Application of OASL in treatment of psoriasis

Abstract

The invention relates to the technical field of biological medicine, in particular to application of OASL in treatment of psoriasis. The disclosure finds that the OASL promotes the generation of inflammatory factors and the disorder of lipid metabolism by promoting an MAPK signal channel so as to promote cell proliferation, and the pathogenic effect of the OASL in psoriasis is proved. And by accurately positioning a treatment target, the side effects in the psoriasis treatment process can be effectively reduced.

Description

Technical Field

[0001] The present disclosure relates generally to the field of biomedical technology, and more particularly to the use of OASL in the treatment of psoriasis. Background Art

[0002] Psoriasis is a prevalent, chronic immune skin disease that affects an estimated 2% to 3% of the global population, placing a significant burden on individual health and healthcare systems. The societal impact of psoriasis is multifaceted and extends beyond healthcare spending to include indirect costs of work disability and reduced quality of life. Existing treatments, including topical, phototherapy, and systemic therapies, are often limited by suboptimal efficacy, significant side effects, and the inability to achieve sustained disease control. The advent of biologics, targeting specific cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-17 (IL-17), and interleukin-23 (IL-23), has marked a major advance in the treatment of moderate to severe psoriasis. However, these agents also carry a higher economic burden and potential safety risks, including the risk of infection and the development of malignancies. This highlights the urgency of developing more effective and safe treatment strategies.

[0003] The JAK-STAT signaling pathway has emerged as a central player in immune dysregulation in psoriasis and has become a potential target for therapeutic intervention. JAK inhibitors have shown promise as an alternative oral treatment option in clinical trials, particularly for patients who do not respond to or are intolerant of conventional therapies. However, the use of JAK inhibitors has been closely scrutinized for safety concerns regarding their potential to induce cardiovascular events and thromboembolic complications. Therefore, a deeper understanding of the mechanism of action of the JAK-STAT pathway in psoriasis is critical for the development of novel and potentially more effective treatments. Summary of the invention

[0004] The purpose of the present disclosure is to provide an application of an OASL antagonist in the preparation of a medicament for treating psoriasis.

[0005] Optionally, in the application as described above, the functions of the antagonist include one or more of the following:

[0006] Reduce the expression of OASL, reduce the translation of OASL, and inhibit the activity of OASL.

[0007] Optionally, as used above, the antagonist does not inhibit STAT1 and / or JAK1.

[0008] Optionally, as described above, the antagonist comprises at least one of antisense oligonucleotides, dsRNA, siRNA, shRNA, miRNA, RNAi and ribozymes, which are designed to reduce the expression of OASL.

[0009] Optionally, as described above, the antagonist comprises at least one of an antibody or antigen-binding fragment thereof, a small molecule compound, and an aptamer to OASL to reduce or offset the activity of OASL.

[0010] Optionally, as used above, the antagonist knocks down or knocks out the OASL gene.

[0011] Optionally, as described above, the antagonist is a CRISPR-CAS gene editing composition comprising gRNA and CRISPR enzyme.

[0012] Optionally, as described above, the antagonist is used alone or in combination with other drugs.

[0013] Optionally, in the application described above, the antagonist and pharmaceutically acceptable excipients constitute a pharmaceutical composition.

[0014] Optionally, for the use described above, the pharmaceutical composition is in a form selected from one or more of solution, injection, spray, nasal drops, aerosol, powder spray, tablet, capsule and granule.

[0015] The beneficial effects of the present disclosure are:

[0016] It was found that OASL promoted the production of inflammatory factors and lipid metabolism disorders by promoting the MAPK signaling pathway, thereby promoting cell proliferation, confirming the pathogenic role of OASL in psoriasis. The addition of the JAK1 selective inhibitor upadacitinib in vitro found that upadacitinib could inhibit the expression of OASL in a dose-dependent manner by regulating the activity of STAT1. Therefore, it was confirmed that OASL is a downstream factor of the JAK-STAT1 pathway and plays a role in the onset of psoriasis; by accurately positioning the therapeutic target, the side effects of psoriasis treatment can be effectively reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the specific embodiments of the present disclosure or the technical solutions in the prior art, the drawings required for use in the specific embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1:AC: Volcano plots of differentially expressed genes in GSE114286, GSE166388, and GSE186117; D: Venn diagrams of GSE114286, GSE166388, and GSE186117; E: PPI networks of the communal DEGs were constructed by Cytoscape software; F: GO and KEGG bubble plots of commonly upregulated genes. G: Immunohistochemistry was used to detect the expression levels of OASL in the skin of psoriasis patients and normal subjects.

[0019] Figure 2 :A, cell proliferation activity of shRNA-control, shRNA-OASL-1, and shRNA-OASL-2; B, G0 / G1, S, and G2 / M phase ratios of shRNA-control, shRNA-OASL-1, and shRNA-OASL-2; C, EUD fluorescence staining of shRNA-control, shRNA-OASL-1, and shRNA-OASL-2 after M5 stimulation and normal cells; D, quantitative analysis of EDU fluorescence staining; E, KRT1 and KRT5 protein expression levels and semi-quantitative analysis after shRNA-control and shRNA-OASL.

[0020] Figure 3 : A. RT-qPCR was used to detect the expression levels of inflammatory factors in shRNA-control and shRNA-OASL after M5 stimulation; B. RT-qPCR was used to detect the lipid metabolism levels in shRNA-control and shRNA-OASL after M5 stimulation; C. FFA kit was used to detect the intracellular FFA content in shRNA-control and shRNA-OASL, as well as after M5 stimulation; D. WB was used to detect the expression of p-ERK, ERK, pp38, p38, pJNK, and JNK2 after M5 stimulation after OASL knockdown; E. Semi-quantitative analysis of MAPK signaling pathway.

[0021] Figure 4:A, OASL overexpression efficiency detection; B, vector and OASL overexpression (OE) EUD fluorescence staining; C, detection of KRT1, KRT5 mRNA expression after OASL overexpression; D, KRT1, KRT5 protein expression after OASL overexpression; E, expression of inflammatory factors in vector and OE groups; F, changes in IL-17A protein expression after OASL overexpression and semi-quantitative analysis; G, changes in cellular lipid metabolism after OASL overexpression; H, ACC activity after OASL overexpression; I, intracellular FFA content after OASL overexpression; J, changes in p-ERK, ERK, pp38, p38, p-JNK, JNK2 pathway protein expression after OASL overexpression; K, semi-quantitative analysis of MAPK pathway-related proteins

[0022] Figure 5 :A, Effect of upadacitinib on cell activity; B, Effect of upadacitinib on OASL mRNA expression; C, Effect of upadacitinib on OASL protein expression; D, Effect of upadacitinib on OASL and STAT1 expression; E, Effect of upadacitinib on the expression of inflammatory factors after M5 stimulation; F, Effect of upadacitinib on lipid metabolism of Hacat cells after M5 stimulation; G, Effect of upadacitinib on intracellular FFA content after M5 stimulation. DETAILED DESCRIPTION

[0023] References to embodiments of the present disclosure will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present disclosure. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present disclosure without departing from the scope or spirit of the present disclosure. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0024] Unless otherwise indicated, the meaning of all terms (including technical and scientific terms) used to disclose the present disclosure is the same as that commonly understood by those of ordinary skill in the art to which the present disclosure belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present disclosure. The terms used herein in the specification of the present disclosure are only for the purpose of describing specific embodiments and are not intended to limit the present disclosure.

[0025] In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation procedures used herein are terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present disclosure, the definitions and explanations of the relevant terms are provided below.

[0026] The terms "and / or", "or / and", and "and / or" used herein have a selection range that includes any one of two or more related listed items, and also includes any and all combinations of related listed items, wherein the any and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in the present disclosure, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0027] As used in this disclosure, the terms "comprising," "including," and "including" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps.

[0028]

[0013] Numerical ranges recited in this disclosure as endpoints include all numbers and fractions subsumed within that range, as well as the recited endpoints.

[0029] As used herein, reference to "about" a value or parameter includes (and describes) embodiments directed to the value or parameter itself. For example, description referring to "about X" includes description of "X".

[0030] The present disclosure relates to concentration values, and the meaning includes fluctuations within a certain range. For example, fluctuations can be allowed within a corresponding precision range. For example, 2%, a fluctuation within the range of ±0.1% can be allowed. For values ​​that are large or do not require too fine control, the meaning is also allowed to include greater fluctuations. For example, 100mM, fluctuations within the range of ±1%, ±2%, ±5%, etc. can be allowed. Involving molecular weight, the meaning is allowed to include fluctuations of ±10%.

[0031] As used herein, the singular articles "a," "an," and "the" include plural referents unless otherwise indicated.

[0032] In the present disclosure, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.

[0033] In the present disclosure, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0034] In the present disclosure, "preferred", "better", "more preferred", and "suitable" are only used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute a limitation on the scope of protection of the present disclosure. In the present disclosure, "optionally", "optional", and "optional" mean optional, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0035] All documents mentioned in the present disclosure are cited as references in the present disclosure, just as each document is cited as a reference individually. Unless they conflict with the invention objectives and / or technical solutions of the present disclosure, the cited documents involved in the present disclosure are cited with all their contents and all their purposes. When the present disclosure involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the present disclosure involves cited documents, the examples and preferred methods of the cited relevant technical features may also be incorporated into the present disclosure as references, but are limited to the implementation of the present disclosure. It should be understood that when the cited content conflicts with the description in the present disclosure, the present disclosure shall prevail or be modified adaptively according to the description of the present disclosure.

[0036] The present disclosure relates to the use of an antagonist of OASL in the preparation of a medicament for treating psoriasis.

[0037] In some embodiments, the functions of the antagonist include one or more of the following:

[0038] Reduce the expression of OASL, reduce the translation of OASL, and inhibit the activity of OASL.

[0039] In some embodiments, the antagonist comprises at least one of an antisense oligonucleotide, a dsRNA, a siRNA, a shRNA, a miRNA, an RNAi, and a ribozyme, which is designed to reduce the expression of OASL.

[0040] siRNA refers to double-stranded RNA that inhibits the expression of a target nucleic acid, which means "RNAi agent", "short-chain interfering RNA", "short-chain interfering nucleic acid" and "siRNA". The nucleic acid molecule is capable of inhibiting or down-regulating gene expression or viral replication through sequence-specific RNA interference (RNAi) or gene silencing. This can consist of RNA alone, or it can be a fusion of DNA and RNA. siRNA can be prepared according to conventional methods based on information obtained by searching the target nucleic acid and the NCBI database.

[0041] Ribozymes are RNA molecules that specifically cleave other single-stranded RNA molecules by a mechanism similar to DNA restriction endonucleases. Ribozymes used to recognize and cleave specific base sequences in single-stranded RNA can be produced by appropriately modifying the nucleic acid sequence of RNA using known techniques (Science, 239, p. 1412-1416, 1988).

[0042] In some embodiments, the antagonist comprises at least one of an antibody or antigen-binding fragment thereof, a small molecule compound, and an aptamer to OASL to reduce or counteract the activity of OASL.

[0043] In one embodiment of the present disclosure, the OASL antagonist may be a substance that specifically binds to the OASL protein. The substance that specifically binds to the OASL protein may be a substance selected from the group consisting of antibodies, antibody fragments, and aptamers.

[0044] Antibodies include, for example, polyclonal antibodies, monoclonal antibodies, chimeric antibodies and humanized antibodies. Examples of antibodies include full-length antibodies, single-chain molecules, bifunctional molecules, scFv, diabodies (Exody), single domain antibodies (VHH), chimeric antibodies and immunoadhesion. "Antibody fragments" include fragments of Fv, Fv', Fab, Fab' and F(ab')2. If it is related to the present disclosure, "antibody" means a polypeptide containing one or more regions that bind to the epitope of the target antigen. Antibodies to OASL can be prepared by any method known in the art. For example, monoclonal antibodies may be produced by immunizing mice with recombinant OASL or one or more synthetic peptides (including epitopes that are considered to be highly antigenic). Alternatively, polyclonal antibodies can also be raised by immunizing rabbits with the above antigens. Once produced using hybridoma technology, it is preferred that the monoclonal antibodies are humanized by exchanging mouse and human frameworks and constant regions.

[0045] "Antibody fragments" can be obtained by treating antibodies with proteases such as enzymes such as papain and pepsin (Morimoto et al., J. Biochem. Biophys. Methods (1992) 24: 107-17; Brennan et al., Science (1985) 229: 81). Antibodies can also be produced by genetic recombination based on the amino acid sequence of antibody fragments.

[0046] A low molecular weight antibody having a modified structure of an "antibody fragment" can be constructed by utilizing an antibody fragment obtained by enzyme treatment or gene recombination. Alternatively, a gene encoding the entire low molecular weight antibody is constructed and introduced into an expression vector and then expressed in a suitable host cell (e.g., Co et al., J. Immunol. (1994) 152: 2968-76; Better and Horvitz, Methods Enzymol. (1989) 178: 476-96; Pluckthun and Skerra, Methods Enzymol. (1989) 178: 497-515; Lamoy, Methods Enzymol. (1986) 121: 652-63; Rousseaux et al., Methods Enzymol. (1986) 121: 663-9; Bird and Walker, Trends Biotechnol. (1991) 9: see 132-7.

[0047] In the present disclosure, an aptamer is a nucleic acid molecule or a peptide molecule that binds to a specific target molecule. Aptamers are usually produced by selecting them from a large random array pool, but natural aptamers are also present on riboswitches. The aptamer can be used as a polymer drug for basic research and clinical purposes. Aptamers can be combined with self-cleaving ribozymes in the presence of target molecules. More specifically, aptamers can be classified as nucleic acid aptamers, such as DNA or RNA aptamers, or classified as peptide aptamers. The former is usually composed of oligonucleotide chains (usually shorter), while the latter is preferably composed of short variable peptide domains connected to both ends of a protein scaffold. In principle, nucleic acid aptamers bind to a variety of molecular targets, including small molecules, proteins, nucleic acids, and even cells, tissues, and even organisms themselves. In vitro selection or equivalent SELEX (systematic evolution of exponentially enriched ligands) is a nucleic acid species manipulated by repeated rounds of enrichment (enrichment). Peptide aptamers are usually peptides or proteins designed to interfere with the interaction of other proteins in cells. They are composed of variable peptide loops connected to both ends of a protein scaffold. This dual structural constraint increases the binding affinity of the peptide aptamer to a level that is large and equal to that of antibodies (nanomolar range). The variable peptide loop typically contains 10-20 amino acids, and the scaffold can be any protein with good solubility properties. Currently, the bacterial protein thioredoxin A is the most commonly used scaffold protein, with -Cys-Gly-Pro-Cys-loop in the wild-type protein. In the embedding of the variable peptide loop in the redox active site, the two cysteines in the side chain can form a disulfide cross-link. The selection of peptide aptamers can be carried out using different systems, but the most widely used one now is the yeast to hybrid system.

[0048] Small molecule compounds can bind to OASL protein to inhibit its enzymatic activity, or block or reduce its binding with its downstream effector molecules.

[0049] In some embodiments, the antagonist knocks down or knocks out the OASL gene.

[0050] Knockdown or knockout can adopt any known existing technology, and examples include "suicide plasmid", RNA interference, zinc finger ribonuclease (ZFN) technology, TALEN (transcription activator-like (TAL) effector nucleases) technology, CRISPR technology, etc. In some embodiments, the antagonist is a CRISPR-CAS gene editing composition, which comprises gRNA and CAS enzyme (such as Cas9). There are two common gene knockout strategies, one is single sgRNA-mediated gene knockout, which causes gene frameshift mutations through the InDels generated in the exons of the coding gene by the CRISPR-Cas9 system, thereby achieving the purpose of gene knockout. Therefore, this gene knockout strategy is limited to the knockout of coding genes; the other is two sgRNA-mediated gene knockout, which acts on both sides of the target fragment by two sgRNAs to achieve the deletion of the target fragment sequence, thereby achieving the knockout of the target gene.

[0051] In some embodiments, the antagonist is used alone or in combination with other drugs. For example, it is used in combination with an inhibitor of MAPK, and in combination with a phosphorylation inhibitor of p38 and / or JNK2. In some embodiments, the antagonist does not inhibit STAT1. In some embodiments, the antagonist does not inhibit JAK1. In some embodiments, the present disclosure also relates to the use of a phosphorylation inhibitor of p38 and / or JNK2 in the treatment of psoriasis. In some embodiments, the present disclosure also relates to the use of an inhibitor of MAPK in the treatment of psoriasis.

[0052] In some embodiments, the antagonist and a pharmaceutically acceptable excipient constitute a pharmaceutical composition.

[0053] A pharmaceutical composition generally refers to a drug used for the treatment or prevention of a disease, or for examination or diagnosis.

[0054] Pharmaceutical composition can be prepared by methods well known to those skilled in the art. For example, it can be used parenterally in the form of sterile solutions or suspensions of pharmaceutically acceptable solutions other than water or water. For example, suitable combinations of pharmacologically acceptable carriers or media, specifically sterile water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative, adhesive, etc. It is considered to be prepared by mixing in the unit dosage form required for the implementation of generally accepted drugs. The amount of active ingredient in these preparations is set to obtain suitable capacity within the range shown.

[0055] The sterile composition for injection can be formulated according to a common preparation using excipients such as distilled water for injection.

[0056] As the aqueous solution for injection, for example, isotonic solution containing physiological saline solution, glucose and other supplements (such as D-sorbitol, D-mannose, D-mannitol, sodium chloride) can be cited. Suitable dissolution aids such as alcohols (ethanol, etc.), polyols (propylene glycol, polyethylene glycol, etc.) and nonionic surfactants (polysorbate 80 (TM), HCO-50, etc.) can be used in combination.

[0057] As the oily liquid, sesame oil and soybean oil can be cited, and benzyl benzoate and / or benzyl alcohol can be used in combination as a dissolution aid. The composition can be mixed with a buffer (e.g., phosphate buffer solution and sodium acetate buffer solution), a painless agent (e.g., procaine hydrochloride), a stabilizer (e.g., benzyl alcohol and phenol) and an antioxidant. The prepared injection is usually contained in an appropriate ampoule.

[0058] The pharmaceutical composition is preferably administered parenterally. For example, in some embodiments, the pharmaceutical composition is in the form of one or more selected from solutions, injections, sprays, nasal drops, aerosols, powder sprays, tablets, capsules, and granules. For example, it can be administered systemically or topically by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc.

[0059] The method of administration can be appropriately selected according to the age and symptoms of the patient. The dosage of the active ingredient can be set within the range of, for example, 0.0001 mg to 1000 mg per kg body weight each time. Alternatively, for example, the dosage can be 0.001-100000 mg / patient, but the disclosure is not necessarily limited to these values. The dosage and method of administration vary according to the patient's weight, age, symptoms, etc., but those skilled in the art can consider these situations to set appropriate dosages and methods of administration.

[0060] The embodiments of the present disclosure will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present disclosure and are not intended to limit the scope of the present disclosure. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance given in the present disclosure, and can also be based on the experimental manuals or conventional conditions in the art, and can also be based on other experimental methods known in the art, or according to the conditions recommended by the manufacturer.

[0061] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0062] Example 1 OASL expression is increased in the skin of patients with psoriasis

[0063] By querying the GEO database, we selected three datasets containing skin samples from psoriasis patients and healthy controls: GSE114286, GSE166388, and GSE186117. Differential gene expression analysis was performed for each dataset, and a volcano plot was generated ( Figure 1 At the same time, we made a Venn diagram to show the commonly up-regulated genes in the three datasets, and the results revealed 129 shared up-regulated genes ( Figure 1 D in the figure). Protein-protein interaction (PPI) network analysis was further performed on these 129 commonly upregulated genes, and the top 10 key genes were identified, including STAT1, CXCL10, CXCL8, OASL, and CXCL1. GO and KEGG enrichment analysis showed that GO was mainly enriched in viral infection, cytoplasmic vesicle cavity, and cytokine activity, while KEGG pathways were enriched in cytokine-cytokine receptor interaction, interaction between viral proteins and cytokines and cytokine receptors, and IL-17 signaling pathway ( Figure 1 EF in ). OASL, as a gene related to interferon, has not been reported in the context of psoriasis. Immunohistochemical staining of skin tissues of psoriasis patients and healthy controls showed that the expression of OASL in the skin of psoriasis patients was significantly increased compared with that of healthy controls ( Figure 1 G in ), and is mainly localized in the epidermis. Therefore, we confirmed that the expression level of OASL in the skin of psoriasis patients is significantly increased.

[0064] Example 2 OASL knockdown inhibits HaCaT cell proliferation and differentiation

[0065] Given that immunohistochemistry results showed that OASL was mainly localized in the epidermis, we further explored the role of OASL in psoriasis. We chose HaCaT cells as a model for this study because they are the most abundantly expressed cells in the epidermis. Using shRNA plasmid transfection technology, we constructed HaCaT cells with OASL knockdown. Pathological changes in the epidermis of psoriatic skin are manifested as hyperkeratosis and parakeratosis, and these changes are due to excessive proliferation of keratinocytes. We performed CCK8 experiments on HaCaT cells with OASL knockdown, and the results showed that cell proliferation was significantly restricted compared with normal HaCaT cells ( Figure 2 Further cell cycle analysis showed that OASL knockdown cells were arrested in the G0 / G1 phase and had a shortened S phase ( Figure 2B in the figure). M5 (composed of IL-22, IL-17A, IL-1α, TNF-α, and tumor growth factor M) is commonly used as an in vitro stimulation model for psoriasis. EDU is a common method for detecting cell proliferation. Our results showed that the proportion of EDU-positive cells in the OASL knockdown group was significantly reduced compared with the shRNA control group. After M5 stimulation, the proportion of EDU-positive cells in the shRNA control group increased, and although the number of positive cells in the OASL knockdown group also increased, its proportion was still significantly lower than that in the control group ( Figure 2 C in the figure). Therefore, OASL knockdown inhibited the proliferation of HaCaT cells in vitro. KRT1 and KRT5 are commonly used as markers of HaCaT cell differentiation and proliferation. We found that OASL knockdown inhibited the expression of KRT5 and KRT1 in HaCaT cells ( Figure 2 Therefore, OASL knockdown inhibited the proliferation and differentiation of HaCaT cells in vitro.

[0066] Example 3 OASL knockdown slowed down the expression of inflammatory factors and lipid metabolism levels in HaCat cells induced by M5

[0067] To further explore the role of OASL in the M5-induced in vitro psoriasis-like model, we detected the expression of inflammatory cytokines in M5-stimulated cells and found that OASL knockdown inhibited the expression of S100A8, S100A9, CCL2, CCL20, IL-6, and IL-8 ( Figure 3 Meanwhile, previous studies have demonstrated that M5 stimulation can induce changes in lipid metabolism in HaCaT cells, and our molecular function (MF) enrichment analysis of public databases highlighted changes in the cytoplasmic vesicle lumen. Therefore, we further investigated the changes in lipid metabolism after M5 stimulation. We found that OASL knockdown reduced lipid metabolism in HaCaT cells and inhibited the M5-induced increase in lipid metabolism, especially lipid uptake and lipid oxidation ( Figure 3 B in Figure 2). Non-esterified fatty acids (FFAs) are fatty acids that are highly sensitive to factors such as lipid metabolism, glucose metabolism, and endocrine function. The uptake of FFAs is affected by transporters on the cell membrane, such as the fatty acid translocase FAT / CD36. OASL knockdown reduced the levels of FFAs in HaCaT cells and inhibited M5-induced lipid uptake ( Figure 3 C in the figure). The mitogen-activated protein kinase (MAPK) signaling pathway is an important downstream target that mediates cellular responses to external stimuli. Cells can sense and respond to their extracellular environment by activating cell surface receptors. OASL knockdown inhibited M5 activation of the MAPK signaling pathway, mainly by reducing the phosphorylation of JNK2 and p38 ( Figure 3 DE in ).

[0068] Example 4 OASL overexpression promotes HaCaT cell proliferation, inflammatory factor expression and lipid metabolism level

[0069] We further constructed HaCaT cells overexpressing OASL ( Figure 4 A), EUD detection of cell proliferation found that the percentage of EUD-positive cells in HaCaT cells overexpressing OASL increased significantly, and was comparable to the result after M5 stimulation ( Figure 4 At the same time, OASL overexpression promoted the expression of KRT1 and KRT5 ( Figure 4 CD in ). It can be seen that OASL overexpression promoted the proliferation of HaCaT cells. Similarly, we detected the levels of inflammatory factors after OASL overexpression and found that the expression of S100A7, S100A8, S100A9, CXCL10, CCL2, CCL20, IL-6, and IL-8 were all increased after OASL overexpression ( Figure 4 E), which is consistent with the increase of inflammatory factors after M5 stimulation. We then detected the increase in IL-17A expression after OASL overexpression ( Figure 4 F in the figure). We detected the changes in lipid metabolism after OASL overexpression and found that lipid metabolism increased after OASL overexpression, including lipid uptake, lipid production, and lipid oxidation ( Figure 4 G in the figure), acetyl-CoA carboxylase (ACC) is the key rate-limiting enzyme in fatty acid synthesis. It catalyzes the conversion of acetyl-CoA (Acetyl-CoA) into malonyl-CoA (Malonyl-CoA), which is the first and rate-limiting step in fatty acid synthesis. ACC activity detection found that ACC activity increased after OASL overexpression ( Figure 4 H in the cells), the intracellular FFA content also increased ( Figure 4 After OASL overexpression, the MAPK signaling pathway is activated, mainly through the increase of phosphorylation of JNK2 and p38 ( Figure 4 JK in ). Therefore, OASL overexpression promoted the proliferation and differentiation of HaCaT cells, and at the same time mimicked the effects of M5 stimulation, promoting the production of inflammatory factors and increased lipid uptake and production through MAPK, and promoting cell proliferation and the production of inflammatory factors through MAPK.

[0070] Example 5 Upadacitinib inhibits the secretion of inflammatory factors and lipid metabolism disorders stimulated by M5 by inhibiting the production of OASL

[0071] The JAK-STAT pathway is a key signaling pathway for cytokine signal transduction and plays an important regulatory role in cell proliferation, differentiation, apoptosis, and immune response. Upadacitinib is a selective JAK inhibitor that has been approved by multiple regulatory agencies, including the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA), for the treatment of various chronic inflammatory diseases, such as rheumatic diseases, skin diseases, and gastrointestinal diseases. Nuclear activation of STAT1 is dependent on JAK kinases. Previous studies have shown that JASPAR prediction identifies STAT1 binding sites in the OASL promoter and that OASL expression in gastric cancer is regulated by the transcription factor STAT1. We first evaluated the effects of different concentrations of upadacitinib on the viability of HaCaT cells. The results showed that different concentrations of upadacitinib had very limited effects on cell viability within 24 and 48 hours ( Figure 5 Next, we pretreated the cells with different concentrations of upadacitinib and then stimulated them with M5. We found that the inhibitory effect of upadacitinib on OASL expression increased with increasing concentration ( Figure 5 BC in ), and upadacitinib also inhibited the activation of STAT1 ( Figure 5 D). Since OASL knockdown can inhibit M5-induced inflammatory cytokine secretion, we pretreated the cells with different concentrations of upadacitinib and subsequently stimulated them with M5, and observed that upadacitinib reduced the M5-induced secretion of S100A7, S100A8, S100A9, CCL2, CCL20, and IL-6 ( Figure 5 E), but had no effect on the expression of CXCL10. Similar to the effect of OASL knockdown, upadacitinib inhibited the increase in lipid metabolism induced by M5 ( Figure 5 and reduced the production of intracellular FFAs ( Figure 5 Thus, upadacitinib inhibits OASL production by inhibiting STAT1 and reduces the production of inflammatory cytokines and lipid metabolism induced by M5.

[0072] discuss

[0073] Our study identified common upregulated molecules in transcriptome samples of different psoriasis patients, and then focused on OASL through PPI network analysis and literature search. The role of OASL in the pathogenesis of psoriasis was verified by intervening in its expression. Inhibition of OASL can inhibit the production of inflammatory factors and lipid metabolism uptake in vitro through the MAPK signaling pathway, thereby inhibiting cell proliferation.

[0074] OASL is a member of the OAS family. Unlike other family members, OASL lacks 2'-5' linked OAS activity. This public study found that knocking down OASL can inhibit the proliferation and differentiation of HaCaT cells induced by M5 in vitro. HaCaT cells are a spontaneously immortalized human keratinocyte cell line that retains the characteristics of differentiation and can be used to study the differentiation state of keratinocytes. In the proliferation and differentiation of keratinocytes, KRT1 and KRT5 play an important regulatory role and can be used as markers of cell proliferation and differentiation. The proliferation and differentiation of keratinocytes are closely related. Calcium ions are considered to be the key inducer of keratinocyte differentiation, and the increase in intracellular calcium ion concentration promotes cell differentiation. It has been reported that OASL can participate in the regulation of intracellular signal transduction through liquid phase separation (LLPS) and play a role in viral infection. Calcium ions can affect the process of LLPS and affect the function of cells. Therefore, OASL may affect the state of intracellular calcium ions through LLPS, thereby promoting the proliferation and differentiation of HaCaT cells.

[0075] We found that after OASL overexpression, cell proliferation accelerated, and was mainly affected by the phosphorylation of p38 and JNK2 in the MAPK family. Knockdown of OASL also inhibited the acceleration of cell proliferation stimulated by M5. Zhao et al. found in their study of gastric adenocarcinoma that OASL knockout can inhibit the proliferation of gastric cancer cells and inhibit the progression of gastric adenocarcinoma. This is also consistent with our research results. They further predicted and experimentally confirmed that STAT1 is the transcription factor of OASL through the JASPAR database. Therefore, we speculate that OASL may be regulated by STAT1 and participate in the occurrence and development of psoriasis. Upadacitinib is a selective JAK1 inhibitor. JAK1 phosphorylates and activates STAT1, thereby activating the JAK-STAT signaling pathway. Therefore, upadacitinib can block the phosphorylation and activation of STAT1. Therefore, we added upadacitinib to the in vitro psoriasis model to inhibit STAT1 transcriptional activation and found that the expression of OASL was inhibited in a dose-dependent manner. At the same time, the expression of inflammatory factors induced by M5 was also significantly reduced. Although clinical trials have found that upadacitinib can significantly improve the skin lesions of psoriasis patients, its clinical application is limited due to its side effects such as severe infection, malignant tumors, cardiovascular events, etc. Our study found that OASL is a downstream factor of upadacitinib and inhibited the M5-induced psoriasis-like model in vitro, which provides a new direction for the treatment of psoriasis.

[0076] In patients with psoriasis, lipid expression and metabolism in the skin and blood are significantly altered. Lipids play an important role in skin inflammation, especially bioactive molecules such as eicosanoids, endocannabinoids, and ceramides. Ceramides are formed by the combination of ceramides and N-acylated fatty acids and show remarkable molecular diversity. In psoriatic lesions, the levels of ceramide phosphate (CerP) and ceramides, both of which are derivatives of ceramides, are increased. Ceramide-1-phosphate can induce the expression of inflammatory factors IL-6, IL-1β, and IL-17A in keratinocytes, activate DNA replication and cell cycle signaling pathways, and increase the involvement of arachidonic acid in the inflammatory process. Our study found that OASL enhanced the level of lipid metabolism in cells. Under M5 stimulation, OASL knockdown inhibited the increase in lipid metabolism induced by M5. OASL may interfere with intracellular lipid metabolism through LLPS properties, leading to dysregulation of intracellular lipid metabolism.

[0077] MAPK signaling pathways play a key role in regulating the proliferation and differentiation of keratinocytes and the expression of proinflammatory and chemokines in psoriasis. In psoriatic lesions, the protein expression levels of JNK, p38, and ERK were significantly higher than those in normal skin. In epidermal keratinocytes, activation of the p38 MAPK pathway promotes IL-17-mediated immune responses and the progression of psoriatic dermatitis. In addition, hyperphosphorylation of p38 can increase the expression of S100A8 in human serum. The JNK signaling pathway is activated by IL-22, which promotes the proliferation and migration of keratinocytes, while downregulating a variety of epidermal barrier proteins, leading to the production of a variety of cytokines and chemokines, thereby recruiting a large number of immune cells and aggravating psoriasis. In addition, lipids can directly and indirectly affect the MAPK signaling pathway. Directly, specific lipid binding can regulate the subcellular localization and kinase activity of MAPK signaling proteins; indirectly, since most MAPK signals occur on intracellular endosomes, lipids can affect the physicochemical properties of membrane lipids by regulating endocytosis and stimulation by growth factors. We found that overexpression of OASL accelerated cell proliferation, mainly mediated by phosphorylation of p38 and JNK2 in the MAPK family. Conversely, knockdown of OASL also inhibited the accelerated cell proliferation induced by M5 stimulation. Therefore, OASL may disrupt intracellular lipid metabolism through its LLPS properties, leading to changes in lipid metabolism levels, thereby regulating cell growth and the production of inflammatory factors through the intracellular MAPK signaling pathway.

[0078] The above-described embodiments only express several implementation methods of the present disclosure, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present disclosure, and these all belong to the protection scope of the present disclosure. Therefore, the protection scope of the patent of the present disclosure shall be based on the attached claims, and the description and drawings may be used to interpret the contents of the claims.

Claims

1. Use of OASL antagonists in the preparation of drugs for treating psoriasis.

2. The use according to claim 1, characterized in that: The functions of the antagonist include one or more of the following: Reduce the expression of OASL, reduce the translation of OASL, and inhibit the activity of OASL.

3. The use according to claim 1, characterized in that: The antagonist does not inhibit STAT1 and / or JAK1.

4. The use according to claim 1, characterized in that: The antagonist comprises at least one of antisense oligonucleotides, dsRNA, siRNA, shRNA, miRNA, RNAi and ribozymes, which are designed to reduce the expression of OASL.

5. The use according to claim 1, characterized in that: The antagonist comprises at least one of an antibody or an antigen-binding fragment thereof, a small molecule compound, and an aptamer to OASL, so as to reduce or offset the activity of OASL.

6. The use according to claim 1, characterized in that: The antagonist knocks down or knocks out the OASL gene.

7. The use according to claim 6, characterized in that: The antagonist is a CRISPR-CAS gene editing composition, which includes gRNA and CRISPR enzyme.

8. The use according to any one of claims 1 to 7, characterized in that: The antagonists are used alone or in combination with other drugs.

9. The use according to any one of claims 1 to 7, characterized in that: The antagonist and pharmaceutically acceptable excipients form a pharmaceutical composition.

10. The use according to claim 9, characterized in that: The pharmaceutical composition is in the form of one or more selected from the group consisting of solution, injection, spray, nasal drops, aerosol, powder spray, tablet, capsule and granule.

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