Application of ALOX15 inhibitor in preparation of medicine for treating atopic dermatitis

Abstract

The invention provides application of an ALOX15 inhibitor in preparation of a medicine for treating atopic dermatitis and belongs to the technical field of biological medicine, high expression of an ALOX15 gene promotes formation of atopic dermatitis skin lesion, the ALOX15 inhibitor is applied to preparation of the medicine for treating atopic dermatitis, and a new thought is provided for manufacturing of the medicine for treating atopic dermatitis. The invention also provides an application of the ALOX15 as a molecular marker in preparation of a detection kit for diagnosing atopic dermatitis.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the use of an ALOX15 inhibitor in the preparation of a drug for treating atopic dermatitis. Background Art

[0002] Atopic dermatitis (AD) is a common chronic recurrent inflammatory skin disease, with severe itching and eczematous skin lesions as the main clinical features, which seriously affects the quality of life of up to 20.1% of children and 2.1% to 4.9% of adults. The pathogenesis of AD involves barrier dysfunction, abnormal activation of immune cells, IgE-mediated hypersensitivity reactions and environmental factors. Recent studies have confirmed that epigenetic modifications driven by environmental factors play a vital role in the occurrence and development of complex inflammatory diseases. Therefore, exploring AD therapeutic targets and studying a new drug or new method for the treatment of AD are of great significance to the development of precision medicine for AD. Summary of the invention

[0003] In order to obtain a new approach to treat atopic dermatitis, the present invention provides the use of an ALOX15 inhibitor in the preparation of a drug for treating atopic dermatitis. The high expression of the ALOX15 gene promotes the formation of atopic dermatitis lesions. The use of an ALOX15 inhibitor in the preparation of a drug for treating atopic dermatitis provides a new idea for the manufacture of atopic dermatitis therapeutic drugs.

[0004] The present invention also provides the use of ALOX15 as a molecular marker in the preparation of a detection kit for diagnosing atopic dermatitis.

[0005] The present invention is achieved through the following technical solutions:

[0006] The present invention provides use of an ALOX15 inhibitor in preparing a medicament for treating atopic dermatitis.

[0007] Optionally, the ALOX15 inhibitor comprises siRNA of the ALOX15 gene.

[0008] Preferably, the nucleotide sequence of the siRNA is as shown in SEQ ID NO.1.

[0009] Based on the same inventive concept, the present invention provides a drug for treating atopic dermatitis, wherein the active ingredient of the drug comprises siRNA of the ALOX15 gene, and the nucleotide sequence of the siRNA is shown in SEQ ID NO. 1. Based on the same inventive concept, the present invention provides the use of ALOX15 as a diagnostic marker or detection marker for atopic dermatitis.

[0010] Based on the same inventive concept, the present invention provides a diagnostic marker for atopic dermatitis, wherein the diagnostic marker comprises the ALOX15 gene or the ALOX15 protein.

[0011] Based on the same inventive concept, the present invention provides the use of ALOX15 as a molecular marker in the preparation of a detection kit for diagnosing atopic dermatitis.

[0012] Based on the same inventive concept, the present invention provides the use of a detection reagent for ALOX15 gene expression level in the preparation of a detection kit for diagnosing atopic dermatitis.

[0013] Based on the same inventive concept, the present invention also provides a detection kit for diagnosing atopic dermatitis, wherein the detection kit comprises a detection reagent for the expression level of the ALOX15 gene.

[0014] Optionally, the detection reagent for the ALOX15 gene expression level includes the primer Human-ALOX15 for amplifying the ALOX15 gene, wherein the nucleotide sequence of the forward primer is shown as SEQ ID NO.2, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO.3.

[0015] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0016] 1. The use of the ALOX15 inhibitor of the present invention in the preparation of a drug for treating atopic dermatitis. The inventors have found through research that WTAP, which is abnormally highly expressed in the skin lesions of AD patients, mediates m 6 A modification promotes the expression of ALOX15, which in turn activates dendritic cells (DCs) to secrete inflammatory factors and arachidonic acid metabolites, thereby causing abnormal lipid metabolism and differentiation and proliferation of keratinocytes (KCs) involved in the occurrence and development of AD. The high expression of ALOX15 in the lesions of patients with atopic dermatitis promotes the formation and development of atopic dermatitis lesions. The present invention applies inhibitors targeting ALOX15 to the preparation of drugs for the treatment of atopic dermatitis, providing a new idea for the manufacture of drugs for the treatment of atopic dermatitis.

[0017] 2. The invention discloses an inhibitor of ALOX15 for the preparation of a drug for treating atopic dermatitis. The invention experimentally proves that the high expression of ALOX15 in DCs is closely related to the pathogenesis of AD, and proposes that WTAP mediates m 6A modification regulates ALOX15 expression to activate DCs cells, which promotes the molecular mechanism of the occurrence and development of AD by affecting the levels of lipid metabolites. Therefore, ALOX15 has the potential to be used as a target site in the preparation of AD therapeutic drugs, and can also be used as a molecular marker for the diagnosis of AD in the preparation of clinical detection kits. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0019] Figure 1 To screen suitable siRNA sequences to silence WTAP and ALOX15 in DC2.4: (ab) WB analysis shows the protein expression level of WTAP after knocking down WTAP using WTAP siRNA with different sequences in DC2.4 (n=3); (cd) WB analysis shows the protein expression level of ALOX15 after knocking down ALOX15 using ALOX15 siRNA with different sequences in DC2.4 (n=3).

[0020] Figure 2 Clinical validation of the differential expression of ALOX15 in atopic dermatitis: (a) Immunofluorescence staining and quantitative analysis of ALOX15 in AD (n=3) and NC (n=3, original magnification ×100); (bc) WB (c, n=4) and RT-qPCR (b, n=16) detected the expression of ALOX15 in AD and NC; (d) Immunofluorescence colocalization of ALOX15 and CD11C in AD and NC.

[0021] Figure 3 Figure 3 Alterations of pathways and DEGs in mouse primary KCs: (a) KEGG pathway analysis of metabolism-related DEGs in mouse primary KCs after indirect co-culture with BMDCs in the EV group and ALOX15 overexpression group; (b) Heat map showing the expression levels of selected DEGs between the two groups of KCs.

[0022] Figure 4Effects of ALOX15 on indirect co-culture of primary BMDCs and primary KCs: (ab) RT-qPCR analysis showed the mRNA expression of ALOX15, ELOVL1, ELOVL6, keratin 1 and filaggrin after indirect co-culture of ALOX15-overexpressing and ALOX15-knockout mouse primary BMDCs with mouse primary KCs (n=3); (cd) ELISA analysis showed the expression of LXA4, LTB4, 12-HETE and 15-HETE in the cell culture supernatant after indirect co-culture of ALOX15-overexpressing and ALOX15-silenced mouse primary BMDCs and primary KCs (n=3); (ef) The effect of indirect co-culture of primary mouse KCs with ALOX15-overexpressing and ALOX15-silenced primary mouse BMDCs on the cell proliferation status was evaluated by CCK-8 (n=3).

[0023] Figure 5 Effects of ALOX15 on co-culture of DC and primary KC: (ab) After indirect co-culture of ALOX15 overexpressed and knocked-out mouse primary BMDCs and mouse primary KCs, RT-qPCR results showed that there was no significant difference in the mRNA expression of CCL2 and TSLP (n=3); (c and e) After Transwell co-culture of overexpressed DC2.4 cells with knocked-out ALOX15 and mouse primary KCs, RT-qPCR results showed that there was no significant difference in the mRNA expression of IL-4 and IL-33 in DC2.4 (n=3); (d and f) After Transwell co-culture of overexpressed DC2.4 cells with knocked-out ALOX15 and mouse primary KCs, RT-qPCR results showed that there was no significant difference in the mRNA expression of CCL2 and TSLP in primary KCs (n=3).

[0024] Figure 6 Effect of ALOX15 on Transwell co-culture of DC2.4 and primary KC: (a and c) RT-qPCR analysis showed the mRNA expression levels of ALOX15, IL-5, IL-12 and TNF-α in DC2.4 after co-culture of overexpressed DC2.4 cells with knockout ALOX15 and mouse primary KCs by Transwell (n=3); (b and d) RT-qPCR analysis showed the mRNA expression levels of ELOVL1, ELOVL6, keratin 1 and filaggrin in mouse primary KCs after co-culture of overexpressed DC2.4 cells with knockout ALOX15 and mouse primary KCs by Transwell (n=3).

[0025] Figure 7Effects of ALOX15 on direct co-culture of DC2.4 and primary KCs: (ab) Immunofluorescence staining and quantitative analysis of CD11C, keratin 1, and filaggrin in mouse primary KCs directly co-cultured with ALOX15 overexpressing DC2.4 (original magnification, ×100); (cd) Immunofluorescence staining and quantitative analysis of CD11C, keratin 1, and filaggrin in mouse primary KCs directly co-cultured with ALOX15 overexpressing DC2.4 (original magnification, ×100).

[0026] Figure 8 ALOX15 regulates the expression levels of arachidonic acid metabolites in BMDCs in vitro: (ab) RT-qPCR (n=3) and Western b lot (=3) showed the expression levels of ALOX15 after overexpression and knockout of ALOX15 in BMDCs; (cd) ELISA analysis showed the expression levels of LXA4, LTB4, 12-HETE and 15-HETE in the cell supernatant after overexpression and knockout of ALOX15 in BMDCs (n=3). DETAILED DESCRIPTION

[0027] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0028] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0029] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0030] The overall idea of ​​the present invention is as follows:

[0031] The present invention aims to provide a new target for the preparation of drugs for treating AD. 6A modification promotes abnormal expression of S100A9 (S100 calcium binding protein A9) and SERPINB3 (serpinpeptidase inhibitor, clade B (ovalbumin), member 3), which may synergistically promote chronic and recurrent skin inflammation, KCs proliferation and abnormal differentiation in AD. In addition, we also found that the mRNA expression level of ALOX15 in AD lesions was significantly increased, but the potential role of WTAP and ALOX15 in AD was not studied in depth.

[0032] Based on the results of chip analysis and bioinformatics analysis of AD patients' skin lesions, in this paper, we first verified the expression location and differences of ALOX15 in AD patients' skin lesions. Then, we used in vitro cultured mouse bone marrow-derived dendritic cells (BMDCs) and mouse dendritic cells (DC2.4) to explore the mRNA expression of ALOX15 by WTAP. 6 A methylation level and expression level and its regulatory effect on lipid metabolites.

[0033] We also performed transcriptomic analysis on mouse primary KCs after indirect co-culture with mouse primary BMDCs overexpressing ALOX15, screened out some differentially expressed genes related to KCs lipid metabolism and differentiation and proliferation, and observed the correlation between DCs and KCs through a series of in vitro experiments. Finally, we also studied the effect of ALOX15 on the content of arachidonic acid metabolites secreted by BMDCs in vitro.

[0034] The inventors found through experiments that high expression of ALOX15 in DCs is closely related to the onset of AD, and proposed that WTAP mediates m 6 A modification regulates ALOX15 expression, which in turn activates DCs to secrete inflammatory factors and arachidonic acid metabolites, thereby causing abnormal lipid metabolism and differentiation and proliferation of KCs, and ultimately promoting the molecular mechanism of the occurrence and development of AD. Our research results support the concept that high expression of ALOX15 in skin lesions promotes the formation of atopic dermatitis lesions, and targeted inhibition of ALOX15 molecules can provide new ideas for the preparation of drugs for the treatment of atopic dermatitis.

[0035] Therefore, the present invention proposes the use of an inhibitor of ALOX15 in the preparation of a drug for treating atopic dermatitis.

[0036] The application of the ALOX15 inhibitor of the present application in the preparation of a drug for treating atopic dermatitis will be described in detail below in combination with examples and experimental data.

[0037] Example 1

[0038] 1. Materials and Methods

[0039] 1.1 Human samples

[0040] AD skin lesion samples and normal skin tissues were obtained from the Third Xiangya Hospital of Central South University, which was approved by the Ethics Committee of the Third Xiangya Hospital of Central South University (NO 2022-S041). All participants signed the informed consent for surgery.

[0041] 1.2WB Analysis

[0042] RIPA lysis buffer (CWBIO, China) containing protease inhibitor cocktail (CWBIO, China) was added to skin tissue or cells, and the total protein solution was extracted after homogenization, lysis, and centrifugation. The total protein concentration in the extract was detected by BCA analysis kit (Thermo Fisher Scientific, USA). Protein electrophoresis was performed using SDS-PAGE with a concentration of 4% to 20%, and then the protein was transferred to a PVDF membrane (Millipore, USA). The PVDF membrane was transferred to a blocking solution (Beyatime, China) and blocked at room temperature for 1 hour. The primary antibodies included mouse anti-β-actin (1:5,000; cat no.60004-1-Ig; Proteintech, USA), rabbit anti-ALOX15 Ab (1:1,000; ab244205; Abcam, USA) and rabbit anti-WTAP Ab (1:1,000; PA6128S; Abmart, China), which were incubated at 4°C overnight. After washing, the membranes were incubated with HRP-labeled goat anti-mouse IgG (H+L) and goat anti-rabbit IgG (H+L) (1:10000; Proteintech, USA) secondary antibodies at room temperature for 1 h. After washing, the bands were developed using G:BOX (SYNGENE, England). The grayscale value of each band was analyzed using Image J software, and β-actin was used as an internal reference.

[0043] 1.3 Immunofluorescence analysis

[0044] After fixation and blocking, the skin sections were incubated with rabbit anti-ALOX15 Ab (1:1,000; ab244205; Abcam, USA) and mouse anti-CD11C Ab (1:100; ab254183; Abcam, USA) primary antibodies at 37°C for 1 h, washed, and then incubated with secondary antibodies and Opal 540fluorophore (PerkinElmer, USA) at 37°C for 20 min, and finally stained with DAPI.

[0045] After the collected cells were fixed and blocked, they were incubated with mouse anti-CD11C Ab (1:100; ab254183; Abcam, USA), rabbit anti-Cytokeratin 1 Ab (1:20000; ab185628; Abcam, USA), mouse anti-filaggrin (1:100; GTX23137; GeneTex, USA) primary antibodies at 4°C overnight, washed, and then incubated with fluorescent secondary antibodies at 37°C for 1 hour, and finally stained with DAPI. Image J software was used to analyze the fluorescence intensity, so as to understand the expression of ALOX15, CD11C, Keratin1 and filaggrin through the fluorescence intensity.

[0046] 1.4 RT-qPCR

[0047] TRIzol reagent (Molecular Research Center, USA) was used to extract cell RNA. Nanodrop2000 spectrophotometer (Thermo Fisher Scientific, USA) was used to determine the concentration of R888885NA. Then, a reverse transcription kit (Accurate Biology, China) was used to synthesize cDNA. A fluorescent quantitative RCR instrument (BIO-RAD, China) was used for amplification. -ΔCt Statistical analysis was performed. The primers used in this study are shown in Table 1.

[0048] Table 1: Primers used in the study

[0049]

[0050]

[0051]

[0052] 1.5 MeRIP-qPCR

[0053] RNA was extracted using TRIzol reagent (Molecular Research Center, USA). Then, MeRIP-qPCR was performed on 10 ug RNA extracted from cells using the EpiQuikCUT&RUN m6A RNA Enrichment (MeRIP) kit (EpiGentek, USA) according to the manufacturer's instructions to analyze the enrichment of m6A mRNA.

[0054] 1.6 Cell culture

[0055] DC2.4 (Millipore, cat no. SCC142) was cultured in DMEM (Gibco, Thermo Fisher Scientific, USA) medium containing 10% fetal bovine serum (ExCell). Mouse primary BMDCs were extracted from the femoral and tibial bone marrow of 5-week-old C57BL / 6j mice and induced for one week in 1640 medium containing 50ng / ml GM-CSF and 20ng / ml IL-4. Then, 1μg / ml LPS was added to stimulate maturation and then cultured in DMEM medium containing 10% fetal bovine serum (Cellmax). Mouse primary KCs were extracted from the skin tissue of 0-2 day newborn C57BL / 6j mice and then cultured in DMEM medium containing 10% fetal bovine serum (Cellmax) until the cells reached 90% confluence for passage.

[0056] 1.7 Cell transfection

[0057] DC2.4 cells were seeded in 6-well cell culture plates and stained with Lipofectamine TM 3000 (Invitrogen, USA) was used to transfect WTAP, ALOX15 siRNA and corresponding negative control (RiboBio, China). TM WTAP overexpression plasmid and ALOX15 plasmid and corresponding negative control (YouBio, China) were transfected with 3000 (Invitrogen, USA). BMDCs were transfected with siRNA and overexpression plasmid using suspension cell transfection enhancement solution (BAIDAI, China). The WTAP siRNA sequence used was si-m-WTAP-2, i.e., 5'-GGCCCAACTGAGATCAACA-3'( Figure 1 ab); the ALOX15 siRNA sequence used was si-m-ALOX15-1, i.e., 5'-AGCGATTTCGAGAGGACAA-3'( Figure 1 cd).

[0058] 1.8 ELISA

[0059] According to the manufacturer's instructions, ELISA kits were used to measure the levels of LXA4 (E-EL-0053c; elabscience, China), LTB4 (ml201802V; mlbio, China), 12-HETE (ml105047V; mlbio, China), and 15-HETE (ml162450V; mlbio, China) in the cell supernatants of mouse primary BMDCs and mouse primary KCs, respectively. The absorbance value was then detected at 450 nm using an enzyme-linked immunosorbent assay (BioTek, USA).

[0060] 1.9 Cell Co-culture

[0061] This example uses three co-culture methods, including indirect co-culture of DC2.4 cells and mouse primary KCs using Transwell cells, direct co-culture of DC2.4 cells and mouse primary KCs, and indirect co-culture of mouse primary BMDCs and mouse primary KCs.

[0062] Transwell culture dishes were used for cell co-culture. DC2.4 cells transfected with ALOX15 plasmid or siRNA were inoculated into the upper chamber of the Transwell, and primary mouse KCs were inoculated into the lower chamber of the Transwell. After 48 h of culture, the cells were collected for RT-qPCR.

[0063] DC2.4 cells were inoculated in culture plates, and DC2.4 cells transfected with ALOX15 plasmid and siRNA were co-inoculated with mouse primary KCs in cell culture plates. After 48 h of co-culture, DC2.4 and mouse primary KCs cells were collected for IF experiments.

[0064] Mouse primary BMDCs were inoculated in a 12-well culture plate and transfected with ALOX15 plasmid and siRNA. After culturing for 48 hours, the cell supernatant was aspirated and centrifuged at 3000r for 10 minutes. The supernatant was then added to the culture plate of mouse primary KCs. After culturing for 48 hours, the cells of mouse primary KCs were collected for RT-qPCR, and the cell supernatant was used for ELISA.

[0065] 1.10 Cell Counting Kit-8

[0066] After treatment, primary mouse KC cells were cultured at 3×10 3The cells were seeded at a density of 10 cells / well in a 96-well cell culture plate and cultured at 37°C. After incubation for 0, 24, 48, and 72 hours, 10 μL of Cell Counting Kit-8 (CCK-8; Dojindo, Japan) solution was added to each well and incubated for 1 hour. The OD values ​​were then measured using a microplate reader (BioTek, USA). 450nm Determine cell viability.

[0067] 1.11 Transcriptome sequencing and bioinformatics analysis

[0068] KCs were collected after indirect co-culture with primary BMDCs from mice in the ALOX15 EV group and overexpression group. RNA was extracted using Trizol reagent, and the RNA quantity and purity were analyzed using Bioanalyzer 2100 and RNA 6000Nano LabChip Kit (Agilent, CA, USA, 5067-1511). High-quality RNA samples with RIN number > 7.0 were used to construct sequencing libraries. Illumina Novaseq TM The library was sequenced with 2×150 bp double-end sequencing (PE150) using LC-Bio 6000 (LC-Bio Technology, HangZhou, China). Differential gene expression analysis between the two groups was performed using DESeq2 software. Genes with a false discovery rate (FDR) parameter lower than 0.05 and an absolute fold change ≥ 2 were defined as differentially expressed genes, and KEGG pathway enrichment analysis was performed on them.

[0069] 1.12 Statistical methods

[0070] Data are presented as mean ± SD of at least three independent experiments. Two-tailed Student's t test or two-tailed Mann-Whitney U test was used to compare statistical differences between the two groups. Statistical significance was determined using GraphPad Prism software 8.0. p < 0.05 was considered statistically significant, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and ns indicates no statistical significance.

[0071] 2. Results and Analysis

[0072] 2.1 ALOX15 is highly expressed in DCs of AD patients

[0073] We performed immunofluorescence staining to locate and quantify the skin tissues of AD patients with lesions and normal controls. The results showed that the fluorescence intensity of ALOX15 in AD patients with lesions was significantly higher than that in normal skin tissues, and ALOX15 was mainly highly expressed in the dermis ( Figure 2a). Based on this result, we separated the epidermis and dermis from the skin lesions of AD patients and normal controls, and used the dermal tissue for RT-qPCR and Western blot (WB) analysis. The results showed that the expression level of ALOX15 in the dermal tissue of AD patients was significantly upregulated ( Figure 2 bc). Combined with the applicant's previous research data analysis results, we found that ALOX15 is mainly expressed in DCs. So we did a co-staining of ALOX15 and CD11C (molecular marker of dendritic cells) and found that the tissue area expressing ALOX15 highly overlapped with the tissue area expressing CD11C, further proving that ALOX15 is expressed in DCs, and the fluorescence intensity of CD11C and ALOX15 in AD patients is higher than that in the normal control group ( Figure 2 d) These results indicate that ALOX15 is mainly upregulated in DCs in the dermis of AD lesions.

[0074] 2.2 ALOX15 activates DCs and affects the biological behavior of primary KCs

[0075] Since abnormal activation of KCs is one of the important characteristics of AD, we further intervened the expression of ALOX15 in DCs and co-cultured them with primary mouse KCs to observe the interaction between DCs and KCs. However, due to the large difference in culture conditions, primary mouse BMDCs can only be indirectly co-cultured with primary mouse KCs to observe the effect on KCs. Consistent with the sequencing results, in vitro studies found that the expression of ELOVL1, ELOVL6, Keratin1 and Filaggrin in primary mouse KCs after indirect co-culture with primary mouse BMDCs overexpressing ALOX15 was significantly reduced ( Figure 4 a), CCL2, TSLP showed no significant difference ( Figure 5 a). In addition, the results of CCK-8 experiments showed that the proliferation of mouse primary KCs was significantly enhanced after indirect co-culture with ALOX15-overexpressing mouse primary BMDCs ( Figure 4 e). In contrast to the overexpression group, the expression of ELOVL1, ELOVL6, Keratin1 and Filaggrin in mouse primary KCs was significantly increased after indirect co-culture with ALOX15 knockdown BMDCs ( Figure 4 b), CCL2 and TSLP also showed no significant difference ( Figure 5 b). Next, we co-cultured DC2.4 cells with ALOX15 overexpression with mouse primary KCs in Transwell culture. Compared with the control group, the secretion of inflammatory factors IL-5, IL-12, and TNF-α by DC2.4 cells with ALOX15 overexpression significantly increased after Transwell co-culture ( Figure 6a), no significant difference was found in IL-4 and IL-33 ( Figure 5 c). At the same time, the expression of ELOVL1, ELOVL6, Keratin1 and Filaggrin in primary mouse KCs was significantly reduced ( Figure 6 b), no significant difference was found in CCL2 and TSLP ( Figure 5 d). In contrast to the overexpression group, the inflammatory factors IL-5, IL-12, and TNF-α secreted by DC2.4 cells with ALOX15 knockdown were significantly reduced ( Figure 6 c), no significant difference was found in IL-4 and IL-33 (Appendix Figure 5 e); Similarly, the expression of ELOVL1, ELOVL6, Keratin1 and Filaggrin in primary mouse KCs was significantly increased ( Figure 6 d), CCL2 and TSLP showed no significant difference ( Figure 5 f). Subsequently, we directly co-cultured DC2.4 with mouse primary KCs and performed immunofluorescence staining. The results showed that compared with the control group, the expression of Keratin1 and Filaggrin in the KCs of the group co-cultured with ALOX15-overexpressing DC2.4 was significantly reduced ( Figure 6 ab); In contrast, the expression of Keratin1 and Filaggrin in KCs co-cultured with ALOX15 knockdown DC2.4 group was significantly increased ( Figure 7 cd). The above results show that overexpression of ALOX15 in DCs can affect the level of DCs lipid metabolites, and after activated DCs are co-cultured with mouse primary KCs, the arachidonic acid metabolic pathway of KCs is significantly affected. In addition, the expression of KCs lipid metabolism-related genes (ELOVL1, ELOVL6), KCs differentiation proteins (Keratin1, Filaggrin) and arachidonic acid metabolite LXA4 were significantly reduced, but the proliferation function of KCs was significantly enhanced.

[0076] 2.3 ALOX15 overexpression affects lipid metabolite levels in DCs

[0077] Previous research results suggest that ALOX15 is mainly enriched in the arachidonic acid pathway. Many studies have found that ALOX15 can participate in the arachidonic acid metabolic pathway and produce a variety of metabolites with physiologically active functions. We took KCs indirectly co-cultured with primary BMDCs of mice overexpressing ALOX15 for transcriptomic analysis. KEGG pathway analysis showed that the differentially expressed genes (DEGs) in the metabolism of primary KCs of mice were mainly enriched in the arachidonic acid metabolic pathway ( Figure 3a). The heat map shows the changes in mRNA levels of some DEGs (ELOVEL6, Keratin1, Filaggrin, and CCL2), and the results are arranged by z-score ( Figure 3 b). We detected the levels of four arachidonic acid metabolites in the supernatant of mouse primary KCs cells after indirect co-culture of the two cells. The results showed that the level of LXA4 decreased, while the levels of the other three metabolites (LTB4, 12-HETE, and 15-HETE) did not show significant differences ( Figure 4 c). Similarly, ELISA cell supernatant detection of arachidonic acid metabolites showed that the content of LXA4 increased, while the contents of the other three metabolites (LTB4, 12-HETE, 15-HETE) remained unchanged ( Figure 4 d).

[0078] To verify whether ALOX15 could affect the content of arachidonic acid metabolic pathway metabolites in DCs, we overexpressed and knocked down ALOX15 in BMDCs ( Figure 8 ab), and the expression of four key arachidonic acid metabolites in the cell supernatant was detected by ELISA. The results showed that the levels of LTB4, 12-HETE, and 15-HETE in the supernatant of ALOX15-overexpressing BMDCs cells were significantly increased, while the level of LXA4 was decreased ( Figure 8 c). On the contrary, the levels of LTB4, 12-HETE, and 15-HETE in the supernatant of ALOX15 knockdown BMDCs cells were reduced, while the level of LXA4 was increased ( Figure 8 d). The above results indicate that the expression level of ALOX15 in BMDCs can affect the content changes of arachidonic acid metabolites LTB4, 12-HETE and 15-HETE.

[0079] In summary, we provide convincing evidence that high expression of ALOX15 in DCs is closely related to the pathogenesis of AD and propose that WTAP mediates the m 6 A modification promotes ALOX15 expression, thereby activating DCs cells, causing abnormal arachidonic acid metabolism and differentiation and proliferation of DCs, leading to the molecular mechanism of AD development. Therefore, ALOX15 has the potential to be used as a target site in the preparation of drugs for the treatment of AD, and can also be used as a molecular marker for the diagnosis of AD in clinical detection kits.

[0080] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0081] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0082] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Application of ALOX15 inhibitors in the preparation of drugs for the treatment of atopic dermatitis.

2. The use according to claim 1, characterized in that: The ALOX15 inhibitor includes siRNA of the ALOX15 gene.

3. The use according to claim 2, characterized in that: The nucleotide sequence of the siRNA is shown in SEQ ID NO.

1.

4. A drug for treating atopic dermatitis, characterized in that: The active ingredient of the drug comprises siRNA of ALOX15 gene, and the nucleotide sequence of the siRNA is shown in SEQ ID NO.

1.

5. Application of ALOX15 as a diagnostic marker or detection marker for atopic dermatitis.

6. A diagnostic marker for atopic dermatitis, characterized in that: The diagnostic marker includes ALOX15 gene or ALOX15 protein.

7. Application of ALOX15 as a molecular marker in the preparation of diagnostic kits for atopic dermatitis.

8. Application of ALOX15 gene expression level detection reagent in the preparation of diagnostic atopic dermatitis detection kit.

9. A detection kit for diagnosing atopic dermatitis, characterized in that: The detection kit comprises a detection reagent for the expression level of the ALOX15 gene.

10. A detection kit for diagnosing atopic dermatitis according to claim 9, characterized in that: The detection reagent for the expression level of the ALOX15 gene includes the primer Human-ALOX15 for amplifying the ALOX15 gene, wherein the nucleotide sequence of the forward primer is shown as SEQ ID NO.2, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO.3.

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