Application of TCN2 inhibitor in treatment of psoriasis

Abstract

The invention relates to the technical field of biological medicine, in particular to application of a TCN2 inhibitor in treatment of psoriasis. The invention discloses the important significance of TCN2 in the psoriasis mechanism for the first time, and the TCN2 becomes a new target for psoriasis treatment.

Description

Technical Field

[0001] The present disclosure relates generally to the field of biomedical technology, and more particularly to the use of TCN2 inhibitors in the treatment of psoriasis. Background Art

[0002] Psoriasis is a chronic inflammatory skin disease mediated by the immune system that is prone to relapse. The number of patients worldwide exceeds 125 million, accounting for about 2%-3% of the world's total population. Mechanistically, psoriasis lesions are infiltrated by a large number of monocytes, neutrophils, and T cells. Under inflammatory stimulation, the differentiation, proliferation, and activation of epidermal stem cells of keratinocytes are abnormal, and excessive growth of keratinocytes leads to the formation of thick scaly plaques in the lesions. A large number of immunological and genetic studies have found that the number of Th17 cells in the blood and lesions of psoriasis patients has increased significantly, and IL-17 has been identified as a key initiator of psoriasis. The IL-17 family consists of 6 members, among which the expression of IL17-A, IL17-C, and IL17-F in psoriasis lesions is significantly increased, and the biological activity of IL17-A is much higher than that of IL17-C and IL17-F. The main targets of IL-17 in psoriasis include keratinocytes, endothelial cells, and innate immune cells. Keratinocytes can secrete a large number of cytokines and chemokines under the stimulation of IL-17A, further aggravating the local inflammatory response. In view of the key pathogenic role of IL-17A in autoimmune and inflammatory diseases, targeted therapy for IL-17 can regulate the expression of multiple cytokines and chemokines and effectively alleviate the inflammatory response of psoriasis. At present, biological agents targeting IL-17 for the treatment of psoriasis, such as secukinumab, have been widely used clinically and have shown good therapeutic effects on patients with moderate to severe psoriasis. As a fully humanized IgG1 monoclonal antibody, secukinumab blocks the pro-inflammatory effect of IL-17A by specifically binding and neutralizing it, thereby reducing the abnormal proliferation and inflammatory response of keratinocytes.

[0003] Transcobalamin II (TCN2) belongs to the vitamin B12 binding protein family and helps cells absorb vitamin B12, making it a biomarker of active vitamin B12. TCN2 has been reported to be involved in a variety of biological processes, including cell proliferation, death, and migration. In addition, studies have reported that it is closely related to the onset of autoimmune-related diseases such as multiple sclerosis, systemic lupus erythematosus, and idiopathic recurrent spontaneous abortion. Summary of the invention

[0004] The present invention covers the following solutions:

[0005] Use of TCN2 inhibitors in preparing drugs for treating psoriasis.

[0006] The present invention reveals for the first time the importance of TCN2 in the pathogenesis of psoriasis, making it a new target for the treatment of psoriasis. Specifically, in the present invention, we observed that TCN2 expression was increased in PBMC and skin tissue of patients with psoriasis. TCN2 expression was significantly reduced in patients with moderate to severe psoriasis treated with succubitus for 6 months. At the same time, in the comparison of the psoriasis mouse model induced by imiquimod, it was found that TCN2 was significantly increased in skin tissue. The psoriasis model was induced in heterozygous and homozygous mice with TCN2 knockout, and it was found that TCN2 silencing could affect the epidermal thickness of mice, the content of immune cells in the skin, and the expression of IL-17A. We constructed Hacat cells with TCN2 knockdown, and used M5 stimulants (IL-1α, IL-17A, IL-22, TNF-α, Oncostatin M) to simulate the psoriasis inflammatory environment to study the effect of TCN2 on inflammation. The results showed that TCN2 can inhibit cell proliferation, affect the cell cycle, and reduce the inflammatory expression of multiple inflammatory factors and STAT3 pathways. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] 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.

[0008] Figure 1 :TCN2 is significantly elevated in psoriasis patient samples and mouse models. A. Representative immunohistochemical images of TCN2 in skin biopsy specimens from psoriasis patients and healthy donors. N=3. Scale bar=100μm B. TCN2 mRNA levels in PBMCs from psoriasis patients (PS, N=30) and healthy donors (HC, N=32). C. TCN2 mRNA levels and PASI scores in PBMCs from moderate-to-severe psoriasis patients (N=14) treated with secukinumab for 6 months at baseline and after treatment. D. Tcn2 mRNA levels in skin samples from wild-type mice (WT, N=5) and psoriasis model mice (N=5). E. Staining with Tcn2 (green) and DAPI (blue). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0009] Figure 2:Tcn2 deficiency can alleviate the psoriasis phenotype. A. WT, Tcn2 knockdown heterozygous mice and Tcn2 knockdown homozygous mice were modeled with imiquimod for 7 consecutive days. B. The figure shows representative H&E stained sections of the three groups of mice after modeling. C. Measurement of skin thickness in the lesion area of ​​the three groups of mice. D. Measurement of spleen weight ratio of the three groups of mice. E. Representative immunofluorescence images of Il-17a (green) and Cd45 (red) expression in the skin samples of modeled mice in each group. F. Western blot verified the expression of Il-17a in the skin of the control group and the modeling group mice. *p<0.05, **p<0.01, ***p<0.001).

[0010] Figure 3 :RNA-seq reveals the mechanism of TCN2 improving IMQ-induced psoriasis. A. Up-regulated and down-regulated genes in the WT group and Tcn2- / - group of IMQ-induced psoriasis model. B. KEGG pathway analysis of DEGs in the control group WT and psoriasis model group (FDR<0.1). C. KEGG pathway analysis of DEGs in the WT group and Tcn2- / - after psoriasis modeling (FDR<0.1).

[0011] Figure 4 :Proliferation-promoting effect of TCN2 in psoriasis. A. Western blot verified the efficiency of TCN2 knockdown. B. CCK-8 detected the proliferation rate of cells in the control group (NC) and TCN2 knockdown strain (TKO). C. Flow cytometry was used to detect the cell cycle in the NC and TKO groups. The percentage of cells in each stage of the cell cycle in the NC and TKO groups was compared. D. Representative immunofluorescence images of Ki-67 (red) expression in the skin samples of modeled mice in each group. *p<0.05, **p<0.01, ***p<0.001)

[0012] Figure 5 :The pro-inflammatory effect of TCN2 in psoriasis. A. qPCR comparison of the changes in inflammatory factors in the skin of WT and Tcn2- / - mice after modeling. B. qPCR comparison of the changes in inflammatory factors in the NC group and TCN2KO group after the use of M5 stimulant. C. Western blot verification of the changes in Stat3 pathway activation in the skin of mice in the control group and modeling group. D. Western blot verification of the changes in STAT3 pathway activation in the NC group and TCN2 KO group after the use of M5 stimulant. E. qPCR comparison of the expression changes of IL-12R and LCN2 in the NC group and TCN2 KO group after the use of M5 stimulant. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 DETAILED DESCRIPTION

[0013] 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.

[0014] 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.

[0015] 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.

[0016] 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").

[0017] 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.

[0018]

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

[0019] 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".

[0020] 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%.

[0021] As used herein, the singular articles "a," "an," and "the" include plural referents unless otherwise indicated.

[0022] In the present disclosure, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.

[0023] 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.

[0024] In the disclosure, "treat" and variations thereof or "ameliorate" refer to therapeutic treatment, wherein the objective is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a disease (e.g., psoriasis), its associated conditions and / or symptoms. The term "treat" includes reducing or alleviating at least one adverse effect or symptom of a disease (e.g., psoriasis). More specifically, it may refer to (1) delaying the onset of psoriasis; (2) slowing or stopping the progression, aggravation or exacerbation of psoriasis symptoms; (3) bringing about relief from psoriasis symptoms; or (4) referring to a method or process for curing psoriasis. Treatment may be performed prior to the onset of a disease or condition as a preventive measure, or treatment may be performed after the onset of the disease.

[0025] As used in this disclosure, "therapeutically effective" and "effective dose" refer to a substance or amount that elicits a desired biological activity or effect.

[0026] Unless otherwise indicated, the terms "subject" or "patient" are used interchangeably and refer to mammals, such as primates (further human patients), and experimental animals, such as rabbits, rats and mice, and other animals. Therefore, the terms "subject" or "patient" used in the present disclosure refer to any mammalian patient or subject to which the compounds of the present disclosure can be administered. In exemplary embodiments of the present disclosure, in order to identify the subject patient to be treated according to the methods of the present disclosure, an acceptable screening method is used to determine the risk factors associated with the target or suspected disease or condition or to determine the subject's existing disease or condition. These screening methods include, for example, routine examinations to determine the risk factors that may be associated with the target or suspected disease or condition. These and other conventional methods allow clinicians to select patients in need of treatment using the methods and compounds of the present disclosure.

[0027] 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.

[0028] 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.

[0029] The present invention relates to the use of a TCN2 inhibitor in preparing a medicine for treating psoriasis.

[0030] In some embodiments, the inhibitor comprises at least one of antisense oligonucleotides, dsRNA, siRNA, shRNA, miRNA, RNAi, and ribozymes, which are designed to reduce or block the expression of the TCN2 gene.

[0031] 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.

[0032] shRNA, or a small hairpin RNA or shorthairpin RNA (shRNA), is an RNA sequence with a tight hairpin turn, which contains a sense strand segment, an antisense strand segment, and a stem-loop structure connecting the sense strand segment and the antisense strand segment, and is often used for RNA interference to silence the expression of target genes. The sequences of the sense strand and the antisense strand are complementary, and the sequence of the antisense strand segment is identical to 10 to 30 consecutive nucleotide sequences in the target mRNA. The hairpin structure of shRNA can be cut into siRNA by the cell mechanism, and then siRNA binds to the RNA-induced silencing complex (RISC), which can bind to the target mRNAs and degrade them.

[0033] 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).

[0034] In one embodiment of the present disclosure, as an inhibitor of TCN2, a substance that specifically binds to the TCN2 protein can be cited. A substance selected from the group consisting of antibodies, antibody fragments, small molecule compounds and aptamers can be cited. They can be designed to reduce or offset the activity of TCN2.

[0035] 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 TCN2 can be prepared by any method known in the art. For example, monoclonal antibodies may be produced by immunizing mice with recombinant TCN2 or one or more synthetic peptides (including epitopes that are considered to be highly antigenic). Alternatively, polyclonal antibodies can also be improved by immunizing rabbits with the above-mentioned antigens. Once produced using hybridoma technology, it is preferred that the monoclonal antibody be humanized by exchanging mouse and human skeletons and constant regions.

[0036] "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.

[0037] In some embodiments, the inhibitor knocks down or knocks out the TCN2 gene. Knockdown or knockout can be performed using any known prior art, including zinc finger ribonuclease (ZFN) technology, TALEN (transcription activator-like effector nucleases) technology, or CRISPR technology.

[0038] In some embodiments, the inhibitor comprises gRNA and Cas enzyme, or a vector capable of expressing them. In some embodiments, the antagonist is a CRISPR-CAS gene editing composition / combination product. In some embodiments, it comprises gRNA and Cas enzyme (e.g., Cas9) or a vector capable of expressing them. There are two common gene knockout strategies, one is single sgRNA-mediated gene knockout, which causes gene frameshift mutations by 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 through two sgRNAs to achieve the deletion of the target fragment sequence, thereby achieving the knockout of the target gene.

[0039] In some embodiments, the inhibitor is used alone or in combination with other drugs (particularly psoriasis treatment drugs).

[0040] In some embodiments, the inhibitor and pharmaceutically acceptable adjuvant constitute a pharmaceutical composition. The 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 a sterile solution or suspension of a pharmaceutically acceptable solution other than water or water. For example, a pharmacologically acceptable carrier or medium is appropriately combined, specifically sterile water, saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative, adhesive, etc. It is considered to be prepared by mixing in a unit dosage form required for generally accepted drug implementation. The amount of active ingredient in these preparations is set to obtain an appropriate capacity within the range shown.

[0041] The sterile composition for injection can be formulated according to a common preparation using excipients such as distilled water for injection.

[0042] As aqueous solutions for injection, isotonic solutions containing, for example, physiological saline solution, lactose, glucose and other supplements (e.g., D-sorbitol, D-mannose, D-mannitol, sodium chloride) can be cited. Appropriate 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.

[0043] 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.

[0044] 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.

[0045] In some embodiments, the subject of the medicament is a mammal.

[0046] In some embodiments, the subject of the medicament is a human.

[0047] 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.

[0048] 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.

[0049] Example 1 TCN2 expression is upregulated in psoriasis lesions

[0050] We first performed immunohistochemical staining on skin sections of healthy controls and psoriasis patients to explore the expression of TCN2 in psoriasis lesions. In normal skin, TCN2 is mainly distributed in the basal layer of the epidermis. In psoriasis lesions, TCN2 is widely expressed in the thickened epidermis, and its expression is increased in the vascular endothelial cells and inflammatory cells of the dermis. We extracted PBMCs from the peripheral blood of 30 psoriasis patients and 32 healthy controls, and used qPCR technology to analyze and found that the expression of TCN2 was significantly increased in psoriasis patients. Then we compared the expression of TCN2 in PBMCs of the peripheral blood of 14 patients with moderate to severe psoriasis treated with secukinumab after 6 months of treatment. We found that the PASI scores of these 14 patients with moderate to severe psoriasis were significantly reduced after treatment, and the expression of TCN2 in PBMCs was significantly reduced. Subsequently, we first IMQ-induced WT mice to establish a psoriasis mouse model and induce skin inflammation in the back skin. We performed qPCR and immunofluorescence techniques to compare the skin tissues of the IMQ-induced group and normal mice and found that the expression of Tcn2 in the skin of the induced group mice was significantly upregulated. Figure 1 This suggests that TCN2 may be involved in the pathogenesis of psoriasis.

[0051] Example 2 Tcn2 deletion can alleviate IMQ-induced psoriasis phenotype

[0052] To further determine the relationship between TCN2 and psoriasis, we established three groups of IMQ-induced psoriasis mouse models: WT, Tcn2+ / -, and Tcn2- / -. We found that 7 days after IMQ induction, the scales and roughness of the back skin of WT mice were more severe than those of Tcn2 knockout mice, and the degree of epidermal hyperplasia in WT, Tcn2+ / -, and Tcn2- / - mice showed a decreasing trend, indicating that the loss of Tcn2 effectively improved the phenotype of psoriasis. We used Tcn2 knockout mice to induce a psoriasis model using imiquimod, and HE staining was performed on the back skin lesions of the three groups of mice. Histological analysis of mouse skin after IMQ treatment showed that each group had obvious psoriasis-like skin lesions, including epidermal hyperplasia, epidermal parakeratosis, and dermal hyperkeratosis. However, we found that the degree of epidermal hyperplasia and inflammatory infiltration in wild-type mice was more severe than that in the knockout group. At the same time, we sampled the spleens of the three groups of mice and found that the ratio of spleen to body weight of the mice in the Tcn2 knockout group was significantly lower than that in the WT group. We used immunofluorescence technology to compare the expression of Cd45 and Il-17a in the skin of the two groups and found that the Cd45 and Il-17a in the Tcn2 knockout group were significantly lower than those in the WT group. We used Western blot to compare the skin tissue proteins of the control group and the three groups of model mice and also verified the effect of Tcn2 on Il-17a. For the relevant results of this example, see Figure 2 The results of the study demonstrated that knockdown of TCN2 could reduce epidermal hyperplasia and skin inflammation, showing a potential therapeutic effect in psoriasis.

[0053] Example 3 Analysis of the effect of TCN2 on psoriasis by single cell transcriptome analysis

[0054] To analyze the function of TCN2 in psoriasis, we used RNA-seq to analyze the expression profiles of psoriasis model group, control group and TCN2-deficient psoriasis mice. RNA-seq results showed that compared with the control group, 45 genes were upregulated and 73 genes were downregulated in the Tcn2+ / - model group, while 441 genes were upregulated and 442 genes were downregulated in the Tcn2- / - model group. Among them, inflammatory-related factors and chemokines such as Cxcr2, S100a8, S100a9, Lcn2, Il1b, and Il6 were significantly downregulated in the Tcn2- / - model group. Secondly, we further analyzed the DEGs using Kyoto Encyclopedia of Gens and Genomes (KEGG) pathway analysis. First, comparing the DEGs of the control group and the model group, we found that most of the DEGs in the two groups were enriched in Cytokine-cytokine receptor interaction, JAK-STAT signalingpathway, and IL17 signaling pathway. At the same time, we compared the Tcn2- / - model group and the psoriasis group, and found that most DEGs were involved in multiple pathways, and found that most of them were enriched in Cytokine-cytokine receptor interaction and Chemkinesignaling pathways. Compared with the enriched pathways in the psoriasis group, it was found that TCN2 also affected cell proliferation and inflammatory pathways. For the relevant results of this example, see Figure 3 .

[0055] Example 4 Proliferative Effect of TCN2 in Psoriasis

[0056] Since skin lesions are the main changes in psoriasis, we focused on the changes in epidermal cells, especially keratinocytes. Therefore, we constructed a Hacat cell line with TCN2 knockdown and verified that TCN2 had been knocked down by western-blot. The CCK-8 experiment showed that the proliferation rate of the TCN2 knockdown cell line was significantly reduced compared with the control group cells. At the same time, we also proved by flow cytometry that the heterozygous knockout of TCN2 regulated the cell cycle through G1 phase arrest of the cell line. In addition, we used immunofluorescence technology to stain and compare the skin tissues of the three groups of mice, and confirmed that silencing Tcn2 can reduce the expression of Ki67 in the epidermis. The above results suggest that the effect of TCN2 on cell proliferation and cycle may be related to the manifestation of psoriatic epidermal cell proliferation. For related results of this example, see Figure 4 .

[0057] Example 5 Proinflammatory effects of TCN2 in psoriasis

[0058] Pro-inflammatory effect of TCN2 in psoriasis. To further explore the role of TCN2 in psoriasis, we extracted RNA from mouse skin to detect the expression changes of inflammatory factors after Tcn2- / - and WT mice were modeled, and found that the expression of Il-1β, S100a8 and S100a9 was significantly reduced. Then we used M5 stimulant 10ng / ml to stimulate the TCN2 knockdown cell line for 24h to simulate the inflammatory environment. We found that under the same stimulation conditions, the mRNA expression of inflammatory factors such as IL-1β, IL-6, TNFα, S100A7, S100A8 and S100A9 in cells knocked down by TCN2 was significantly lower than that in the NC group. This means that knocking down TCN2 can alleviate the inflammatory response, and this result also verifies the result that the inflammatory response of mice is reduced after Tcn2 knockdown. Afterwards, combined with the RNA-seq results of mice and existing studies, we believe that TCN2 may affect the activation of the STAT3 pathway through the secretion of IL-1β. Therefore, we extracted proteins from the skin of mice after IMQ modeling for Western blot experiments and found that knocking down Tcn2 could significantly reduce the activation of the Stat3 pathway. Similarly, we verified the same conclusion in in vitro cell experiments. In addition, we extracted RNA from the cell lines after TCN2 knockdown, detected the expression of IL-12R and LCN2, and found that the expression of the knockdown group was significantly reduced. This result is consistent with the mouse RNA-seq results. Combined with the changes in inflammatory factors and data analysis results, it suggests the correlation between TCN2 and the STAT signaling pathway and the IL-17 signaling pathway. For related results of this example, see Figure 5 .

[0059] Psoriasis is a chronic inflammatory disease prone to recurrence, characterized by excessive growth of keratinocytes leading to thick, scaly papules and plaques. Its pathogenesis is the dysregulation of keratinocyte proliferation, and the release of cytokines by local T cells and dendritic cells further aggravates the cascade of inflammatory responses. TCN2 has been reported to play an important role in many autoimmune diseases. Our study showed that TCN2, a protein that had not been paid attention to in psoriasis before, can regulate the proliferation of psoriatic keratinocytes. We first found that TCN2 was significantly upregulated in the lesional tissues and peripheral blood PBMCs of psoriasis patients, and significantly decreased after treatment with secukinumab. Therefore, we further verified it in mice and found that Tcn2- / - mice showed a milder psoriasis phenotype than the WT group, further proving that TCN2 can regulate the proliferation of keratinocytes both in vivo and in vitro. Our RNA-Seq analysis of Tcn2- / - mice showed that silencing Tcn2 significantly affected the intercellular factor exchange and the regulation of inflammatory pathways. Moreover, genes such as Clec4e, Ccl3, Il-1b, Areg, and Cxcl2 were significantly downregulated by Tcn2 silencing, among which Ccl3 and Cxcl2 were most significantly downregulated. Ccl3 plays a role in inflammatory response by binding to receptors CCR1, CCR4, and CCR5. It has been reported that its expression is elevated in the serum of patients with psoriasis, and reducing the release of Ccl3 in chronic inflammatory diseases helps to relieve inflammatory response. Cxcl2, as a common inflammatory factor indicator in psoriasis, is a secretory protein involved in immune regulation and inflammatory process. At present, a large number of studies have proved its important role in pathogenesis, and it can relieve inflammatory response and keratinocyte proliferation by reducing Cxcl2. In addition, we mainly focus on the relationship between IL-1β, IL17 and TCN2. Previous studies have shown that in psoriasis, IL-1β not only activates dermal γδT cells to secrete IL-17, but also stimulates keratinocytes to secrete chemokines, further promoting γδT17 cells to secrete IL-17, and can also affect STAT3 and NF-κB pathways through chemokines. In addition, studies have shown that the differentiation, proliferation and activation of epidermal stem cells in patients with psoriasis are significantly abnormal. We found that TCN2 protein is highly expressed in the basal layer and the entire epidermis of psoriatic lesions, suggesting that TCN2 may regulate the proliferation of psoriatic keratinocytes. Our results further demonstrate that TCN2 regulates the proliferation of keratinocytes in vivo and in vitro. Moreover, under the same inflammatory conditions, we observed that the inflammatory response of Tcn2 knockout mice was milder. In addition to being associated with psoriasis, we also observed that the weight of Tcn2 knockout mice was significantly lower than that of the control group, suggesting that Tcn2 plays an important role in growth and development. In summary, we demonstrated that a previously unreported TCN2 is associated with the excessive proliferation and inflammatory response of psoriatic keratinocytes.

[0060] 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 TCN2 inhibitors in the preparation of drugs for treating psoriasis.

2. The use according to claim 1, wherein the inhibitor comprises at least one of antisense oligonucleotides, dsRNA, siRNA, shRNA, miRNA, RNAi and ribozymes, which are designed to reduce or block the expression of the TCN2 gene.

3. The use according to claim 1, wherein the inhibitor comprises at least one of an antibody or an antigen-binding fragment thereof, a small molecule compound, and an aptamer, which is designed to reduce or offset the activity of the TCN2 protein.

4. The use according to claim 1, wherein the inhibitor knocks down or knocks out the TCN2 gene through ZFN technology, TALEN technology or CRISPR technology.

5. The use according to claim 4, wherein the inhibitor comprises gRNA and Cas enzyme, or a vector capable of expressing them.

6. The use according to any one of claims 1 to 5, wherein the inhibitor is used alone or in combination with other drugs.

7. The use according to any one of claims 1 to 5, wherein the inhibitor and pharmaceutically acceptable excipients constitute a pharmaceutical composition.

8. The use according to claim 7, wherein 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.

9. The use according to any one of claims 1 to 5 and 8, wherein the subject of the drug is a mammal.

10. The use according to claim 9, wherein the subject of the medicine is a human.

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