Use of CD300e gene or expression product thereof as a therapeutic target in the preparation of a medicament for treating psoriasis

Abstract

This invention provides the application of the CD300e gene or its expression product as a therapeutic target in the preparation of drugs for treating psoriasis. The invention uses RNA sequencing to screen for significantly high expression of the CD300e gene in the psoriasis group. In vitro studies validated the high expression of CD300e and sphingomyelin in the blood and tissues of the psoriasis group. In vivo experiments revealed the mechanism by which CD300e / sphingomyelin activates imiquimod-induced psoriatic dermatitis, and based on this mechanism, a targeted therapy for psoriasis was developed.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of the CD300e gene or its expression product as a therapeutic target in the preparation of drugs for treating psoriasis. Background Technology

[0002] Psoriasis is a common immune-mediated chronic inflammatory skin disease characterized by itchy, scaly, erythematous plaques, leading to hyperkeratosis, parakeratosis, acanthosis, inflammatory infiltration of dermal cells, and angiogenesis. Psoriasis affects multiple organ systems, including the skin, nails, joints, and metabolic systems. Based on the morphology of the lesions and clinical manifestations, the disease is mainly divided into four categories: plaque psoriasis (including chronic plaque psoriasis and guttate psoriasis), pustular psoriasis, erythrodermic psoriasis, and psoriatic arthritis, with plaque psoriasis being the most common.

[0003] Although immunological and genetic studies have identified IL-17 and IL-23 as key drivers in the pathogenesis of psoriasis, no definitive and complete cure for skin lesions has yet been found. Furthermore, little is known about the molecular mechanisms controlling the initial stages of psoriasis. Therefore, researching novel immune mechanisms of psoriasis and screening for new therapeutic targets is of great significance. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide the application of the CD300e gene or its expression product as a therapeutic target in the preparation of drugs for treating psoriasis. This invention uses RNA sequencing to screen for the CD300e gene, and simultaneously employs biological methods such as cellular immunity, Western blotting (WB), and gene knockout to further explore the immunological functions of the CD300e gene and its ligand sphingomyelin at both cellular and animal immune levels, as well as their immunological effects and targets in psoriasis. The CD300e gene holds promise as a novel therapeutic target for psoriasis.

[0005] To achieve this objective, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides the application of the CD300e gene or its expression product as a therapeutic target in the preparation of a drug for treating psoriasis.

[0007] This invention provides a novel immune mechanism for psoriasis, involving CD300e and its ligand sphingomyelin participating in the development of psoriasis through the IL-23A axis. Specifically, this mechanism involves CD300e regulating the IL-23A axis and participating in the psoriasis immune response by binding to its ligand sphingomyelin.

[0008] Preferably, the drug comprises a reagent that inhibits CD300e gene expression or a reagent that reduces the content of CD300e gene expression products.

[0009] Preferably, the expression product of the CD300e gene includes: RNA transcribed from the CD300e gene or CD300e protein.

[0010] Preferably, the reagent for inhibiting CD300e gene expression includes any one of: interfering RNA targeting the CD300e gene, hairpin RNA targeting the CD300e gene, or guide RNA for CD300e gene knockout.

[0011] Preferably, the reagent that inhibits CD300e gene expression reduces protein expression in psoriatic dermatitis.

[0012] Preferably, the protein in the psoriatic dermatitis includes at least one of IL-23A, IL-12A, or IL-17A.

[0013] In this invention, inhibiting CD300e expression can alleviate psoriatic dermatitis.

[0014] Secondly, the present invention provides the use of sphingomyelin inhibitors in the preparation of medicaments for treating psoriasis.

[0015] In this invention, it was found that CD300e is highly expressed in psoriasis and that it, along with its ligand sphingomyelin, participates in the occurrence and development of psoriasis.

[0016] Preferably, the sphingomyelin inhibitor reduces protein expression in psoriatic dermatitis.

[0017] Preferably, the protein in the psoriatic dermatitis includes at least one of IL-23A, IL-12A, or IL-17A.

[0018] Thirdly, the present invention provides the use of at least one of a CD300e gene inhibitor, a CD300e gene expression product inhibitor, or a sphingomyelin inhibitor in the preparation of a medicament for treating psoriasis.

[0019] Preferably, the CD300e gene inhibitor includes an agent that inhibits CD300e gene expression.

[0020] Preferably, the CD300e gene expression product inhibitor includes an agent that reduces the content of the CD300e gene expression product.

[0021] Preferably, the formulation for inhibiting CD300e gene expression includes any one of the following: interfering RNA targeting the CD300e gene, hairpin RNA targeting the CD300e gene, or guide RNA for CD300e gene knockout.

[0022] The numerical range described in this invention includes not only the point values ​​listed above, but also any point values ​​within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific point values ​​included in the range.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) Clarify the relationship between CD300e and severe chronic plaque psoriasis.

[0025] (2) Inhibition of CD300E can improve psoriatic dermatitis in mice.

[0026] (3) CD300e is highly expressed in psoriasis and, together with its ligand sphingomyelin, participates in the occurrence and development of psoriasis. Attached Figure Description

[0027] Figure 1 To screen for the expression levels of the differentially expressed gene CD300e mRNA and the expression level of the psoriasis-associated inflammatory factor IL-17A.

[0028] Figure 2 The expression levels of psoriasis-related inflammatory factors IL-23A, IL-12A, and TNF-α mRNA.

[0029] Figure 3 Association analysis of CD300e mRNA with IL-17A and IL-23A.

[0030] Figure 4 Association analysis of CD300e mRNA with IL-12A and TNF-α mRNA, respectively.

[0031] Figure 5 Sphingomyelin (SM) stimulates CD14 + mRNA levels in monocytes and dendritic cells 24 h later.

[0032] Figure 6 The skin lesions in mice were modeled using petrolatum, PBS, sphingomyelin, imiquimod, and a combination of sphingomyelin and imiquimod.

[0033] Figure 7 Line graphs showing the PASI score, infiltration score, scaling score, and erythema score of mice.

[0034] Figure 8 Histopathological analysis of skin lesions induced in mice by petrolatum, PBS, sphingomyelin, imiquimod, and sphingomyelin combined with imiquimod.

[0035] Figure 9The flowchart shows the experimental procedure in CD300E-KO mice.

[0036] Figure 10 The image shows the results of Western blotting (WB) detection of protein expression in psoriatic dermatitis in mice.

[0037] Figure 11 Western blot analysis was performed to detect the relative band intensity of protein expression in psoriatic dermatitis in mice. Detailed Implementation

[0038] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0039] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0040] Example 1

[0041] 1. Sample collection.

[0042] This embodiment investigated and recorded clinical information of patients with psoriasis vulgaris, collected imaging data, and used trephine drilling to obtain skin lesion tissue from 20 untreated patients with progressive psoriasis vulgaris and 12 patients with stable psoriasis vulgaris treated with drugs such as acitretin and Tripterygium wilfordii. RNA was extracted and sequenced. Central tissue of skin lesions from 7 untreated patients with progressive psoriasis vulgaris, unaffected skin tissue adjacent to skin lesions from 7 untreated patients with progressive psoriasis vulgaris, skin lesions from 14 healthy controls, as well as blood samples from 111 patients with progressive psoriasis vulgaris, 78 blood samples from stable psoriasis vulgaris, and 67 blood samples from healthy controls were used for validation and screening of highly expressed genes.

[0043] 2. Transcriptome sequencing analysis.

[0044] Transcriptome sequencing was performed on RNA from skin biopsies taken during active and quiescent phases of psoriasis. Differentially expressed genes (DEGs) were initially screened using the Illumina HiSeq X Ten platform. Genes with a log2-fold change (FC) ≥1 and a q value <0.05 were selected as differentially expressed genes. Subsequently, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to explore the biological functions and pathways associated with differentially expressed genes. ImuUCC and GSVA were used to predict immune cell composition. qRT-PCR experiments were performed to validate the results.

[0045] Experimental results:

[0046] (1) RNA sequencing analysis revealed that CD300e was significantly highly expressed in the psoriasis group.

[0047] In our RNA sequencing study, we collected 20 tissue samples from active psoriasis lesions and 12 tissue samples from quiescent psoriasis lesions. Principal component analysis was performed on all samples and their corresponding gene expression levels to observe clustering associations based on gene expression levels. A total of 20,522 differentially expressed genes were identified between the active and quiescent phases, of which 323 genes were upregulated and 465 genes were downregulated. The corrected p-values ​​for all differentially expressed genes were <0.05. Notably, the expression of genes such as CD300e, CLEC4C, SH2D5, PTX3, TDO2, and PI15 was higher in the active phase than in the quiescent phase. Regarding signaling pathways, we observed upregulation of immune cell differentiation and adaptive immune responses, and the interaction between CD300e and CLEC4C proteins reflected the obtained signaling pathway results.

[0048] Example 2

[0049] Molecular level verification.

[0050] Based on the top 50 highly expressed genes selected for RNA seq analysis, RT-qPCR primers for CD300e, IL-17A, IL-23A, IL-12A, and TNF-α were designed. Simultaneously, blood RNA was extracted from 111 patients with severe chronic plaque psoriasis in the progressive phase, 78 patients in the regressive phase, and 67 healthy controls. RNA was also extracted from skin lesions of 7 pairs of patients in the progressive and regressive phases of severe chronic plaque psoriasis and from skin tissue specimens of 13 healthy controls. Each specimen included a matched adjacent tissue control from psoriasis lesions. RNA from blood samples and skin lesions was extracted using the Qiagen RNA blood kit and the Qiagen RNeasy plus mini kit, and reverse transcribed into cDNA. RT-qPCR was then used to further validate the transcriptional expression of DEGs closely related to immunity.

[0051] Experimental results:

[0052] In vitro studies have found high expression of CD300e in the blood and tissues of psoriasis patients.

[0053] In RT-PCR testing, among 111 blood samples with active psoriasis, 78 blood samples with quiescent psoriasis, and 67 control samples, the mRNA levels of CD300e, IL-17A, and IL-23A in the active phase samples were approximately 4.917±3.443 times, 51.852±22.490 times, and 5.165±2.832 times, respectively. The mRNA levels of CD300e, IL-17A, and IL-23A in the active phase were higher than those in the stable phase and the control group. Correlation analysis between CD300e mRNA and IL-17A, IL-23A, or IL-12A in the skin lesions showed a positive result. Figures 1-4 Compared with healthy individuals, the expression of CD300e mRNA in peripheral blood of psoriasis patients was increased by 1.5-fold (n=111, P<0.005). Similar to IL-23A, the expression of IL-17A in the lesions of psoriasis patients was similar to that in the lesions of healthy controls. Correlation analysis between CD300e mRNA and IL-17A mRNA in peripheral blood was positive.

[0054] Figure 1 To screen for the expression levels of CD300e mRNA, a gene with specific differential expression, and IL-17A mRNA, an inflammation-related factor in psoriasis.

[0055] Figure 2 The expression levels of psoriasis-related inflammatory factors IL-23A, IL-12A, and TNF-α mRNA.

[0056] Figure 3 Association analysis of CD300e mRNA with IL-17A and IL-23A.

[0057] Figure 4 Association analysis of CD300e mRNA with IL-12A and TNF-α mRNA, respectively.

[0058] Figure 1-4 In this context, PP represents the central tissue of the lesion; PN represents the unaffected skin tissue adjacent to the lesion; and Control represents the healthy control group.

[0059] Example 3

[0060] The level of sphingomyelin in the psoriasis group was measured.

[0061] 1. Experimental methods.

[0062] (1) Sample information: 6 normal individuals in the progression phase and 6 in the stable phase, and 10 normal control group individuals.

[0063] (2) Prepare the basic solution and purchase SM standard.

[0064] (3) Sample processing.

[0065] 1) Perform the procedure on ice, and pipette 50 μL of sample into a 2 mL QSP tube.

[0066] 2) Next, add 225 μL of methanol and 75 μL of sodium methoxide solution (sodium methoxide is soluble in methanol and has a concentration of 1M).

[0067] 3) After vortexing for 1 min, add 1 mL of MTBE and vortex at 37℃ for 2 h.

[0068] 4) After cooling, add 250 μL of pure water and 25 μL of 20% acetic acid / water solution to neutralize the pH and prevent hydrolysis of SM.

[0069] 5) After centrifuging at 12000 rpm for 15 min, take 200 μL of the supernatant and centrifuge to concentrate.

[0070] 6) Before performing the analysis, the centrifuged and concentrated extract sample was redissolved in 100 μL of MeOH / IPA / H2O=65:30:5 (v / v / v) solution for positive and negative ion mode analysis.

[0071] 7) LC / MS parameters:

[0072] a) Chromatographic conditions: Column: Nanospectral analysis, ChromCore C8 column (3μm, 2.1×100mm); Column temperature: 50℃; Injection volume: 2μL; Flow rate: 0.26mL / min.

[0073] Mobile phase A: Acetonitrile: Water = 60:40, 10 mM ammonium acetate.

[0074] Mobile phase B: Acetonitrile: Isopropanol = 10:90, 10 mM ammonium acetate.

[0075] b) Gradient elution procedure (Table 1).

[0076] Table 1

[0077]

[0078] c) Mass spectrometry conditions: positive ion mode (Table 2).

[0079] Table 2

[0080]

[0081] All samples were coupled to an AB Sciex 5500 triple quadrupole trap mass spectrometer (Q-TRAP-MS) in a Sciex LC system, with electrospray ionization operated in positive mode. Compound dependence of two standard compounds was optimized using direct infusion mass spectrometry to obtain the maximum multiple reaction monitoring signal intensity. General instrument parameters for all compounds, such as different gases, heat, and ion spray voltage, were optimized using MPLC flow injection analysis. These instrument parameters were compromised for a given flow rate of all compounds within one cycle. Instrument control and data integration were performed using analysis software version 1.6.2. Sample metabolite concentrations were calculated.

[0082] 2. Experimental results.

[0083] Sphingomyelin levels were high in the psoriasis group.

[0084] Serum SM levels in PsV patients and healthy donors were measured by LS / MS. The SM concentration in the active phase of psoriasis (n=12) was 262.9±49.23 ng / mL, which was significantly higher than that in the quiescent phase (n=12) (134.9±21.36 ng / mL) and the control group (n=16) (136.3±20.05 ng / mL) (P<0.05). DCs and CD14 were also observed. + Cells upregulate sphingomyelin-induced IL-23A via CD300e. To investigate whether sphingomyelin can stimulate dendritic cells (DCs) or CD14... + Cells secrete IL-23A and TNF-α cytokines. Peripheral blood CD14 levels in psoriasis patients were stimulated with sphingomyelin. + Cells 24 hours ( Figure 5 The results showed that CD14 levels were higher in the sphingomyelin group and the LPS group. + The secretion of IL-23A and TNF-α in cells increased significantly.

[0085] Figure 5 Sphingomyelin (SM) stimulates CD14 + mRNA levels in monocytes and dendritic cells 24 h later. Figure 5 In the image, (A) CD300e mRNA; (B) IL-23A mRNA; (C) IL-12A mRNA; (D) TNF-α mRNA. Ethanol was used as the solvent; SM (200 μg / mL) was used; and LPS (1 μg / mL) was used as the positive control. Blue represents CD14. + Cells, red represents DCs. ns, no statistically significant difference.

[0086] Example 4

[0087] In vivo animal-level verification.

[0088] To establish an imiquimod-induced psoriasis mouse model, 5% imiquimod cream was applied to the back skin daily for 6 days. Disease activity was assessed using the PASI (Pain and Scale Indication System), and the cumulative score (considering erythema, scaling, and thickness) also served as an indicator of inflammation severity, ranging from 0 to 12. Multiple subcutaneous and intraperitoneal injections of sphingomyelin were administered to the skin area, followed by application of 5% imiquimod ointment. CD300e expression was assessed; inhibition of CD300e effectively improved psoriatic dermatitis in mice.

[0089] Verification results:

[0090] In vivo experiments revealed high expression of CD300e in imiquimod-induced psoriatic dermatitis. Imiquimod-induced mice were injected with sphingomyelin and phosphate-buffered saline. Six days later, the dorsal skin of mice in the Vaseline, PBS, and sphingomyelin groups was pink and smooth from day 1 to day 7. However, the skin of mice in the imiquimod group and sphingomyelin + imiquimod groups began to show erythema, scaling, and thickening after 2 to 3 days of continuous IMQ administration. After 4 to 7 days of imiquimod administration, the erythema gradually deepened, and the scales became increasingly thick. By day 7, the skin lesions were covered with a large amount of white scales that could be easily peeled off with a light touch, exhibiting more typical psoriatic changes. Figure 6 The Psoriasis-like Area and Severity Index (PASI) scores and infiltration scores in mice began to increase from day one and gradually reached their peak. The hypertrophy and erythema scores began to increase from day two and reached their peak levels. The mRNA levels of CD300E, IL-23A, IL-17A, IL-12A, and TNF-α in the dorsal skin of mice in the imiquimod group and the sphingomyelin + imiquimod group were significantly higher than those in the petrolatum group, PBS group, and sphingomyelin group.

[0091] Figures 6-8 The study showed imiquimod-induced psoriatic dermatitis in mice and its molecular expression. Figure 6 In the figure, (AE) represent the skin lesions in mice induced by petrolatum, PBS, sphingomyelin, imiquimod, and sphingomyelin combined with imiquimod, respectively. Figure 7 In the middle, (AD) are line graphs of the mouse PASI score, infiltration score, scaling score and erythema score, respectively; Figure 8 In the figures, (AE) represent the histopathological tissues of skin lesions induced by Vaseline, PBS, sphingomyelin, imiquimod, and sphingomyelin combined with imiquimod in mice, respectively.

[0092] Example 5

[0093] To verify the effect of cd300e gene knockout on improving imiquimod-induced psoriatic dermatitis in mice.

[0094] 1. Experimental procedures.

[0095] (1) WT mice were randomly divided into two groups of three each; CD300E-KO mice were also randomly divided into two groups of three each. After the mice had adapted, a 2cm wide pet shaver was used to shave a 2×3cm hairless area on the back of each mouse. Treatment details for each group are as follows. Figure 9 , Figure 9 The flowchart shows the experimental procedure in CD300E-KO mice.

[0096] 1) WT Vaseline: Apply 62.5 mg of Vaseline cream to the back daily for 6 consecutive days.

[0097] 2) WT IMQ group: Apply 62.5 mg of 5% IMQ cream to the back daily for 6 consecutive days.

[0098] 3) cd300e-KO Vaseline: Apply 62.5 mg of Vaseline cream to the back daily for 6 consecutive days.

[0099] 4) cd300e-KO IMQ group: Apply 62.5 mg of 5% IMQ cream to the back daily for 6 consecutive days.

[0100] (2) Before applying the medication each day, photographs were taken with a camera and micrometers were used to measure changes in skin lesions on the backs of mice. The PASI scores of the mice were evaluated by three independent experimenters (see Table 3 for details). Finally, the scores for erythema, infiltration, and scaling were calculated, and the sum of the three was used as a relatively objective indicator to assess the severity of the mouse skin lesions. Line graphs were plotted and statistical analysis was performed. Table 3 shows the PASI score table for psoriatic dermatitis in mice, and the results are shown in Table 4.

[0101] Table 3

[0102]

[0103] Table 4

[0104]

[0105] 2. Experimental results.

[0106] cd300e gene knockout improves imiquimod-induced psoriatic dermatitis in mice.

[0107] Six days after CD300e gene knockout, experiments were conducted. The dorsal skin of both wild-type Vaseline and knockout mice was pink and smooth from day 1 to day 7. However, histopathological examination of the skin of imiquimod mice showed epidermal thickening, hyperkeratosis, parakeratosis, a significant reduction in the granular layer, and acanthosis. In immunohistochemical studies, CD300e-positive cells were present in imiquimod mice, but not in Vaseline mice. Furthermore, the levels of CD300e, IL-23a, and IL-17a proteins in the dorsal skin of imiquimod mice were significantly higher than those in the knockout imiquimod, Vaseline, and Vaseline knockout groups. Figure 10-11 ).

[0108] Figure 10 and Figure 11 Western blot analysis was performed to detect protein expression in psoriatic dermatitis in mice. Figure 10 The images show the development of β-actin, CD300e, IL-23A, IL-12A, and IL-17A proteins. Figure 11 This is a statistical analysis chart of the corrected grayscale values. Blue represents wild-type mice, and red represents knockout mice. WT Vaseline (Vaseline), wild-type mice treated with Vaseline; WT IMQ, wild-type mice treated with imiquimod; KO Vaseline (Vaseline), knockout mice treated with Vaseline; KO IMQ, knockout mice treated with imiquimod; WT IMQ / Vaseline (Vaseline), the ratio of wild-type mice treated with imiquimod to wild-type mice treated with Vaseline; KO IMQ / Vaseline (Vaseline), the ratio of knockout mice treated with imiquimod to knockout mice treated with Vaseline. *P<0.05, ***P<0.001.

[0109] In summary, this invention reveals a novel immune mechanism for psoriasis, involving CD300e and its ligand sphingomyelin in the development of psoriasis via the IL-23A axis. This newly established model will facilitate the development of targeted therapies capable of preventing or reversing the overexpression of CD300e / sphingomyelin, suggesting that CD300e has potential breakthrough significance in the field of molecular immunology and could serve as a new target for psoriasis treatment.

[0110] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. Application of the CD300e gene or its expression product as a therapeutic target in the preparation of drugs for treating psoriasis.

2. The application according to claim 1, characterized in that, The drug includes reagents that inhibit CD300e gene expression or reagents that reduce the content of CD300e gene expression products.

3. The application according to claim 2, characterized in that, The expression products of the CD300e gene include: RNA transcribed from the CD300e gene or CD300e protein.

4. The application according to claim 2 or 3, characterized in that, The reagents for inhibiting CD300e gene expression include any one of the following: interfering RNA targeting the CD300e gene, hairpin RNA targeting the CD300e gene, or guide RNA for CD300e gene knockout.

5. The application according to any one of claims 2-4, characterized in that, The reagent that inhibits CD300e gene expression reduces protein expression in psoriatic dermatitis.

6. Application of sphingomyelin inhibitors in the preparation of drugs for treating psoriasis.

7. The application according to claim 6, characterized in that, The sphingomyelin inhibitor reduces protein expression in psoriatic dermatitis.

8. The use of at least one of CD300e gene inhibitors, CD300e gene expression product inhibitors, or sphingomyelin inhibitors in the preparation of drugs for treating psoriasis.

9. The application according to claim 8, characterized in that, The CD300e gene inhibitor includes agents that inhibit CD300e gene expression; Preferably, the CD300e gene expression product inhibitor includes an agent that reduces the content of the CD300e gene expression product; Preferably, the formulation for inhibiting CD300e gene expression includes any one of the following: interfering RNA targeting the CD300e gene, hairpin RNA targeting the CD300e gene, or guide RNA for CD300e gene knockout.

10. The application according to claim 5, characterized in that, The proteins in the psoriatic dermatitis include at least one of IL-23A, IL-12A, or IL-17A.

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