Application of N-acetyltransferase 10 or its encoding gene as a target in the preparation of drugs for treating psoriasis

Abstract

The present invention discloses the application of N-acetyltransferase 10 or its coding gene as a target in the preparation of a drug for treating psoriasis, and belongs to the field of biomedicine technology. The present invention, through experiments, confirms for the first time that N-acetyltransferase 10 is highly expressed in psoriasis patient lesion tissue, and expression is elevated in a psoriasis keratinocyte model, and N-acetyltransferase 10 deficiency causes psoriasis keratinocyte proliferation to slow down, and the generation of inflammatory factors and chemokines is reduced. In animal experiments, N-acetyltransferase 10 inhibitor Remodelin can alleviate imiquimod-induced psoriasis mouse skin inflammation, including improving scales and lesion thickness, reducing the generation of inflammatory factors and chemokines, etc. Therefore, the present invention proposes to prepare a new target N-acetyltransferase 10 for treating psoriasis drugs, and N-acetyltransferase 10 or its coding gene as an intervention target has a clear effect on the treatment of psoriasis, and has good safety.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to the use of N-acetyltransferase 10 or its encoding gene as a target in the preparation of a drug for treating psoriasis. Background Art

[0002] Psoriasis is a chronic inflammatory disease with typical rash manifestations of scaly erythema or plaques. The course of the disease is often prolonged and recurrent. The disease is characterized by excessive proliferation, abnormal differentiation and inflammatory cell infiltration of keratinocytes. Histopathological manifestations include epidermal hyperkeratosis with incomplete keratinization, visible Munro microabscesses, reduced or absent granular layer, thickened stratum spinosum, neat downward extension of epidermal processes, upward extension of dermal papillae, dilated and tortuous capillaries, and inflammatory cell infiltration around them. The pathogenesis of psoriasis is complex and there is currently no cure. Treatments include traditional glucocorticoids, vitamin D analogs, and biological agents targeting TNF-α, IL-23, IL-17, etc. in recent years. These therapies have problems such as poor efficacy in some patients, increased risk of infection, formation of immune tolerance and relapse. Therefore, it is still imperative to find new treatment strategies for psoriasis.

[0003] N-acetyltransferase 10 (NAT10) is an acetylase with both an acetylase domain and an RNA binding domain. It is identified as a key enzyme for ac4C catalytic modification because it can catalyze the modification of N4-acetylcytidine (ac4C) on mRNA and participate in the stability and translation regulation of mRNA. Studies have found that NAT10 is overexpressed in various tumor types such as hepatocellular carcinoma, colorectal cancer and melanoma. The ac4C acetylation modification mediated by NAT10 can regulate the proliferation, migration and invasion of various tumor cells and is closely related to the tumor immune microenvironment. Remodelin is the most studied NAT10 selective inhibitor. It inhibits NAT10 activity and has excellent effects on inhibiting tumor growth in xenograft mouse models and prolonging healthy lifespan in early aging syndrome mouse models. So far, the role of NAT10 in psoriasis is unknown, and there has been no report on the use of NAT10 or its encoding gene as a target in the preparation of drugs for the treatment of psoriasis. Summary of the invention

[0004] In order to solve the deficiencies of the prior art, the present invention proposes the use of N-acetyltransferase 10 or its encoding gene as a target in the preparation of a drug for treating psoriasis, which solves the problems of poor efficacy and insufficient safety of biological preparations in the prior art in treating psoriasis. The technical solution provided by the present invention is as follows:

[0005] In one aspect, the present invention provides the use of N-acetyltransferase 10 or its encoding gene as a target in the preparation of a drug for treating psoriasis.

[0006] Furthermore, the drug treats psoriasis by targeting N-acetyltransferase 10 at the gene level and / or protein level.

[0007] Furthermore, the drug is selected from at least one of NAT10 siRNA, a small molecule inhibitor of N-acetyltransferase 10 or its encoding gene, and a specific antibody.

[0008] The carriers for siRNA to inhibit N-acetyltransferase 10 include, but are not limited to, phosphate buffer, water, and the like.

[0009] Furthermore, the small molecule inhibitor is Remodelin. As an inhibitor of N-acetyltransferase 10, the embodiments of the present invention prove that it has good therapeutic effect and safety for psoriasis.

[0010] In some embodiments, the drug may be in the form of a solution, granules, powder, suspension, emulsion, etc. The drug may be administered by topical application, injection, oral administration, etc.

[0011] As a second aspect of the present invention, it provides the use of overexpressed N-acetyltransferase 10 as a psoriasis marker.

[0012] In an embodiment of the present invention, a product for diagnosing, detecting, monitoring or predicting the development of psoriasis is also provided, and the product can be used to detect the expression of N-acetyltransferase 10. The product can be a detection kit, a colloidal gold test strip, a gene chip or a protein chip.

[0013] In some embodiments, psoriasis progression includes keratinocyte proliferation and production of inflammatory factors.

[0014] In an embodiment of the present invention, a drug for treating psoriasis is also provided, wherein the drug is Remodelin, an inhibitor of N-acetyltransferase 10.

[0015] The study of the embodiments of the present invention shows that the expression of N-acetyltransferase 10 in the lesions of psoriasis patients is higher than that in the normal skin of healthy controls; the expression of N-acetyltransferase 10 is increased in the mixed cytokine-induced psoriasis keratinocyte model, and after knocking down N-acetyltransferase 10 using NAT10 siRNA, the proliferation activity of psoriasis keratinocytes is inhibited, and the production of inflammatory factors and chemokines is reduced; based on the in vitro experimental results, the embodiments of the present invention further use the psoriasis animal model for in vivo experiments, and confirm from the three levels of clinical manifestations, tissue pathology and molecular experiments that the N-acetyltransferase 10 inhibitor Remodelin has a good therapeutic effect on psoriasis mice and good safety.

[0016] In general, the present invention provides preclinical evidence for the use of N-acetyltransferase 10 or its encoding gene as a target in the preparation of drugs for treating psoriasis. The embodiments provide the use of N-acetyltransferase 10 as a drug target at the gene level and / or protein level, a vector comprising siRNA to inhibit N-acetyltransferase 10 and an inhibitor that targets and inhibits N-acetyltransferase 10 in the preparation of psoriasis drugs, providing a new strategy for the treatment of psoriasis.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention, through experiments, confirms for the first time that N-acetyltransferase 10 is highly expressed in the skin lesions of psoriasis patients. In the psoriasis keratinocyte model, the expression of N-acetyltransferase 10 is elevated, and the lack of N-acetyltransferase 10 leads to a slow proliferation of psoriasis keratinocytes and a reduction in the production of inflammatory factors and chemokines. In animal experiments, the N-acetyltransferase 10 inhibitor Remodelin can alleviate imiquimod-induced skin inflammation in psoriasis mice, including improving the thickness of scales and lesions, reducing the production of inflammatory factors and chemokines, etc. The present invention proposes a new target N-acetyltransferase 10 for preparing drugs for treating psoriasis, and using N-acetyltransferase 10 or its encoding gene as an intervention target has a clearer effect on the treatment of psoriasis and good safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0020] Figure 1 The expression of NAT10 in normal skin tissue of healthy controls and skin lesion tissue of psoriasis patients was detected by immunohistochemistry; (a) is normal skin tissue, (b) is psoriasis skin lesion tissue, and the scale bar is 50 μm.

[0021] Figure 2 After stimulating HEKa cells with M5 for 24 h, total RNA and protein were extracted from the cells, and the mRNA and protein expressions of NAT10 were detected by real-time fluorescence quantitative PCR and immunoblotting, respectively; (a) is the relative expression of NAT10 mRNA, and (b) is the protein expression. **p<0.01.

[0022] Figure 3After adding M5 stimulation to HEKa cells and knocking down NAT10 using NAT10 siRNA for 48 h, the expression of NAT10 mRNA and protein was detected by real-time fluorescence quantitative PCR and immunoblotting, respectively; (a) is the relative expression of NAT10 mRNA, and (b) is the protein expression. ***p<0.001.

[0023] Figure 4 Figure 48 h after M5 stimulation in HEKa cells and NAT10 knockdown using NAT10 siRNA, the proliferation activity of cells was detected by CCK-8 method. ***p<0.001.

[0024] Figure 5 After adding M5 stimulation to HEKa cells and knocking down NAT10 with NAT10 siRNA for 48 h, the mRNA expression levels of psoriasis-related inflammatory factors in cells were examined by real-time fluorescence quantitative PCR; (a) is the relative expression of IL1β mRNA, (b) is the relative expression of IL6 mRNA, (c) is the relative expression of IL8 mRNA, (d) is the relative expression of TNF mRNA, (e) is the relative expression of CCL20 mRNA, (f) is the relative expression of CXCL10 mRNA, (g) is the relative expression of IL22 mRNA, (h) is the relative expression of IL23A mRNA, and (i) is the relative expression of IL17A mRNA. *p<0.05, **p<0.01, ***p<0.001.

[0025] Figure 6 These are photos of the efficacy of topical NAT10 inhibitor Remodelin in treating imiquimod-induced psoriasis in mice; among them, (a) is the base cream group, (b) is the base cream + 50 mg / kg Remodelin group, (c) is the imiquimod group, and (d) is the imiquimod + 50 mg / kg Remodelin group.

[0026] Figure 7 The histopathological changes of skin lesions in mice with imiquimod psoriasis were detected by HE staining after the topical NAT10 inhibitor Remodelin was used to treat the mice; (a) is the base cream group, (b) is the base cream + 50 mg / kg Remodelin group, (c) is the imiquimod group, and (d) is the imiquimod + 50 mg / kg Remodelin group. The scale bar is 100 μm.

[0027] Figure 8The mRNA expression level of inflammatory factors in the skin lesions of mice with imiquimod psoriasis was detected by RT-qPCR method after the topical NAT10 inhibitor Remodelin was used to treat the skin lesions; (a) is the relative expression of Il1b mRNA, (b) is the relative expression of Il6 mRNA, (c) is the relative expression of Il8 mRNA, (d) is the relative expression of Tnf mRNA, (e) is the relative expression of Ccl20 mRNA, (f) is the relative expression of Cxcl10 mRNA, (g) is the relative expression of Il22 mRNA, (h) is the relative expression of Il23a mRNA, (i) is the relative expression of Il17a mRNA. **p<0.01, ***p<0.001, ns indicates no statistical difference. DETAILED DESCRIPTION

[0028] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0029] In order to provide a new treatment strategy for psoriasis, the present invention provides the use of targeting N-acetyltransferase 10 in the treatment of psoriasis.

[0030] In this embodiment, the base cream uses medical pure white vaseline (brand: Lierkang) as a drug carrier.

[0031] Imiquimod cream, trade name: Mingxinlidi, manufacturer: Sichuan Mingxin Pharmaceutical Co., Ltd.

[0032] In this example, the expression levels of inflammatory markers were detected in mouse and human skin, including cytokines interleukin 1b (Il1b) / interleukin 1β (IL1β), interleukin 6 (Il6 / IL6), interleukin 8 (Il8 / IL8), tumor necrosis factor (Tnf / TNF), C-C motif chemokine ligand 20 (Ccl20 / CCL20), C-X-C motif chemokine ligand 10 (Cxcl10 / CXCL10), interleukin 22 (Il22 / IL22), interleukin 23A (Il23a / IL23A), and interleukin 17A (Il17a / IL17A).

[0033] Example 1: Application of targeting N-acetyltransferase 10 in the treatment of psoriasis

[0034] 1. Materials and Methods

[0035] (1) Immunohistochemistry

[0036] 1) The skin tissue obtained during the biopsy operation was rinsed with physiological saline to remove blood and other contaminants, and then placed in 4% paraformaldehyde; 2) The skin tissue was embedded in paraffin and sliced; 3) Slice dewaxing: The paraffin slices were immersed in xylene and gradient ethanol in sequence, in order of xylene for 20 min, xylene for 20 min, 100% ethanol for 20 min, 95% ethanol for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, 75% ethanol for 5 min, and distilled water for 5 min; 4) Antigen repair: Prepare Tris-EDTA antigen repair solution with pH 9.0, heat it to boiling in a pressure cooker, put the slices in, cover the pot, stop heating after 1.5 min after the jet, cool naturally, and wash in PBS with pH 7.4 for 3 times, each time for 5 min; 5) Block endogenous peroxidase: After washing, the slices were incubated in 3% H2O2 for 20 min, and then washed in PBS with pH 7.4 for 3 times, each time for 5 min; 6) Blocking: Add 3% BSA evenly covers the tissue and incubates at room temperature for 30 min; 7) Primary antibody incubation: remove the blocking solution, add 50 μL of primary antibody working solution diluted with PBS (the dilution ratio of NAT10 antibody is 1:200), and place the slices in a humidified box at 4°C overnight; 8) Secondary antibody incubation: wash the slices in PBS at pH 7.4 for 3 times, 5 min each time, remove the water stains on the slices, add immunohistochemistry rabbit and mouse universal secondary antibody, and incubate at room temperature for 1 h; 9) Color development: wash the slices in PBS at pH 7.4 for 3 times, 5 min each time, remove the water stains on the slices, add the freshly prepared DAB working solution, strictly control the color development time under the microscope, and stop the color development reaction by washing with tap water; 10) Counterstaining: stain the nucleus with hematoxylin for 3 min, wash with tap water, differentiate with differentiation solution for 3-8 s and then wash with tap water, wash with hematoxylin blueing solution and then wash with tap water; 11) Dehydration and sealing: put the slices into 75%, 80%, 90%, and 100% ethanol for 5 minutes each in sequence After 5 min, place in xylene for transparentization, let it dry slightly and then seal with sealing glue; 12) Observation and analysis: After sealing, scan the slices with a slice scanner to observe the staining intensity of NAT10.

[0037] (2) Recovery and culture of HEKa keratinocytes

[0038] 1) Cell recovery: Take out the frozen cells from the liquid nitrogen tank, quickly place the cells in a 37°C water bath, transfer the cells to a 15 mL centrifuge tube with a pipette after the cells are completely dissolved, centrifuge at 1000 rpm for 5 min, discard the supernatant, add 1 mL of culture medium to the centrifuge tube to resuspend the cells, and transfer them to the cell culture dish. Continue to add 5 mL of culture medium to the culture dish, shake evenly, and place in a cell culture incubator at 37°C with 5% carbon dioxide; 2) Cell culture: Place the cells in an incubator and replace with fresh culture medium every 2 days; 3) Cell passaging: When the cell density in the cell culture dish is 80%-90%, discard the culture medium in the dish, wash the cells 1-2 times, add trypsin to cover the cells, and place in the incubator for 2-4 min. Take out the culture dish and observe under a microscope that the cells have become round and partially fallen off. Add the same amount of culture medium as trypsin to terminate the digestion, gently blow with a pipette to make all the cells on the bottom of the dish fall off, collect the cells in a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 After 10 min, the supernatant was discarded, fresh culture medium was added to resuspend the cells, and the cells were inoculated into a new culture dish at a subculture ratio of 1:4.

[0039] (3) Construction of psoriasis keratinocyte model

[0040] 1) After digesting the cells into a single cell suspension, the cells were counted and inoculated into a six-well plate at a cell density of 4×10 5 cells in each well, shaken and placed in a cell culture incubator; 2) observed under a microscope, when the growth density of the cells reached 60% after attachment, the medium was replaced with serum-free medium and starved for 12 h; 3) the old medium was discarded, and complete medium containing M5 (a mixture of five cytokines, IL-17A, IL-22, IL-1α, TNF-α and OSM) with a final concentration of 10 ng / mL was added to the cells in the experimental group, and the cells were placed in a cell culture incubator for continued culture. After M5 stimulation for a specified time, total cell RNA and total protein were extracted.

[0041] (4) RNA extraction from cells and mouse skin lesions

[0042] 1) Mouse skin lesion tissue was homogenized with a tissue homogenizer, and the subsequent steps for RNA extraction from tissues were the same as those for RNA extraction from cells; 2) 1 mL of TRIzol reagent was added to the homogenized tissue or 6-well plate cells, and after the tissue or cells were lysed, they were transferred to a 1.5 mL enzyme-free EP tube and allowed to stand for 5 min; 3) 200 μl of chloroform was added, and the tube was allowed to stand for 10 min after vigorous shaking, and then centrifuged at 12,000 rpm and 4°C for 15 min; 4) The supernatant was aspirated into a new 1.5 mL enzyme-free EP tube, 400 μl of isopropanol was added, and the tube was mixed by inversion, and then allowed to stand for 10 min, and then centrifuged at 12,000 rpm and 4°C for 15 min; 5) The supernatant was discarded, 1 mL of 75% DEPC-ethanol was added, and the tube was washed once by centrifugation; 6) After natural drying, 20-40 μL of Dissolved in DEPC water; 7) RNA quantification: Use a spectrophotometer to detect RNA concentration. When the OD260 / OD280 value is between 1.9-2.0, it indicates that the extracted RNA is of good quality. The RNA concentration of each sample is uniformly adjusted to 500 μg / mL; 8) Reverse transcription: Add each component to the PCR tube according to the system in Table 1, with 20 μL of the system in each tube. Mix the system and centrifuge. Put the PCR tube into the PCR instrument and perform reverse transcription according to the program in Table 2. After the reverse transcription is completed, dilute the reverse transcription product cDNA with 80 μL ddH2O and store it at -20℃ for later use.

[0043] Table 1 Reverse transcription system

[0044]

[0045] Table 2 Reverse transcription procedures

[0046]

[0047] (5) Real-time fluorescence quantitative PCR method

[0048] 1) The primer sequences of the genes involved are shown in Table 3 (SEQ ID NO.1~SEQ ID NO.22) and Table 4 (SEQ ID NO.23~SEQ ID NO.42); 2) Add the reaction system in Table 5 to 0.1 mL sterile PCR eight-tube strips; 3) Mix the added samples evenly and centrifuge; 4) Put the PCR eight-tube strips into the PCR instrument and perform real-time quantitative PCR reaction according to Table 6; 5) After the reaction is completed, observe the amplification curve and melting curve, and record the Ct value of each gene, using β-actin as the internal reference and 2 -ΔΔCt Methods The transcription level of the gene to be tested was calculated and then statistically analyzed.

[0049] Table 3 PCR primer sequences (human origin)

[0050]

[0051] Table 4 PCR primer sequences (mouse)

[0052]

[0053] Table 5 RT-qPCR reaction system

[0054]

[0055] Table 6 Real-time quantitative PCR reaction program

[0056]

[0057] (6) Total cell protein extraction

[0058] Protein extraction: 1) discard the cell culture medium and wash the cells twice with PBS; 2) add RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors; 3) scrape the cells with a cell scraper and transfer them to a 1.5 mL EP tube, shake vigorously and let stand for 15 minutes; 4) centrifuge at 12000 rpm and 4℃ for 15 minutes, and pipette the supernatant, i.e., the protein sample, into a new 1.5 mL EP tube. b. Protein quantification: take 5 μl of the supernatant and use the BCA protein quantification kit to quantify the protein. All protein samples are adjusted to the same concentration with RIPA according to the lowest concentration, and 5× Loading buffer is added according to 1 / 4 of the total volume of the protein sample. Mix well and boil at 100℃ for 10 minutes.

[0059] (7) Immunoblotting method

[0060] a.SDS-PAGE gel electrophoresis: 1) Prepare SDS-PAGE gel: Prepare the separation gel and the concentration gel system respectively, and pour them into the gel glass plate gel holder in turn, insert the comb, and use it for subsequent electrophoresis after the gel solidifies; 2) Electrophoresis: Install the gel plate into the electrophoresis tank, pour in 1× electrophoresis buffer, remove the comb, add different group protein samples into the gel wells in turn, add protein markers on both sides, connect the power supply, and keep the voltage constant at 80 V until the protein sample enters the separation gel, and stop the electrophoresis at 120 V when the bromophenol blue runs to the bottom of the gel. b. Transfer: 1) Prepare 1× transfer solution, cut the SDS-PAGE gel, place the sponge pad, 2 layers of filter paper, SDS-PAGE gel, nitrocellulose membrane, 2 layers of filter paper, and sponge pad on the transfer clip in turn, put it into the transfer tank, add ice packs and transfer solution; 2) Connect the power supply, and transfer the membrane at 300 mA constant current for 90-120 min. c. Antibody incubation: 1) After transfer, place the nitrocellulose membrane in a fast blocking solution for 10 min; 2) After washing the membrane with 1×TBST, add the diluted primary antibody and incubate at 4°C overnight; 3) The next day, wash the membrane with 1×TBST 3 times, 10 min each time, add the diluted secondary antibody, and incubate at room temperature for 1 h; 4) Wash the membrane with 1×TBST 3 times, 10 min each time. d. Luminescence: Prepare ECL luminescent solution, evenly cover the membrane, place it in a chemiluminescence imager for luminescence and collect images.

[0061] (8) Cell transfection

[0062] 1) Before transfection, cells were plated at a density of 4×10 5 1) 5 μL of RNAimax was added to 100 μL of reduced serum medium Opti-MEM, shaken upside down to mix well, and incubated at room temperature for 5 min; 2) 5 μL of NAT10 siRNA and negative control siRNA (see Table 7 SEQ ID NO.43~SEQ ID NO.45 for the targeted sequences of each siRNA) were added to 100 μL of reduced serum medium Opti-MEM, mixed well, shaken upside down to mix well, and incubated at room temperature for 5 min; 3) 5 μL of NAT10 siRNA and negative control siRNA (see Table 7 SEQ ID NO.43~SEQ ID NO.45 for the targeted sequences of each siRNA) were added to 100 μL of reduced serum medium Opti-MEM, mixed well, shaken upside down to mix well, and incubated at room temperature for 5 min; 4) The liquids in 2) and 3) were mixed, mixed well by inversion, and incubated at room temperature for 15 min; 5) The transfection mixture obtained in 4) was added dropwise to the wells of the six-well plate, and the volume was supplemented to 2 mL / well with culture medium. The six-well plate was gently shaken to mix well, and the six-well plate was placed in a cell culture incubator after marking.

[0063] Table 7 NAT10 siRNA targeting sequences

[0064]

[0065] (9) CCK-8 method

[0066] 1) Human primary keratinocytes in the logarithmic growth phase were counted and inoculated into 96-well plates at a density of 8,000 cells / well for culture; 2) After the cells adhered to the wall, NAT10 was knocked down using NAT10 siRNA and M5 was added to stimulate the cells to simulate the state of psoriasis cells; 3) 48 h after NAT10 siRNA knockdown and M5 addition, 10 μL of CCK-8 solution was added to each well, incubated in a cell culture incubator in the dark for 2 h, and the OD value of each well at a wavelength of 450 nm was detected and recorded using a microplate reader.

[0067] (10) Topical Remodelin for the treatment of imiquimod-induced psoriasis in mice

[0068] 1) Twenty wild-type C58BL / 6 mice aged 6-8 weeks were selected and randomly divided into matrix cream group, matrix cream + 50 mg / kg Remodelin group, imiquimod group, and imiquimod + 50 mg / kg Remodelin group, with 5 mice in each group, and the backs of the mice were depilated; 2) The mice in the matrix cream group were applied with 62.5 mg matrix cream daily, and then with dimethyl sulfoxide (DMSO) 4 hours later; the mice in the matrix cream + 50 mg / kg Remodelin group were applied with 62.5 mg matrix cream daily, and then with 50 mg / kg Remodelin dissolved in DMSO 4 hours later; the mice in the imiquimod group were applied with 62.5 mg imiquimod cream daily, and then with DMSO 4 hours later; the mice in the imiquimod + 50 mg / kg Remodelin group were applied with 62.5 mg imiquimod cream daily, and then with 50 mg / kg Remodelin dissolved in DMSO 4 hours later; 3) The mice were euthanized on the 7th day, and the back skin tissues of the mice were collected for subsequent detection.

[0069] (11) HE staining

[0070] 1) Collect the skin tissue from the back of the mouse, embed it in paraffin and slice it, and dewax the sliced ​​tissue; 2) Hematoxylin staining: stain the sliced ​​tissue with hematoxylin staining solution for 3 min, wash with tap water, differentiate with differentiation solution for 3-8 s and then wash with tap water, and then wash with tap water after bluing with hematoxylin bluing solution; 3) Eosin staining: dehydrate the slices in 85% and 95% gradient alcohol for 5 min each, and stain with eosin staining solution for 5 min; 4) Dehydration and sealing: put the slices in 75%, 80%, 90%, and 100% ethanol for 5 min each, then put them in xylene for 5 min, dry them slightly and seal them with sealing glue; 5) Observation and analysis: scan the slices with a slice scanner after sealing, and analyze the tissue pathological changes.

[0071] (12) Statistical analysis

[0072] All statistical analyses were performed using SPSS 23.0 and Graphpad Prism 9.5 software. Measurement data were expressed as mean ± standard deviation. The differences between groups were determined by t-test or one-way analysis of variance (ANOVA). P < 0.05 was considered statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001, ns indicated no statistical difference.

[0073] 2. Results

[0074] (1) NAT10 expression is elevated in skin lesions of patients with psoriasis

[0075] This example first evaluated the expression of NAT10 in the skin lesions of psoriasis patients and the normal skin of healthy controls. According to the results of immunohistochemistry, NAT10 is mainly expressed in the nuclei of skin tissue. In normal skin, NAT10 is weakly expressed in keratinocytes, while in the skin lesions of psoriasis patients, NAT10 is strongly expressed in keratinocytes of the entire thickened epidermis. Therefore, the expression of NAT10 in the skin lesions of psoriasis patients is significantly higher than that in normal tissues ( Figure 1 ).

[0076] (2) NAT10 expression is elevated in psoriasis keratinocytes

[0077] In this example, a psoriasis keratinocyte model was constructed to evaluate the expression of NAT10 in the psoriasis cell model. The inventors used mixed cytokine M5 to stimulate keratinocytes to simulate the psoriasis cell model, and used real-time fluorescence quantitative PCR to detect the mRNA level of NAT10, and used immunoblotting to detect the protein expression of NAT10. Compared with normal cells, the mRNA and protein expression levels of NAT10 in the psoriasis cell model were increased ( Figure 2 ).

[0078] (3) NAT10 deficiency can inhibit the proliferation activity of psoriatic keratinocytes

[0079] In order to further detect the biological function of NAT10, this example used siRNA-mediated gene knockdown of NAT10, and the real-time fluorescence quantitative PCR method and immunoblotting method were used to verify the knockdown efficiency of NAT10 at the mRNA level and protein level ( Figure 3 In the psoriasis cell model treated with M5, the cell proliferation activity increased significantly, which is consistent with the excessive proliferation of keratinocytes in psoriasis. Negative control siRNA and NAT10 siRNA were used in M5-treated cells, and the proliferation activity of psoriasis-like keratinocytes treated with NAT10 siRNA decreased ( Figure 4), indicating that NAT10 deficiency can inhibit the proliferation activity of psoriatic keratinocytes.

[0080] (4) NAT10 deficiency can inhibit the production of inflammatory factors in psoriasis keratinocytes

[0081] The disease state of psoriasis includes not only keratinocyte proliferation, but also the secretion of inflammatory factors. Therefore, the inventors further tested the effect of NAT10 deficiency on the secretion of inflammatory factors. In the psoriasis cell model constructed using M5, the relative mRNA expression of IL1β, IL6, IL8, TNF, CCL20, CXCL10, IL22, IL23A and IL17A increased significantly, while in NAT10 siRNA-mediated NAT10 deficiency cells, the production of the above inflammatory factors decreased significantly ( Figure 5 ), indicating that NAT10 deficiency can comprehensively inhibit the production of multiple inflammatory factors in psoriatic keratinocytes.

[0082] (5) NAT10 inhibitor Remodelin can treat psoriasis mice with good safety

[0083] After the cell model experiment, in order to further illustrate the feasibility of NAT10 as a target for the treatment of psoriasis, this example further constructed an imiquimod-induced psoriasis animal model, and treated psoriasis mice with topical NAT10 inhibitor Remodelin. The clinical photos of psoriasis mice with different treatments showed that the skin appearance of mice in the topical matrix cream group and the matrix cream + 50 mg / kg Remodelin group did not change, and imiquimod successfully induced psoriasis skin inflammation, including scaling and epidermal thickening, while topical Remodelin treatment of psoriasis mice could significantly improve the scaling and epidermal thickening of mice ( Figure 6 ). This example further detected the histopathological changes of the back skin of mice. The skin appearance of mice in the topical matrix cream group and the matrix cream + 50 mg / kg Remodelin group did not change. The skin of mice in the imiquimod group showed pathological changes similar to those of psoriasis patients, such as hyperkeratosis, parakeratosis, basal hypertrophy and inflammatory cell infiltration. The topical application of 50 mg / kg Remodelin to treat psoriasis mice significantly improved pathological changes such as hyperkeratosis, parakeratosis, basal hypertrophy and inflammatory cell infiltration ( Figure 7). This example further detected the levels of inflammatory factors in the skin lesions of mice. Consistent with the appearance and pathological changes, the expression levels of skin inflammatory factors in the mice in the topical matrix cream group and the matrix cream + 50 mg / kg Remodelin group were basically the same. The expression levels of Il1b, Il6, Il8, Tnf, Ccl20, Cxcl10, Il22, Il23a and Il17a in the mice in the imiquimod group were significantly increased. The topical application of 50 mg / kg Remodelin to treat psoriasis mice can significantly reduce the production of the above inflammatory factors ( Figure 8 ). Figure 6-8 Clinical manifestations, tissue pathology, and molecular experiments showed that there was no significant difference between the topical matrix cream group and the topical matrix cream + 50 mg / kg Remodelin group, indicating that Remodelin has good safety. The topical imiquimod group successfully induced a psoriasis mouse model, and topical Remodelin had a therapeutic effect on imiquimod-induced psoriasis mice.

[0084] Embodiment 2,

[0085] This example provides the use of overexpressed N-acetyltransferase 10 as a psoriasis marker.

[0086] Example 3, a product for diagnosing, detecting, monitoring or predicting the development of psoriasis

[0087] The product can be used to detect the expression of N-acetyltransferase 10.

[0088] In some embodiments, psoriasis progression includes keratinocyte proliferation and production of inflammatory factors.

[0089] In some embodiments, the product can be a detection kit, a colloidal gold test strip, a gene chip, or a protein chip.

[0090] Example 4: Use of an inhibitor of N-acetyltransferase 10 or its encoding gene in the preparation of a drug for treating psoriasis

[0091] The drug that inhibits N-acetyltransferase 10 or its encoding gene can be NAT10 siRNA, small molecule inhibitors, specific antibodies, etc. The dosage form of the drug can be a solution, granules, powder, suspension, emulsion, etc. It can be administered by topical application, injection, oral administration, etc.

[0092] Example 5: Use of a siRNA vector for inhibiting NAT10 in the preparation of a drug for treating psoriasis

[0093] The carrier includes, but is not limited to, phosphate buffer, water, and the like.

[0094] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of small molecule inhibitor Remodelin in the preparation of drugs for the treatment of psoriasis.

2. Use of a product for detecting the expression of N-acetyltransferase 10 in the preparation of a product for diagnosing, detecting, monitoring or predicting the development of psoriasis.

Images