Application of CREB3L1 in diagnosis and treatment of systemic sclerosis

Abstract

The invention discloses application of CREB3L1 in diagnosis and treatment of systemic sclerosis, and belongs to the technical field of biological medicine. The invention discovers that CREB3L1 has the function of regulating and controlling fibrosis of skin and lung tissues of a patient with systemic sclerosis for the first time, and plays an important role in the progress of fibrosis of systemic sclerosis. The CREB3L1 can be used as a biomarker for diagnosis and auxiliary diagnosis of systemic sclerosis, and the CREB3L1 inhibitor can be used as an effective component for controlling and / or reversing fibrosis of systemic sclerosis, and has a good clinical application prospect. A new target is provided for diagnosis and treatment of systemic sclerosis, and a solid research foundation is laid for development of drugs for resisting systemic sclerosis fibrosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an application of CREB3L1 in diagnosing and treating systemic sclerosis. Background Art

[0002] Systemic Sclerosis (SSc) is a rare, intractable autoimmune disease that can lead to hardened and contracted skin and extensive fibrosis of internal organs, such as pulmonary fibrosis, pulmonary hypertension, heart damage and renal failure, which endanger the patient's life. The difficulty in the clinical treatment of systemic sclerosis lies in the fact that the existing treatment methods are only immunosuppressants and symptomatic treatment, and there is no effective cure. Long-term immunosuppression not only brings a series of side effects, but also increases the risk of infection in patients. As the patient ages, the side effects of immunosuppression are particularly significant. Secondly, patients need to regularly evaluate their condition throughout their lives. Every six months or every year, they need to undergo a large number of examinations and tests such as blood routine, liver and kidney function, myocardial enzymes, autoantibodies, KL-6, lung function, high-resolution chest CT, cardiac ultrasound, abdominal organ ultrasound, etc. to comprehensively evaluate the disease involvement, which imposes a heavy medical and psychological burden on patients. Therefore, it is particularly important to find biological markers that can truly reflect the severity of the patient's disease and new therapeutic targets that do not rely on immunosuppression.

[0003] Fibrosis is the most basic pathological manifestation of SSc. The abnormal activation of fibroblasts produces too much collagen fibers, resulting in remodeling and hardening of the intercellular matrix, which is the direct cause of SSc visceral fibrosis. However, the factors that determine abnormal activation and the effects of enhanced activation are still unclear. Studies have shown that after culturing fibroblasts in the reticular dermis and papillary dermis of SSc patients, the production of collagen and other extracellular matrix components by fibroblasts increases; this phenomenon has been confirmed in vivo, in biopsy samples, and in vitro cultures of fibroblasts from SSc skin lesions. Normal skin fibroblasts are in a relatively static state, and in the absence of positive stimulation, they are unlikely to proliferate or produce extracellular matrix.

[0004] CREB3L1 (CAMP Responsive Element Binding Protein 3Like 1): It is a precursor of a transcription factor. Upon ER stress or DNA damage, it is transported to the Golgi apparatus, where it is cleaved in a site-specific manner by the resident proteases S1P / MBTPS1 and S2P / MBTPS2. The released N-terminal cell membrane domain is transported to the nucleus, where it activates the transcription of specific target genes involved in the inhibition of cell cycle progression.

[0005] The present invention first discovered that CREB3L1 has the function of regulating fibrosis of the skin and lung tissue of Ssc patients. Specifically, the present invention found that compared with healthy people, CREB3L1 was significantly highly expressed in the skin and skin fibroblasts of Ssc patients, and in the model mice with scleroderma and pulmonary fibrosis, high expression of CREB3L1 promoted the progression of skin and lung fibrosis, while low expression of CREB3L1 inhibited skin and lung fibrosis. Based on the above research, the present invention believes that CREB3L1 can be used as a biomarker for the diagnosis and auxiliary diagnosis of systemic sclerosis, and CREB3L1 can be used as a target for the development of drugs for the treatment of systemic sclerosis fibrosis. Summary of the invention

[0006] One object of the present invention is to provide an application of CREB3L1 as a biomarker in the diagnosis or auxiliary diagnosis of systemic sclerosis; a second object of the present invention is to provide an application of CREB3L1 as a target in the preparation of a drug for the treatment of systemic sclerosis, specifically, the drug is a CREB3L1 inhibitor; a further object of the present invention is to provide a pharmaceutical composition comprising the CREB3L1 inhibitor.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] In a first aspect, the present invention provides a use of CREB3L1 as a biomarker in at least one of the following:

[0009] a1) Use in the preparation and / or screening of products for diagnosis or auxiliary diagnosis of systemic sclerosis;

[0010] a2) Application in the preparation and / or screening of products for evaluating or assisting in evaluating the therapeutic efficacy of systemic sclerosis;

[0011] a3) Use in the preparation and / or screening of products for evaluating or assisting in the evaluation of the degree of skin and / or pulmonary fibrosis in systemic sclerosis;

[0012] a4) Use in the preparation and / or screening of products for determining or assisting in determining the prognosis of systemic sclerosis.

[0013] The products include but are not limited to reagents, test kits, chips, test strips, membrane strips or detection platforms.

[0014] In a second aspect, the present invention provides a use of a substance for detecting CREB3L1 in at least one of the following:

[0015] b1) Use in the preparation and / or screening of products for diagnosis or auxiliary diagnosis of systemic sclerosis;

[0016] b2) Application in the preparation and / or screening of products for evaluating or assisting in evaluating the therapeutic efficacy of systemic sclerosis;

[0017] b3) Use in the preparation and / or screening of products for evaluating or assisting in the evaluation of the degree of skin and / or pulmonary fibrosis in systemic sclerosis;

[0018] b4) Use in the preparation and / or screening of products for determining or assisting in determining the prognosis of systemic sclerosis.

[0019] The products include but are not limited to reagents, test kits, chips, test strips, membrane strips or detection platforms.

[0020] The substances for detecting CREB3L1 include any reagents required for detecting the expression level of CREB3L1 protein or the expression level of CREB3L1 gene by RT PCR, RT qPCR, biochip detection, Southern blotting, Western Blot, in situ hybridization, immunohistochemistry (IHC), enzyme-linked immunosorbent assay, and immunofluorescence technology.

[0021] In some embodiments of the present invention, the substance for detecting CREB3L1 is a reagent for detecting CREB3L1 protein by immunofluorescence technique.

[0022] In some embodiments of the present invention, the substance for detecting CREB3L1 is a reagent for detecting CREB3L1 protein by Western Blot.

[0023] In some embodiments of the present invention, the substance for detecting CREB3L1 is a reagent for detecting CREB3L1 protein by immunohistochemistry.

[0024] In a third aspect, the present invention provides a kit, the kit comprising the substance for detecting CREB3L1 provided in the second aspect of the present invention. The kit can be used to detect the expression level of CREB3L1 in a sample to be tested, which can be used for diagnosing or assisting in diagnosing whether a patient has systemic sclerosis.

[0025] In a fourth aspect, the present invention provides a use of CREB3L1 as a target in at least one of the following:

[0026] c1) Use in the preparation and / or screening of drugs for preventing systemic sclerosis;

[0027] c2) Use in the preparation and / or screening of drugs for the treatment or adjuvant treatment of systemic sclerosis.

[0028] In a fifth aspect, the present invention provides a use of a CREB3L1 inhibitor in at least one of the following:

[0029] d1) Use in the preparation and / or screening of drugs for preventing systemic sclerosis;

[0030] d2) Use in the preparation and / or screening of drugs for the treatment or adjuvant treatment of systemic sclerosis.

[0031] The treatment or adjuvant treatment described in the present invention includes controlling and / or reversing systemic sclerosis skin and / or lung tissue fibrosis.

[0032] In the present invention, the CREB3L1 inhibitor includes a substance that reduces the expression of CREB3L1 protein, the content of CREB3L1 protein, or the activity of the protein, or a substance that inhibits the expression of the CREB3L1 gene, including but not limited to one or more of nucleic acid molecules, small molecule compounds, antibodies, polypeptides, proteins, gene editing vectors, lentiviruses, or adeno-associated viruses.

[0033] In some embodiments of the present invention, the CREB3L1 inhibitor is short hairpin RNA (shRNA), small interfering RNA (siRNA) or CREB3L1 low-expressing adenovirus, and the inhibitor can reduce the expression of the gene encoding the CREB3L1 protein.

[0034] In some embodiments of the present invention, the CREB3L1 inhibitor is a molecule that binds to the CREB3L1 protein, such as one or a combination of two or more of an antibody, a peptide, or a small molecule, including but not limited to a humanized monoclonal antibody, a receptor antagonist with CREB3L1 as a ligand, and the inhibitor can reduce the content or activity of the CREB3L1 protein, or interfere with the binding of CREB3L1 to its receptor.

[0035] In a sixth aspect, the present invention provides a pharmaceutical composition comprising an effective amount of the CREB3L1 inhibitor provided in the fifth aspect of the present invention.

[0036] The CREB3L1 inhibitor includes a substance that reduces the expression of CREB3L1 protein, the content of CREB3L1 protein, or the activity of the protein, or a substance that inhibits the expression of the CREB3L1 gene, including but not limited to one or more of nucleic acid molecules, small molecule compounds, antibodies, polypeptides, proteins, gene editing vectors, lentiviruses, or adeno-associated viruses.

[0037] In some embodiments of the present invention, the CREB3L1 inhibitor is short hairpin RNA (shRNA), small interfering RNA (siRNA) or CREB3L1 low-expressing adenovirus, and the inhibitor can reduce the expression of the gene encoding the CREB3L1 protein.

[0038] In some embodiments of the present invention, the CREB3L1 inhibitor is a molecule that binds to the CREB3L1 protein, such as one or a combination of two or more of an antibody, a peptide, or a small molecule, including but not limited to a humanized monoclonal antibody, a receptor antagonist with CREB3L1 as a ligand, and the inhibitor can reduce the content or activity of the CREB3L1 protein, or interfere with the binding of CREB3L1 to its receptor.

[0039] In a specific embodiment of the present invention, the CREB3L1 inhibitor is a CREB3L1 low-expression adenovirus that inhibits the expression of CREB3L1 protein-related genes.

[0040] Furthermore, the pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0041] The term "pharmaceutically acceptable" as used herein means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the mammal to be treated with it. Preferably, the term "pharmaceutically acceptable" means approved by a federal regulatory agency or a national government or listed in the United States Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, especially humans. The pharmaceutically acceptable excipients described herein may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for a specific target dosage form.

[0042] The pharmaceutical composition of the present invention can be used for the treatment of diseases and can also be used for in vitro cell culture experiments. When used for the treatment of diseases, the pharmaceutical composition generally refers to a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical field. All methods include the step of combining the active ingredient with an excipient that constitutes one or more auxiliary ingredients.

[0043] The disease treatment refers to administering a therapeutically effective amount of the CREB3L1 inhibitor or pharmaceutical composition of the present invention to a subject in need thereof.

[0044] Furthermore, the disease treatment also includes administering the CREB3L1 inhibitor or pharmaceutical composition of the present invention in combination with other conventional agents or conventional treatment methods in the art.

[0045] The technical solution provided by the present invention contributes to the diagnosis and treatment of systemic sclerosis in the following ways:

[0046] 1. The present invention found that the expression of CREB3L1 protein in the skin and skin fibroblasts of SSc patients was increased through immunofluorescence staining technology and Western Blot experiments, indicating that by detecting the level of CREB3L1 protein in the skin or skin fibroblasts of patients, it is possible to diagnose or assist in the diagnosis of whether the patient suffers from systemic sclerosis;

[0047] 2. When adenovirus expressing high CREB3L1 was injected into mice with scleroderma and pulmonary fibrosis, collagen fibrosis in the mouse skin and lungs was significantly increased; when adenovirus expressing low CREB3L1 was injected into mice with scleroderma and pulmonary fibrosis, collagen fibrosis in the mouse skin and lungs was significantly reduced. Therefore, reducing the expression or activity of CREB3L1 protein, or inhibiting CREB3L1 gene expression can control and / or reverse the fibrosis of the skin and / or lung tissues in systemic sclerosis.

[0048] The conclusions proposed in the present invention suggest that CREB3L1 plays an important role in the progression of systemic sclerosis fibrosis, and CREB3L1 can be used as a biomarker or target for the diagnosis and treatment of systemic sclerosis. CREB3L1 inhibitors can be used as active ingredients to control and / or reverse systemic sclerosis fibrosis, and have good clinical application prospects. The present invention provides a new target for the diagnosis and treatment of systemic sclerosis, and lays a solid research foundation for the development of drugs against systemic sclerosis fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Immunofluorescence staining detection of CREB3L1 expression level in skin tissue; Figure 1 (a) is the microscopic result of healthy subjects. Figure 1 (b) is the microscopic result of Ssc patients. Figure 1 (c) is the statistical result of CREB3L1 expression.

[0050] Figure 2 WB detection of CREB3L1 and COL1A1 expression levels in skin fibroblasts; Figure 2 (a) is the WB detection image, Figure 2 (b) is the relative expression level of CREB3L1, Figure 2 (c) is the relative expression level of COL1A1.

[0051] Figure 3 WB detection of mitochondrial related indicators and CREB3L1 expression levels in mouse skin and lung tissues; Figure 3 (a) is the WB detection image of skin tissue. Figure 3 (b) is the WB detection image of lung tissue.

[0052] Figure 4 Masson staining and immunohistochemistry results of mouse skin and lung tissue to detect collagen 1 expression levels. DETAILED DESCRIPTION

[0053] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0054] The technicians of the present invention performed single-cell sequencing on skin tissues of healthy subjects and Ssc patients, and performed pseudo-time analysis on fibroblasts, and found that there was a group of unique differentiation trajectories in the skin of Ssc patients' fibroblasts, which was called Fate1. Further in-depth analysis of Fate1 revealed that CREB3L1 was its TOP-specific highly expressed transcription factor. Therefore, the present invention speculates that CREB3L1 is closely related to the unique fibroblast group existing in Ssc. The present invention further designed the following series of experiments to verify this hypothesis.

[0055] 1. CREB3L1 expression is elevated in the skin and skin fibroblasts of Ssc patients

[0056] 1. CREB3L1 is highly expressed in the skin of Ssc patients

[0057] Purpose of the experiment: To detect the expression level of CREB3L1 in the skin tissues of healthy subjects and Ssc patients by immunofluorescence staining.

[0058] Experimental method: Skin tissues of healthy subjects and Ssc patients were obtained, and the expression levels of CREB3L1 in the skin tissues to be tested were detected by immunofluorescence staining technology using commercially available kits and statistically analyzed.

[0059] Test results: After immunofluorescence staining of skin tissue, the results under the microscope are as follows Figure 1 (a) and Figure 1 (b) shows the statistical results of CREB3L1 expression. Figure 1 (c) As shown in the immunofluorescence staining results and statistical results, it can be seen that the expression level of CREB3L1 in the skin of Ssc patients is significantly higher than that in healthy subjects.

[0060] 2. CREB3L1 is highly expressed in skin fibroblasts of Ssc patients

[0061] Purpose of the experiment: To detect the expression levels of CREB3L1 and collagen 1 (COL1A1) in skin fibroblasts (Fb) of healthy subjects and Ssc patients by WB (western blot) experiments.

[0062] Experimental method: Skin fibroblasts from healthy subjects and Ssc patients were isolated, and the expression levels of CERB3L1 and collagen 1 (COL1A1) in skin fibroblasts were detected by WB experiment and statistically analyzed.

[0063] Test results: WB test results are as follows Figure 2 As shown in (a), the relative expression of CREB3L1 is Figure 2 (b) shows that the relative expression of COL1A1 is Figure 2 As shown in (c), the expression levels of CREB3L1 and COL1A1 in skin fibroblasts of Ssc patients were significantly higher than those in healthy subjects.

[0064] Based on the above detection results of CERB3L1 expression levels in skin and skin fibroblasts, it can be seen that the expression level of CERB3L1 in the skin and skin fibroblasts of Ssc patients is significantly higher than that of healthy people. Therefore, those skilled in the art can diagnose or assist in the diagnosis of whether a patient is an Ssc patient by detecting the expression level of CERB3L1 in the skin or skin fibroblasts.

[0065] 2. Overexpression of CREB3L1 promotes bleomycin-induced skin and lung fibrosis in Ssc mice, while underexpression of CREB3L1 inhibits bleomycin-induced skin fibrosis in Ssc mice

[0066] 1. Preparation of Scleroderma-related Pulmonary Fibrosis Model Mice

[0067] 1.1 Three types of adenovirus were customized, namely AAV blank control, AAV-oeCREB3L1 (for high expression of CREB3L1), and AAV-shCREB3L1 (for knocking down CREB3L1);

[0068] 1.2 The mice were randomly divided into 4 groups, namely control group, experimental group 1, experimental group 2, and experimental group 3;

[0069] 1.3 Before the first injection of bleomycin, the mice in the experimental group were anesthetized with sodium pentobarbital and 75 μl of adenovirus (AAV) (AAV blank control, AAV-oeCREB3L1, AAV-shCREB3L1) were injected at a rate of 1*10 ^ 11 viral genome concentrations were injected into the mouse trachea for overexpression of CREB3L1 or knockdown of CREB3L1;

[0070] 1.4 Thereafter, the upper back (about 2 cm) of the experimental group mice was 2) were injected with 100 μl (100 ug) bleomycin (produced by Kayaku Co., Ltd., Japan) for 3 consecutive weeks to induce scleroderma with pulmonary fibrosis model mice. The control group mice were injected with 100 μl phosphate-balanced saline (PBS) on the upper back. In this experiment, they were represented by HC, BLM-blank, BLM-oe, and BLM-si respectively;

[0071] 1.5 On day 22, all experimental mice were euthanized and skin and lung tissues were taken for further experiments.

[0072] 2. WB detection of fibrosis-related indicators

[0073] The skin and lung tissues of mice were taken for WB experiments to detect fibrosis-related indicators such as FN1, COL1A1, Periostin and CREB3L1. The WB experimental results of mouse skin and lung tissues are as follows: Figure 3 (a) and Figure 3 (b) as shown.

[0074] As can be seen from the figure, compared with healthy control mice, the skin and lung fibrosis indicators of the modeling mice were increased. Compared with the modeling control group (i.e., the blank control adenovirus transfected group), the CREB3L1 expression of the CREB3L1 high expression group mice increased, indicating that the transfection was successful, and the fibrosis indicators also increased, indicating that high expression of CREB3L1 would promote fibrosis; while the CREB3L1 expression of the CREB3L1 low expression group mice decreased, indicating that the transfection was successful, and the fibrosis indicators also decreased, indicating that low expression of CREB3L1 would inhibit fibrosis.

[0075] 3. Masson staining to observe tissue fibrosis

[0076] Masson staining can make collagen fibers appear blue, so it is used to observe tissue fibrosis. Masson staining was performed on the lungs and skin tissues of mice in the HC, BLM-blank, BLM-oe, and BLM-si groups. The results and statistics of Masson staining are shown in the figure. Figure 4 As shown. Compared with healthy control mice, the collagen fibrosis in the skin and lungs of the model mice was more obvious. Compared with the model control group mice, the collagen fibrosis in the skin and lungs of the CREB3L1 high expression group mice was significantly increased, indicating that high expression of CREB3L1 would promote fibrosis; while the collagen fibrosis level in the skin and lungs of the CREB3L1 low expression group mice was reduced, indicating that low expression of CREB3L1 would inhibit fibrosis.

[0077] The above experimental results can be combined to prove that high expression of CREB3L1 will promote bleomycin-induced skin and lung fibrosis in Ssc mice, and low expression of CREB3L1 will inhibit bleomycin-induced skin and lung fibrosis in Ssc mice.

[0078] The AAV-shCREB3L1 used in the embodiment of the present invention is prepared by a method for constructing an adenovirus expression vector well known in the art, wherein the vector is pHBAAV-U6-MCS-CMV-ZsGreen, the host cell is an AAV293 cell, and the shRNA sequence nucleotide information is as follows:

[0079]

[0080]

[0081] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of CREB3L1 as a biomarker in at least one of the following: a1) Use in the preparation and / or screening of products for diagnosis or auxiliary diagnosis of systemic sclerosis; a2) Application in the preparation and / or screening of products for evaluating or assisting in evaluating the therapeutic efficacy of systemic sclerosis; a3) Use in the preparation and / or screening of products for evaluating or assisting in the evaluation of the degree of skin and / or pulmonary fibrosis in systemic sclerosis; a4) Use in the preparation and / or screening of products for determining or assisting in determining the prognosis of systemic sclerosis.

2. Use of a substance for detecting CREB3L1 in at least one of the following: b1) Use in the preparation and / or screening of products for diagnosis or auxiliary diagnosis of systemic sclerosis; b2) Application in the preparation and / or screening of products for evaluating or assisting in evaluating the therapeutic efficacy of systemic sclerosis; b3) Use in the preparation and / or screening of products for evaluating or assisting in the evaluation of the degree of skin and / or pulmonary fibrosis in systemic sclerosis; b4) Use in the preparation and / or screening of products for determining or assisting in determining the prognosis of systemic sclerosis; The products include reagents, test kits, chips, test strips, membrane strips or detection platforms.

3. The use according to claim 2, characterized in that: The substances for detecting CREB3L1 include any reagents required for detecting the expression level of CREB3L1 protein or the expression level of CREB3L1 gene by RT PCR, RT qPCR, biochip detection, Southern blotting, protein blotting, in situ hybridization, immunohistochemistry, enzyme-linked immunosorbent assay, and immunofluorescence technology.

4. The use according to claim 3, characterized in that: The substance for detecting CREB3L1 is a reagent for detecting CREB3L1 protein by immunofluorescence technique; or, The substance for detecting CREB3L1 is a reagent for detecting CREB3L1 protein by Western blotting; or, The substance for detecting CREB3L1 is a reagent for detecting CREB3L1 protein by immunohistochemistry.

5. A kit comprising the substance for detecting CREB3L1 according to any one of claims 2 to 4.

6. Use of CREB3L1 as a target in at least one of the following: c1) Use in the preparation and / or screening of drugs for preventing systemic sclerosis; c2) Use in the preparation and / or screening of drugs for the treatment or adjuvant treatment of systemic sclerosis.

7. Use of a CREB3L1 inhibitor in at least one of the following: d1) Use in the preparation and / or screening of drugs for preventing systemic sclerosis; d2) Application in the preparation and / or screening of drugs for the treatment or adjuvant treatment of systemic sclerosis; The CREB3L1 inhibitor includes a substance that reduces the expression level of CREB3L1 protein or the content of CREB3L1 protein or the activity of the protein, or a substance that inhibits the expression of the CREB3L1 gene, and the substance includes one or more of nucleic acid molecules, small molecule compounds, antibodies, polypeptides, proteins, gene editing vectors, lentiviruses or adeno-associated viruses.

8. The use according to claim 7, characterized in that: The CREB3L1 inhibitor is short hairpin RNA, small interfering RNA or CREB3L1 low-expressing adenovirus; or, The CREB3L1 inhibitor is a molecule that binds to the CREB3L1 protein, including a humanized monoclonal antibody and a receptor antagonist with CREB3L1 as a ligand.

9. A pharmaceutical composition comprising an effective amount of the CREB3L1 inhibitor according to any one of claims 7 to 8.

10. The pharmaceutical composition according to claim 9, characterized in that The CREB3L1 inhibitor is a CREB3L1 low-expression adenovirus that inhibits the expression of CREB3L1 protein-related genes.

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