Use of smoc2 protein in the preparation of a medicament for treating psoriasis

Abstract

This invention belongs to the field of biomedical technology, specifically relating to the application of SMOC2 protein in the preparation of psoriasis treatment drugs. The amino acid sequence of the SMOC2 protein is shown in SEQ ID NO.1. This invention has discovered that SMOC2 protein can treat psoriasis, and therefore provides the application of SMOC2 protein in the preparation of psoriasis treatment drugs. Using SMOC2 protein can treat or improve the scaling, erythema, and thickening phenotypes of the skin, as well as inhibit the abnormal proliferation of keratinocytes and the expression of inflammatory factors IL1β, IL23, cAMP, TNFα, CXCL2, CXCL3, S100A8, and S100A9. By inhibiting the infiltration of neutrophils in inflammatory cells in psoriatic lesions, it exerts a therapeutic or alleviating effect on inflammatory skin diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of SMOC2 protein in the preparation of drugs for the treatment of psoriasis. Background Technology

[0002] Psoriasis is a global chronic inflammatory skin disease characterized by abnormal proliferation of keratinocytes and dysregulation of the immune system. The onset of psoriasis has seriously affected patients' quality of life, mental health and social function. The pathological mechanism and public health research have become important research topics in the medical field.

[0003] The pathological mechanism of psoriasis is complex. Keratinocytes in the skin are both target cells and drivers of disease progression. They primarily activate Th17 and other immune cells to release various cytokines, such as IL-17 and IL-23, which in turn promote keratinocyte proliferation and create a positive feedback inflammatory response, exacerbating skin inflammation. Simultaneously, in psoriatic skin, various immune cells, such as dendritic cells, T cells, T helper cells, and neutrophils, interact to form a complex immune regulatory network. Targeting the cytokines IL-23 / IL-17 secreted by immune cells has become a clinical target for psoriasis treatment. Biologics such as secukinumab can significantly improve the clinical symptoms of psoriasis patients by blocking IL-17A. However, biologic cytokine monoclonal antibodies still face multiple limitations in clinical treatment, including individual differences in efficacy with poor response in about 20% of patients, residual lesions in special areas such as the scalp and nails, secondary failure caused by long-term use, high relapse rate after discontinuation of the drug, immune-related adverse reactions, and infection risks. Therefore, there is an urgent clinical need to find new therapeutic targets for psoriasis.

[0004] Currently, clinical treatment targets for psoriasis include TNFα and cytokines IL-23 / IL-17, which target keratinocytes and immune cells and have significantly improved the clinical symptoms of psoriasis patients. However, biologic cytokine monoclonal antibodies still face limitations in multiple clinical treatments, and there is still a need to find new target cells and molecules.

[0005] Extracellular matrix protein SMOC2 (SPARC-related modular calcium binding protein 2, Entrez gene: 64094) participates in important biological processes such as embryonic development, tissue fibrosis, and angiogenesis. Specifically, SMOC2 regulates bone development and differentiation through the BMP signaling pathway, promotes keratinocyte adhesion and wound healing by binding to integrin family molecules, regulates lung fibrosis through the TGFβ signaling pathway, and regulates endothelial cell proliferation and angiogenesis through the MAPK signaling pathway. However, its role in psoriasis remains unknown. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides the application of SMOC2 protein in the preparation of drugs for treating psoriasis.

[0007] The application of SMOC2 protein in the preparation of psoriasis treatment drugs, wherein the amino acid sequence of the SMOC2 protein is: MLLPQLCWLPLLAGLLPPVPAQKFSALTFLRVDQDKDKDCSLDCAGSPQKPLCASDGRTFLSRCEFQRAKCKDPQLEIAYRGNCKDVSRCVAERKYTQEQARKEFQQVFIPECNDDGTYSQVQCHSYTGYCWCVTPNGRPISGTAVAHKTPRCPGSVNEKLPQREGTGKTDDAAAPALETQPQGDEEDIASRYPTLWTEQVKSRQN KTNKNSVSSCDQEHQSALEEAKQPKNDNVVIPECAHGGLYKPVQCHPSTGYCWCVLVDTGRPIPGTSTRYEQPKCDNTARAHPAKARDLYKGRQLQGCPGAKKHEFLTSVLDALSTDMVHAAS DPSSSSGRLSEPDPSHTLEERVVHWYFKLLDKNSSGDIGKKEIKPFKRFLRKKSKPKKCVKKFVEYCDVNNDKSISVQELMGCLGVAKEDGKADTKKRHTPRGHAESTSNRQPRKQG, recorded as SEQ IDNO.1, 446 amino acids, molecular weight 49,674Da.

[0008] This invention uses an animal model of psoriasis to discover that the SMOC2 protein can treat or improve the scaling, erythema, and thickening phenotypes of the skin, as well as inhibit the abnormal proliferation of keratinocytes and the expression of inflammatory factors IL1β, IL23, cAMP, TNFα, CXCL2, CXCL3, S100A8, and S100A9. Therefore, it provides an application of extracellular matrix protein in the preparation of psoriasis treatment drugs using SMOC2.

[0009]

[0010] Preferably, the therapeutic agent is made from the SMOC2 protein and an excipient, wherein the excipient is a pharmaceutically acceptable carrier.

[0011] Preferably, the pharmaceutically acceptable carrier is an excipient or a diluent.

[0012] Preferably, the dosage form of the therapeutic drug is an injection or an oral preparation.

[0013] Preferably, the concentration of SMOC2 protein in the injection is 100 μg / mL to 1000 μg / mL.

[0014] Preferably, the oral formulation is a capsule or a tablet.

[0015] Preferably, the therapeutic agent is used to improve scaling, erythema, and thickening of the skin.

[0016] Preferably, the therapeutic agent is used to reduce skin inflammation in psoriasis.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] This invention has discovered that the SMOC2 protein can treat psoriasis, and therefore provides an extracellular matrix protein SMOC2 or its encoding gene. SMOC2 (Entrez gene: 64094) is used as a novel target molecule in the preparation of drugs for the treatment of psoriasis.

[0019] Using SMOC2 protein can treat or improve the scaling, erythema and thickening phenotypes of the skin, as well as inhibit the abnormal proliferation of keratinocytes and the expression of inflammatory factors IL1β, IL23, cAMP, TNFα, CXCL2, CXCL3, S100A8 and S100A9.

[0020] This invention exerts its therapeutic or alleviating effect on inflammatory skin diseases by inhibiting the infiltration of inflammatory centrioles in psoriatic lesion tissue. Attached Figure Description

[0021] Figure 1 To illustrate how SMOC2 protein improves the phenotype of imiquimod-induced psoriasis in mice, A shows a schematic diagram of establishing a mouse model of imiquimod-induced psoriasis treated with subcutaneous injection of SMOC2 protein; B shows the skin lesion phenotypes on the backs of mice in the negative control group (Vaseline cream), model group (5% IMQ), control group (PBS), and treatment group (SMOC2); and C shows the PASI scores for the severity of psoriasis in each group (** represents...). p <0.01).

[0022] Figure 2 To investigate how SMOC2 protein improves the pathological structural changes in the skin lesions of an imiquimod-induced mouse model of psoriasis, A shows the pathological structural changes in the dorsal skin lesions of mice in the negative control group (Vaseline cream), model group (5% IMQ), control group (PBS), and treatment group (SMOC2) analyzed by H&E staining (scale bar is 100 μm). B shows the changes in dorsal epidermal thickness in each group in the H&E staining results (*** represents...). p <0.001).

[0023] Figure 3 To inhibit the mRNA expression of inflammatory factors in imiquimod-induced mouse psoriasis model, SMOC2 protein was used. A represents IL1β, B represents IL23, C represents cAMP, D represents TNFα, E represents CXCL2, F represents CXCL3, G represents S100A8, and H represents S100A9 (** represents...). p <0.01, *** represents p <0.001).

[0024] Figure 4 To investigate the effect of SMOC2 protein on the inhibition of imiquimod-induced neutrophil infiltration in skin lesions and the proportion of neutrophils in peripheral blood in a mouse model of skin psoriasis, the following data were used: A) Immunofluorescence staining showing neutrophil infiltration in skin lesions of each group (scale bar 100 μm); B) Immunofluorescence staining showing the number of infiltrated neutrophils in skin lesions of each group; C) Flow cytometry showing changes in the proportion of neutrophils in peripheral blood of each group of mice; D) CD45 levels in peripheral blood of each group. + CD11b + Ly6G + The proportion of neutrophils (*** represents) p <0.001). Detailed Implementation

[0025] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods.

[0026] To illustrate the in vivo therapeutic effect of extracellular matrix protein SMOC2 in a mouse psoriasis model, this invention provides specific implementation methods and results:

[0027] 1. Establishment of an IMQ-induced mouse model of dorsal psoriasis

[0028] Eighteen C57BL / 6J mice aged 8–10 weeks were randomly divided into three groups: a negative control group, a model group, a control group (PBS), and a treatment group (SMOC2), with six mice in each group. The day before model establishment (-1), the mice's backs were shaved, creating a bare area of ​​approximately 2 cm × 3 cm. On days 0, 1, 2, and 3, commercially available 5% IMQ cream was applied evenly to the bare skin area on the backs of the model group mice at a dose of 62.5 mg / day / mouse. The negative control group mice received an equal amount of petroleum jelly cream applied evenly to their bare skin area. The skin condition of the bare skin area on the backs of the mice was photographed daily, and the severity of the psoriasis model was comprehensively assessed using the Psoriasis Area and Severity Index (PASI). The model was terminated on day 4, and tissue samples from the bare skin area on the backs were collected for subsequent experimental analysis.

[0029] 2. The effect of SMOC2 protein in treating psoriasis in mice

[0030] In the established mouse dorsal psoriasis model treatment group, SMOC2 protein was dissolved in PBS to obtain SMOC2 protein solution. This solution was subcutaneously injected into the exposed skin area of ​​the mouse's back at a dose of 75 μg / kg on days -1, 1, and 3. The control group received an equal volume of PBS subcutaneously. Figure 1 As shown in A in the diagram.

[0031] The results showed that, compared with the control group, the psoriasis symptoms such as infiltration, scaling, and erythema in the exposed skin areas on the backs of mice in the treatment group were significantly reduced. Figure 1 As shown in B. Furthermore, PASI scores also confirmed that the treatment group's PASI score began to decrease on day 3 and was significantly lower than the control group on day 4, as shown in Figure B. Figure 1 As shown in C. The above results indicate that treatment with SMOC2 protein can significantly reduce psoriasis symptoms on the back of mice.

[0032] Subsequently, the efficacy of SMOC2 protein in treating psoriasis in mice was pathologically verified using H&E staining. The experimental model mice were euthanized using CO2 asphyxiation. The mice were fixed, and skin tissue from the dorsal lesion area was collected using sterile scissors and forceps and thoroughly fixed in pre-cooled 4% PFA fixative. The fixed skin tissue was placed in an embedding cassette and subjected to a gradient dehydration process, passing through 70% ethanol, 80% ethanol, 95% ethanol I, 95% ethanol II, anhydrous ethanol I, and anhydrous ethanol II for 1 hour each. After clearing with xylene I and xylene II, the skin tissue was embedded in paraffin. The embedded paraffin-embedded skin tissue was sectioned and then passed through xylene I, xylene II, anhydrous ethanol I, anhydrous ethanol II, 95% ethanol, 90% ethanol, and 80% ethanol for 5 minutes each, followed by rinsing with running water. The tissue was then stained with hematoxylin for 5 minutes, rinsed with running water for 2 minutes, and then immersed in 1% hydrochloric acid ethanol for a few seconds for differentiation, followed by rinsing with running water for 10 minutes. Immerse in 0.5% ammonia solution for 30 seconds to achieve blue reversion, then rinse with running water for 1 minute. Immerse in eosin staining solution for 3 minutes, then rinse quickly with water for a few seconds. Pass through 80% ethanol, 95% ethanol I, 95% ethanol II, anhydrous ethanol I, and anhydrous ethanol II for 2 minutes each, then mount with neutral resin. Observe the changes in pathological structure of skin lesions in each group of mouse psoriasis models under an optical microscope, photograph and record the H&E staining results of skin lesions in each group, and use software to measure the thickness of the epidermis in the dorsal skin lesions of each group of mice, thereby indicating the proliferation of keratinocytes in psoriatic disease.

[0033] The results showed that, compared with the negative control group (Vaseline cream), the epidermis of the dorsal skin lesions in the model group (PBS) mice was significantly thickened, with parakeratosis and more inflammatory cell infiltration, indicating excessive proliferation and abnormal differentiation of keratinocytes, aggravated skin inflammation, and successful establishment of the IMQ mouse psoriasis model. However, the dorsal skin lesions in the treatment group (SMOC2) mice in the model group showed reduced epidermal thickening and parakeratosis compared with the control group, and also reduced inflammatory cell infiltration, such as... Figure 2 As shown in A in the figure. Statistical analysis of the epidermal thickness of the dorsal skin lesions in mice revealed that, compared with the control group, the epidermal thickness of the dorsal skin lesions in the treatment group was significantly reduced, as shown in Figure A. Figure 2 As shown in B in the figure. This part of the results shows that treatment with SMOC2 protein can significantly inhibit the abnormal proliferation and differentiation of keratinocytes in psoriatic lesions on the back of mice and reduce skin inflammation.

[0034] 3. Effects of SMOC2 protein on the expression of inflammatory factors in the skin of psoriatic mice.

[0035] The inflammatory response of psoriasis skin can release a large number of inflammatory factors, exacerbating the disease. H&E staining results showed that treatment with SMOC2 protein reduced the infiltration of inflammatory cells. Therefore, Real-time qPCR was used to verify the effect of SMOC2 protein on the expression of inflammatory factors in the skin of psoriatic mice. Approximately 10 mg of fresh skin lesion tissue was collected as described above, minced, and placed in an RNase-free EP tube. Lysis buffer from a commercial total RNA extraction kit was added, the tissue was homogenized, and an organic solvent was added to separate the lysis buffer. The aqueous phase was added to a centrifuge column, and after centrifugation, washing, DNA removal, washing, and drying, 30 μL of RNase-free water was added to elute the total RNA from the tissue. The concentration of total RNA was quantified using a UV spectrophotometer. 1 μg of total RNA was reverse transcribed into cDNA using a commercial reverse transcription kit and stored. Subsequently, the mRNA expression levels of inflammatory factors in the skin lesion tissue were detected using a commercial real-time quantitative PCR kit.

[0036] The specific primers are as follows:

[0037] Gapdh: The upstream primer is AGGTCGGTGTGAACGGATTTG, denoted as SEQ ID NO.2, and the downstream primer is TGTAGACCATGTAGTTGAGGTCA, denoted as SEQ ID NO.3;

[0038] Il1b: The upstream primer is GCAACTGTTCCTGAACTCAACT, denoted as SEQ ID NO.4, and the downstream primer is ATCTTTTGGGGTCCGTCAACT, denoted as SEQ ID NO.5;

[0039] Il23a: The upstream primer is ATGCTGGATTGCAGAGCAGTA, denoted as SEQ ID NO.6, and the downstream primer is ACGGGGCACATTATTTTTAGTCT, denoted as SEQ ID NO.7;

[0040] Camp: The upstream primer is GCTTGTGGCGGTCACTATCAC, denoted as SEQ ID NO.8, and the downstream primer is TGTCTAGGGACTGCTGGTTGA, denoted as SEQ ID NO.9;

[0041] Tnf: The upstream primer is GACTGGAACTGGCAGAAGAG, denoted as SEQ ID NO.10, and the downstream primer is TTGGTGGTTTGTGAGTGTGAG, denoted as SEQ ID NO.11;

[0042] Cxcl2: The upstream primer is CCAACCACCAGGCTACAGG, denoted as SEQ ID NO.12, and the downstream primer is GCGTCACACTCAAGCTCTG, denoted as SEQ ID NO.13;

[0043] Cxcl3: The upstream primer is AGGACCTTGATTATGCCAGCAA, denoted as SEQ ID NO.14, and the downstream primer is GGGTTAGAGCCGGTTAACTTCA, denoted as SEQ ID NO.15;

[0044] S100a8: The upstream primer is AAATCACCATGCCCTCTACAAG, denoted as SEQ ID NO.16, and the downstream primer is CCCACTTTTATCACCATCGCAA, denoted as SEQ ID NO.17;

[0045] S100a9: The upstream primer is ATACTCTAGGAAGGAAGGACACC, denoted as SEQ ID NO.18, and the downstream primer is TCCATGATGTCATTTATGAGGGC, denoted as SEQ ID NO.19.

[0046] The results showed that, compared with the control group, the mRNA expression levels of inflammatory factors IL1β, IL23, cAMP, TNFα, CXCL2, CXCL3, S100A8, and S100A9 in the dorsal skin lesions of mice in the treatment group were significantly reduced. Figure 3 As shown in the figure. This result demonstrates that treatment with SMOC2 protein can significantly inhibit the expression of inflammatory factors in psoriatic lesions on the back of mice, thereby alleviating the inflammatory response of psoriasis.

[0047] 4. Effects of SMOC2 protein on neutrophil infiltration in the skin of psoriatic mice

[0048] Early-stage psoriasis involves chemotaxis of neutrophils to the lesion area via CXCL family molecules, activating and releasing inflammatory factors that exacerbate skin inflammation. Therefore, neutrophils are the main pathogenic cell type in psoriasis and an important therapeutic target cell. Since the levels of neutrophil chemotactic factors Cxcl2 and Cxcl3 were significantly reduced in the skin lesions of mice treated with SMOC2 protein, and the release of inflammatory factors S100a8 and S100a9 from neutrophils was also significantly reduced, this invention uses immunofluorescence technology to detect neutrophil infiltration in the dorsal skin lesions of a SMOC2 protein-treated mouse psoriasis model.

[0049] The mouse dorsal skin lesions fixed in the above experiments were sequentially immersed in a gradient of sucrose / PBS solutions for dehydration, and then rapidly frozen and preserved in an OCT embedding cassette. The embedded tissue was sectioned using a cryostat, and after drying at room temperature for 1 hour, it was washed twice with PBS for 5 minutes each time, permeabilized with 0.5% Triton X-100 for 10 minutes, and washed three times with PBS for 5 minutes each time. The slides were blocked with 5% goat serum at room temperature for 1 hour. The neutrophil marker molecule MPO antibody was diluted 1:200 and incubated overnight in a humidified chamber at 4°C. The slides were washed three times with PBST for 5 minutes each time, and the secondary antibody (goat anti-rabbit IgG) was diluted 1:500 and added to the slides. The slides were incubated at room temperature in the dark for 1 hour. The slides were washed three times with PBST for 5 minutes each time, and the cell nuclei were stained with DAPI. After washing, the slides were mounted with anti-fluorescence quenching mounting medium, and photographed using a fluorescence microscope.

[0050] Analysis of immunofluorescence results of neutrophil infiltration in skin tissue of a mouse psoriasis model revealed that the fluorescence intensity of MPO-positive neutrophils in the skin lesions of the treatment group was significantly lower than that in the control group. Figure 4 As shown in A, the number of MPO-positive neutrophil infiltrations was also significantly reduced, as... Figure 4 As shown in B in the diagram.

[0051] To verify the effect of SMOC2 protein on neutrophil chemotaxis in the peripheral blood of psoriatic mice, this invention used flow cytometry to detect the proportion of neutrophils in the peripheral blood of model mice. Before sacrifice, blood was collected from the mice using EDTA / heparin anticoagulant tubes. 50 μL of whole blood was added to 500 μL of pre-chilled 1×ACK lysis buffer, vortexed, and lysed on ice for 5 minutes. Lysis was then terminated by adding 10 volumes of pre-chilled PBS. The cells were centrifuged at 400g for 5 minutes at 4°C, the supernatant was discarded, and erythrocytes were repeatedly lysed until the cell pellet was white. Cell counting was performed using a hemocytometer. A certain number of cells were added to commercially available fluorescently labeled CD45, CD11b, Ly6G, and isotype control antibodies, mixed thoroughly, and incubated at 4°C in the dark for 30 minutes. The cells were washed with pre-chilled PBS and finally resuspended in 200 μL of pre-chilled PBS for flow cytometry analysis of the labeled cells.

[0052] Flow cytometry results showed that CD45 levels in the peripheral blood of mice in the SMOC2 protein treatment group of a mouse psoriasis model were significantly increased. + CD11b + Ly6G + The proportion of neutrophils was significantly lower than that in the control group, such as Figure 4 As shown in C and D in the figure. The above results indicate that the SMOC2 protein exerts a therapeutic effect on psoriasis by inhibiting neutrophil chemotaxis and inflammatory factor expression, thereby reducing skin inflammation and phenotype in psoriasis.

[0053] Psoriatic lesions contain not only keratinocytes and immune cells, but also the extracellular matrix (ECM) supporting these cells. Abnormal changes and deposition of collagen and laminin are frequently observed in psoriatic lesions, affecting not only cell-cell and cell-ECM interactions, but also promoting inflammatory responses through the integrin signaling pathway. Furthermore, hyaluronic acid fragments, ECM degradation products, have been reported as biomarkers for psoriasis activity. Therefore, in-depth research into ECM proteins in psoriatic lesions can provide new ideas and methods for disease diagnosis and treatment, possessing significant scientific and applied value.

[0054] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0055] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0056] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of SMOC2 protein in the preparation of drugs for treating psoriasis, characterized in that, The amino acid sequence of the SMOC2 protein is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The gene sequence encoding the SMOC2 protein is shown in SEQ ID NO.

20.

3. The application according to claim 1, characterized in that, The therapeutic agent is made from the SMOC2 protein and excipients, wherein the excipients are pharmaceutically acceptable carriers.

4. The application according to claim 3, characterized in that, The pharmaceutically acceptable carrier is an excipient or a diluent.

5. The application according to claim 1, characterized in that, The therapeutic drug is in the form of an injection or an oral preparation.

6. The application according to claim 5, characterized in that, The concentration of SMOC2 protein in the injection is 100 μg / mL to 1000 μg / mL.

7. The application according to claim 5, characterized in that, The oral preparation is a capsule or tablet.

8. The application according to claim 1, characterized in that, The therapeutic drug is used to improve scaling, erythema, and thickening of the skin.

9. The application according to claim 1, characterized in that, The treatment medication is used to reduce skin inflammation associated with psoriasis.

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