according to any one of claims 1 to 3, characterized in that: the specific application mode is microneedle administration, nanoparticle administration or drug microsphere administration.

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