Application of CHI3L1 as target spot in treatment of systemic sclerosis

Abstract

The invention discloses application of CHI3L1 as a target spot in treatment of systemic sclerosis, and belongs to the technical field of biological medicine. The invention finds that the expression of Chi3L1 protein in the skin and serum of SSc patients and SSc mouse models is increased; the Chi3L1 is used for stimulating the SSc-DFs to be transformed into the myofibroblast; chi3L1 gene knockout and Chi3L1 monoclonal antibody treatment relieve the fibrosis degree of an SSc mouse model. The result shows that the Chi3L1 plays an important role in promoting the occurrence process of SSc fibrosis, and the Chi3L1 can be used for diagnosing and treating systemic sclerosis as a biomarker or a target spot. The invention provides a new target for treating systemic sclerosis and lays a solid research foundation for preparing the medicine for resisting systemic sclerosis fibrosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of CHI3L1 as a target in the treatment of systemic sclerosis. Background Art

[0002] Systemic sclerosis (SSc), commonly known as "scleroderma", is a rare autoimmune disease characterized by fibrosis of multiple organs such as the skin and lungs. It has the highest mortality rate among all rheumatic diseases due to its irreversible and extensive fibrosis. It is estimated that there are about 2 million systemic sclerosis patients worldwide, and up to 90% may develop varying degrees of pulmonary fibrosis, which is associated with a very poor prognosis and accounts for about 35% of all deaths.

[0003] For a long time, there has been no specific therapeutic drug for systemic sclerosis on the market, and the clinical treatment mainly focuses on treating complications of various organ systems and relieving specific symptoms. For example, a variety of agents are used to treat skin fibrosis, such as D-penicillamine, mycophenolate mofetil, colchicine, psoralen + ultraviolet A exposure (PUVA), relaxin, cyclosporine and EPA (ω-3 oil derivative). It was not until 2019 that Boehringer Ingelheim's investigational drug Ofev (nintedanib) was approved by the European Commission (EC) and the U.S. Food and Drug Administration (FDA) for the treatment of systemic sclerosis, confirming its orphan drug status for the treatment of systemic sclerosis.

[0004] Systemic sclerosis is a rare connective tissue disease characterized by widespread fibrosis throughout the body. Controlling or even reversing fibrosis in systemic sclerosis is a key goal of treatment. However, the mechanism of abnormal activation of fibroblasts in systemic sclerosis has not been fully elucidated. Therefore, controlling or even reversing fibrosis in systemic sclerosis remains an unsolved technical challenge, and the preparation of drugs to treat systemic sclerosis is still urgently needed.

[0005] Chi3L1 (Chitinase-3-like-1 protein) is secreted by a variety of immune cells and structural cells. A large number of studies have shown that Chi3L1 plays a vital role in various inflammatory reactions, tissue remodeling and fibrosis. The research team of the present invention unexpectedly found that Chi3L1 can activate SSc-DFs activation, and the absence of Chi3L1 or the use of Chi3L1 monoclonal antibody treatment can reduce fibrosis in SSc model mice. Based on the above basic research, the present invention provides a new treatment idea for systemic sclerosis fibrosis, and at the same time, lays a research foundation for the development of specific drugs for the treatment of systemic sclerosis. Summary of the invention

[0006] One object of the present invention is to provide Chi3L1 as a biomarker for the diagnosis and efficacy evaluation of systemic sclerosis; a second object of the present invention is to provide the use of Chi3L1 as a target in the preparation of a drug for the treatment of systemic sclerosis, specifically, the drug is a Chi3L1 inhibitor, and the drug can effectively control and / or reverse systemic sclerosis fibrosis.

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

[0008] In a first aspect of the present invention, the present invention provides a use of a biomarker Chi3L1 and / or a substance for detecting Chi3L1 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] The products include but are not limited to reagents, test kits, chips, test strips, membrane strips or detection platforms.

[0012] The substance for detecting Chi3L1 includes any reagent required for detecting Chi3L1 protein expression or gene expression level by RTPCR, RTqPCR, biochip detection, Southern blotting, Western Blot, in situ hybridization, immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), and spatial transcriptome technology.

[0013] In some embodiments of the present invention, the substance for detecting Chi3L1 is a reagent for detecting Chi3L1 protein by immunohistochemistry.

[0014] In some embodiments of the present invention, the substance for detecting Chi3L1 is a reagent for detecting Chi3L1 protein by Western Blot.

[0015] In some embodiments of the present invention, the substance for detecting Chi3L1 is a reagent for detecting Chi3L1 protein by enzyme-linked immunosorbent assay (ELISA).

[0016] In some embodiments of the present invention, the substance for detecting Chi3L1 is a reagent for detecting the expression of nucleic acid related to Chi3L1 protein, and the reagent includes but is not limited to antibodies, polypeptides, proteins or nucleic acid molecules that bind to Chi3L1 protein.

[0017] In a second aspect of the present invention, the present invention provides a use of Chi3L1 as a target in at least one of the following:

[0018] b1) Use in the preparation and / or screening of drugs for preventing systemic sclerosis;

[0019] b2) Use in the preparation and / or screening of drugs for the treatment or adjuvant treatment of systemic sclerosis;

[0020] b3) Use in the preparation and / or screening of drugs for controlling and / or reversing systemic sclerosis fibrosis.

[0021] In the third aspect of the present invention, the present invention provides a use of a Chi3L1 inhibitor in at least one of the following:

[0022] c1) Use in the preparation and / or screening of drugs for preventing systemic sclerosis;

[0023] c2) Use in the preparation and / or screening of drugs for the treatment or adjuvant treatment of systemic sclerosis;

[0024] c3) Use in the preparation and / or screening of drugs for controlling and / or reversing systemic sclerosis fibrosis.

[0025] In the present invention, the Chi3L1 inhibitor includes a substance that reduces the expression of Chi3L1 protein, the content of Chi3L1 protein, or the activity of the protein, or a substance that inhibits the expression of the Chi3L1 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.

[0026] In some embodiments of the present invention, the Chi3L1 inhibitor is a molecule that binds to the Chi3L1 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 Chi3L1 as a ligand, and the inhibitor can reduce the content or activity of the Chi3L1 protein, or interfere with the binding of Chi3L1 to its receptor.

[0027] In some embodiments of the present invention, the Chi3L1 inhibitor is a short hairpin RNA (shRNA) or a small interfering RNA (siRNA), and the inhibitor can reduce the expression of a gene encoding a Chi3L1 protein.

[0028] In the fourth aspect of the present invention, the present invention provides a pharmaceutical composition, wherein the pharmaceutical composition comprises an effective amount of the Chi3L1 inhibitor according to the third aspect of the present invention.

[0029] The Chi3L1 inhibitor includes a substance that reduces the expression of Chi3L1 protein, the content of Chi3L1 protein, or the activity of the protein, or a substance that inhibits the expression of the Chi3L1 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.

[0030] In some embodiments of the present invention, the Chi3L1 inhibitor is a molecule that binds to the Chi3L1 protein, such as one or a combination of two or more of an antibody, a peptide, or a small molecule. The inhibitor can reduce the content or activity of the Chi3L1 protein, or interfere with the binding of Chi3L1 to its receptor.

[0031] In some embodiments of the present invention, the Chi3L1 inhibitor is a short hairpin RNA (shRNA) or a small interfering RNA (siRNA), and the inhibitor can reduce the expression of a gene encoding a Chi3L1 protein.

[0032] In a specific embodiment of the present invention, the Chi3L1 inhibitor is a humanized monoclonal antibody that specifically binds to Chi3L1.

[0033] In a specific embodiment of the present invention, the Chi3L1 inhibitor is a receptor antagonist with Chi3L1 as a ligand.

[0034] In a specific embodiment of the present invention, the Chi3L1 inhibitor is a small interfering RNA (siRNA) that inhibits the expression of Chi3L1-related genes.

[0035] Furthermore, the pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0036] 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.

[0037] 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.

[0038] The disease treatment refers to administering a therapeutically effective amount of the Chi3L1 inhibitor or pharmaceutical composition of the present invention to a subject in need thereof.

[0039] Furthermore, the disease treatment also includes administering the Chi3L1 inhibitor or pharmaceutical composition of the present invention in combination with other conventional agents or conventional treatment methods in the art.

[0040] The present invention confirms the following conclusions through scientific experiments: 1. It is found that the expression of Chi3L1 protein is increased in the skin and serum of SSc patients. Similarly, the expression is also increased in the skin and serum of the SSc mouse model induced by bleomycin; 2. The Chi3L1 recombinant protein can stimulate the transformation of SSc-DFs into myofibroblasts, increase collagen synthesis, and increase the migration and proliferation ability of SSc-DFs; 3. In the bleomycin-induced SSc mouse model, after the Chi3L1 gene was knocked out by CRISPR-Cas9 gene editing technology, the mice showed a reduction in the degree of fibrosis; 4. After the SSc mouse model was treated with humanized Chi3L1 monoclonal antibody, the degree of fibrosis was reduced; 5. The knockout of the Chi3L1 gene and the Chi3L1 monoclonal antibody treatment did not affect tissue homeostasis.

[0041] The results suggest that Chi3L1 plays an important role in promoting the occurrence of SSc fibrosis, and Chi3L1 can be used as a biomarker or target for the diagnosis and treatment of systemic sclerosis. And CHI3L1 inhibitors can be used as an effective ingredient for the treatment of systemic sclerosis, specifically for controlling and / or reversing systemic sclerosis fibrosis, and have good clinical application prospects. The present invention provides a new target for the treatment of systemic sclerosis, lays a solid research foundation for the preparation of anti-systemic sclerosis fibrosis drugs, in order to solve the problem of the lack of effective anti-systemic sclerosis fibrosis therapeutic drugs in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Serum Chi3L1 concentrations in SSc patients and healthy subjects.

[0043] Figure 2 WB representation of Chi3L1 protein concentration in skin tissues of SSc patients and healthy subjects (left) and quantification (right), *** represents p < 0.001.

[0044] Figure 3WB representative graph (left) and quantitative graph (right) of Chi3L1 expression levels in mouse skin tissue in PBS control-wild type group (CTR, WT), PBS control-Chi3L1 gene KO group (CTR, KO), BLM-SSc model-wild type group (BLM, WT), and BLM-SSc model-Chi3L1 gene KO group (BLM, KO), *** represents p < 0.001.

[0045] Figure 4 Chi3L1 concentration in the serum of mice in the PBS control-wild type group (CTR, WT), PBS control-Chi3L1 gene KO group (CTR, KO), BLM-SSc model-wild type group (BLM, WT), and BLM-SSc model-Chi3L1 gene KO group (BLM, KO), *** represents p < 0.001.

[0046] Figure 5 Representative WB images (left) and quantitative images (right) of α-SMA, POSTN, COL1, and COL3 in SSc-DFs stimulated with different concentrations of Chi3L1 for 24 hours. ** represents p<0.01, and *** represents p<0.001.

[0047] Figure 6 The CCK8 method was used to measure the absorbance of SSc-DF at a wavelength of 450 nm on a microplate reader after stimulation of SSc-DF with different concentrations of Chi3L1 for 24 hours. * represents p<0.1 compared with the unstimulated group, *** represents p<0.001 compared with the unstimulated group, and ns represents no significant difference.

[0048] Figure 7 The scratch assay was used to measure the cell migration ratio of SSc-DFs after stimulation with different concentrations of Chi3L1 for 24 hours. ** represents p<0.01, *** represents p<0.001.

[0049] Figure 8 Transwell assay was used to measure the cell migration ratio of SSc-DFs after stimulation with different concentrations of Chi3L1 for 24 hours. ** represents p<0.01, *** represents p<0.001.

[0050] Fig. 9 Mice were grouped Figure 3, 6 mice in each group. A. Ultrasound representative images (left) and quantitative images (right) show the changes in skin thickness of mice in each group; B. Quantitative images of hydroxyproline content in skin (left) and lung (right) of mice in each group; C. Representative images of HE and MASSON staining of skin and lung of mice in each group (left) and quantitative images of dermal thickness and lung fibrosis score (right); D. WB representative images (left) and quantitative images (right) of the expression levels of α-SMA, POSTN, COL1, and COL3 in skin tissue of mice in different groups; E, COL1 concentration in lung tissue of mice in different groups. ** represents p<0.01, *** represents p<0.001, and ns represents no significant difference.

[0051] Fig.10 The mice were divided into PBS control-IgG control group, PBS control-Chi3L1 monoclonal antibody treatment group, BLM model-IgG control group, and BLM model-Chi3L1 monoclonal antibody treatment group, with 6 mice in each group. A. Representative images of HE and MASSON staining of the skin of mice in each group (left) and quantification of dermal thickness (right). B. Representative images of HE and MASSON staining of the lungs of mice in each group (left) and lung fibrosis scores (right). DETAILED DESCRIPTION

[0052] 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.

[0053] The present invention confirms the following conclusions through experiments:

[0054] 1. Chi3L1 protein expression is elevated in the skin and serum of SSc patients

[0055] 1. Detection of Chi3L1 protein concentration in the serum of SSc patients

[0056] Purpose of the experiment: To detect the concentration of Chi3L1 protein in the serum of SSc patients and healthy subjects by ELISA (enzyme-linked immunosorbent assay).

[0057] Experimental method: Peripheral blood was drawn from 48 SSc patients and 24 healthy subjects to determine the Chi3L1 concentration in serum. Specifically, the steps for determining the Chi3L1 concentration in serum are as follows:

[0058] S1) Draw peripheral blood from the subject, centrifuge the blood sample at 3000×g for 10 minutes, and take the supernatant;

[0059] S2) Prepare 8 tubes of standard protein according to the instructions of the kit (ABCAM, catalog number ab255719), and reduce the concentration by half from the first tube to the seventh tube. Only add the sample diluent to the eighth tube, that is, the standard protein concentration is 0 pg / mL;

[0060] S3) Take out the kit ELISA plate, add 8 different concentrations of standard protein solutions and samples, set two duplicate wells, stick a sealing film, and incubate in a 37°C oven for 1 hour;

[0061] S4) Pour out the liquid in the wells, tap on absorbent paper several times without washing, prepare biotin antibody working solution according to the instructions, add 100 μL to each well, apply sealing film, and incubate in a 37°C oven for 1 hour;

[0062] S5) Pour out the liquid in the wells, prepare washing solution according to the instructions, add 200 μL of washing solution to each well, pour out after 2 minutes, and gently tap on absorbent paper to dry, and wash three times in this way;

[0063] S6) Add HRP-coupled avidin working solution to each well, apply sealing film, and incubate in a 37°C oven for 30 minutes. Then add 200 μL of washing solution to each well, pour it out after 2 minutes, and gently tap it on absorbent paper to dry it. Wash it 3 times in this way;

[0064] S7) adding the reaction substrate 3,3',5,5'-tetramethylbenzidine (TBM), incubating at 37°C for about 15 minutes in the dark, and adding the stop solution to terminate the reaction after sufficient reaction;

[0065] S8) The absorbance value of each well at 450 nm was measured using an ELISA instrument, and a concentration-absorbance standard curve was drawn according to the concentration and absorbance of the standard, and finally the protein concentration of Chi3L1 in the serum sample was calculated according to the standard curve.

[0066] The statistical results of Chi3L1 concentration in the serum of 48 SSc patients and 24 healthy subjects measured by the above method are as follows Figure 1 As shown, the results showed that the Chi3L1 concentration in the serum of SSc patients was significantly higher than that in healthy subjects.

[0067] 2. Detection of Chi3L1 protein expression level in skin tissue of SSc patients

[0068] Purpose of the experiment: To detect the expression level of Chi3L1 in skin tissues of SSc patients and healthy subjects by WB (Western Blot, immunofixation electrophoresis).

[0069] Experimental method: Skin tissues were extracted from 6 SSc patients and 6 healthy subjects, and the expression level of Chi3L1 in the skin tissues was detected according to the conventional WB (immunofixation electrophoresis) operation method.

[0070] The results of WB analysis of Chi3L1 protein in skin tissues of SSc patients and healthy subjects are as follows Figure 2 (left) shows the quantitative statistical results. Figure 2 As shown in (right), the expression level of Chi3L1 in the skin tissue of SSc patients was significantly higher than that in healthy subjects.

[0071] 2. Chi3L1 protein expression is elevated in the skin and serum of SSc model mice

[0072] SSc model mouse modeling method: 6-week-old female C57 mice were subcutaneously injected with 0.5 mg / ml bleomycin (diluted with PBS) every day for 21 consecutive days to obtain SSc model mice. At the same time, a control group of mice was set up: 6-week-old female C57 mice were subcutaneously injected with the same volume of PBS every day for 21 consecutive days to obtain control group mice.

[0073] Construction of Chi3L1 knockout mice by CRISPR-Cas9 gene editing technology 1 According to the above modeling method: wild-type PBS control group mice were marked as (CTR, WT), Chi3L1 gene knockout control group mice were marked as (CTR, KO), wild-type BLM modeling group mice were marked as (CTR, SSc), and wild-type Chi3L1 gene knockout mice were marked as (SSc, KO).

[0074] 1. Detection of the expression level of Chi3L1 protein in the skin tissue of the above mouse models

[0075] Experimental purpose: To detect the expression level of Chi3L1 in the skin tissue of the above four mouse models by WB (Western Blot, immunofixation electrophoresis).

[0076] Experimental method: The skin tissues of mice in the (SSc, KO) group, (SSc, WT) group, (CTR, KO) group, and (CTR, WT) group were obtained, with 6 mice in each group, and the expression level of Chi3L1 in the skin tissues was detected according to the conventional WB (immunofixation electrophoresis) operation method.

[0077] WB results are as follows Figure 3 (left) shows the quantitative statistical results. Figure 3 The results showed that Chi3L1 was not expressed in the skin tissue of Chi3L1 knockout mice, and the expression level of Chi3L1 in the skin tissue of (SSc, WT) group mice was significantly higher than that in (CTR, WT) group mice, indicating that the expression level of Chi3L1 in the skin tissue of SSc mice was increased.

[0078] 2. Detection of Chi3L1 protein concentration in the serum of the above mouse models

[0079] Experimental purpose: To detect the Chi3L1 protein concentration in the serum of the above four mouse models by ELISA (enzyme-linked immunosorbent assay).

[0080] Experimental method: Peripheral blood was drawn from mice in the (SSc, KO) group, (SSc, WT) group, (CTR, KO) group, and (CTR, WT) group, with 12 mice in each group, and the Chi3L1 concentration in the serum was determined.

[0081] The statistical results of Chi3L1 concentration in the serum of the above four groups of mouse models are as follows: Figure 4 As shown, the results showed that: similar to the expression trend of Chi3L1 in mouse skin tissue, Chi3L1 was not expressed in the serum of Chi3L1 knockout mice, and the Chi3L1 concentration in the serum of SSc mice (SSc, WT) was significantly higher than that of healthy mice (CTR, WT).

[0082] 3. Chi3L1 recombinant protein induces SSc-DFs activation

[0083] The "SSc-DFs" described in this example refers to DFs (dermal fibroblasts) extracted from the skin of SSc patients. The skin tissue of SSc patients is collected, and the dermal fibroblasts of the patients are extracted by enzymatic hydrolysis and passaged.

[0084] 1. Chi3L1 stimulation increases the expression of SSc-DFs markers α-SMA, POSTN, COL1, and COL3

[0085] The Chi3L1 recombinant protein (MedChemExpress, catalog number HY-P70030) was prepared into solutions with concentrations of 50, 100, and 200 ng / ml with PBS, and the SSc-DFs myofibroblasts were incubated with the solutions for 24 hours. The expression levels of SSc-DFs cell markers α-SMA, POSTN, COL1, and COL3 were detected by WB (Western Blot, immunofixation electrophoresis).

[0086] WB results are as follows Figure 5 (left) shows the quantitative statistical results. Figure 5 (Right) As shown, WB results showed that Chi3L1 stimulation increased the expression of SSc-DFs myofibroblast markers α-SMA and POSTN, suggesting the promotion of myofibroblast transformation; in addition, Chi3L1 stimulation increased the expression of SSc-DFs myofibroblast markers COL1 and COL3, suggesting increased collagen synthesis.

[0087] 2. CCK-8 Experiment

[0088] The Chi3L1 recombinant protein was prepared into solutions with concentrations of 10, 20, 50, 100, 200, 400, and 800 ng / ml using PBS, and SSc-DFs myofibroblasts were incubated respectively. CCK-8 experiments were performed to verify its effect on cell proliferation.

[0089] The results are as follows Figure 6 As shown, with the increase of Chi3L1 concentration, the survival rate of SSc-DFs myofibroblasts increased, suggesting that Chi3L1 stimulation increased the proliferation capacity of SSc-DFs.

[0090] 3. Cell Scratch Healing and Migration Assay

[0091] The Chi3L1 recombinant protein was prepared into solutions with concentrations of 50, 100, and 200 ng / ml using PBS, and the SSc-DFs myofibroblasts were incubated with the solutions for 24 hours. The cell migration ratio was verified by a cell scratch healing migration experiment.

[0092] The results of cell migration ratio are as follows Figure 7 (left) shows the quantitative statistical results. Figure 7 (Right) The results suggest that Chi3L1 stimulation increases the migration ability of SSc-DFs.

[0093] 4. Cell Transwell Invasion Assay

[0094] The Chi3L1 recombinant protein was prepared into solutions with concentrations of 50, 100, and 200 ng / ml using PBS, and the SSc-DFs myofibroblasts were incubated with the solutions for 24 hours. The cell migration ratio was verified by a cell Transwell invasion assay.

[0095] The results of cell migration ratio are as follows Figure 8 (left) shows the quantitative statistical results. Figure 8 (Right) The results suggest that Chi3L1 stimulation increases the migration ability of SSc-DFs.

[0096] 4. Skin and lung tissue detection of Chi3L1 knockout mice

[0097] Similar to Experiment 2 of the present invention, C57 mice were modeled using bleomycin to obtain SSc model mice, and the mice were divided into four groups: (SSc, KO), (SSc, WT), (CTR, KO), and (CTR, WT).

[0098] 1. Ultrasonic testing

[0099] The skin of the above four groups of mouse models was examined by ultrasound. The ultrasound results were as follows: Fig. 9 A (left) shows the quantitative results. Fig. 9A (right) shows the changes in skin thickness of each group of mice. The control group mice with Chi3L1 knockout (CTR, KO) showed no tissue homeostasis effects, specifically, ultrasound showed no significant changes in skin thickness. Compared with the SSc model group mice (SSc, WT), the SSc model group mice with Chi3L1 knockout (SSc, KO) showed reduced fibrosis, specifically, ultrasound showed reduced skin thickness.

[0100] 2. Detection of Hydroxyproline Content in Skin and Lung

[0101] The skin and lung hydroxyproline levels of the above four groups of mouse models were tested. The results were as follows Fig. 9 As shown in B, the left figure is a quantitative graph of mouse skin hydroxyproline content, and the right figure is a quantitative graph of mouse lung hydroxyproline content. The results show that the skin and lung hydroxyproline content of the control group mice with Chi3L1 knockout gene (CTR, KO) did not change significantly compared with the blank control group mice (CTR, WT). Compared with the SSc model group mice (SSc, WT), the skin and lung hydroxyproline content of the SSc model group mice with Chi3L1 knockout gene (SSc, KO) decreased, indicating a decrease in collagen content.

[0102] 3. Skin and Lung Tissue Staining

[0103] HE and MASSON staining were performed on the skin and lungs of the above four groups of mouse models. The HE and MASSON staining of the mouse skin and lungs were as follows: Fig. 9 C (left), quantification of dermal thickness and lung fibrosis scores were shown in Fig. 9 C (right). The results showed that the skin and lung staining results of the Chi3L1 knockout control group mice (CTR, KO) were not significantly different from those of the blank control group mice (CTR, WT). Compared with the SSc model group mice (SSc, WT), HE and MASSON staining of the skin and lungs of the Chi3L1 knockout SSc model group mice (SSc, KO) indicated a reduced degree of fibrosis.

[0104] 4. Western Blot Experiment

[0105] The expression levels of α-SMA, POSTN, COL1, and COL3 in the skin tissues of the above four groups of mouse models were detected by WB (Western Blot, immunofixation electrophoresis). The WB results are as follows Fig. 9 D (left) shows the quantitative statistical results. Fig. 9D (right). The results showed that the expression of α-SMA, POSTN, COL1, and COL3 in the skin tissue of the Chi3L1 knockout control group mice (CTR, KO) was not significantly different from that of the blank control group mice (CTR, WT). Compared with the SSc model group mice (SSc, WT), the expression of α-SMA, POSTN, COL1, and COL3 in the skin tissue of the Chi3L1 knockout SSc model group mice (SSc, KO) was reduced.

[0106] 5. ELISA Experiment

[0107] The expression of COL1 in the lung tissues of the above four mouse models was detected by ELISA (enzyme-linked immunosorbent assay). Fig. 9 As shown in E, the results indicate that the expression of COL1 in the lung tissue of the Chi3L1 knockout control group mice (CTR, KO) was not significantly different from that of the blank control group mice (CTR, WT). Compared with the SSc model group mice (SSc, WT), the expression of COL1 in the lung tissue of the Chi3L1 knockout SSc model group mice (SSc, KO) was reduced.

[0108] 5. Chi3L1 monoclonal antibody treatment of SSc model mice

[0109] As in Experiment 2 of the present invention, bleomycin was used to model C57 mice to obtain SSc model mice, which are marked as SSc in the figure. At the same time, a control group of mice was set up, which is marked as CTR in the figure.

[0110] The SSc group mice were randomly divided into two groups, with 6 mice in each group. One group of mice was treated with Chi3L1 monoclonal antibody (0.2 mg / kg, subcutaneous injection, once a week, on the 1st, 8th and 15th days, for a total of 3 times), marked as SSc+Anti-Chi3L1 in the figure; the untreated mouse group was still marked as SSc.

[0111] The mice in the CTR group were randomly divided into two groups, with 6 mice in each group. One group of mice was treated with Chi3L1 monoclonal antibody, marked as Anti-Chi3L1 in the figure; the group of mice that did not receive treatment was still marked as CTR.

[0112] HE and MASSON staining were performed on the skin and lungs of the four groups of mice. Fig.10 As shown in A (left), the quantitative results of dermal thickness are as follows Fig.10 A (right). HE and MASSON staining of mouse lungs Fig.10 B (left) shows the quantitative results of dermal thickness. Fig.10B (right). The above results show that the control group mice treated with humanized Chi3L1 monoclonal antibody (Anti-Chi3L1) showed no tissue homeostasis effects, specifically, no significant changes in HE and MASSON staining of the skin and lungs. Compared with SSc model mice, the SSc model mice treated group (SSc+Anti-Chi3L1) showed reduced fibrosis, as shown by HE and MASSON staining of the skin and lungs, indicating a reduced degree of fibrosis.

[0113] 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.

[0114] References:

[0115] 1. Kim KC, Yun J, Son DJ, et al. Suppression of metastasis through inhibition of chitinase 3-like1expression by miR-125a-3p-mediated up-regulation of USF1. Theranostics 2018;8:4409-28.

Claims

1. Use of the biomarker Chi3L1 and / or a substance for detecting Chi3L1 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; The products include reagents, test kits, chips, test strips, membrane strips or detection platforms.

2. The use according to claim 1, characterized in that: The substance for detecting Chi3L1 includes any reagent required for detecting Chi3L1 protein expression or gene expression level by RT-PCR, RT-qPCR, biochip detection, Southern blotting, protein blotting, in situ hybridization, immunohistochemistry, enzyme-linked immunosorbent assay, and spatial transcriptome technology.

3. The use according to claim 2, characterized in that: The substance for detecting Chi3L1 includes a reagent for detecting Chi3L1 protein by immunohistochemistry; or a reagent for detecting Chi3L1 protein by Western blotting; or a reagent for detecting Chi3L1 protein by enzyme-linked immunosorbent assay; or a reagent for detecting the expression amount of nucleic acid related to Chi3L1 protein.

4. Use of Chi3L1 as a target in at least one of the following: b1) Use in the preparation and / or screening of drugs for preventing systemic sclerosis; b2) Use in the preparation and / or screening of drugs for the treatment or adjuvant treatment of systemic sclerosis; b3) Use in the preparation and / or screening of drugs for controlling and / or reversing systemic sclerosis fibrosis.

5. Use of Chi3L1 inhibitors 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; c3) Use in the preparation and / or screening of drugs for controlling and / or reversing systemic sclerosis fibrosis.

6. The use according to claim 5, characterized in that: The Chi3L1 inhibitor includes a substance that reduces the expression amount of Chi3L1 protein or the Chi3L1 protein content or protein activity, or a substance that inhibits the expression of the Chi3L1 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.

7. A pharmaceutical composition comprising an effective amount of the Chi3L1 inhibitor according to any one of claims 5 to 6.

8. The pharmaceutical composition according to claim 7, characterized in that The Chi3L1 inhibitor includes one or a combination of two or more of an antibody, a polypeptide or a small molecule that binds to the Chi3L1 protein; or the Chi3L1 inhibitor is a short hairpin RNA or a small interfering RNA that reduces the expression of a gene encoding the Chi3L1 protein.

9. The pharmaceutical composition according to claim 8, characterized in that The Chi3L1 inhibitor includes a humanized monoclonal antibody that specifically binds to Chi3L1; or a receptor antagonist that uses Chi3L1 as a ligand; or a small interfering RNA that inhibits the expression of Chi3L1-related genes.

10. The pharmaceutical composition according to any one of claims 7 to 9, characterized in that The pharmaceutical composition also includes pharmaceutically acceptable excipients.

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