Application of NCF2 in treatment of psoriasis

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of NCF2 in treatment of psoriasis. In order to solve the technical problem of how to treat the psoriasis, the invention provides a new application of a key component NCF2 (Neurophil Cytological Factor 2) of an NADPH oxidase compound in the treatment of the psoriasis. Experiments prove that after being activated, NCF2 / NOX2 plays a role through the following pathways: (i) promoting expression and functions of p53 and activating AMPK; (ii) reducing lactic acid accumulation to block HIF1 alpha stabilization; and (iii) inhibiting GSDME expression and activation through ROS space-time specific regulation and control. And by accurately positioning a treatment target, the side effects in the psoriasis treatment process can be effectively reduced. According to the invention, the limitation of the existing single-channel targeted therapy is broken through, and an innovative scheme of multi-dimensional intervention is provided for psoriasis treatment.

Description

Technical Field

[0001] The present disclosure belongs to the field of biomedical technologies, and more particularly, relates to the use of NCF2 in the treatment of psoriasis. Background Art

[0002] Psoriasis is an immune-mediated disease characterized by abnormal proliferation of epidermal keratinocytes and chronic inflammatory responses, with a global prevalence of approximately 2-3%. Its pathological mechanism involves multi-dimensional interactions of genetic susceptibility, immune dysregulation, and metabolic disorders. The clinical manifestations are erythematous and scaly skin lesions, often accompanied by systemic complications such as arthritis and cardiovascular diseases, causing a heavy socio-economic burden. Existing treatment methods mainly include topical treatment (glucocorticoids / vitamin D3 derivatives), phototherapy, and systemic immunosuppressants (methotrexate / cyclosporine A). Although they can relieve symptoms in the short term, long-term use is prone to adverse reactions such as skin atrophy, liver and kidney toxicity, and opportunistic infections. Moreover, about 30% of patients have a risk of treatment resistance or recurrence. Biological agents (TNF-α / IL-17 / IL-23 inhibitors) have significantly improved the efficacy in moderate-to-severe patients, but there are clinical limitations such as high treatment costs, an increased risk of infection (such as tuberculosis recurrence / opportunistic infection) with long-term use, primary or secondary failure (efficacy attenuation) in some patients, and a potential risk of cardiovascular events, highlighting the urgent need for precise intervention in the core driving mechanism of the disease.

[0003] At the molecular mechanism research level, it is currently believed that the epidermis of psoriasis has characteristics such as abnormal increase in glycolysis and oxidative stress disorder. Metabolomics research reveals a significant phenomenon of glycolytic reprogramming in psoriatic skin lesions, manifested as enhanced glycolysis (Warburg effect). This metabolic abnormality not only provides bioenergy for keratinocyte proliferation but also promotes the secretion of pro-inflammatory factors such as IL-1β / IL-6 through the accumulation of lactic acid microenvironment, forming a "metabolism-inflammation" vicious cycle. Recent studies have shown that the hyperactive glycolysis driven by the Warburg effect in psoriatic skin lesions is related to the abnormal activation of the mTOR-HIF1α pathway, and the imbalance of ROS metabolism can simultaneously exacerbate oxidative damage and the NF-κB-mediated inflammatory cascade reaction. However, there is currently no synergistic target that can simultaneously correct metabolic disorders and inflammatory responses. Existing metabolic intervention drugs (such as the hexokinase inhibitor 2-DG) cause systemic energy metabolism disorders due to lack of tissue specificity.

[0004] NCF2 (Neutrophil Cytosolic Factor 2), as the core regulatory subunit of the NADPH oxidase complex (NOX2), is mainly responsible for mediating the activation of the NOX2 enzyme and the generation of reactive oxygen species (ROS). Previous studies have mostly focused on its role in primary immunodeficiency diseases such as chronic granulomatous disease (CGD). Previous studies have found that NOX2 is a negative regulatory protein in autoimmune diseases including psoriasis. However, existing NOX2 activators often non-specifically activate the NFκB pathway, limiting their clinical application value. Its biological effects in epidermal keratinocytes and its mechanism of action on the metabolism-inflammation axis need to be elucidated urgently. Summary of the Invention

[0005] The technical problem to be solved by the present invention is how to treat psoriasis. To this end, the present invention provides the use of an NCF2 agonist, and the use can be at least one of the following: A1) Use in the treatment of psoriasis, A2) Use in inhibiting the activation of GSDME, A3) Use in enhancing the activity of the p53 signaling pathway, A4) Use in activating AMPK, A5) Use in inhibiting the expression of inflammatory factors, A6) Use in inhibiting the expression of IL-1β, A7) Use in inhibiting the expression of CXCL10, A8) Use in reducing the level of HIF-1α, A9) Use in reducing the production of lactic acid.

[0006] The present invention also provides the use of an NCF2 agonist in the preparation of a drug, and the use can be at least one of the following: B1) Use in the preparation of a drug for the treatment of psoriasis, B2) Use in the preparation of a drug for inhibiting the activation of GSDME, B3) Use in the preparation of a drug for enhancing the activity of the p53 signaling pathway, B4) Use in the preparation of a drug for activating AMPK, B5) Use in the preparation of a drug for inhibiting the expression of inflammatory factors, B6) Use in the preparation of a drug for inhibiting the expression of IL-1β, B7) Use in the preparation of a drug for inhibiting the expression of CXCL10, B8) Use in the preparation of a drug for reducing the level of HIF-1α, B9) Use in the preparation of a drug for reducing the production of lactic acid.

[0007] Furthermore, the dosage forms of the drug include: liquid preparations, solid preparations, emulsions, etc.

[0008] Furthermore, the drug also contains a pharmaceutically acceptable carrier.

[0009] It is understandable that based on the different dosage forms of the drug, the types and dosages of the carriers or excipients selected are also adjusted adaptively. The above carriers or excipients are all conventional reagents in the art.

[0010] The above-mentioned "pharmaceutically acceptable salts" are acid addition salts formed by the compound and an acid, or base addition salts formed by the compound and a base; wherein, the acids include but are not limited to: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, acetic acid, tartaric acid, salicylic acid, citric acid, malic acid, methanesulfonic acid, p-toluenesulfonic acid, lactic acid, pyruvic acid, maleic acid, succinic acid; the base addition salts include salts derived from inorganic bases and salts derived from organic non-toxic bases. The salts derived from inorganic bases include but are not limited to: aluminum, ammonium, potassium, sodium, iron, copper, calcium, ferrous, magnesium, lithium, manganese, and the salts derived from organic non-toxic bases include but are not limited to: primary, secondary, and tertiary amine salts, substituted amines including natural substituted amines, cyclic amines, ethanolamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine.

[0011] The above-mentioned "pharmaceutically acceptable carrier" is one or more compatible solid or liquid fillers or cream-like substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. The above-mentioned pharmaceutically acceptable carriers are all conventional adjuvants in the art.

[0012] In the above application, the functions of the agonist may include one or more of the following: C1) Up-regulating the expression of the NCF2 gene, C2) Enhancing the translation efficiency of the NCF2 protein, C3) Activating the activity of the NCF2 / NOX2 complex.

[0013] In the above application, the agonist does not activate NFκB.

[0014] Optionally, in the above application, the agonist may include at least one of the NCF2 protein, the NCF2 gene expression vector, and the NCF2 / NOX2 agonist, which is designed to increase the expression of NCF2 and / or enhance the activity of NCF2 / NOX2.

[0015] A ribozyme is an RNA molecule that specifically cleaves other single-stranded RNA molecules through a mechanism similar to that of DNA restriction endonucleases. A ribozyme for recognizing and cleaving a specific base sequence in a single strand of RNA can be produced by appropriately modifying the nucleic acid sequence of RNA using known techniques (Science, 239, p. 1412-1416, 1988).

[0016] In some embodiments, the agonist can be a small molecule compound, an antibody, an aptamer, or a gene editing tool.

[0017] 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 immunoadhesins. "Antibody fragments" include fragments of Fv, Fv′, Fab, Fab′, and F(ab′)2. If the present disclosure is involved, "antibody" means a polypeptide containing one or more regions that bind to the epitope of the antigen of interest. Antibodies against OASL can be prepared by any method known in the art. For example, monoclonal antibodies can be generated by immunizing mice with recombinant OASL or one or several synthetic peptides, including epitopes considered to be highly antigenic. Alternatively, polyclonal antibodies can also be raised by immunizing rabbits with the above antigens. Once generated using hybridoma technology, the monoclonal antibody is preferably humanized by exchanging the mouse and human frameworks and constant regions.

[0018] "Antibody fragments" can be obtained by treating antibodies with proteases such as enzymes, for example, 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 sequences of antibody fragments.

[0019] In some embodiments, the gene editing tool can be a CRISPR activation system.

[0020] In the above applications, the agonist can be used alone or in combination with other drugs.

[0021] In the above applications, the agonist and a pharmaceutically acceptable excipient form a pharmaceutical composition. The pharmaceutical composition is a topical preparation and contains a transdermal absorption promoter, preferably a liposome or nanoemulsion carrier.

[0022] Optionally, for the applications described above, the form of the pharmaceutical composition is selected from one or more of solutions, injections, sprays, nasal drops, aerosols, powder aerosols, tablets, capsules, and granules.

[0023] A pharmaceutical composition generally refers to a drug used for the treatment or prevention of diseases, or for examination or diagnosis.

[0024] The pharmaceutical composition can be formulated by methods well-known to those skilled in the art. For example, it can be used parenterally in the form of an injection of a sterile solution or suspension with water or a pharmaceutically acceptable solution other than water. For example, a pharmacologically acceptable carrier or medium is appropriately combined, specifically sterile water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative, binder, etc. It is considered to be formulated by mixing in a unit dosage form required for generally accepted pharmaceutical practice. The amount of the active ingredient in these preparations is set to obtain an appropriate volume within the indicated range.

[0025] The sterile composition for injection can be prescribed according to a usual formulation using an excipient such as distilled water for injection.

[0026] As an aqueous injection solution, an isotonic solution containing, for example, a physiological saline solution, glucose, and other supplements (such as D-sorbitol, D-mannose, D-mannitol, sodium chloride) can be cited. Appropriate solubilizing aids can be used in combination, such as alcohols (ethanol, etc.), polyols (propylene glycol, polyethylene glycol, etc.), and nonionic surfactants (polysorbate 80 (TM), HCO-50, etc.).

[0027] As an oily liquid, sesame oil and soybean oil can be cited, and benzyl benzoate and / or benzyl alcohol can be used in combination as a solubilizing aid. The composition can be mixed with a buffer (for example, phosphate buffer solution and sodium acetate buffer solution), a painless agent (for example, procaine hydrochloride), a stabilizer (for example, benzyl alcohol and phenol), and an antioxidant. The prepared injection is usually filled in an appropriate ampoule.

[0028] The pharmaceutical composition is preferably administered by parenteral administration. For example, in some embodiments, the form of the pharmaceutical composition is selected from one or more of solutions, injections, sprays, nasal drops, aerosols, powder aerosols, tablets, capsules, and granules. For example, it can be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc.

[0029] The present invention also provides a drug for treating psoriasis, and the drug contains the agonist described above.

[0030] In some embodiments, the agonist may be the LXA4 analog BML-111 (CAS# 78606-80-1). The BML-111 is a synthetic lipoxin A4 agonist with antioxidant and anti-inflammatory properties and can be administered intraperitoneally at 1 mg / kg / day or topically on the skin (5 mg / kg / day) in mice.

[0031] The research of the present disclosure reveals the key role of NCF2 in regulating keratinocyte inflammation and proliferation through metabolic reprogramming and epigenetic regulatory networks in inflammatory skin diseases. We found that the expression levels of the NOX2 family were significantly increased in the skin of inflammatory skin diseases - psoriasis and atopic dermatitis, and the function of NCF2 regulated the metabolism and proliferation of keratinocytes through p53-AMPK. Collectively, these results indicate that NCF2 is a negative regulator of psoriasis in the epidermis, and its normal function can maintain epidermal metabolism and control epidermal inflammation.

[0032] NOX2 as the "molecular sensor" of metabolic homeostasis: the balance hub of the pentose phosphate pathway and anaerobic respiration. Our results show that the expression and activation of NOX2 are dependent on the metabolic state of keratinocytes. Upon stimulation with inflammatory factors, enhanced glycolysis and anaerobic respiration are accompanied by upregulation of the expression of NOX2 family genes (CYBB, NCF2, NCF1), while inhibition of the pentose phosphate pathway significantly reduces its activity. This phenomenon is consistent with the "metabolism-oxidative stress coupling" model proposed in recent studies. Mice deficient in Ncf1 have metabolic resistance, which may explain why the expression level of NOX2 decreases when the pentose phosphate pathway is inhibited - cells regulate the content of the important anabolic raw material NADPH through the expression level of NOX2. Notably, the glycolysis inhibitor 2-DG further exacerbates NOX2 expression, suggesting that when glucose metabolism is limited, cells may activate the pentose phosphate pathway by upregulating NOX2 to maintain NADPH homeostasis and thus resist oxidative damage. However, knockout of NCF2 leads to lactate accumulation and overactivation of HIF1α, indicating that NOX2 inhibits excessive glycolytic shift by regulating the P53-AMPK axis, and this mechanism may be the core pathway for it to maintain metabolic homeostasis.

[0033] Bidirectional regulation of the NOX2-P53-AMPK signaling axis: A molecular bridge connecting metabolism and inflammation. We found that the deletion of NCF2 led to downregulation of P53 and its downstream targets (MDM2, AIFM2), and at the same time, the phosphorylation level of AMPK decreased, which echoed the classical theory that P53 affects cell metabolism by regulating mitochondrial function. Notably, as a transcriptional regulator of NCF2, the functional antagonism between TP63 and P53 may explain the contradictory phenomenon of high expression of NOX2 but inhibited P53 activity in psoriasis. In addition, the inhibition of AMPK may exacerbate inflammation by activating the NLRP3 inflammasome or enhancing the GSDME-dependent pyroptosis pathway, which provides a mechanistic explanation for the aggravation of the phenotype in the IMQ mouse model by NCF2 deletion. In summary, the NOX2-P53-AMPK axis can establish a dynamic balance between keratinocyte proliferation and inflammatory response by integrating metabolic signals and epigenetic regulation. Currently, there is a lack of drugs targeting and activating NCF2 / NOX2. According to previous reports, FPR2 activation regulates oxidative phosphorylation through NOX2 without activating NFκB. BML-111 significantly improves inflammation by activating the FPR2-NOX2 axis, which is in sharp contrast to the pro-inflammatory function of NOX2 in phagocytes. This cell type-dependent effect may stem from the unique metabolic microenvironment of keratinocytes - high glycolytic activity and limited mitochondrial respiration make them more dependent on NOX2 to maintain redox homeostasis. Recent studies suggest that FPR2 agonists may coordinate metabolic and inflammatory signals by remodeling the mitochondrial-endoplasmic reticulum contact sites (MERCs), which is consistent with our observation that BML-111 alleviates the glycolytic disorder in keratinocytes.

[0034] The beneficial effects of the present disclosure are as follows: It is found that inhibiting NCF2 / NOX2 promotes the disorder of glucose metabolism by inhibiting the p53-AMPK signaling pathway, thereby promoting cell proliferation and the generation of inflammatory factors, and confirming the protective role of NCF2 / NOX2 in psoriasis. After activation, NCF2 / NOX2 exerts its effects through the following pathways: (i) promoting the expression and function of p53 and activating AMPK; (ii) reducing lactate accumulation to block the stabilization of HIF1α; (iii) inhibiting the expression and activation of GSDME through spatio-temporally specific regulation of ROS. By precisely targeting the therapeutic target, the side effects during the treatment of psoriasis can be effectively reduced.

[0035] Based on the new mechanism analysis of the NCF2 / NOX2 / P53 axis, the present invention breaks through the limitations of existing single-pathway targeted therapies and provides an innovative solution for multi-dimensional intervention in the treatment of psoriasis. The present invention belongs to the field of biomedical technology, and specifically relates to a new use of NCF2, a component of the NADPH oxidase complex, in the treatment of psoriasis, and particularly relates to the application of an intervention strategy based on the NOX2 / P53 signaling pathway regulated by NCF2 in the preparation of drugs for the treatment of psoriasis. Brief Description of the Drawings

[0036] To more clearly illustrate the specific embodiments of the present disclosure or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present disclosure. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0037] Figure 1 : A-C show the expression levels of lactic acid / inflammatory factors and NOX2 family genes in normal HaCaT cells and after stimulation with TI (TNF-α / IFN-γ), 2-DG, and FX-11 respectively; D: Western blot (WB) was used to detect the expression of CYBB, NCF4, NCF1, p-NCF1, and Pan-Kla after stimulation with TI, 2-DG, LDC7559, and FX-11; E: Schematic diagram of the mechanism of action of three glucose metabolism regulators.

[0038] Figure 2 : A: Volcano plot showing differentially expressed genes between NCF2 overexpression (OE) and NCF2 knockdown (shNCF2); B: KEGG enrichment analysis showing differentially expressed genes between NCF2 OE and NCF2 shNCF2; C: Expression levels of total ROS and mitochondrial ROS in cells. D-E: Expression of lactic acid and HIF1A, MDM2, and AIFM2 in control group (Ctrl), 2-DG, nutlin-3a-stimulated, and unstimulated shNCF2 cells; E: WB was used to detect the expression of p-AMPK, AMPK, CDK1 / 2, p21, and Pan-Kla in Ctrl, 2-DG, nutlin-3a-stimulated, and unstimulated shNCF2 cells, and after TI stimulation; F: CCK-8 assay was used to detect the proliferation of Ctrl, shNCF2, and NCF2 OE cells; G: qPCR was used to detect the mRNA expression levels of TP53 and TP63 in Ctrl, shNCF2, and NCF2 OE cells.

[0039] Figure 3 : A: Correlation analysis of GSEA-KEGG enrichment pathways and NCF2 expression levels in bulk-seq data of lesional tissues of AD (atopic dermatitis) patients in the GSE121212 database; B: Correlation analysis of GSEA-KEGG enrichment pathways and NCF2 expression levels in bulk-seq data of lesional tissues of PS (psoriasis) patients in the GSE121212 database.

[0040] Figure 4: A: Immunofluorescence analysis of the expression level of NCF2 in normal controls (HC) and lesional skin areas of psoriasis patients (PS). B: Expression levels of NOX2 family genes in normal skin and lesional skin areas of psoriasis patients in the GSE121212 database; C: Immunofluorescence detection of the expression of NCF2, TNF-α, and KRT5 in skin tissues of control group (Ctrl) and imiquimod (IMQ)-induced skin inflammation mouse models.

[0041] Figure 5 : A: Effects of BML-111 on the expression levels of p-NCF1 and NCF2; B: Effects of BML-111 on lactate expression in Ctrl and shNCF2 cells after TI stimulation; C: qPCR detection of the mRNA expression levels of TP63, TP53, MDM2, AIFM2, IL1B, CXCL10, and HIF1A in Ctrl, NCF2 OE, shNCF2 cells and after treatment with BML-111 and nutlin-3a; D: WB detection of the expression of p-AMPK, AMPK, CDK1 / 2, and p21 in Ctrl and shNCF2 cells after TI stimulation with BML-111.

[0042] Figure 6 : A: WB analysis of the expression level of GSDME protein in control group (Ctrl), shNCF2, and NCF2 OE cells with or without TI stimulation; B: WB analysis of the expression of GSDME and caspase-3 in control group, BML-111 (1, 2, 5, 10 μM) treatment groups, and NCF2 OE cells after TI stimulation. Detailed implementation manners

[0043] The present invention will be further described in detail below in conjunction with the specific implementation manners. The examples given are only for clarifying the present invention, rather than limiting the scope of the present invention. The following examples provided can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0044] Reference 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 by way of explanation and not limitation of the present disclosure. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the present disclosure without departing from the scope or spirit of the present disclosure. For example, features illustrated or described as part of one embodiment can be used in another embodiment to yield a still further embodiment.

[0045] In the experimental methods of the following embodiments, unless otherwise specified, they are all conventional methods, and are carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0046] Unless otherwise specified, the meanings of all terms (including technical and scientific terms) used to disclose the present disclosure are the same as those commonly understood by those of ordinary skill in the art to which the present disclosure pertains. Through further guidance, the following definitions are used to better understand the teachings of the present disclosure. The terms used in the specification of the present disclosure herein are only for the purpose of describing specific embodiments and are not intended to limit the present disclosure.

[0047] 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. Moreover, the terms and laboratory operation procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and conventional procedures in the corresponding fields. At the same time, to better understand the present disclosure, the definitions and explanations of related terms are provided below.

[0048] The term "and / or", "or / and", "and / or" as used herein includes any one of two or more related listed items, and also includes any and all combinations of the related listed items. The said any and all combinations include combinations of any two related listed items, any more related listed items, or all related listed items. It should be noted that when at least two conjunctions selected from "and / or", "or / and", "and / or" are used to connect at least three items, it should be understood that in the present disclosure, this technical solution undoubtedly includes the technical solution connected by "logical AND", and also undoubtedly includes the technical solution connected by "logical OR". For example, "A and / or B" includes three parallel solutions: A, B, and A + B. Another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, D (that is, the technical solution connected by "logical OR"), and also includes any and all combinations of A, B, C, D, that is, it includes combinations of any two or any three of A, B, C, D, and also includes the four-item combination of A, B, C, D (that is, the technical solution connected by "logical AND").

[0049] The terms "comprising", "containing", and "including" used in the present disclosure are synonyms, which are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps.

[0050] The numerical ranges expressed by endpoints in the present disclosure include all the numerical values and fractions included in the range, as well as the recited endpoints.

[0051] As used herein, the term "about" in reference to a value or parameter includes (and describes) embodiments directed to the value or parameter itself. For example, a description of "about X" includes a description of "X".

[0052] In the present disclosure, concentration values are meant to include fluctuations within a certain range. For example, fluctuations may occur within the corresponding precision range. For example, for 2%, fluctuations within the range of ±0.1% are allowed. For larger values or values that do not require overly precise control, larger fluctuations are also allowed. For example, for 100 mM, fluctuations within the ranges of ±1%, ±2%, ±5%, etc. are allowed. When referring to molecular weight, fluctuations within the range of ±10% are allowed.

[0053] As used herein, unless otherwise indicated, the singular forms of the articles "a", "an", and "the" include plural referents.

[0054] In the present disclosure, descriptions such as "a plurality of" and "a variety of", unless otherwise specified, mean greater than or equal to 2 in number.

[0055] In the present disclosure, among the technical features described in an open-ended manner, closed technical solutions composed of the recited features are included, as well as open technical solutions containing the recited features.

[0056] In the present disclosure, "treatment" and its variations or "amelioration" refer to therapeutic treatment, wherein the aim is to reverse, alleviate, improve, inhibit, slow down or stop the progression or severity of a disease (such as psoriasis), its related disorders and / or symptoms. The term "treatment" includes reducing or alleviating at least one adverse effect or symptom of a disease (such as psoriasis). More specifically, it may refer to (1) delaying the onset of psoriasis; (2) slowing down or stopping the progression, exacerbation or aggravation of psoriasis symptoms; (3) bringing about remission of psoriasis symptoms; or (4) referring to a method or process for curing psoriasis. As a preventive measure, treatment can be carried out before the onset of the disease or condition, or treatment can be carried out after the onset of the disease.

[0057] As used in the present disclosure, "therapeutically effective" and "effective dose" refer to a substance or amount that elicits a desired biological activity or effect.

[0058] Unless otherwise specified, the terms "subject" or "patient" are used interchangeably and refer to mammals, such as primates (more particularly human patients), as well as laboratory animals, such as rabbits, rats, and mice, and other animals. Thus, the terms "subject" or "patient" as used in this disclosure refer to any mammalian patient or subject to whom the compounds of this disclosure can be administered. In an exemplary embodiment of this disclosure, in order to identify the target patient to be treated according to the methods of this disclosure, acceptable screening methods are employed to determine risk factors associated with a target or suspected disease or disorder or to determine the existing disease or disorder of the subject. These screening methods include, for example, routine examinations to determine risk factors that may be associated with a target or suspected disease or disorder. These and other routine methods allow clinicians to select patients in need of treatment using the methods and compounds of this disclosure.

[0059] In this disclosure, "preferred", "better", "more preferred", and "preferably" are only used to describe embodiments or examples with better effects. It should be understood that they do not constitute a limitation on the scope of protection of this disclosure. In this disclosure, "optionally", "optional", and "option" mean that it can be either present or absent, that is, it refers to any one of the two parallel options of "present" or "absent". If "optional" appears multiple times in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "optional" is independent of each other.

[0060] All documents mentioned in this disclosure are incorporated herein by reference as if each document were individually incorporated by reference. Unless it conflicts with the inventive purpose and / or technical solution of this disclosure, the cited documents related to this disclosure are incorporated by reference in their entirety and for all purposes. When this disclosure refers to cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also incorporated by reference. When this disclosure refers to cited documents, the examples and preferred ways of the relevant technical features cited can also be incorporated as references into this disclosure, but only to the extent that this disclosure can be implemented. It should be understood that when the cited content conflicts with the description in this disclosure, this disclosure shall prevail or be modified adaptively according to the description of this disclosure.

[0061] The following examples used GraphPad Prism 10 statistical software to process the data. The experimental results were expressed as mean ± standard deviation. Student t, One-way ANOVA, or two-way ANOVA tests were used. P < 0.05 (*) indicates significant difference, P < 0.01 (**) indicates extremely significant difference, and P < 0.001 (***) indicates extremely significant difference.

[0062] Example 1: NOX2 expression increased significantly when the pentose phosphate pathway / anaerobic respiration was enhanced and oxidative phosphorylation was inhibited To clarify the function of NOX2 in the regulation of keratinocyte metabolism, we first established an inflammation model induced by TNF-α / IFN-γ. HaCat cells are commonly used in the study of human keratinocyte function. The cell lactate level of the supernatant of HaCat cells cultured for 24 hours was detected using a lactate detection analysis kit (L-lactic acid (L-LA) content detection kit, Solarbio, BC2235), and the expression of inflammatory factors was detected by real-time fluorescence quantitative PCR (qPCR). The results showed that the cell lactate level under the inflammatory state was 1.185 μmol / ml, which was significantly higher than that of the unstimulated group (0.755 μmol / ml) ( Figure 1 in A), accompanied by upregulation of HIF1A and inflammatory factor expression ( Figure 1 in B), and enhanced protein lactylation modification of the cells ( Figure 1 in D). After treatment with the glycolysis inhibitor 2-DG and the lactate dehydrogenase inhibitor FX-11, the above indicators were significantly alleviated, confirming that the glycolysis and anaerobic respiration metabolic pathways of keratinocytes were activated under the inflammatory state.

[0063] Notably, the expression of the NOX2 complex subunits CYBB, NCF2, and NCF1 showed specific regulation of the metabolic pathway: cells or tissue samples treated with glycolysis / pentose phosphate pathway inhibitors were taken, and total protein was extracted using RIPA lysis buffer. After quantification by BCA method, it was separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane; blocked with 5% non-fat milk for 1 hour, incubated successively with rabbit anti-human NCF2 primary antibody (1:1000) and HRP-labeled secondary antibody (1:20000), and developed by ECL analysis. The relative expression of NCF2 was calculated using β-actin as an internal reference to clarify its expression changes under metabolic inhibition. qPCR and WB detection showed that the expression of NCF2 was upregulated when glycolysis was inhibited, while it decreased when lactate dehydrogenase and the pentose phosphate pathway were inhibited ( Figure 1 in C and D). The change in the level of phosphorylated NCF1 (p-NCF1) indicated that the activation state of NOX2 was positively correlated with the expression level of its subunits ( Figure 1 in E). The expression of NOX2 increased significantly when the pentose phosphate pathway / anaerobic respiration was enhanced and oxidative phosphorylation was inhibited, and the regulatory amplitude of NCF2 was the most significant.

[0064] Example 2. Construction of stable transfected HaCaT cell lines with NCF2 knockdown (shNCF2) and overexpression (NCF2-OE) Based on the vector PGMLV-6751 (purchased from GeneCopoeia), two effective shNCF2 sequences (target sequences shNCF2-1-CGGGACATGGTGTCTAAGAAA, shNCF2-2-GCTATCAAAGACCTTAAAGAA) were constructed, and plasmid religation and screening were completed. The NCF2 overexpression plasmid was also based on PGMLV-6751, and the target gene sequence was the sequence provided in the NCBI database (https: / / www.ncbi.nlm.nih.gov / gene / 4688), and plasmid religation and screening were completed. The above plasmids were used to package lentiviruses in 293T cells using the second-generation lentiviral system. The concentrated viruses were transfected into keratinocytes and screened with puromycin. Based on this, we successfully constructed stable HaCaT cell lines with NCF2 knockdown (shNCF2) and overexpression (NCF2-OE), which can also be called shNCF2 cells and NCF2-OE cells in the following text.

[0065] Example 3. Regulation of p53-p21 axis and AMPK signaling pathway by NCF2 We performed transcriptome sequencing on NCF2-edited cells and used a nested design to compare NCF2 knockdown (shNCF2) and overexpression (NCF2-OE) with the control cell line (shCtrl). There were 1,496 differentially expressed genes (DEGs) ( Figure 2 in A). KEGG enrichment analysis showed that DEGs were significantly enriched in the cell cycle, glycolysis, p53, and IL-17 signaling pathways ( Figure 2 The methods in Example 1 were used to measure the intracellular lactate level, protein lactylation modification, and gene expression level. The verification experiments showed that NCF2 knockdown led to a significant increase in the intracellular lactate level and protein lactylation modification ( Figure 2 in D and F), and at the same time, the expression of HIF1A was upregulated while the expression of p53 target genes MDM2 and AIFM2 was downregulated ( Figure 2 in E). 2-DG and the p53 agonist (Nutlin-3a) could reverse the metabolic abnormalities caused by shNCF2, suggesting that NCF2 maintains metabolic homeostasis by regulating glycolysis and the p53 pathway. Further, NCF2 deficiency led to a decrease in the phosphorylation level of AMPK, a decrease in the expression of the cell cycle inhibitor p21, and an increase in the activity of CDK1 / 2 ( Figure 2 in F). The CCK-8 experiment confirmed that shNCF2 significantly promoted the proliferation of keratinocytes ( Figure 2G). Through the analysis of the KnockTF2.0 database (http: / / www.licpathway.net / KnockTF / index.html), we found that TP63 is the common transcription factor regulating NCF2, NCF1, and CYBA. qPCR verification showed that shNCF2 led to a significant decrease in the transcriptional levels of TP53 / TP63 ( Figure 2 H), revealing a bidirectional regulatory mechanism between NCF2 and the p53 pathway. The above results indicate that NCF2 maintains the metabolic homeostasis of keratinocytes and inhibits abnormal proliferation by regulating the p53-p21 axis and the AMPK signaling pathway.

[0066] Example 4. The expression level of NCF2 is positively correlated with the JAK-STAT pathway and negatively correlated with the glycolysis pathway By querying the GEO database (https: / / www.ncbi.nlm.nih.gov / geo / ), based on the GSE121212 database (https: / / www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE121212) containing the transcriptome data of lesional skin of patients with atopic dermatitis (AD) and psoriasis (PS), we conducted a KEGG pathway correlation analysis. The results showed that the expression of NOX2 was significantly positively correlated with the glycolysis, HIF-1α, and JAK-STAT pathways in AD / psoriasis lesions. Notably, the expression level of NCF2 was positively correlated with the JAK-STAT pathway (PS, R = 0.75) and positively correlated with the glycolysis pathway (R = 0.51) ( Figure 3 ), suggesting that NCF2 may play a balancing regulatory role between epidermal inflammatory responses and metabolic imbalance. Cryo-embedded sections were taken from the skin of human psoriasis patients, and immunofluorescence was used to detect the expression level of NCF2. The expression of NCF2 in the lesional skin area (PS) of patients was significantly increased in the epidermis compared with that in normal human controls (HC) ( Figure 4 A). Further comparison showed that the expression of NCF2 was higher in the lesional area of psoriasis than in the non-lesional area ( Figure 4 B). According to the method described in Example 1, the expression levels of NCF2, CYBB, and TNF-α in the skin of mice induced by imiquimod (Mingxin Lidi, H20030128) were detected. Cryo-embedded sections were taken from the epidermis of the model mice, and immunofluorescence was used to detect the expression level of KRT5. The mouse model induced by imiquimod also showed more expression of NCF2, CYBB, TNF-α, and KRT5 in the epidermis ( Figure 4 C).

[0067] Example 5: NCF2 expression is activated by BML-111, and NCF2 knockout blocks the improvement effect of BML-111 on glycolytic dysregulation and inflammatory response To explore the NOX2-targeted treatment strategy, we detected the mechanism of action of the LXA4 analog BML-111. BML-111 is a synthetic lipoxin A4 agonist with antioxidant and anti-inflammatory properties. In mice, it can be administered intraperitoneally at a dose of 1 mg / kg / day or topically on the skin (5 mg / kg / day). In vitro experiments showed that BML-111 treatment significantly upregulated NCF2 expression and activated NOX2 ( Figure 5 as shown in A of The Ctrl treatment was a blank treatment group in which HaCaT cells were cultured in a conventional medium.

[0068] The NCF2-OE treatment was a treatment group in which a HaCaT stable cell line overexpressing (NCF2-OE) NCF2 was cultured in a conventional medium.

[0069] The shNCF2 treatment was a treatment group in which a HaCaT stable cell line with knocked-down NCF2 (shNFC2) was cultured in a conventional medium.

[0070] The Ctrl+BML-111 treatment was a treatment group in which HaCaT cells were cultured in a medium with a BML-111 concentration of 10 µMol / L.

[0071] The shNCF2+BML-111 treatment was a treatment group in which a HaCaT stable cell line with knocked-down NCF2 (shNFC2) was cultured in a medium with a BML-111 concentration of 10 µMol / L.

[0072] The shNCF2+nutlin was a treatment group in which a HaCaT stable cell line with knocked-down NCF2 (shNFC2) was cultured in a medium with a nutlin concentration of 10 µMol / L.

[0073] The formula of the conventional medium was: DMEM + 10% FBS; the culture conditions were: 37 degrees Celsius, 5% CO2.

[0074] It was found that BML-111 treatment simultaneously enhanced the activity of the p53 signaling pathway (upregulation of MDM2, p21, and AIFM2 expression) ( Figure 5In B-D). The intracellular lactate level, protein lactylation modification, and gene expression level were measured in the same manner as described in Example 1. The verification experiment showed that NCF2-OE and the compound could effectively inhibit the expression of inflammatory factors such as IL1B and CXCL10, and reduce the levels of HIF1A and lactate production ( Figure 5 In B-D). Notably, NCF2 knockdown could block the improvement effect of BML-111 on glycolytic dysregulation and inflammatory response ( Figure 5 In B-D), confirming that its pharmacological effect depends on the NCF2 / NOX2 pathway.

[0075] Example 6. Overexpression of NCF2 inhibits GSDME activation GSDME is a key execution protein during pyroptosis of keratinocytes. It is cleaved and activated by the upstream Caspase-3 protein, and forms a destructive pore-like structure on the cell membrane, causing cell death, rupture, and release of inflammatory factors. In cells undergoing apoptosis, if there is high expression of endogenous GSDME, the cell death mode will change from apoptosis to pyroptosis. The increase in GSDME activation induced by TNF-α leading to increased release of inflammatory factors in keratinocytes has been well reported in psoriasis. Based on the new mechanism of GSDME-mediated pyroptosis in psoriasis, we detected the regulatory effects of BML-111 and NCF2. Using the method in Example 1 to detect protein expression levels, NCF2-OE significantly inhibited GSDME protein expression and its cleavage activation ( Figure 6 In A-B). Treatment with BML-111 dose-dependently reduced the activation level of GSDME, Figure 6 1, 2, 5, and 10 in B are the treatment groups with 1 μM, 2 μM, 5 μM, and 10 μM of BML-111 respectively, indicating that the NCF2 / NOX2 pathway participates in the pathological regulation of psoriasis by inhibiting GSDME activation.

[0076] The present invention has been described in detail above. For those skilled in the art, without departing from the gist and scope of the present invention, and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any changes, uses, or improvements to the present invention, including changes made using conventional techniques known in the art that are outside the scope disclosed in this application.

Claims

1. Use of an NCF2 agonist, said use being at least one of the following: A1) Use in the treatment of psoriasis, A2) Use in inhibiting GSDME activation, A3) Use in enhancing the activity of the p53 signaling pathway, A4) Use in activating AMPK, A5) Use in inhibiting the expression of inflammatory factors, A6) Use in inhibiting IL-1β expression, A7) Use in inhibiting CXCL10 expression, A8) Use in reducing the level of HIF-1α, A9) Use in reducing lactic acid production.

2. Use of an NCF2 agonist in the preparation of a drug, said use being at least one of the following: B1) Use in the preparation of a drug for the treatment of psoriasis, B2) Use in the preparation of a drug for inhibiting GSDME activation, B3) Use in the preparation of a drug for enhancing the activity of the p53 signaling pathway, B4) Use in the preparation of a drug for activating AMPK, B5) Use in the preparation of a drug for inhibiting the expression of inflammatory factors, B6) Use in the preparation of a drug for inhibiting IL-1β expression, B7) Use in the preparation of a drug for inhibiting CXCL10 expression, B8) Use in the preparation of a drug for reducing the level of HIF-1α, B9) Use in the preparation of a drug for reducing lactic acid production.

3. The application according to claim 1 or 2, characterized in that, The functions of the agonist include one or more of the following: C1) Up-regulating the expression of the NCF2 gene, C2) Enhancing the protein translation efficiency of NCF2, C3) Activating the activity of the NCF2 / NOX2 complex.

4. The application according to any one of claims 1 to 3, characterized in that, The agonist does not activate NFκB.

5. The application according to any one of claims 1-4, characterized in that The agonist comprises at least one of an NCF2 protein, an NCF2 gene expression vector, and an NCF2 / NOX2 agonist, and is designed to increase the expression of NCF2 and / or enhance the activity of NCF2 / NOX2.

6. The application according to any one of claims 1 to 5, characterized in that The agonist is a small molecule compound, an antibody, an aptamer, or a gene editing tool.

7. The application according to any one of claims 1-6, characterized in that, The agonist is used alone or in combination with other drugs.

8. The application according to any one of claims 1 to 7, characterized in that, The agonist and a pharmaceutically acceptable excipient form a pharmaceutical composition. The pharmaceutical composition is a topical preparation and contains a transdermal absorption promoter, preferably a liposome or a nanoemulsion carrier.

9. The application according to claim 8, characterized in that, The form of the pharmaceutical composition is selected from one or more of a solution, an injection, a spray, a nasal drop, an aerosol, a powder aerosol, a tablet, a capsule, and a granule.

10. A drug for treating psoriasis, characterized in that, The drug contains the agonist described in claim 1.

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