KR101963501B1 - Method For Diagnosis of Diabetic Skin Diseases using the Concentration Of Skin Lipid Metabolites and Expression Level of Skin Lipid Biosynthesis Enzymes, and Screening Method thereby - Google Patents

Abstract

The present invention relates to a method for providing information for diagnosis or treatment of diabetic skin disease and a method for screening a composition for treating diabetic skin disease. The present invention relates to a method for screening a composition for treating diabetic skin disease, Expression and activity of the protein, and the fatty acid and the sphingosine concentration can be confirmed and used as a marker for the information providing method and the screening method.

Description

TECHNICAL FIELD The present invention relates to a method for diagnosing diabetic skin diseases using skin lipid metabolism substances and lipid biosynthetic enzymes and a screening method using the same. thereby}

The present invention relates to a method for diagnosing a diabetic skin disease using a skin lipid metabolite and an expression amount of biosynthetic enzyme, and a method for screening a therapeutic agent for diabetic skin disease using the same.

Type 2 diabetes mellitus (DM) is a frequently occurring disease that accounts for more than 80% of all diabetic patients. Skin-related problems are known to occur in 30% of diabetic patients, and thus skin-related problems can be used as an abnormality in glycemic control function or a symptom of a new disease.

Symptoms such as xerosis, pruritus, skin infections and delays in wound restoration are caused by damage to skin barriers and skin barriers may be caused by diabetes-induced skin metabolic disorders or by blood vessels Can be damaged by diabetic complications such as vasculopathy and neuropathy.

The skin barrier is mainly in the stratum corneum (SC), and the uppermost epidermal layer is composed of corneocytes and SC intercellular lipid lamellae. SC lipids are mainly composed of ceramides, cholesterol and free fatty acids. Among them, ceramide is the most important lipid because it is present in more than 50%.

The present inventors analyzed the skin tissue of a diabetic animal model and found that the expression and activity of ceramidase were decreased and the levels of total free fatty acids and sphingosine were decreased. Completed.

1. Korean Patent No. 10-1570143

It is an object of the present invention to provide a method for the treatment and prophylaxis of at least one selected from the group consisting of ceramidase activity, ceramidase expression level, fatty acid concentration and sphingosine concentration in each skin tissue isolated from a normal group and a subject And comparing the measured results of the normal group and the subject to determine whether or not the diabetic skin disease is caused. The present invention also provides a method for providing information for diagnosing or treating a diabetic skin disease.

Another object of the present invention is to provide a pharmaceutical composition comprising ceramidase activity, ceramidase expression level, fatty acid concentration and sphingosine concentration in each skin tissue isolated from a subject to which a drug candidate substance is not administered and a drug candidate substance, And determining the effectiveness of the drug candidate substance by comparing the measurement result of the drug candidate substance-administered subject with the measurement result of the drug candidate substance-administered subject, And to provide a screening method for a composition for treating a diabetic skin disease.

In one aspect of the present invention, there is provided a pharmaceutical composition comprising at least one compound selected from the group consisting of ceramidase activity, ceramidase expression level, fatty acid concentration and sphingosine concentration in each skin tissue isolated from a normal group and a subject, And comparing the measurement results of the normal group and the subject to determine whether or not the diabetic skin disease is present. The present invention also provides a method of providing information for diagnosing or treating a diabetic skin disease.

As used herein, the term "subject" includes animals, such as primates, rodents, canines, felines, horses, sheep and pigs, and the like, preferably human.

As used herein, the term " normal group " means an individual not having diabetes of the same species, same sex, and similar age group as the subject.

As used herein, the term " diabetic skin disease " refers to a complication of diabetes mellitus, which refers to a skin disease caused by long-term maintenance of high glucose levels in the blood. Specifically, there is provided a method for treating diabetic rheumatoid arthritis, which comprises scleredema diabeticorum, vitiligo, acanthosis nigricans, necrobiosis lipoidica diabeticorum, diabetic blisters and disseminated granuloma annulare ), But is not limited thereto.

According to one embodiment of the present invention, the determination of whether or not diabetic skin disease is caused by the information providing method may be determined by measuring the activity of ceramidase activity, ceramidase expression level, fatty acid concentration and sphingosine concentration measured in skin tissue isolated from normal group If the measurement result of the skin tissue isolated from the subject is further reduced based on at least one result selected from the group consisting of the subjects, it can be determined that the subject has diabetic skin disease.

According to one embodiment of the present invention, the ceramidase to be measured may be an acidic ceramidase or an alkaline ceramidase. Ceramidase is an enzyme that hydrolyzes ceramide to sphingosine and fatty acids. It is an acid ceramidase (N-acylsphingosine amidohydrolase, ASAH1), neutral ceramidase (ASAH2, ASAH2B and ASAH2C) And alkaline ceramidase 1 to 3 (ACER 1 to 3).

Methods for measuring the activity and expression changes of ceramidase can be performed by methods known in the art, for example, enzyme-substrate reaction using isotopes, chemical luminescence immunoassay, Western blot, etc. have.

According to an embodiment of the present invention, the sphingosine to be measured may be sphingosine having 16 or 18 carbon atoms, and the sphingosine concentration may be measured by a method known in the art, for example, LC-MS / MS (liquid chromatography-mass spectrometry ), Multiple reaction monitoring (MRM mode), and the like.

Another aspect of the present invention is a pharmaceutical composition comprising a combination of ceramidase activity, ceramidase expression level, fatty acid concentration, and sphingosine concentration in each skin tissue isolated from a subject to which a drug candidate substance has not been administered and a subject to which a drug candidate substance has been administered And determining the effectiveness of the drug candidate substance by comparing the measurement result of the drug candidate substance-administered subject with the measurement result of the drug candidate substance-administered subject, There is provided a screening method for a composition for treating a diabetic skin disease.

In the screening method, the drug candidates are administered to a subject, and after a predetermined time has elapsed, the skin tissue is collected from the subject to compare the ceramidase activity, ceramidase expression level, fatty acid concentration, and / or sphingosine concentration before and after administration , It is possible to confirm whether the drug candidate substance is useful for the treatment of diabetic skin disease and the degree of the effect.

The drug candidates may be administered in a manner customary in the art depending on their characteristics, and oral administration is generally used, but injections and suppositories may be used.

According to an embodiment of the present invention, the step of judging the effectiveness of the drug candidates may include measuring the activity of ceramidase activity, ceramidase expression level, fatty acid concentration, Wherein the drug candidates are effective as a composition for the treatment of diabetic skin diseases when the result of measurement of the skin tissue isolated from a subject administered with the drug candidate substance is further increased based on at least one result selected from the group consisting of It can be judged or decided.

According to one embodiment of the present invention, there is provided a method for providing information for diagnosis or treatment of diabetic skin disease by measuring the expression and activity of ceramidase in skin tissue isolated from a subject, confirming fatty acid and sphingosine concentration, And can be utilized as a marker for a screening method of a composition for treating a skin disease.

FIG. 1 is a graph showing the results of measuring the expression level and activity level of ceramidase in the skin of a diabetic animal model.
FIG. 2 is a graph showing the results of measuring the concentration of total free fatty acids and sphingosine in the skin of a diabetic animal model. FIG.

Hereinafter, one or more embodiments will be described in more detail by way of examples. However, these embodiments are intended to illustrate one or more embodiments, and the scope of the present invention is not limited to these embodiments.

1. Materials and Experimental Methods

1-1. Compounds and reagents

Methanol (LC-MS grade) was purchased from Merck (Darmstadt, Germany) and ammonium acetate, chloroform and dichloromethane were purchased from Sigma-Aldrich (St. Louis, Mo., USA).

Anti-ASAH1 (PA5-20574), anti-ASM (PA1-12756), anti-ASAH3L (PA5-39016) and anti-DEGS2 (PA5-24082) were purchased from Thermo Fischer scientific , anti-ACER1 (GTX48935) were purchased from GeneTex International Corporation (USA). Goat anti-rabbit IgG (sc-2004) and goat anti-mouse igG (sc-2005) were purchased from Santa Cruz biotechnology (CA, USA).

The standard ceramide d18: 1/12: 0 (860512p) used for lipid profiling was purchased from Avanti Polar Lipids (Alabaster, AL) and Matreya (Pleasant Gap, PA).

1-2. Type 2 diabetic animal model

(C57BLKS / J- db / db ) (hereinafter referred to as the experimental group) and the control group (C57BL / 6J- + / + mice) And cultivated in standard raising conditions (temperature 22 ± 0.5 ° C, relative humidity 60 ± 5% and 12 hour light period). Weight, fasting blood glucose and glycated hemoglobin (glycated hemoglobin; HbA1c) were measured at 8 and 14 weeks of age using methods known in the art to identify metabolic changes. Diabetes mellitus was diagnosed when the peak blood glucose level was over 300 ㎎ / dl. Transepidermal water loss (TEWL), stratum corneum (SC) hydration and skin pH were measured by Tewameter (TM 300 ^® , Courage & Khazaka, Cologne, Germany), Corneometer (CM 825 ^® , Courage & Khazaka) and pH meter (WTW, Weilheim, Germany). Basic TWEL, keratin moisture content, skin surface pH, SC integrity and barrier recovery rate were measured in the dorsal skin of the mice.

1-3. Western blotting

The skin was separated from the control and experimental groups, and the skin tissues were immersed in 10 mM Na 2 -EDTA solution (37 ° C) for 1 hour. When the dermis and epidermis are separated, the epidermis is rinsed with PBS and used for immunoblotting and lipid profiling.

The isolated mouse epidermis was washed with PBS (1 mM EDTA, 0.25 M sucrose and protease inhibitors (Complete ^TM , Roche Biochemical) at 30 Hz (1 / s) for 30 seconds using a tissue crusher (Tissue Lyser, Lt; / RTI > The homogenized skin samples were centrifuged at 4 and 5,000 rpm for 15 minutes, and only the protein-containing supernatant was taken and quantified with BCA Protein Assay Kit (Thermo Scientific Pierce, IL, USA).

After 10 μg of epidermis lysate was electrophoresed on a 10% SDS-polyacrylamide gel, the proteins were transferred to a PVDF membrane (polyvinylidene fluoride; Millipore, Germany). Then, the PVDF membrane was washed with TBST buffer (10 mM Tris-HCl pH 7.6, 150 mM NaCl and 0.1% Tween-20) containing tween-20 and washed with 5% And blocked for 1 hour at room temperature. After 1 hour, each primary antibody was diluted (1: 1,000) and reacted with PVDF membrane at 4 ° C for 12 hours. After the reaction, the membrane was washed with TBST buffer. Next, HRP (horseradish peroxidase) -binding secondary antibody (goat anti-rabbit or goat anti-mouse IgG) was diluted (1: 5,000) and further reacted with PVDF membrane at 4 ° C for 12 hours. Protein band signals were confirmed by enhanced chemiluminescence detection system (Thermo Scientific, USA) according to the manufacturer's protocol.

1-4. Ceramidase  Assay of ceramidase  activity)

(1.8 mmol) of palmitoylsphingosine (1 μmol) and [ ¹⁴ C] palmitoyl sphingosine (1.8 nmol) were dissolved in a mixture of sodium cholate (16 mg / ml) and Triton X-100 Mg / ml) and Tween 80 (4 mg / ml) in PBS). Then, the reaction was started by adding an alkaline ceramidase (pH 8) or a Tris-HCl buffer (acid ceramidase; pH 4) and adding a skin lysate containing 50 μg protein (final volume 100 μl). After the reaction was carried out at 37 ° C for 8 hours, 200 μl of chloroform / methanol (1: 2, v / v) mixed solution was added to terminate the reaction. The reaction solution was dried in the presence of nitrogen gas and 50 μl of a mixed solution of chloroform / methanol (2: 1, v / v) was added. The lipid mixture was purified using a high performance thin layer chromatography (HPTLC) (silica gel 60, Merck, Millipore, Germany) and a development system (Hexane: Ethyl ether: Acetic acid, 60: 40: . The palmitate fraction (Rf 0.38) was discarded from the plate and radioactivity was measured with a liquid scintillation counter.

1-5. Sphingosine  analysis

Sphingosine was isolated using the modified Masson method. First, 2 mg of the lyophilized skin sample was homogenized in 75% ice-cold methanol with a tissue crusher (30 Hz (1 / s), 4 times), MTBE (Methyl Tertiary-Butyl Ether) And vortexed at room temperature for 1 hour. Water was then added for phase sepration, left for 10 minutes, and centrifuged at 14,000 rpm and 4 ° C for 15 minutes. 220 μl of the upper phase and 110 μl of the lower phase were mixed and evaporated and then re-dissolved in methanol and used for LC-MS / MS analysis.

LC-MS / MS analysis was performed in a negative mode with a Nexera2 LC system (Shimadzu Corporation, Kyoto, Japan) connected with a triple quadrupole mass spectrometer (LC-MS 8040; Shimadzu). 0.1% formic acid in H2O) and B (0.1% formic acid in MeOH) were used as the mobile phase: Kinetex C18 cloumn (50 x 2.1 mm, 2.6 ㎛, Phenomenex, CA, USA) The mobile phase gradient condition is A 30%, B 70% for 0 to 1 minute; A 20%, B 80% for 2 to 2.5 minutes; A 1%, B 99% for 3 to 4 minutes; And 30% for A and 70% for 4 to 9 minutes. We used multiple reaction monitoring (MRM mode).

1-6. Quantification of total free fatty acids

The upper and lower skin samples from 1-3 were mixed and dried, and then 1 ml of chloroform, 100 μl of water and 1 ml of Copper reagent (1 M Triethanolamine: 1 N acetic acid: 6.45% Copper (II) nitrate trihydrate = 9: 1: 10, v / v / v). After thorough mixing for 2 minutes and centrifugation for 10 minutes, the supernatant was removed. To the remaining lower layer, 100 μl of diethyldithiocarbamate solution (0.1% sodium diethyldithiocarbamate in 2-butanol, w / v) was added to induce color development. The degree of color development was confirmed at 440 nm using a UV-visible spectrophotometer (Shimadzu Corporation, Kyoto, Japan), and nonadecenoic acid was used as a standard sample.

2. Experimental results

2-1. Comparison of metabolic activity and skin condition of control and experimental groups

Compared with the control group, the experimental group ( db / db mouse) showed fast body weight gain, high body weight gain, fasting blood glucose level and glycated hemoglobin level, which was similar to that of diabetic patients. In addition, the experimental group showed delayed recovery of skin barrier and increased skin surface pH. However, the degree of transdermal water loss and keratin - containing ability were similar to those of the control group.

2-2. Identification of ceramidase expression and activity changes in skin of diabetic animal models

Ceramides are one of the major constituents of keratin and various enzymes are known to be involved in the synthesis and degradation of ceramides.

The expression level of ceramidase was confirmed by western blot. As a result, the expression of alkaline ceramidase 1 (ACER1) and acid ceramidase (N-acylsphingosine amidohydrolase 1, ASAH1) was significantly decreased in the experimental group compared to the control group there was. On the other hand, the expression of acid sphingomyelin phosphodiesterase 1, acid sphingomyelinase (ASM), and alkaline ceramidase 2 (ACER2, ASAH3L) was not significantly different between the two groups.

FIG. 1A shows the change in expression of ceramidase. As a result, the expression of ACER1 and ASAH1 was significantly decreased in the experimental group as compared with the control group.

In addition, the activity of ceramidase was measured, and it was confirmed that the activity of acidic ceramidase and alkaline ceramidase was significantly reduced in the experimental group as compared with the control group.

FIG. 1B is a graph showing the activity of ceramidase, showing that the activity of acidic and alkaline ceramidase was reduced to about 50% in the experimental group as compared with the control group.

2-3. Identify total fats and sphingosine concentrations in skin of diabetic animal models

From the above results, it was confirmed that the expression and activity of ceramidase decreased in the experimental group. Therefore, the concentration of total free fatty acid and sphingosine was confirmed.

As a result, it was confirmed that the total free fatty acid concentration was significantly decreased in the experimental group as compared with the control group. It was also found that the sphingosine concentration of carbon atoms 18 and 20 decreased significantly in the case of sphingosine.

From these results, it can be seen that ceramide expression and activity are decreased in the skin of the diabetic animal model, and the concentration of free fatty acid and sphingosine is decreased. Therefore, the expression or activity change of ceramide, the concentration of free fatty acid and the concentration of sphingosine can be confirmed to be used for diagnosing and predicting diabetic skin disease, or for screening for a therapeutic agent for diabetic skin disease.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (6)

Measuring at least one selected from the group consisting of ceramidase activity, ceramidase expression level, fatty acid concentration, and sphingosine concentration in each skin tissue isolated from the normal group and the subject; and
Comparing the measurement results of the normal group and the subject to determine whether or not the diabetic skin disease is present, the method comprising:
The step of determining whether or not the diabetic skin disease is selected is selected from the group consisting of ceramidase activity, ceramidase expression level, fatty acid concentration, and sphingosine concentration measured in skin tissue isolated from a normal group When the result of measurement in the skin tissue separated from the target based on one or more results is further decreased, it is determined that the subject has diabetic skin disease,
The ceramidase is an acid ceramidase (ASAH1) or an alkaline ceramidase 1 (ACER1)
Wherein the sphingosine has 18 or 20 carbon atoms.
delete delete delete The level of expression of ceramidase, the level of expression of ceramidase, the concentration of fatty acid and the concentration of sphingosine in each of the skin tissues separated from the subject to which the drug candidate substance is not administered and the subject to which the drug candidate substance is administered, Measuring steps: and
And comparing the measurement result of the subject with the drug candidate material to the measurement result of the subject to which the drug candidate material has not been administered and determining the effectiveness of the drug candidate substance, the screening method for a composition for treating diabetic skin disease ,
The step of determining the efficacy of the drug candidate substance may be selected from the group consisting of ceramidase activity, ceramidase expression level, fatty acid concentration, and sphingosine concentration measured in skin tissue isolated from a subject to which the drug candidate substance is not administered The candidate drug is judged to be effective as a composition for the treatment of diabetic skin disease, and the drug candidates are judged to be effective as a composition for the treatment of diabetic skin disease,
The ceramidase is an acid ceramidase (ASAH1) or an alkaline ceramidase 1 (ACER1)
Wherein the sphingosine has 18 or 20 carbon atoms.

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