Application of secoisolariciresinol diglucoside in preparation of medicine for treating Treg cell related autoimmune diseases

Abstract

The invention discloses an application of secoisolariciresinol diglucoside in preparation of a medicine for treating Treg cell related autoimmune diseases. The invention proves that the secoisolariciresinol diglucoside can be combined with AIM2, the protein stability of the secoisolariciresinol diglucoside is improved, the differentiation of Treg cells is promoted, and the adaptive immune response is effectively inhibited. Therefore, the secoisolariciresinol diglucoside can be used as an effective target for regulating and controlling the Treg cells and relieving Treg cell related inflammatory diseases, can be used for treating Treg cell related autoimmune diseases, and particularly has a remarkable curative effect on systemic lupus erythematosus. The invention provides application potential of secoisolariciresinol diglucoside targeted AIM2 in treatment of Treg cell related inflammatory diseases, and the secoisolariciresinol diglucoside targeted AIM2 has important significance in drug development and prevention and treatment of the diseases in the future.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to an application of flax lignan in the preparation of a medicine for treating Treg cell-related autoimmune diseases. Background Art

[0002] Autoimmune diseases usually originate from an imbalance of immune tolerance, which causes the immune system to mistakenly attack and destroy normal tissues. The occurrence and development of the disease is often accompanied by an increase in the proportion of dysfunctional cells and a relative decrease in the number of regulatory T cells (Treg), which further leads to immune regulation disorders. Treg cells play an important role in maintaining the balance of the immune system and preventing autoimmune reactions. They usually account for 60% of the peripheral blood CD4 + 5%-10% of the total number of T lymphocytes. Most Treg cells highly express interleukin-2 receptor (IL-2R) α chain (CD25) and FoxP3 transcription factor, and exert their inhibitory function by secreting inhibitory cytokines, mediating cell-to-cell contact inhibition, and interfering with target cell energy metabolism. Extensive studies have shown that Treg cells play a key role in a variety of autoimmune diseases, including systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, Sjögren's syndrome, inflammatory bowel disease, type I diabetes, and idiopathic thrombocytopenic purpura. In these diseases, the number of Treg cells is usually reduced or their function is impaired, resulting in an overactive immune system and exacerbated disease symptoms.

[0003] Therefore, effectively monitoring and regulating the expression and function of Treg can not only guide the rational use of immunosuppressants, but also control abnormal immune hyperfunction and significantly improve the prognosis of patients with autoimmune diseases.

[0004] Currently, there is no effective therapeutic drug for Treg cell-related autoimmune diseases. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing a drug for treating autoimmune diseases related to Treg cells by using flax lignans in order to address the deficiencies of the prior art. Flax lignans are used as an effective target for regulating Treg cells and alleviating Treg cell-related inflammatory diseases to treat Treg cell-related autoimmune diseases.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] Absent in melanoma 2 (AIM2) is an important component of the inflammasome and, as a sensor for double-stranded DNA, plays an important role in antiviral and antibacterial defense.

[0008] The present invention screened and identified that flax lignan (Secoisolariciresinoldiglucoside, SDG) can bind to AIM2 and increase the stability of AIM2 protein, thereby promoting Treg cell differentiation, inhibiting adaptive immune response, and improving the symptoms of pristane-induced lupus mice.

[0009] Therefore, the present invention provides the use of flax lignans in preparing drugs for treating Treg cell-related autoimmune diseases, and in particular, flax lignans can be used as the only effective ingredient in preparing drugs for treating Treg cell-related autoimmune diseases.

[0010] Preferably, the Treg cell-related autoimmune disease is any one or a combination of systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, systemic sclerosis, Sjögren's syndrome, inflammatory bowel disease, myasthenia gravis, psoriasis, type I diabetes, Graves' disease, Hashimoto's thyroiditis and idiopathic thrombocytopenic purpura.

[0011] Most preferably, the Treg cell-related autoimmune disease is systemic lupus erythematosus.

[0012] Flax lignans can improve the stability of AIM2 protein and promote the differentiation of Treg cells by binding to AIM2 protein. The flax lignans can be used as AIM2 protein conjugate stabilizers in the preparation of drugs for treating systemic lupus erythematosus.

[0013] Furthermore, the flax lignans have at least any of the following functions: the drug can delay the disease progression of pristane-induced lupus, increase the proportion and number of Treg cells in the spleen of lupus-like mice, reduce the level of serum-specific anti-double-stranded DNA-IgG, reduce renal inflammatory cell infiltration, and reduce renal deposition of IgG and C3. Therefore, the drug prepared from flax lignans can treat Treg cell-related autoimmune diseases, especially can significantly slow down the disease progression of lupus mice. Flax lignans can be used in the preparation of drugs for alleviating the progression of systemic lupus erythematosus.

[0014] Furthermore, the present invention verifies through experiments that flax lignans can also be used in the preparation of drugs for treating systemic lupus erythematosus-related renal damage. Flax lignans can be used in the preparation of drugs for reducing immune complex deposition. Flax lignans can be used in the preparation of drugs for reducing the level of anti-double-stranded DNA-IgG in serum.

[0015] Furthermore, the present invention provides a drug for treating Treg cell-related autoimmune diseases, wherein the drug contains flax lignans.

[0016] The medicine is a preparation made of flax lignan and a pharmaceutically acceptable carrier, and the dosage form includes any one of injection, infusion, powder, tablet, capsule, gel or paste.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention screens and identifies flax lignans as Treg cell regulators for the first time, effectively binds to AIM2 protein and maintains its stability, and promotes Treg cell differentiation. Experiments have shown that flax lignan treatment significantly improves the disease progression of the pristane-induced lupus model, increases the proportion and number of Treg cells in the spleen and lymph nodes, reduces serum-specific antibody levels and alleviates lupus kidney damage. It has been confirmed that flax lignans can be used as an effective target for regulating Treg cells and alleviating Treg cell-related inflammatory diseases, and can treat Treg cell-related autoimmune diseases, especially systemic lupus erythematosus, with significant therapeutic effects. The present invention provides the application potential of flax lignans targeting AIM2 in the treatment of Treg cell-related inflammatory diseases, which is of great significance for the future drug development and preventive treatment of such diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1-A : are the top 10 natural products after virtual screening based on the mouse AIM2 structure in the embodiments of the present invention;

[0019] Figure 1-B :The binding ability of 10 compounds to AIM2 protein was verified in human GM12878 cell line and mouse BAF3 cell line using CETSA technology in the examples of the present invention;

[0020] Figure 1-C : In the examples of the present invention, the DARTS technology was used to verify the effects of 10 compounds on the stability of AIM2 protein in human GM12878 cell line and mouse BAF3 cell line.

[0021] Figure 2-A This is the result of the docking of flax lignan and human AIM2 protein molecule in the example of the present invention.

[0022] Figure 2-B This is the result of the docking of flax lignan and mouse AIM2 protein molecule in the examples of the present invention.

[0023] Figure 3-A In the present embodiment, under the conditions of activation with anti-CD3 and anti-CD28, different concentrations of SDG (1 μM, 10 μM, 100 μM) were added, CD4 + Treg cells (CD25 hi CD127 low ) ratio changes;

[0024] Figure 3-B : In the present invention, under the conditions of Treg cell differentiation, different concentrations of SDG treatment have an effect on human T cell differentiation by FOXP3 + CD4 + Effects of T cells.

[0025] Figure 4-A : Splenic CD4 + CXCR5 in T cells + PD1 + Tfh cell frequency and CD25 + FOXP3 + ratio of Treg cell frequencies;

[0026] Figure 4-B : In the embodiment of the present invention, the draining lymph node CD4 + CXCR5 in T cells + PD1 + Tfh cell frequency and CD25 + FOXP3 + The ratio of Treg cell frequency.

[0027] Figure 5-A : Determination of anti-double-stranded DNA antibody levels in mouse serum in the embodiments of the present invention;

[0028] Figure 5-B : Determination of mouse urine protein level in the embodiment of the present invention;

[0029] Figure 5-C : Representative H&E staining images of kidneys in the present invention, showing pathological changes in renal tissue;

[0030] Figure 5-D : Representative immunofluorescence staining images of IgG and C3 in the kidneys of the present invention, showing the deposition of immune complexes;

[0031] Figure 5-E : The statistical results of the proportion of Treg cells in the spleen analyzed by flow cytometry in the embodiment of the present invention;

[0032] Figure 5-F : The statistical results of the proportion of Treg cells in the draining lymph nodes analyzed by flow cytometry in the embodiments of the present invention;

[0033] Figure 5-G : Splenic CD4 + Mean fluorescence intensity analysis of FOXP3 in T cells;

[0034] Figure 5-H : In the embodiment of the present invention, the draining lymph node CD4+ Analysis of mean fluorescence intensity of FOXP3 in T cells. DETAILED DESCRIPTION

[0035] The present invention is further described below in conjunction with specific examples, which are carried out under the preferred conditions of the present invention. The methods are conventional methods unless otherwise specified, and the raw materials can be obtained from public commercial channels unless otherwise specified.

[0036] The experimental methods described in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. described in the following examples are all commercially available unless otherwise specified.

[0037] In the following examples, the protein immunoblotting detection, the flow cytometry, the hematoxylin-eosin staining, the immunofluorescence analysis and other experimental methods are all conventional experimental methods well known to those skilled in the art. If the experimental method does not specify specific conditions, it is usually performed under conventional conditions.

[0038] Experimental animals:

[0039] The mice used in this example were provided by Hangzhou Ziyuan Experimental Animal Technology Co., Ltd. The feeding conditions were as follows: room temperature 18-20°C, humidity 50-60%, alternating light and dark (12h), moderate light intensity, good ventilation and cleanliness. All experiments were approved and conducted in accordance with the guidelines of the Ethics Committee of the Dermatology Hospital of the Chinese Academy of Medical Sciences (Institute of Dermatology, Chinese Academy of Medical Sciences).

[0040] Example 1: The interaction between SDG and AIM2 protein and its effect on the stability of AIM2 protein were verified by cell thermal shift assay (CETSA) and drug affinity response target stability (DARTS) experiments.

[0041] Based on the crystal structure of mouse AIM2 protein, virtual screening was performed from a commercial natural compound library containing more than 4,600 natural small molecules, and the screening was based on molecular docking scores (see Example 2 for the specific method). Figure 1-AThe table shows the top ten natural compounds with the highest docking scores, among which number 6 is flax lignan (SDG). To further verify the interaction between these compounds and AIM2 protein, the human GM12878 B cell line and the mouse BAF3 pro-B cell line were used to evaluate these ten compounds using CETSA and DARTS experiments. CETSA and DARTS are experimental methods for verifying the interaction between small molecule compounds and target proteins based on changes in protein thermal stability and resistance to protease digestion, respectively. The specific steps are as follows: The cell lysate containing AIM2 protein was divided into 10 groups, and compounds numbered 1 to 10 were added to each group. In the CETSA experiment, each group of samples was exposed to heating at 66°C for 10 minutes to induce protein denaturation; in the DARTS experiment, each group of samples was digested at room temperature for 10 minutes with the addition of low-concentration protease. Subsequently, soluble proteins and precipitated proteins were separated by high-speed centrifugation. The soluble part was extracted for SDS-PAGE electrophoresis separation, and the presence of AIM2 protein was detected by Western Blot.

[0042] The experimental results are as follows Figure 1-B (CETSA trial) and Figure 1-C (DARTS experiment) shows that by comparing the treatment effects of 10 compounds, compound 6 (SDG) showed the effect of stabilizing AIM2 protein in both experiments. SDG can effectively inhibit the heat-induced degradation of AIM2 protein and enhance its resistance to protease digestion, confirming that SDG is a potential binding ligand of AIM2 protein.

[0043] The above virtual screening, cell thermal migration analysis (CETSA) and drug-binding protein degradation (DARTS) experiments confirmed that flax lignans can increase the protein stability of AIM2.

[0044] Example 2: Analysis of the binding between SDG and AIM2 protein using molecular docking technology

[0045] The binding mode of SDG and AIM2 protein was analyzed by molecular docking technology using AutoDock and Discovery Studio software. The docking process calculates the binding free energy based on the scoring function and simulates the interaction between the ligand and the target protein.

[0046] Molecular docking results showed that SDG could form four hydrogen bonds with human AIM2 protein, with binding sites including LYS160, ALA161, LYS162 and ARG311, and the docking score was -5.699 (see Figure 2-ASDG forms eight hydrogen bonds with mouse AIM2 protein, with binding sites including LYS117, ARG95, ARG99, GLU96, ALA94, and LYS92, and the docking score is -8.1777 (see Figure 2-B ). The above results indicate that SDG exhibits good binding ability to both mouse and human AIM2 proteins, providing an important theoretical basis for the study of SDG as an AIM2 target.

[0047] Example 3: Flow cytometry detection of SDG promoting the expression of human naive CD4 + T( The ability of T cells to differentiate into Treg cells.

[0048] Peripheral blood was collected from healthy subjects, peripheral blood mononuclear cells (PBMC) were isolated, and EasySep TM Human CD4 + T Cell Isolation KitⅡ(Stemcell) extraction CD4 + T cells. CD4 + T cells were activated with 5 μg / mL Anti-CD3 and 2 μg / mL Anti-CD28 and treated with three concentrations of SDG (1 μM, 10 μM, 100 μM) for 5 days under neutral culture conditions. CD4 + T cell viability and analysis of Treg cells (CD25 hi CD127 low ) ratio. The results are as follows Figure 3-A As shown, the three concentrations of SDG had an effect on CD4 + There was no significant effect on the activity of T cells, but the proportion of Treg cells gradually increased with the increase of SDG concentration. To further confirm the induction effect of SDG on Treg cells, TGF-β (2ng / mL, Peprotetin) and IL-12 (40U / mL, Peprotetin) were further added to the above culture system to induce Treg cell differentiation. At the same time, the cells were treated with SDG (1μM, 10μM, 100μM) respectively, and flow cytometry was used to detect the effect of SDG on Treg cells (CD4 + CD25 + FOXP3 + ) differentiation. Figure 3-B As shown in the figure, under Treg cell induction conditions, treatment with different concentrations of SDG did not affect CD4 + The activity of T cells was decreased, but the proportion of Treg cells was significantly increased.

[0049] The above results confirm that flax lignans promote the expression of human naive CD4 + T cells differentiate into Treg cells.

[0050] Example 4: Flow cytometry was used to detect the ability of SDG to inhibit the ratio of Tfh / Tfr cells in the spleen and lymph node germinal centers in KLH immune model mice.

[0051] Eight-week-old female C57BL / 6J mice were selected and randomly divided into three groups (n=8): control group, KLH immunization group, and KLH immunization plus SDG treatment group. All mice received subcutaneous immunization with 400 μL of KLH (concentration 0.5 mg / ml, Sigma) on days 0 and 14. KLH was emulsified in complete Freund's adjuvant (CFA), and booster immunization was performed on day 7. Mice in the SDG treatment group were given SDG by intragastric injection from day 0, with a daily dose of 100 mg / kg. All mice were euthanized on day 14, and tissues such as serum, spleen, and draining lymph nodes were collected and ground to obtain single cell suspensions. Flow staining analysis was performed to detect serum antibody levels and changes in the ratio of Tfh / Tfr in the spleen and lymph nodes. The results showed that the level of anti-KLH-IgG in the serum of the KLH immunization group increased, while the SDG treatment group significantly inhibited the production of anti-KLH-IgG, which showed a significant downward trend compared with the KLH immunization group. At the same time, the total number of spleen and lymph node cells in the SDG-treated group was significantly reduced compared with the KLH-immunized group, suggesting that SDG inhibited cell proliferation caused by immune activation (see Figure 4-A ). CD4 + The Tfh / Tfr ratio of T cells was significantly increased, while that of the SDG-treated group was significantly decreased (see Figure 4-B ), indicating the potential role of SDG in regulating the immune response of germinal centers. The above provides the results of flax lignans inhibiting the immune response induced by KLH.

[0052] Example 5: Evaluation of the effect of SDG on alleviating disease progression in SLE model mice.

[0053] Eight-week-old female C57BL / 6J mice were selected and intraperitoneally injected with 500 μL of pristane (Sigma) for the first treatment, and then a 500 μL booster injection was performed 8 weeks later. At 20 weeks of age, the mice with successful modeling were randomly divided into three groups (n=8) according to urine protein levels and body weight: model group, low-dose SDG treatment group (100 mg / kg / d), high-dose SDG treatment group (200 mg / kg / d), and C57BL / 6J mice not treated with pristane were used as negative control group (Control group). The treatment groups were gavaged from day 0 for 8 weeks. Body weight and urine protein levels were monitored weekly to evaluate renal injury, and serum anti-double-stranded DNA-IgG levels were detected by ELISA every two weeks. At the end of the eighth week of treatment, the mice were euthanized, and spleen, lymph nodes, and kidney tissues were collected. Immunofluorescence staining (IF) was used to analyze the deposition of IgG and C3 in renal tissue, and H&E staining was used to evaluate the morphological changes of renal tissue. Flow cytometry was used to detect the proportion of immune cells in the spleen and lymph nodes, especially CD4 + Changes in the proportion of Treg cells in T cells.

[0054] The experimental results showed that compared with the Model group, the anti-double-stranded DNA-IgG levels in the serum of the low-dose and high-dose SDG treatment groups were significantly reduced (see Figure 5-A ). The urine protein level in the SDG-treated group was significantly lower than that in the Model group, indicating that SDG has a protective effect on lupus-related renal injury (see Figure 5-B ). H&E staining and immunofluorescence staining were performed on the kidney tissues of mice in each group. The glomeruli of mice in the Model group showed significant swelling and inflammatory cell infiltration, and the deposition of IgG and C3 in the kidney tissue was significantly enhanced. The glomerular swelling and inflammatory infiltration of the SDG-treated group were alleviated, the deposition of IgG and C3 in the kidney was reduced, and the immunofluorescence intensity was weakened (see Figure 5C-D). Flow cytometry results showed that compared with the Model group, the CD4 + The ratio of Treg cells in T cells increased significantly (see Figure 5E-H). The above results confirmed that flax lignans can effectively improve the symptoms of pristane-induced lupus mice.

[0055] Overall, SDG showed significant therapeutic effects in the pristane-induced lupus mouse model. It effectively alleviated the progression of the disease by reducing the level of anti-double-stranded DNA antibodies, improving renal function, reducing immune complex deposition, and increasing the proportion of Treg cells, providing experimental support for its potential application in the treatment of lupus.

[0056] The present invention provides a concept and method for using AIM2 as a target in the preparation of drugs for treating Treg-related autoimmune diseases. There are many methods and approaches to specifically implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention, and these improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of flax lignans in the preparation of drugs for the treatment of Treg cell-related autoimmune diseases.

2. The use according to claim 1, characterized in that The Treg cell-related autoimmune disease is any one or a combination of systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, systemic sclerosis, Sjögren's syndrome, inflammatory bowel disease, myasthenia gravis, psoriasis, type I diabetes, Graves' disease, Hashimoto's thyroiditis and idiopathic thrombocytopenic purpura.

3. The use according to claim 1, characterized in that The Treg cell-related autoimmune disease is systemic lupus erythematosus.

4. Application of flax lignans in the preparation of drugs for alleviating the progression of systemic lupus erythematosus.

5. Application of flax lignans in the preparation of drugs for treating systemic lupus erythematosus-related renal damage.

6. Application of flax lignans in the preparation of drugs for reducing immune complex deposition.

7. Application of flax lignans in the preparation of drugs for reducing the level of anti-double-stranded DNA-IgG in serum.

8. A drug for treating Treg cell-related autoimmune diseases, characterized in that: The medicament contains flax lignans.

9. The drug according to claim 8, characterized in that The medicine is a preparation made of flax lignan and a pharmaceutically acceptable carrier.

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