CN114949174B - Application of CST-14 in the preparation of drugs for treating diabetic skin ulcers and damage - Google Patents

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

Application of CST-14 in preparing medicine for treating diabetic skin ulcer and damage

Technical Field

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

Background

At present, diabetic skin ulcers, particularly diabetic feet, are clinically common, the general drug treatment effect of patients is poor, surgical debridement is the final choice of such patients, and surgical failure cases caused by surgical related risks and complications are continuously generated. The current conservation treatment method mainly comprises physiotherapy such as blood sugar stabilization maintenance, baking lamp irradiation and the like, and oral administration of the vegetative nerve medicine, but the curative effect is still limited. In recent years, research has found that local antioxidant stress is a potential treatment method for diabetic skin ulcers, and CST-14 is a neurotrophic polypeptide, and has stronger anti-inflammatory and antioxidant effects. The application and research of CST-14 through early experiments show that the traditional Chinese medicine composition has the function of improving the diabetic skin ulcer process and can provide a potential method for reducing the diabetic skin ulcer condition after long-term use.

Disclosure of Invention

The invention aims to provide an application of an oxidative stress antagonist CST-14 in preparing a medicament for treating diabetic skin ulcers, wherein the CST-14 is used as an ROS antagonistic molecule, has the effects of relieving oxidative stress reaction and improving the conditions of diabetic skin ulcers such as diabetic feet, and has no toxicity to cells and tissues as shown by in vivo and in vitro experiments, so that the medicament is safer to use for a long time.

The technical scheme of the invention is as follows:

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

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-Cys 13), and the preparation is simple, which is the advantage compared with other oxidative stress antagonistic drugs. Meanwhile, the synthetic purity of the traditional Chinese medicine reaches over 99.5 percent, and the purity meets the preparation requirement of the medicine.

The invention provides application of an oxidative stress antagonist CST-14 in preparing a medicament for treating diabetic skin ulcers, and particularly relates to application of the oxidative stress antagonist CST-14 serving as an oxidative stress antagonist to antagonize ROS.

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

Also comprises the application in preparing other medicines for treating diabetic skin ulcers and damages, such as diabetic dermatitis, diabetic gangrene and the like, which are all related to skin tissue damage caused by diabetes, and the CST can also produce corresponding treatment effects.

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

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

The pharmaceutical form is selected from one of spray, lotion, gel, ointment, paste, emulsion, etc.

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

The administration of drugs by oral administration or injection can also be adopted without considering the economic cost.

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

Drawings

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

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

FIG. 3 is a chart 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 vascular endothelial cell oxidative stress.

Detailed Description

The experimental animals, reagents, media and buffer sources referred to in the examples below were 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-Cys 13)) (Jil Biochemical Co., ltd.)

Streptozotocin (STZ), (Melin Corp.)

SD male rats (Shandong university animal center)

PBS buffer, (Biyun Tian biological reagent Co.)

RIPA cell protein extract lysate (Thermo Fisher, pierce)

Protease inhibitor (Beijing Soy Bao technology Co., ltd.)

BCA protein quantitative kit (Shanghai inflammatory Xi Biotechnology Co., ltd.)

Complete EDTA-Free (Rogowski biological medicine)

Xylene (national medicine group chemical reagent Co., ltd.)

Neutral gum (Shanghai Taitan technology Co., ltd.)

Concentrated hydrochloric acid (national medicine group chemical reagent Co., ltd.)

Eosin (Shanghai Taitan science and technology Co., ltd.)

Hematoxylin (Shanghai Taitan science and technology Co., ltd.)

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

Citrate buffer (0.01 m, ph=6.0) (Shanghai Co., ltd., bioengineering)

10% NGS (Shanghai stock Co., ltd., bioengineering)

Hydrogen peroxide (H2O 2) (national medicine group chemical Co., ltd.)

BSA (Shanghai share of bioengineering Co., ltd.)

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

Cell counter was purchased from Thermo Fisher, inc. of America

Microscope was purchased from Shanghai Cai Kang optical instruments Co., ltd

Centrifuge from Nanfu Laibo medical instruments Co., ltd

Electronic balance is purchased from ataxia south European Lebo medical instruments Co., ltd

An ELISA (enzyme-labeled) was purchased from Beijing America Instrument technologies Co., ltd

Flow cytometry was purchased from BD corporation

Ice machine available from Miao Nanfu Laibo medical instruments Inc

Ultra-pure water systems were purchased from atanan European Lebo medical instruments Co., ltd

Vortex mixers were purchased from ataxia, south european and leibo medical instruments limited

1. Construction of murine model of skin inflammation

Streptozotocin (STZ) -induced diabetic rat models (14 total) were established in 14 week old wild type SD male rats. First, the back skin of all rats was shaved off with a razor and depilatory cream. The STZ was injected intraperitoneally at 65mg/kg body weight, blood glucose was detected by tail cutting after three days, and random blood glucose was greater than 16.7mmol/L for inclusion in the experiment. After shaving and sterilizing, perforating with a puncher with the diameter of 1cm, establishing a full-layer skin defect ulcer model, adding 25 mu l PBS into the wound surface as a control, locally using 25 mu lCST-14 (500 mu g/ml) of the wound surface of a treatment group for once every two days, and carrying out aseptic covering on the wound surface. Back skin photographs were taken on the day 7 and 14 after the operation, respectively, and statistical analysis of the ulcer wound healing ratio was performed. After 14 days, all groups of rats were euthanized and skin specimens of the back skin lesion area were collected for subsequent testing.

FIGS. 1 and 2 are graphs showing that diabetic skin ulcers in rats are greatly relieved under CST-14 treatment by visual photographs and skin defect regeneration ratios.

2. Tissue slice preparation

The skin from the dorsal skin lesions of rats from all groups was fixed in 10% formalin for at least 72 hours at room temperature. The tissue is dehydrated by sequentially carrying out the dehydration of 50 percent ethanol (60 minutes), 70 percent ethanol (60 minutes), 85 percent ethanol (60 minutes), 95 percent ethanol (60 minutes), 100 percent ethanol (30 minutes) and 100 percent ethanol (30 minutes), sequentially treating by ethanol, dimethylbenzene (60 minutes) and dimethylbenzene (60 minutes), then using dimethylbenzene and paraffin (60 minutes), and enabling paraffin (80 minutes) to be transparent, putting the tissue into a box, filling the tissue with paraffin, and then placing the tissue on a cold table of a paraffin embedding machine. The embedded tissue paraffin block was placed on a microtome and sectioned to a tissue thickness of about 4 μm, and the paraffin sheet containing the tissue was gently smeared in water at 42 ℃. After complete flattening, the sections were gently plucked with clean glass sheets, placed on slides, numbered, and baked in a 68 ℃ oven for at least 6 hours.

3. Hematoxylin/eosin staining

The sections were dewaxed with conventional fat-soluble solvents 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 stain for 5 min, rinsed with clear water, then stained with eosin stain 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), then air-dried after sections were sealed with neutral resin, and observed and analyzed under an optical microscope.

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

4. Human umbilical vein endothelial cell HUVEC culture and stimulation

HUVEC cells were resuscitated and nuclear cells (95% air, 5% CO2,37 ℃) were cultured simultaneously in DMEM/F-12 medium (Hyclone, thermo CO) with 10% bovine serum (FBS, gibco, USA), 1%100u/ml penicillin and 100mg/ml streptomycin (Hyclone, USA). The medium was changed every 3 days and the cells were passaged until 80-90% confluence was achieved. Second or third generation cells were used for the indicated experiments. Oxidative stress activation was achieved by stimulating HUVEC with 600. Mu.M hydrogen peroxide for 24 hours, and treatment was performed with PBS (control group) or CST-14 polypeptide (experimental group).

Western Blot

The HUVEC cells of each group after in vitro culture and stimulation were placed on ice and washed with ice water after treatment. Proteins were extracted by addition to lysis buffer (p 0013c, beyotime biotechnology) after collection, cultured HUVECs were added to RIPA lysis buffer (p 0013c, beyotime biotechnology), and total protein was collected for each sample. The protein in the loading buffer was heated at 100 ℃ to 10min (thermofisher). Protein electrophoresis (30 g per lane) was performed using a 10% SDS-PAGE gel (beyontimebiotechnology), after which the protein electrophoresis was transferred to nitrocellulose membrane. Blocking with Tween 20 (10 mm tris-hcl, ph8.0;150mm nacl;0.5%tween 20) in 5% skim dry milk for 2h, incubating with specific primary antibody (Caspase-3, bax, bcl-2, GAPDH) at 37℃for 1h, washing 3 times with PBS overnight at 4℃and incubating with horseradish peroxidase secondary antibody (diluted 1:2000) at room temperature for 1h. The membrane was removed with blunt forceps and rinsed at least three times with PBS. 1ml of working fluid (p 0018s, beiotime Biotechnology) was added to each membrane and tested (Amersham Life sciences, elington, illinois, USA). Statistical analysis of the band gray values was performed using Image J software.

FIG. 4 is a graph showing the results of this experiment, in which the abnormality of apoptosis marker levels caused by hydrogen peroxide stimulation was observed by using Western blot, while CST-14 treatment reduced the abnormality of HUVEC cells under oxidative stress stimulation.

5. Statistical analysis

All data are expressed as the mean and standard deviation of at least three independent experiments. Statistical analysis for two sets of data uses paired t-test and statistical analysis for more than two sets of data uses one-factor analysis of variance.

Through the experiment, the CST-14 can directly antagonize ROS production and tissue cell damage caused by oxidative stress, has definite curative effect on diabetic skin ulcer wound animal models, and effectively reduces 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, which 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, and can be directly synthesized, so that the cost is reduced, and the economic burden of a patient is reduced.

The foregoing describes one embodiment of the present invention in detail, but the description is only a preferred embodiment of the present invention and should not be construed as limiting the scope of the invention. All equivalent changes and modifications within the scope of the present invention are intended to be covered by the present invention.

Claims (6)

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 comprises a pharmaceutically acceptable carrier. 4. The use according to any one of claims 1-2, characterized in that the drug is in a form selected from 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.