Application of SMOC2 protein in preparation of medicine for treating psoriasis

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of SMOC2 protein in preparation of a medicine for treating psoriasis, the amino acid sequence of the SMOC2 protein is shown as SEQ ID NO.1, it is found that the SMOC2 protein can treat psoriasis, and therefore the application of the SMOC2 protein in preparation of the medicine for treating psoriasis is provided. The SMOC2 protein can be used for treating or improving scales, erythema and thickening phenotypes of skin, inhibiting abnormal proliferation of keratinocytes and expression of inflammatory factors IL1beta, IL23, CAMP, TNF alpha, CXCL2, CXCL3, S100A8 and S100A9, and playing a role in treating or relieving skin inflammatory diseases by inhibiting infiltration of inflammatory cell neutrophils in psoriasis skin lesion tissues.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of SMOC2 protein in preparation of a psoriasis treatment drug. BACKGROUND

[0002] Psoriasis is a global chronic inflammatory skin disease, which is mainly characterized by abnormal proliferation of keratinocytes and disorder of the immune system. The pathogenesis of psoriasis has seriously affected the quality of life, mental health and social function of patients, and has become an important research topic in the medical field.

[0003] The pathogenesis of psoriasis is relatively complex. Keratinocytes in the skin are both target cells of psoriasis and drivers of disease progression. They mainly release cytokines such as IL-17 and IL-23, activate Th17 immune cells to release various cytokines, promote the proliferation of keratinocytes on the one hand, and form a positive feedback inflammatory reaction to aggravate skin inflammation on the other hand. At the same time, in the skin of psoriasis, various immune cells such as dendritic cells, T cells, T helper cells and neutrophils interact to form a complex immune regulation network. Targeting the cytokines IL-23 / IL-17 secreted by immune cells has become a target for the clinical treatment of psoriasis. Biological agents such as secukinumab can significantly improve the clinical symptoms of psoriasis patients by blocking IL-17A. However, biological agents such as secukinumab still face multiple limitations in clinical treatment, including poor response in about 20% of patients, individual differences in efficacy, residual lesions in special parts such as scalp and nails, secondary failure caused by long-term drug use, high recurrence rate after drug withdrawal, immune-related adverse reactions and infection risk, etc. Therefore, there is an urgent need to find new therapeutic targets for psoriasis.

[0004] Current clinical treatment targets for psoriasis are TNFα and cytokines IL-23 / IL-17 targeting keratinocytes and immune cells, which have significantly improved the clinical symptoms of psoriasis patients. However, biological agents such as secukinumab still face multiple limitations in clinical treatment, and new target cells and target molecules still need to be found.

[0005] Extracellular matrix protein SMOC2 (SPARC-related modular calcium binding protein 2, Entrez gene: 64094) is involved in important biological processes such as embryonic development, tissue fibrosis and angiogenesis. Among them, 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 tissue fibrosis through the TGFβ signaling pathway, and regulates endothelial cell proliferation and angiogenesis through the MAPK signaling pathway. However, its role in psoriasis is unknown. SUMMARY

[0006] In order to solve the above technical problems, the present application provides the application of SMOC2 protein in the preparation of psoriasis treatment drugs.

[0007] The application of SMOC2 protein in the preparation of psoriasis treatment drugs, the amino acid sequence of the SMOC2 protein is: MLLPQLCWLPLLAGLLPPVPAQKFSALTFLRVDQDKDKDCSLDCAGSPQKPLCASDGRTFLSRCEFQRAKCKDPQLEIAYRGNCKDVSRCVAERKYTQEQARKEFQQVFIPECNDDGTYSQVQCHSYTGYCWCVTPNGRPISGTAVAHKTPRCPGSVNEKLPQREGTGKTDDAAAPALETQPQGDEEDIASRYPTLWTEQVKSRQNKTNKNSVSSCDQEHQSALEEAKQPKNDNVVIPECAHGGLYKPVQCHPSTGYCWCVLVDTGRPIPGTSTRYEQPKCDNTARAHPAKARDLYKGRQLQGCPGAKKHEFLTSVLDALSTDMVHAASDPSSSSGRLSEPDPSHTLEERVVHWYFKLLDKNSSGDIGKKEIKPFKRFLRKKSKPKKCVKKFVEYCDVNNDKSISVQELMGCLGVAKEDGKADTKKRHTPRGHAESTSNRQPRKQG, recorded as SEQ ID NO. 1, 446 amino acids, molecular weight 49,674 Da.

[0008] The present application uses a psoriasis animal model to find that SMOC2 protein can treat or improve the scaling, erythema and thickening phenotype of the skin, and inhibit the abnormal proliferation of keratinocytes and the expression of inflammatory factors IL1β, IL23, CAMP, TNFα, CXCL2, CXCL3, S100A8 and S100A9, thus providing the application of an extracellular matrix protein in the preparation of SMOC2 psoriasis treatment drugs.

[0009]

[0010] Preferably, the therapeutic drug is made of the SMOC2 protein and an excipient, which is a pharmaceutically acceptable carrier, excipient or diluent.

[0011] Preferably, the therapeutic drug is in the form of an injection or an oral preparation.

[0012] Preferably, the concentration of the SMOC2 protein in the injection is 100 μg / mL to 1000 μg / mL.

[0013] Preferably, the oral preparation is a capsule or a tablet.

[0014] Preferably, the therapeutic drug is used to improve the scaling, erythema and thickening of the skin.

[0015] Preferably, the therapeutic drug is used to reduce the skin inflammation of psoriasis.

[0016] Compared with the prior art, the present application has the following advantages: The present application discloses that the SMOC2 protein can treat psoriasis, and therefore provides an application of the extracellular matrix protein SMOC2 or its encoding gene SMOC2 (Entrez gene: 64094) as a novel target molecule in the preparation of a psoriasis treatment drug.

[0017] The use of the SMOC2 protein can treat or improve the scaling, erythema and thickening of the skin, and inhibit the abnormal proliferation of keratinocytes and the expression of inflammatory factors IL1β, IL23, CAMP, TNFα, CXCL2, CXCL3, S100A8 and S100A9.

[0018] The present application can treat or relieve skin inflammatory diseases by inhibiting the infiltration of inflammatory cells, neutrophils, in the psoriasis lesion tissue. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The SMOC2 protein improves the phenotype of imiquimod-induced psoriasis in mice, wherein A is a schematic diagram of establishing a treatment of imiquimod-induced psoriasis model in mice by subcutaneous injection of SMOC2 protein, B is the skin lesion phenotype of mice in the negative control group (vaseline cream), model group (5% IMQ), control group (PBS) and treatment group (SMOC2) on the back, and C is the PASI score of the severity of psoriasis in the skin of mice in each group (** represents p <0.01).

[0020] Figure 2To improve the pathological structure changes of the lesion area of the mouse psoriasis model induced by imiquimod, wherein A is the H&E staining analysis of the negative control group (vaseline cream), the model group (5% IMQ): the control group (PBS) and the treatment group (SMOC2) pathological structure changes of the lesion area of the mouse back (the scale is 100 μm), B is the change of the epidermis thickness in the H&E staining results of each group (*** represents p <0.001).

[0021] Figure 3 To inhibit the mRNA expression of inflammatory factors in the lesion tissue of the mouse psoriasis model induced by imiquimod, A is IL1β, B is IL23, C is CAMP, D is TNFα, E is CXCL2, F is CXCL3, G is S100A8 and H is S100A9 (*** represents p <0.01, *** represents p <0.001).

[0022] Figure 4 To inhibit the infiltration of neutrophils in the lesion tissue of the mouse psoriasis model induced by imiquimod and the proportion of peripheral blood, wherein A is the immunofluorescence staining to show the infiltration of neutrophils in the lesion tissue of each group (the scale is 100 μm), B is the number of neutrophil infiltration in the lesion tissue of each group by immunofluorescence staining, C is the change of the proportion of neutrophils in the peripheral blood of each group of mice by flow cytometry, D is the proportion of CD45 + CD11b + Ly6G + The proportion of neutrophils (*** represents p <0.001). DETAILED DESCRIPTION

[0023] The specific embodiments of the present application are described in detail below, but it should be understood that the scope of protection of the present application is not limited by the specific embodiments. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without creative labor are within the scope of protection of the present application. The experimental methods described in each embodiment of the present application are conventional methods unless otherwise specified.

[0024] To illustrate the in vivo therapeutic effect of extracellular matrix protein SMOC2 in the mouse psoriasis model, the present application provides specific implementation methods and results: 1. Establishment of IMQ-induced mouse psoriasis model on the back 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.

[0025] 2. The effect of SMOC2 protein in treating psoriasis in mice 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.

[0026] 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.

[0027] The therapeutic effect of SMOC2 protein on psoriasis in mice was verified at the pathological level by H&E staining. The experimental model mice were sacrificed by CO2 asphyxiation. The mice were fixed, and the skin tissue of the model mice was taken from the back lesion area using sterile scissors and forceps and was placed in pre-cooled 4% PFA fixing solution for sufficient fixation of the tissue. The fixed skin tissue was placed in an embedding box and was subjected to gradient dehydration treatment by 70% ethanol, 80% ethanol, 95% ethanol I, 95% ethanol II, anhydrous ethanol I, and anhydrous ethanol II for 1 hour each. After xylene I and xylene II transparency treatment, the skin tissue was subjected to paraffin embedding. The embedded skin paraffin tissue was sectioned and was then subjected to xylene I, xylene II, anhydrous ethanol I, anhydrous ethanol II, 95% ethanol, 90% ethanol, and 80% ethanol for 5 minutes each, followed by water flushing. The tissue was dyed in hematoxylin dye solution for 5 minutes, was washed with water for 2 minutes, was immersed in 1% hydrochloric acid ethanol for several seconds, and was washed with water for 10 minutes. The tissue was immersed in 0.5% ammonia water for blue returning for 30 seconds and was washed with water for 1 minute. The tissue was dyed in eosin dye solution for 3 minutes and was quickly washed with water for several seconds. The tissue was subjected to 80% ethanol, 95% ethanol I, 95% ethanol II, anhydrous ethanol I, and anhydrous ethanol II for 2 minutes each, was mounted using neutral balsam, and was observed under an optical microscope. The pathological structure changes of the skin lesion tissue of each group of mice were photographed and recorded, and the thickness of the epidermis of the skin lesion tissue of each group of mice was measured using software to determine the proliferation of keratinocytes in the psoriasis model.

[0028] The results showed that, compared with the negative control group (vaseline cream), the epidermis of the skin lesion tissue of the control group (PBS) mice in the model group was significantly thickened, the keratinization was incomplete, and there were more inflammatory cell infiltrations, indicating that the keratinocytes were excessively proliferated and abnormally differentiated, the skin inflammatory reaction was aggravated, and the IMQ mouse psoriasis model was successfully established. However, the skin lesion tissue of the treatment group (SMOC2) mice in the model group showed reduced epidermal thickening and incomplete keratinization phenotype, and the inflammatory cell infiltration was reduced, as shown in A of Figure 2 . Statistical analysis of the epidermal thickness of the skin lesion tissue of the mice showed that, compared with the control group, the epidermal thickness of the skin lesion tissue of the treatment group mice was significantly reduced, as shown in B of Figure 2 . This result showed that the treatment with SMOC2 protein could significantly inhibit the abnormal proliferation and differentiation of keratinocytes in the skin lesion tissue of the mice and reduce the skin inflammatory reaction.

[0029] 3. Effect of SMOC2 protein on the expression of inflammatory factors in the skin of psoriasis mice The skin inflammatory response of psoriasis can release a large amount of inflammatory factors to aggravate the disease, and the H&E staining results show that the treatment of SMOC2 protein can reduce the infiltration of inflammatory cells, so the effect of SMOC2 protein on the expression of inflammatory factors in the skin of psoriasis mice is verified by Real-time qPCR technology. About 10 mg of fresh lesion tissue is taken according to the above method, cut into pieces and put into an RNAse-free EP tube. Add the lysis buffer of the commercial column total RNA extraction kit, homogenize the tissue, add organic solvent to separate the lysis solution into layers, and add the aqueous phase into a centrifugal column. Through the steps of centrifugation, washing, DNA removal, washing and drying, 30 μL of RNAse-free water is added to elute the total RNA of the tissue, and the concentration of the total RNA is quantified by ultraviolet spectrophotometry. 1 μg of total RNA is reverse transcribed into cDNA for storage using a commercial reverse transcription kit, and then the mRNA expression of inflammatory factors in the lesion tissue is detected using a commercial real-time fluorescent quantitative PCR kit.

[0030] The specific primers are as follows: Gapdh: the upstream primer is AGGTCGGTGTGAACGGATTTG, denoted as SEQ ID NO. 2, and the downstream primer is TGTAGACCATGTAGTTGAGGTCA, denoted as SEQ ID NO. 3; Il1b: the upstream primer is GCAACTGTTCCTGAACTCAACT, denoted as SEQ ID NO. 4, and the downstream primer is ATCTTTTGGGGTCCGTCAACT, denoted as SEQ ID NO. 5; Il23a: the upstream primer is ATGCTGGATTGCAGAGCAGTA, denoted as SEQ ID NO. 6, and the downstream primer is ACGGGGCACATTATTTTTAGTCT, denoted as SEQ ID NO. 7; Camp: the upstream primer is GCTGTGGCGGTCACTATCAC, denoted as SEQ ID NO. 8, and the downstream primer is TGTCTAGGGACTGCTGGTTGA, denoted as SEQ ID NO. 9; Tnf: the upstream primer is GACGTGGAACTGGCAGAAGAG, denoted as SEQ ID NO. 10, and the downstream primer is TTGGTGGTTTGTGAGTGTGAG, denoted as SEQ ID NO. 11; Cxcl2: the upstream primer is CCAACCACCAGGCTACAGG, denoted as SEQ ID NO. 12, and the downstream primer is GCGTCACACTCAAGCTCTG, denoted as SEQ ID NO. 13; Cxcl3: the upstream primer is AGGACCTTGATTATGCCAGCAA, denoted as SEQ ID NO. 14, and the downstream primer is GGGTTAGAGCCGGTTAACTTCA, denoted as SEQ ID NO. 15; S100a8: the upstream primer is AAATCACCATGCCCTCTACAAG, denoted as SEQ ID NO. 16, and the downstream primer is CCCACTTTTATCACCATCGCAA, denoted as SEQ ID NO. 17; S100a9: the upstream primer is ATACTCTAGGAAGGAAGGACACC, denoted as SEQ ID NO. 18, and the downstream primer is TCCATGATGTCATTTATGAGGGC, denoted as SEQ ID NO. 19.

[0031] The detection results show that, compared with the control group, the mRNA expression levels of inflammatory factors IL1β, IL23, CAMP, TNFα, CXCL2, CXCL3, S100A8 and S100A9 in the back skin lesion tissues of the treatment group mice are significantly reduced, as shown in the following table. Figure 3 The results prove that the treatment of SMOC2 protein can significantly inhibit the expression of inflammatory factors in the skin lesion tissues of the mouse psoriasis, and further reduce the inflammatory reaction of psoriasis.

[0032] 4. Effect of SMOC2 protein on neutrophil infiltration in the skin of psoriasis mice Early psoriasis attracts neutrophils to the lesion area through CXCL family molecules, activates and releases inflammatory factors to aggravate skin inflammation, so neutrophils are the main pathogenic cell type of psoriasis and an important therapeutic target cell of psoriasis. Since the neutrophil chemotactic factors Cxcl2 and Cxcl3 in the skin lesion tissues of the mice treated with SMOC2 protein for psoriasis are significantly reduced, and the inflammatory factors S100a8 and S100a9 released by neutrophils are also significantly reduced, the present application detects the infiltration of neutrophils in the skin lesion tissues of the mice treated with SMOC2 protein for psoriasis model by immunofluorescence technology.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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 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.

[0037] Psoriasis lesion tissue not only includes keratinocytes and immune cells, but also contains extracellular matrix (ECM) supporting these cells. ECM often occurs abnormal changes and deposition of collagen and laminin in psoriasis lesion tissue, which not only affects the interaction between cells and cells, cells and ECM, but also promotes inflammatory response through integrin signaling pathway. At the same time, the ECM degradation product hyaluronic acid fragment has been reported to be a biomarker of psoriasis disease activity. Therefore, the in-depth exploration of ECM proteins in psoriasis lesion tissue can provide new ideas and methods for disease diagnosis and treatment, and has very important scientific and application value.

[0038] It should be noted that when the present application claims involving numerical ranges, both endpoints of each numerical range and any number between the two endpoints can be selected, and in order to prevent repetition, the present application describes preferred embodiments.

[0039] Although preferred embodiments of the present application have been described, those skilled in the art, once they know the basic creative concept, can make additional changes and modifications to these embodiments. Therefore, the appended claims are intended to include the preferred embodiments and all changes and modifications falling within the scope of the present application.

[0040] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, the present application 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.

2.

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, excipients, or diluents.

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

5. The application according to claim 4, characterized in that, The concentration of SMOC2 protein in the injection is 100 μg / mL to 1000 μg / mL.

6. The application according to claim 4, characterized in that, The oral preparation is a capsule or tablet.

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

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

Images