CN116804675A - Biomarkers for atopic dermatitis and uses thereof - Google Patents

Biomarkers for atopic dermatitis and uses thereof Download PDF

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CN116804675A
CN116804675A CN202310643066.1A CN202310643066A CN116804675A CN 116804675 A CN116804675 A CN 116804675A CN 202310643066 A CN202310643066 A CN 202310643066A CN 116804675 A CN116804675 A CN 116804675A
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lta4h
atopic dermatitis
protein
assessment
product
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陈达灿
于晓波
莫秀梅
张凯
刘俊峰
晏烽根
王赛娅
叶思祺
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BEIJING PROTEOME RESEARCH CENTER
Guangdong Hospital of Traditional Chinese Medicine
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BEIJING PROTEOME RESEARCH CENTER
Guangdong Hospital of Traditional Chinese Medicine
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Abstract

The invention discloses a biomarker for atopic dermatitis and application thereof. The inventor of the invention adopts the liquid chromatography-mass spectrometry and data independent acquisition technology to detect, discovers that the expression of leukotriene A-4 hydrolase (LTA 4H) in the blood of patients with atopic dermatitis is obviously reduced, can be well used as a marker for the diagnosis and the reaction severity of the atopic dermatitis, can provide objective laboratory indexes for the diagnosis of diseases, is beneficial to realizing accurate treatment, and has important significance for improving clinical curative effect.

Description

Biomarkers for atopic dermatitis and uses thereof
Technical Field
The invention relates to the technical field of inspection medicine, in particular to a biomarker of atopic dermatitis, LTA4H and application thereof.
Background
Atopic dermatitis (Atopic Dermatitis, AD) is a common chronic inflammatory skin disease, has the characteristics of relapse, intractable and refractory, the global prevalence rate increases year by year in the past 30 years, the prevalence rate of children in developed countries is 10-20%, the prevalence rate of adults is 2-10%, and the prevalence rate of children in 1-7 years in China is 12.94%. Psychological problems often occur to patients due to long-term repeated illness, severe itching and sleep disturbance, severe patients are accompanied with anxiety, depression and even suicidal tendency, and the life quality of the patients and the whole family is seriously affected. At present, the international belief is that no radical cure method exists, the hormone is still the current first-line therapeutic drug, but the application of the hormone, immunosuppressant, biological agent and the like can temporarily relieve the illness state, but the long-term use has obvious toxic and side effects, and causes heavy economic burden to patients and families, the annual conservation estimated cost of American AD patients in 2015 is 52.97 hundred million dollars, and the international survey shows that the atopic dermatitis is first in disease burden in non-fatal skin diseases.
The cause and mechanism of atopic dermatitis are not completely known, and it is considered that the cause is related to various factors such as heredity, environment, abnormal skin barrier function, and immune imbalance, and the cause is a result of interaction between intrinsic factors and extrinsic factors, and is often multifaceted. Atopic dermatitis has strong heterogeneity, lacks laboratory biological indexes in diagnosis and objective indexes of disease evaluation, does not have a treatment scheme suitable for all patients in treatment, does not have biological preparations effective for all patients, and AD patients need to formulate different treatment schemes according to individual conditions to obtain optimal curative effects, so that the atopic dermatitis already starts to enter a 'layered medical' period, and the core idea is 'individual treatment based on biomarkers'.
Leukotriene a-4 hydrolase (LTA 4H), a bifunctional zinc metalloenzyme comprising Epoxide Hydrolase (EH) and aminopeptidase activities. As epoxide hydrolase catalyzes the conversion of LTA4 to leukotriene B4 (LTB 4), a pro-inflammatory mediator, and also has aminopeptidase activity with high affinity for the N-terminal arginine of various synthetic tripeptides. In addition to its Pro-inflammatory EH activity, inflammation can be counteracted by its aminopeptidase activity, which is inactivated by cleavage of the other neutrophil attractant tripeptide Pro-Gly-Pro (PGP), a biologically active collagen fragment-9 (MMP 9) and prolyl peptidase (PREPL) produced by the action of matrix metalloproteinases. Ma TT, cao MD, yu RL, shi HY, yan WJ, liu JG, pan C, sun J, wei QY, wang DY, wei JF, wang XY, yin JS. Leukotriene A4 Hydrolase Is a Candidate Predictive Biomarker for Successful Allergen immunology. Front immunol.2020Nov 24;11:559746.Doi:10.3389/fimmu.2020.559746.PMID:33329520; PMCID PMC7732448 serum LTA4H is disclosed as a candidate predictive biomarker for Allergen Immunotherapy (AIT). LTA4H has not been reported as a biomarker for atopic dermatitis.
Disclosure of Invention
In a first aspect of the invention there is provided the use of LTA4H as a marker in the manufacture of a product for diagnosis, prevention, treatment, assessment of severity of illness, assessment of efficacy, prognosis, drug screening of atopic dermatitis.
In one embodiment of the invention, the use is the use of LTA4H as a marker in the preparation of a diagnostic product for atopic dermatitis.
In another embodiment of the invention, the use is the use of LTA4H as a marker in the manufacture of a product for the severity assessment of atopic dermatitis.
In particular, the indicators of the severity assessment of atopic dermatitis include the atopic dermatitis integral index (scoring atopic dermatitis index, SCORAD), eczema area and severity index (eczema area and severity index, EASI).
Specifically, the above marker is the LTA4H gene or LTA4H protein, in particular LTA4H protein.
In particular, LTA4H expression levels were significantly reduced in atopic dermatitis patients relative to healthy controls.
Specifically, the above-mentioned product may be a reagent, a kit, a test paper, a gene chip, or the like, which may contain a substance (e.g., an antibody or a fragment thereof) capable of binding to LTA4H protein; instrument platforms such as high throughput sequencing platforms, proteomic analysis products; the proteomic analysis product may comprise a measurement module (for measuring the content of LTA4H protein in the sample to be tested) and an analysis module (for analyzing the difference in the content of LTA4H protein in the sample to be tested and the reference sample).
In particular, the measurement module may be based on mass spectrometry, such as DIA-MS, wherein DIA acquisition ranges from 300 to 1200 mass to charge ratios (m/z). In one embodiment of the invention, the DIA-MS parameters are set as follows: the spray voltage of the ion source is set to be 2.1kV; the scanning range of the first stage is 300-1400m/z, the resolution is 60K (m/z 200), and the AGC target is 3e6,Maximum IT:80ms; the resolution of the second level is 15K (m/z 200), AGC target is 5e4, maximum IT is 40ms, resolution energy is 30, charge state is 2-6, and dynamic exclusion time is 30s.
Specifically, when the product is an instrument platform, the sample to be detected is subjected to pretreatment before detection, and the pretreatment may include: washing a sample to be detected by using a buffer solution (such as Ammonium Bicarbonate (ABC) solution), performing enzymolysis, washing, centrifugally separating and drying; more specifically, the pretreatment may include: washing a sample to be tested with an ABC solution, carrying out enzymolysis at 37 ℃ by trypsin, washing by pure water, carrying out centrifugal separation, drying, and then dissolving in an aqueous solution containing 0.2% acetonitrile.
Specifically, the pretreatment further comprises: the solution obtained after the treatment was separated by an analytical column to obtain a quantitative peptide for DIA-MS analysis.
Specifically, the mobile phase separated by the analytical column comprises a mobile phase aqueous phase (A) and a mobile phase organic phase (B); more specifically, mobile phase A may be 0.05-0.2% (v/v) aqueous formic acid, in particular 0.1% aqueous formic acid; more specifically, mobile phase B may contain 0.1% formic acid, 80% acetonitrile, and in one embodiment of the invention, mobile phase B has a composition of 0.1% formic acid, 80% acetonitrile, 20% mass spectrometry water.
Specifically, the gradient of analytical column separation is: 0min-15min, the mobile phase B linearly rises from 7% to 15%; 15min-85min, the mobile phase B rises linearly from 15% to 30%; from 85min to 110min, mobile phase B rose linearly from 30% to 50%, then mobile phase B rose to 95% and remained for 120min in 2 min.
Specifically, a test sample of the above-mentioned product may use, for example, a tissue sample or fluid obtained from a biopsy subject, for example, tissue, blood, plasma, serum, lymph, urine, serosal cavity fluid, spinal fluid, synovial fluid, aqueous humor, tears, saliva, or the like or fractions thereof or treated materials; in particular, blood (in particular peripheral blood) or a fraction thereof (e.g. serum), preferably serum.
Specifically, LTA4H is used as a unique marker or in combination with other markers as a companion diagnostic marker.
In a second aspect of the invention, there is provided the use of an agent for detecting LTA4H in the manufacture of a product for diagnosis, prevention, treatment, severity assessment, efficacy assessment, prognosis, drug screening of atopic dermatitis.
Specifically, the above-mentioned detection reagent includes a reagent for detecting the expression level of the LTA4H gene or LTA4H protein.
In particular, the reagent for detecting the LTA4H gene may comprise a nucleic acid capable of binding to the LTA4H gene.
Specifically, reagents for detecting LTA4H gene may perform their function based on known methods using nucleic acid molecules: for example, polymerase Chain Reaction (PCR), southern blot hybridization, northern blot hybridization, dot hybridization, fluorescence In Situ Hybridization (FISH), DNA microarray, high throughput sequencing platform, etc., may be employed, in particular, PCR methods such as real-time fluorescent quantitative PCR method. The use of the reagent allows qualitative, quantitative, or semi-quantitative analysis.
Specifically, the above-mentioned nucleic acid can be obtained by chemical synthesis, or by preparing a gene containing a desired nucleic acid from a biological material and then amplifying it using a primer designed for amplifying the desired nucleic acid.
In particular, the reagent for detecting LTA4H protein may comprise a substance (e.g. an antibody or fragment thereof) capable of binding to LTA4H protein.
Specifically, reagents for detecting LTA4H proteins may perform their function based on known methods of using proteins: for example, ELISA, radioimmunoassay, immunohistochemistry, western blot, proteomics (e.g., antibody chips, mass spectrometry (e.g., data independent acquisition (Data Independent Acquision, DIA) mass spectrometry), etc. can be employed.
In particular, a sample for LTA4H detection may use, for example, a tissue sample or fluid obtained from a biopsy subject, e.g., tissue, blood, plasma, serum, lymph, urine, serosal cavity fluid, spinal fluid, synovial fluid, aqueous humor, tears, saliva, etc., or fractions or treated materials thereof; in particular, blood (in particular peripheral blood) or a fraction thereof (e.g. serum), preferably serum.
Specifically, the above-mentioned products may be reagents, kits, test papers, gene chips, high throughput sequencing platforms, proteomic analysis products (e.g., antibody chips, DIA-MS), etc.
In a third aspect of the invention there is provided the use of an agent that affects LTA4H expression level or activity in the manufacture of a product for the prophylaxis, treatment, efficacy assessment of atopic dermatitis.
Specifically, the effect is to increase LTA4H expression levels or protein activity, in particular LTA4H expression levels or protein activity in serum.
Specifically, the agent may be a gene expression inducer (which induces LTA4H gene expression, increases its expression level) or a protein agonist (which enhances LTA4H protein molecular activity).
In particular, the above-mentioned products are pharmaceutical preparations for the prevention or treatment of atopic dermatitis.
Specifically, the above pharmaceutical formulations may further comprise one or more pharmaceutically acceptable excipients, for example, fillers, binders, humectants, disintegrants, lubricants, flavoring agents, sweeteners, antioxidants, preservatives, etc.
Specifically, the above pharmaceutical preparation may be an oral dosage form (e.g., tablet, pill, powder, granule, capsule, etc.), an injection (e.g., subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection), a skin external dosage form (e.g., ointment, cream, lotion, film-coated preparation, etc.).
Specifically, the above-described various dosage forms may be prepared according to conventional production methods in the pharmaceutical field. For example by mixing the active ingredient with one or more pharmaceutically acceptable excipients and then formulating it into the desired dosage form.
In a fourth aspect of the invention, there is provided a product for diagnosis, severity of disease assessment, efficacy assessment, prognosis, drug screening of atopic dermatitis comprising an agent for detecting LTA 4H.
Specifically, the above-mentioned detection reagent includes a reagent for detecting the expression level of the LTA4H gene or LTA4H protein.
In particular, the reagent for detecting the LTA4H gene may comprise a nucleic acid capable of binding to the LTA4H gene.
Specifically, reagents for detecting LTA4H gene may perform their function based on known methods using nucleic acid molecules: for example, polymerase Chain Reaction (PCR), southern blot hybridization, northern blot hybridization, dot hybridization, fluorescence In Situ Hybridization (FISH), DNA microarray, high throughput sequencing platform, etc., may be employed, in particular, PCR methods such as real-time fluorescent quantitative PCR method. The use of the reagent allows qualitative, quantitative, or semi-quantitative analysis.
Specifically, the above-mentioned nucleic acid can be obtained by chemical synthesis, or by preparing a gene containing a desired nucleic acid from a biological material and then amplifying it using a primer designed for amplifying the desired nucleic acid.
In particular, the reagent for detecting LTA4H protein may comprise a substance (e.g. an antibody or fragment thereof) capable of binding to LTA4H protein.
Specifically, reagents for detecting LTA4H proteins may perform their function based on known methods of using proteins: for example, ELISA, radioimmunoassay, immunohistochemistry, western blot, proteomics (e.g., antibody chips, mass spectrometry (e.g., data independent acquisition (Data Independent Acquision, DIA) mass spectrometry), etc. can be employed.
Specifically, the above-mentioned products may be reagents, kits, test papers, gene chips, high throughput sequencing platforms, proteomic analysis products (e.g., antibody chips, DIA-MS), etc.
In a fifth aspect of the invention, there is provided a medicament for the prophylaxis and/or treatment of atopic dermatitis comprising an agent which affects the expression level or activity of LTA 4H.
Specifically, the effect is to increase LTA4H expression levels or protein activity.
Specifically, the agent may be a gene expression inducer (which induces LTA4H gene expression, increases its expression level) or a protein agonist (which enhances LTA4H protein molecular activity).
In a sixth aspect of the invention, there is provided a diagnostic, prognostic method for atopic dermatitis comprising the step of detecting LTA 4H.
Specifically, the method may include the steps of:
(1) Obtaining a subject sample;
(2) Detecting the expression level of a gene or protein of LTA4H in a sample of the subject;
(3) The measured expression level is correlated with the presence or absence of a disease or risk of a disease in the subject.
In particular, if the expression level of the LTA4H gene or protein is decreased as compared to a normal control, the subject may be diagnosed with or at high risk of developing atopic dermatitis, or the subject may be determined to have a poor prognosis. It should be noted that, the risk, severity and prognosis of a specific disease also require comprehensive assessment by a clinician in combination with other detection indicators of the subject.
Specifically, the subject is a human.
In particular, the sample may use, for example, a tissue sample or fluid obtained from a biopsy subject, e.g., tissue, blood, plasma, serum, lymph, urine, serosal cavity fluid, spinal fluid, synovial fluid, aqueous humor, tears, saliva, etc., or fractions or treated materials thereof; in particular, blood (in particular peripheral blood) or a fraction thereof (e.g. serum), preferably serum.
Specifically, step (2) is to detect the expression level of LTA4H protein in a sample of the subject.
In particular, the detection described in step (2) may be based on mass spectrometry, such as DIA-MS, wherein DIA acquisition ranges from 300 to 1200 mass to charge ratios (m/z). In one embodiment of the invention, the DIA-MS parameters are set as follows: the spray voltage of the ion source is set to be 2.1kV; the scanning range of the first stage is 300-1400m/z, the resolution is 60K (m/z 200), and the AGC target is 3e6,Maximum IT:80ms; the resolution of the second level is 15K (m/z 200), AGC target is 5e4, maximum IT is 40ms, resolution energy is 30, charge state is 2-6, and dynamic exclusion time is 30s.
Specifically, when the product is an instrument platform, the sample to be detected is subjected to pretreatment before detection, and the pretreatment may include: washing a sample to be detected by using a buffer solution (such as Ammonium Bicarbonate (ABC) solution), performing enzymolysis, washing, centrifugally separating and drying; more specifically, the pretreatment may include: washing a sample to be tested with an ABC solution, carrying out enzymolysis at 37 ℃ by trypsin, washing by pure water, carrying out centrifugal separation, drying, and then dissolving in an aqueous solution containing 0.2% acetonitrile.
Specifically, the pretreatment further comprises: the solution obtained after the treatment was separated by an analytical column to obtain a quantitative peptide for DIA-MS analysis.
Specifically, the mobile phase separated by the analytical column comprises a mobile phase aqueous phase (A) and a mobile phase organic phase (B); more specifically, mobile phase A may be 0.05-0.2% (v/v) aqueous formic acid, in particular 0.1% aqueous formic acid; more specifically, mobile phase B may contain 0.1% formic acid, 80% acetonitrile, and in one embodiment of the invention, mobile phase B has a composition of 0.1% formic acid, 80% acetonitrile, 20% mass spectrometry water.
Specifically, the gradient of analytical column separation is: 0min-15min, the mobile phase B linearly rises from 7% to 15%; 15min-85min, the mobile phase B rises linearly from 15% to 30%; from 85min to 110min, mobile phase B rose linearly from 30% to 50%, then mobile phase B rose to 95% and remained for 120min in 2 min.
In a seventh aspect of the invention, there is provided a method of assessing the severity of an atopic dermatitis comprising the step of detecting LTA 4H.
Specifically, the method may include the steps of:
(1) Obtaining a subject sample;
(2) Detecting the expression level of a gene or protein of LTA4H in a sample of the subject;
(3) The measured expression level is correlated with an index for evaluating the severity of the atopic dermatitis.
Specifically, the evaluation index includes an atopic dermatitis integral index (scoring atopic dermatitis index, SCORAD), eczema area and severity index (eczema area and severity index, EASI).
In an eighth aspect of the invention there is provided a method of prevention and/or treatment of atopic dermatitis comprising administering to a subject in need thereof an agent affecting LTA4H expression level or activity.
Specifically, the effect is to increase LTA4H expression levels or protein activity.
Specifically, the agent may be a gene expression inducer (which induces LTA4H gene expression, increases its expression level) or a protein agonist (which enhances LTA4H protein molecular activity).
The latest research considers that atopic dermatitis is a systematic disease, the proteomics of blood shows systematic inflammation signals, and the proteomics research can be used as a potential biomarker to provide a sensitive choice for the diagnosis of the atopic dermatitis, the occurrence of the disease, the severity of the disease, the prediction of the curative effect of the medicine, the evaluation of the curative effect and the like. The inventor of the invention adopts the liquid chromatography-mass spectrometry and data independent acquisition technology to detect, and finds that the expression of LTA4H in the blood of atopic dermatitis patients is obviously reduced, the P value of LTA4H and EASI is 0.0299, and the correlation coefficient R2 is 0.8354; LTA4H and SCORAD have a P value of 0.0075 and a correlation coefficient R≡2 of 0.9330. The method shows that LTA4H in serum of patients with atopic dermatitis can be well used as a marker for diagnosing the atopic dermatitis and diagnosing the occurrence and the development of the atopic dermatitis by detecting the expression of LTA4H in the blood of the patients with the atopic dermatitis, provides objective laboratory indexes for diagnosing diseases, is beneficial to realizing accurate treatment, and has important significance for improving clinical curative effects.
Drawings
Fig. 1 shows the PCA of serum proteins of the atopic dermatitis, urticaria, and healthy control group.
FIG. 2 shows a differential protein cluster heat map of serum samples from atopic dermatitis, urticaria, and healthy control groups.
FIG. 3 shows the results of a correlation analysis of LTA4H with the severity index EASI of atopic dermatitis.
FIG. 4 shows the results of a correlation analysis of LTA4H with the atopic dermatitis severity index SCORAD.
Detailed Description
Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention relates.
In the present invention, "expression level" refers to a measurable amount of LTA4H gene product in a sample, wherein the gene product may be a transcription product or a translation product. Thus, expression levels are related to nucleic acid gene products (e.g., mRNA or cDNA) or polypeptide gene products (e.g., LTA4H protein).
In the present invention, "LTA4H gene" includes the LTA4H gene itself as well as polynucleotides of any functional equivalent of LTA4H gene, for example, DNA sequences that have 70% or more (e.g., 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more) homology with the DNA sequences of LTA4H genes in current international public nucleic acid sequence databases genebanks, and encode the same functional proteins.
In the present invention, "diagnosing atopic dermatitis" includes both determining whether a subject has already suffered from atopic dermatitis and determining whether a subject is at risk of suffering from atopic dermatitis.
Various publications, patents, and published patent specifications cited herein are incorporated by reference in their entirety.
The technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
1. Sample collection
Atopic dermatitis, urticaria group blood samples were from patients with atopic dermatitis and urticaria at the clinic of Dade in the middle of Guangdong province, and healthy control group blood samples were from volunteers recruited. The general case for three groups of patients is as follows:
atopic dermatitis patients 5, men 2, women 3, average 10.8 years old, minimum 7 years old, maximum 12 years old;
5 patients with urticaria, 3 men and 2 women, average 10.2 years old, minimum 7 years old and maximum 14 years old;
healthy volunteers 5, men 2, women 3, average 9.0 years old, minimum 7 years old, maximum 12 years old.
2. Sample preparation
Preparing a sample, taking out the sample from the temperature of minus 80 ℃ to melt, centrifuging for 3min at the temperature of 4 ℃ and 10000r, taking the supernatant to a 1.5ml EP tube, and marking for later use. The components were transferred to an ultrafiltration tube, marked on the outside of the upper portion of the ultrafiltration tube, and the ultrafiltration tube was placed in a collection tube, and centrifuged at 12000g for 15min at room temperature. 200ul of 50mM Ammonium Bicarbonate (ABC) solution was added and the mixture was centrifuged at 12000g for 15min at room temperature and repeated 3 times. The collection tube was replaced, trypsin (0.02 ug/ul, 50mM ABC) was added to 200ul, and the mixture was digested in a 37℃water bath for 13-16h.12000g was centrifuged at room temperature for 15min. 200ul of pure water was added to each, and the mixture was centrifuged at 12000g for 15min at room temperature. The filtrate in the collecting tube was transferred to a new 1.5mL EP tube, and the EP tube was put into a centrifugal concentration system in a state of being opened, dried by hot blowing, and dissolved in 30ul (2% acetonitrile+98% pure water) of A solution. After sample treatment (determination of protein concentration), the samples were stored at-20 ℃.
3. Chromatographic mass spectrometry method setup
The polypeptide sample obtained in the 2 nd step was dissolved in 30ul of solution A (0.1% formic acid in water), 5. Mu.L was sampled, and loaded onto a pre-column on an EASY-nano-LC1200 chromatography system at a flow rate of 4.5ul/min, followed by separation on an analytical column at a flow rate of 600 nl/min. The gradient of the chromatographic separation is: from 0min to 15min, the liquid B (composition: 0.1% formic acid, 80% acetonitrile, 20% water by mass spectrum) rises linearly from 7% to 15%; 15-85 min, wherein the liquid B is linearly increased from 15% to 30%; liquid B rose linearly from 30% to 50% for 85min-110min, then liquid B rose to 95% and remained for 120min in 2 min.
Acquisition of mass spectrometry data using a Thermo Q Exactive HF mass spectrometer (Thermo Scientific), specific parameters were set as follows: the spray voltage of the ion source was set to 2.1kV. The scanning range of the first stage is 300-1400m/z, the resolution is 60K (m/z 200), and the AGC target is 3e6,Maximum IT:80ms; the resolution of the second level is 15K (m/z 200), AGC target is 5e4, maximum IT is 40ms, resolution energy is 30, charge state is 2-6, and dynamic exclusion time is 30s.
4. Statistical analysis
Spectronaut software exports the search data, missing value identification, filtered substitution to NA, and storing the xls file in CSV (comma separated) format. And importing the CSV file into the WUWUYUN platform to check the table data, wherein the table data comprises row names, column names, data types and data contents, changing according to error reporting information, deleting all NA rows, and repeatedly checking until the data format requirement is met.
The missing value data processing, integrally considering, normalizing the data median, pulling to the same scale for comparison, and filling the missing value, wherein the method comprises the following steps: the K-nearest neighbor method, the number of nearest neighbors k=5. The data Log2 is converted, so that the data has good normalization. And obtaining high-quality data, and carrying out subsequent statistical analysis.
And comparing the differences among groups by adopting analysis of variance, screening differential proteins according to p value < 0.05 and fold change > 1.2, and then carrying out principal component analysis, layer clustering analysis and Pearson correlation analysis.
5. Experimental results
(1) Serum LTA4H of atopic dermatitis patients has remarkable separation trend and obvious difference between healthy controls and urticaria and atopic dermatitis
The separation degree of different color points of the PCA result in the figure 1 shows that the separation trend of the main proteomics components between healthy people and nettle rash patients is obvious, so that the difference of serum proteomes between the nettle and healthy control groups is obvious, and no cross exists.
The tendency of separation of the major proteomic component between atopic dermatitis and healthy controls was evident, indicating that the difference in serum proteome between atopic dermatitis and healthy controls was evident.
The trend of separation of the proteomic principal components between the atopic dermatitis and urticaria control group was evident, indicating that the difference in serum proteome between the atopic dermatitis and urticaria groups was evident.
(2) Differential protein cluster analysis of serum samples from atopic dermatitis, healthy control group, urticaria group
As can be seen from fig. 2, the expression trend of both LTA4H protein molecules in atopic dermatitis was significantly reduced relative to the healthy control, nettle group.
(3) Correlation analysis of LTA4H with atopic dermatitis severity indices EASI and SCORAD the inventors studied the relationship between serum LTA4H levels and EASI and SCORAD, the severity assessment index of atopic dermatitis currently used, atopic dermatitis integral index (scoring atopic dermatitis index, SCORAD), eczema area and severity index (eczema area and severity index, EASI). As shown in FIGS. 3 and 4, LTA4H and EASI have P values of 0.0299 and correlation coefficients R2 of 0.8354; LTA4H and SCORAD have a P value of 0.0075 and a correlation coefficient R≡2 of 0.9330. The above results indicate that LTA4H in serum of patients with atopic dermatitis can be well used as a marker for diagnosis of atopic dermatitis and severity of reaction.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is to be construed as including any modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
The foregoing embodiments and methods described in this invention may vary based on the capabilities, experience, and preferences of those skilled in the art.
The listing of the steps of a method in a certain order in the present invention does not constitute any limitation on the order of the steps of the method.

Claims (10)

  1. The application of LTA4H as a marker in the preparation of products for diagnosis, prevention, treatment, disease severity assessment, curative effect assessment, prognosis and drug screening of atopic dermatitis.
  2. 2. The use according to claim 1, wherein the marker is the LTA4H gene or LTA4H protein, in particular LTA4H protein.
  3. 3. The application of the reagent for detecting LTA4H in the preparation of products for diagnosis, prevention, treatment, disease severity assessment, curative effect assessment, prognosis and drug screening of atopic dermatitis.
  4. 4. The use of claim 3, wherein the detection reagent comprises a reagent that detects the expression level of LTA4H gene or LTA4H protein.
  5. 5. The use according to any one of claims 1 to 4, wherein the product is a diagnosis of atopic dermatitis, a disease severity assessment, a efficacy assessment product, which is a reagent, a kit, a test paper, a gene chip, a high throughput sequencing platform or a proteomic analysis product.
  6. 6. The use according to any one of claims 1 to 4, wherein the index of severity assessment comprises an atopic dermatitis integral index, eczema area and severity index.
  7. 7. Use according to any one of claims 1 to 4, wherein the test sample of the product is blood or a fraction thereof, preferably serum.
  8. 8. Use of an agent that affects LTA4H expression level or activity in the manufacture of a product for the prevention, treatment, efficacy assessment of atopic dermatitis;
    preferably, the effect is an increase in LTA4H expression level or protein activity.
  9. 9. The use of claim 8, wherein the agent is an LTA4H gene expression inducer or LTA4H protein agonist.
  10. 10. The use according to claim 8 or 9, wherein the product is a pharmaceutical formulation, further comprising one or more pharmaceutically acceptable excipients;
    preferably, the pharmaceutical preparation is an oral dosage form, an injection or a skin external dosage form.
CN202310643066.1A 2023-04-19 2023-06-01 Biomarkers for atopic dermatitis and uses thereof Pending CN116804675A (en)

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