Application of aesculetin in preparation of medicine for inhibiting T cell activation, preventing and / or treating SLE (systemic lupus erythematosus) and medicine composition

Abstract

The invention relates to the technical field of biological medicines, and discloses application of aesculetin in preparation of CD4 + T cell activation inhibition drugs on the first aspect, and the aesculetin reduces CD4 + T cell activation and proliferation by inhibiting T cell receptor (TCR) signal channels so as to inhibit generation of B cell mediated autoantibodies. On the second aspect, the invention discloses an application of aesculetin in preparation of a medicine for preventing and / or treating SLE, and the application is used for preventing and / or treating SLE by inhibiting CD4 + T cell activation. On the third aspect, the invention discloses a pharmaceutical composition which comprises a therapeutically effective amount of aesculetin and one or more pharmaceutically acceptable carriers or auxiliary materials and is used for preventing and / or treating systemic lupus erythematosus or inhibiting CD4 + T cell activation. It is found that aesculetin can effectively inhibit TCR-mediated CD4 + T cell activation, shows a remarkable treatment effect in an SLE model, and fills the blank in the prior art.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of fraxin in the preparation of drugs for inhibiting T cell activation, preventing and / or treating SLE, and a pharmaceutical composition thereof. Background Technology

[0002] Systemic lupus erythematosus (SLE) is a chronic, complex autoimmune disease characterized by immune system dysregulation, producing large amounts of autoantibodies, such as anti-dsDNA antibodies, leading to multi-organ damage. Its core pathogenic mechanism is CD4+. + Dysregulation of T lymphocyte activation is a mechanism also seen in other autoimmune diseases, such as inflammatory bowel disease. In these diseases, abnormal helper T cell responses drive the production of inflammatory cytokines, the production of pathogenic autoantibodies by B cells, and subsequent tissue infiltration and damage.

[0003] Currently, the treatment of SLE mainly relies on glucocorticoids (such as prednisone), antimalarial drugs (such as hydroxychloroquine), immunosuppressants (such as mycophenolate mofetil and cyclophosphamide), and biologics (such as belimumab). However, these drugs have significant side effects, such as metabolic disorders and osteoporosis caused by long-term use of hormones, and bone marrow toxicity and increased risk of infection caused by immunosuppressants. In addition, some patients do not respond well to existing treatment regimens or develop drug resistance.

[0004] Fraxin B, the main active ingredient of the traditional Chinese medicine Fraxinus rhizome, is known to possess anti-inflammatory, antioxidant, antibacterial, and vascular-protective pharmacological activities. It is commonly used to treat intestinal inflammation and hemorrhoids, but current technology has not disclosed or suggested its potential use in treating SLE (spleen and stomach lesions). The pathogenesis of SLE involves CD4+. + Abnormal activation of T cells drives B cells to produce autoantibodies, but therapeutic strategies that directly target this process are still lacking. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide the application of fraxetine in the preparation of drugs for inhibiting T cell activation, preventing and / or treating SLE, and a pharmaceutical composition thereof. It has been found that fraxetine can effectively inhibit TCR-mediated CD4+. + T-cell activation showed significant therapeutic effects in the SLE model, filling a gap in existing technologies.

[0006] The present invention solves the above-mentioned technical problems through the following technical means:

[0007] In a first aspect, this invention discloses the use of fraxin in the preparation of CD4. + Application of drugs that inhibit T-cell activation.

[0008] Furthermore, the fraxetin reduces CD4+ by inhibiting the T-cell receptor TCR signaling pathway. + T cell activation and proliferation, thereby inhibiting B cell-mediated autoantibody production.

[0009] Through extensive creative experiments, the applicant discovered that fraxetin is an effective broad-spectrum CD4 inhibitor. + T-cell activation inhibitors, by targeting the TCR signaling pathway, have shown significant therapeutic effects in various autoimmune models. The applicant's research results are related to CD4. + The treatment of T-cell-mediated autoimmune diseases offers a novel therapeutic strategy and promising lead compounds.

[0010] Secondly, the present invention also discloses the use of fraxin in the preparation of drugs for the prevention and / or treatment of SLE, wherein the use is achieved by inhibiting CD4. + T-cell activation to prevent and / or treat SLE. In this application, SLE refers to systemic lupus erythematosus.

[0011] Furthermore, the fraxetine prevents and / or treats SLE through one or more of the following pathways: inhibiting autoantibody production, regulating immune cell function, and inhibiting the release of inflammatory factors.

[0012] Thirdly, the present invention also discloses a pharmaceutical composition comprising a therapeutically effective amount of fraxetine and one or more pharmaceutically acceptable carriers or excipients for the prevention and / or treatment of systemic lupus erythematosus, or for inhibiting CD4. + T cell activation

[0013] Furthermore, the dosage form of the drug is an oral preparation or an injection.

[0014] Furthermore, the oral formulation is selected from tablets, capsules, granules, or oral liquids.

[0015] Furthermore, the injection is selected from injectable solutions or lyophilized powder injections.

[0016] Furthermore, the pharmaceutical composition is a tablet, and the tablet comprises, by weight, 40-60 parts of fraxin, 30-50 parts of microcrystalline cellulose, 3-7 parts of sodium carboxymethyl starch, and 0.5-1.5 parts of magnesium stearate.

[0017] Furthermore, the pharmaceutical composition is an injection solution, and the components of the injection solution, by mass percentage, include 0.05%-0.15% fraxetine, 0.8%-1.1% sodium chloride for injection, and the balance being water for injection.

[0018] Fraxin is a natural coumarin derivative and a potent immunosuppressive compound that significantly inhibits TCR-mediated CD4+ in vitro. + T cell activation and proliferation. Fraxinus ethylsin also effectively inhibits the production of T cell-dependent antibodies in TB cell co-culture systems. Notably, fraxinus ethylsin showed broad therapeutic effects in two autoimmune models with different mechanisms: in the BM12 metastatic lupus model, fraxinus ethylsin improved autoantibody production and glomerulonephritis; in the RAG1- / - receptor T cell metastatic colitis model, fraxinus ethylsin reduced weight loss, colonic pathology, and inflammatory cytokine responses. Flow cytometry analysis confirmed that pathogenic T cell proliferation and activation were inhibited in target tissues. Mechanistic studies using RNA sequencing revealed that fraxinus ethylsin broadly inhibits the transcriptional program of the TCR signaling pathway. Fraxinus ethylsin is a naturally derived TCR signaling inhibitor that inhibits CD4+. + T-cell-mediated autoimmune diseases have broad therapeutic potential, providing a novel strategy and promising lead compounds for the intervention of autoimmune diseases.

[0019] In summary, this application has the following beneficial effects:

[0020] 1. This invention is the first to discover that fraxetine has significant preventive and / or therapeutic effects on systemic lupus erythematosus (SLE), which completely exceeds the expectations of those skilled in the art and provides a novel treatment option for SLE. The pathogenesis of SLE involves a complex immune regulatory network, which is fundamentally different from simple local inflammation. Therefore, those skilled in the art cannot directly infer from the known anti-inflammatory effects of fraxetine that it can effectively prevent and / or treat SLE.

[0021] 2. This invention demonstrates through animal experiments that fraxetine inhibits the production of autoantibodies in SLE model mice, reduces the titer of anti-ds-DNA antibodies in serum, and improves renal pathological damage. Its efficacy is comparable to that of the positive control drug and even superior in some indicators.

[0022] 3. The fraxin used in this invention is a natural product with low toxicity. Compared with traditional hormones and immunosuppressants, it is expected to have better safety and fewer side effects, potentially improving patient medication adherence and quality of life.

[0023] 4. The fraxin B used in this invention can be extracted from the plant Fraxinus chinensis or obtained in large quantities through chemical synthesis. The raw material sources are wide-ranging, which provides a guarantee for drug development. Attached Figure Description

[0024] Figure 1 The figure shows the experimental results of fraxetine directly inhibiting TCR-dependent helper T cell activation and proliferation;

[0025] Figure 2 The figure shows the experimental results of fraxetine inhibiting helper T cell-mediated B cell proliferation and antibody production;

[0026] Figure 3 The figure shows the experimental results of fraxetine inhibiting the production of autoantibodies in SLE model mice;

[0027] Figure 4 The figure shows the experimental results of fraxetine inhibiting the accumulation of antibodies in the kidneys of SLE model mice. Detailed Implementation

[0028] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0029] Natural products have historically been a valuable resource for drug discovery, particularly in immunology. Their complex chemical structures, refined over thousands of years of biological optimization, often possess unique pharmacological properties, making them an excellent starting point for therapeutic development. Notably, several successful immunomodulatory drugs, including rapamycin and cyclosporine, are derived from natural products. Screening natural compound libraries offers unique advantages for identifying novel immunomodulators: (1) providing a scaffold of chemical diversity with evolutionary biological activity; (2) the potential for novel mechanisms of action that transcend traditional drug discovery methods; and (3) generally exhibiting better toxicity profiles compared to synthetic compounds.

[0030] Based on these considerations, the applicant hypothesizes that systematically screening natural product libraries can identify novel CD4 molecules with the potential to treat autoimmune diseases. + T cell activation inhibitors. To verify this hypothesis, the applicant pursues the following specific objectives: (1) Screening a diverse library of more than 1,000 natural compounds for TCR-mediated CD4 activation. + (2) Inhibitors of T cell activation; (3) Characterize the effects of HIT compounds on T cell function and T cell-dependent antibody production; (4) Evaluate the efficacy of various preclinical autoimmune models; (5) Investigate and identify the molecular mechanisms of the immunomodulatory effects of the compounds.

[0031] The applicant adopted a comprehensive, multi-stage approach to achieve these goals. The applicant's research first utilized primary CD4... +High-throughput screening with T cells was followed by detailed in vitro mechanism studies. The applicant then validated the therapeutic potential in two established but mechanistically different autoimmune models (a bm12-induced lupus model and a T-cell metastatic colitis model) to ensure broad relevance of the findings. Finally, the applicant employed transcriptomics to elucidate the molecular targets of the identified compounds. This rigorous, multifaceted strategy ensured not only the identification of a novel immunomodulatory compound but also a thorough determination of its therapeutic potential and mechanism of action.

[0032] Through this method, the applicant discovered that fraxin is a natural coumarin derivative and an effective broad-spectrum CD4 inhibitor. + T-cell activation inhibitors, by targeting the TCR signaling pathway, have shown significant therapeutic effects in various autoimmune models. The applicant's research results are related to CD4. + The treatment of T-cell-mediated autoimmune diseases offers a novel therapeutic strategy and promising lead compounds.

[0033] Below, the applicant first used fraxetine to directly inhibit TCR-dependent helper T cell activation and proliferation, and evaluated its effect on naïve CD4+. + The effect of T cell activation, the results are as follows Figure 1 As shown.

[0034] By coating culture plates with specific monoclonal antibodies targeting CD3 and CD28, the natural process of antigen presentation can be effectively mimicked, thereby simultaneously activating the vast majority of T cells in the culture plate. Microscopic observation revealed that naive T cells showed a significant increase in cell volume 24 hours after activation, while the T cell volume gradually decreased with increasing fraxetine concentration. At a dosage of 20 μm, the T cell size was the same as in the naive group, indicating inhibited T cell activation. Figure 1 A). Especially after 48 hours, the dose-dependent inhibition of T cell number and size by fraxetine was most evident. To further clarify the selective inhibition of TCR signaling by fraxetine, the applicant used flow cytometry to assess T cell proliferation using CTV and Ki67 indicators. It was found that with increasing fraxetine dosage, T cell proliferation was significantly inhibited, manifested as a decrease in CTV passage number and a reduction in Ki67 positivity rate. These experimental results indicate that fraxetine inhibits CD4+. + T cell proliferation ( Figure 1 B and 1C).

[0035] Next, the applicant conducted an experiment using fraxetine to inhibit helper T cell-mediated B cell proliferation and antibody production, with the following results: Figure 2 As shown.

[0036] Because B cells form germinal centers, perform class switching, and produce high-affinity antibodies, they depend on "second signals" and cytokines provided by activated helper T cells. Fraxinol has been shown to directly inhibit TCR-dependent helper T cell activation. Figure 1 Following this, the applicant investigated whether this T-cell suppression would impair the T-cell's ability to assist B-cell responses. The applicant constructed an in vitro T-cell and B-cell co-culture model and treated it with different concentrations of fraxetine to simulate the effect of fraxetine on the immune response of B cells. Figure 2 A). The results showed that in the co-culture system, increasing fraxetin concentration significantly inhibited the clonal expansion of B cells, specifically manifested as smaller clusters of B cells under the microscope. Figure 2 B). The co-stimulatory signals provided by activated helper T cells (Th cells) determine when B cells truly begin clonal expansion. Flow cytometry analysis revealed that fraxetin significantly inhibited T cell (Th cells) proliferation in the co-culture system. Figure 2 C) and B cell proliferation ( Figure 2 D). This manifests as a decrease in CTV generation. The sole purpose of TB interaction is to guide B cells to produce high-affinity, high-specificity, and long-lasting protective antibodies. To further verify the differences in antibody production by B cells, the applicant detected changes in LGG subtypes using ELISA. The results showed that fraxetine (20µm) significantly inhibited the production of total IgG and major subtypes (IgG1, IgG2b, IgG2c, IgG3) by B cells. Figure 2 E). This study reveals the great potential of fraxetine as a highly effective and broad-spectrum immunosuppressant.

[0037] Furthermore, the applicant conducted an experiment on the inhibition of autoantibody production in SLE model mice by fraxetine, and the results were as follows: Figure 3 As shown.

[0038] The applicant has demonstrated in vitro that fraxetine can effectively inhibit T cell-dependent antibody production. Figure 2 The applicant then investigated whether it could improve the disease condition in a mouse model of systemic lupus erythematosus (SLE), in which pathogenic autoantibodies (especially IgGs) play a central role in the pathogenesis. Specifically, the applicant chose bm12-induced SLE model, which is a CD4+... + A T-cell-driven model of autoimmune lupus disease ( Figure 3 A). Compared with the control group, treatment with fraxetine reversed splenomegaly induced in the SLE model. Consistently, administration of fraxetine reduced spleen weight in SLE mice. Figure 3B). The above results suggest that fraxetine can effectively inhibit the progression of SLE. In SLE, the number of apoptotic cells in tissues increases, and these apoptotic cells release nuclear antigens, such as double-stranded DNA (dsDNA), histones, and ribonucleoproteins. B cells can recognize nuclear antigens, and with the help of activated T cells, these B cells proliferate, differentiate into plasma cells, and produce a large number of pathogenic autoantibodies, especially anti-dsDNA antibodies and antinuclear antibodies (ANA). To visually evaluate the effect of fraxetine on the level and specificity of autoantibodies in a mouse model of systemic lupus erythematosus (SLE), the applicant used human laryngeal cancer epithelial cells (Hep2 cells) as the antigen matrix and performed indirect immunofluorescence detection on antinuclear antibodies in mouse serum. As shown in Figure 3C, compared with the control group, the fluorescence signal intensity of serum from SLE model mice treated with fraxetine on Hep2 cells was significantly reduced. The serum of control mice showed bright, typical nuclear fluorescence staining, predominantly in a homogeneous or speckled pattern, indicating that their serum contained high titers of IgG antinuclear antibodies capable of broadly binding to various self-antigens. Figure 3 C). ELISA results showed that treatment with fraxetine significantly reduced serum levels of anti-dsDNA, IgG, IgM, and anti-nucleosome IgG and IgM in SLE mice. Figure 3 (D) The above results indicate that fraxetine treatment significantly reduced the overall level of pathogenic IgG autoantibodies in the serum of SLE model mice. The decrease in fluorescence intensity is highly consistent with the decrease in the level of specific autoantibodies (such as anti-dsDNA IgG) detected by ELISA in Figure 3D, further confirming from a morphological perspective that fraxetine can effectively inhibit autoimmune responses and reduce the production of pathogenic autoantibodies.

[0039] Furthermore, the applicant conducted an experiment on the inhibition of antibody accumulation in the kidneys of SLE model mice by fraxetine, and the results were as follows: Figure 4 As shown.

[0040] Autoantibodies form immune complexes that deposit in organs such as the kidneys and skin, causing inflammation and tissue damage (e.g., lupus nephritis), a typical symptom of SLE. Immunofluorescence labeling of autoantibodies IgG and DNA indicates a decrease in fluorescence intensity related to… Figure 3 The decrease in the levels of specific autoantibodies (such as anti-dsDNA IgG) detected by ELISA was highly consistent, further confirming from a morphological perspective that fraxetine can effectively inhibit autoimmune responses and reduce the production of pathogenic autoantibodies. Figure 4A). HE staining revealed significant pathological changes in the morphology and structure of the kidney tissue in the control group mice. Multiple glomerular capillary walls showed thickening, exhibiting a "wire loop" appearance, and eosinophilic hemoglobin deposition (suggesting immune complex deposition) was observed. Inflammatory cell infiltration (mainly neutrophils and monocytes) was visible within the glomerular capsule. Figure 4 B). The above results indicate that fraxetine treatment can effectively reduce kidney pathological damage in SLE model mice and has significant protective and therapeutic effects on lupus nephritis.

[0041] Furthermore, the applicant conducted a therapeutic effect study of fraxetine on a BM12 mouse SLE model, as detailed below:

[0042] 1. Methods and steps for inducing a systemic lupus erythematosus (SLE) model using bm12.

[0043] This is a very classic CD4 + The core principle of the T-cell-driven autoimmune lupus model is to induce continuous activation of allogeneic reactive T cells through a single amino acid point mutation (bm12) in MHC class II molecules, which in turn drives B cells to produce a variety of autoantibodies, ultimately leading to SLE-like lesions.

[0044] 2. Introduction to the core mechanism of the model

[0045] Donor mice: bm12 mutant mice with a C57BL / 6J (B6) background. Their MHC-II (IA) molecules have point mutations at positions 68 and 71, differing from wild-type B6 mice by only 3 amino acids.

[0046] 3. Recipient mice: Wild-type C57BL / 6J (B6) mice.

[0047] 4. Driving Factors: When lymphocytes from bm12 mice (as donors) are infused into syngeneic wild-type B6 mice (as recipients), the recipient mouse's T cells recognize the donor's bm12 MHC-II molecules as "non-self" (despite genetic similarity), thus becoming strongly activated. These activated CD4+ cells... + T cells, in turn, help B cells activate and proliferate, and produce a large number of autoantibodies against nuclear antigens (such as dsDNA and histones), forming immune complexes that deposit in organs such as the kidneys, causing diseases such as lupus nephritis.

[0048] 5. Specific methods and steps

[0049] Phase 1: Cell Preparation

[0050] Prepare donor mice: Select healthy bm12 mice (usually 8-12 weeks old).

[0051] Lymphocytes were obtained: bm12 mice were sacrificed, and the spleens were aseptically removed. The spleens and lymph nodes were ground in RPMI-1640 medium and passed through a cell sieve to prepare a single-cell suspension.

[0052] Red blood cell lysis: Spleen cell suspension was treated with red blood cell lysis buffer to remove red blood cell interference. CD4 was obtained by magnetic bead sorting. + T cells.

[0053] Phase Two: Model Induction

[0054] Prepare recipient mice: Select age-matched female wild-type B6 mice as recipients.

[0055] Cell infusion (adoptive transfer): The prepared bm12 lymphocyte suspension is usually injected into the recipient B6 mouse via tail vein injection.

[0056] Phase 3: Fraxin B Treatment

[0057] Injected via the tail vein.

[0058] The methodology of this model can be summarized as follows: lymphocytes are prepared from bm12 donor mice → adopted and transferred to syngeneic female wild-type B6 recipient mice → after a 4-16 week autoimmune response development period → the occurrence of SLE-like disease is verified by detecting autoantibody levels and kidney pathological changes.

[0059] The advantage of this model lies in its clearly defined pathogenesis involving CD4. + T-cell-driven mechanisms can effectively mimic various immunological and pathological features of human lupus erythematosus (SLE), making them a classic tool for studying the pathogenesis of lupus and screening therapeutic drugs.

[0060] 6. Test Results

[0061] Aescin, a natural coumarin derivative, is a small molecule compound known to possess antioxidant and anti-inflammatory properties, but it has limited effect on CD4. +The role of T cells in T cell activity is poorly understood. While esculin (6,7-dihydroxycoumarin) is known to inhibit the proliferation of human T cells stimulated by PHA or phorbol ester plus iodomycin in a dose-dependent manner, the specific mechanism remains unclear. The applicant's research clearly demonstrates that fraxetine is not a broad-spectrum cytotoxic agent or a universal antiproliferator. Instead, it precisely targets the initial activation signal (first signal) triggered by the TCR, without affecting subsequent IL-2-driven clonal expansion and survival. This "source" inhibitory characteristic distinguishes it mechanistically from current mainstream immunosuppressants. For example, calcineurin inhibitors (such as cyclosporine A) act on the calcium signaling pathway downstream of the TCR, inhibiting the transcription of cytokines such as IL-2; while mTOR inhibitors (such as rapamycin) directly interfere with signal transduction downstream of the IL-2 receptor, blocking cell cycle progression. In contrast, fraxetine intervenes at a more upstream stage, fundamentally preventing the initiation of the activation process. The potential advantage of this mode of action lies in its ability to more precisely interrupt the initial pathogenic phase of the autoimmune response while relatively preserving subsequent events mediated by IL-2 that are crucial for maintaining immune memory and executing initiated immune protective functions, such as memory T-cell responses against pathogens. This could potentially achieve a better balance between efficacy and safety. The applicant's research has fully validated the efficacy of fraxetine in a BM12-induced SLE model of autoimmune disease. In this model, fraxetine treatment significantly reduced autoantibody levels (such as anti-dsDNA IgG), alleviated splenomegaly, and reduced renal immune complex deposition. More importantly, even when intervention was initiated in the late stages of the disease (after the emergence of autoantibodies), fraxetine effectively suppressed follicular helper T cell (Tfh) and germinal center (GC) responses, and reduced plasma cell differentiation. This indicates that fraxetine not only prevents initial immune system dysregulation but also actively intervenes in and dismantles established pathological immune responses. This characteristic has significant clinical implications for treating patients with chronic, relapsing autoimmune diseases. The therapeutic effect of fraxetine may be mechanistic, potentially through the regulation of the Tfh-GC-B cell axis. Tfh cells are key regulators of B cell activation and antibody class switching, and their abnormal activation is closely related to the pathogenesis of SLE. After fraxetine intervention, the proportion of Tfh cells decreased, and the number of GC B cells and plasmablasts significantly decreased. This suggests that fraxetine may exert its therapeutic effect by directly inhibiting CD4+. + Activation of T cells indirectly weakens their helper function on B cells, thereby breaking the vicious cycle in SLE pathology.

[0062] The applicant below will prepare fraxin into fraxin tablets, specifically as follows:

[0063] Tablet Formula 1: Fraxinus 40g, microcrystalline cellulose 30g, sodium carboxymethyl starch 3g, magnesium stearate 0.5g, made into 1000 tablets.

[0064] Tablet prescription 2: 50g of fraxin, 40g of microcrystalline cellulose, 5g of sodium carboxymethyl starch, and 1g of magnesium stearate, to make 1000 tablets.

[0065] Tablet Formula 3: Fraxin 60g, microcrystalline cellulose 50g, sodium carboxymethyl starch 7g, magnesium stearate 1.5g, made into 1000 tablets.

[0066] The preparation process of the above tablets is as follows: Fraxinus chinensis, microcrystalline cellulose, and sodium carboxymethyl starch are sieved and mixed evenly. An appropriate amount of ethanol is used as a wetting agent to prepare a soft mass. The mass is sieved and granulated, dried, sized, magnesium stearate is added, mixed evenly, and compressed into tablets to obtain the final product.

[0067] The applicant will prepare fraxetine into a fraxetine injection, as follows:

[0068] Injection prescription 1: Fraxinus 5g, sodium chloride for injection 80g, add water for injection to 10000ml.

[0069] Injection prescription 2: Fraxinus 10g, sodium chloride for injection 90g, add water for injection to 10000ml.

[0070] Injection prescription 3: Fraxinus 15g, sodium chloride for injection 110g, add water for injection to 10000ml.

[0071] The preparation process of the above-mentioned injection solution is as follows: Add approximately 80% of the total amount of water for injection to the mixing tank, add fraxetin and sodium chloride, stir until completely dissolved, adjust the pH to 6.5-7.5, add water for injection to the total volume, filter, fill into ampoules, and autoclave at 115°C for 30 minutes to obtain the solution.

[0072] Experimental Example: The therapeutic effect of tablet formulation 2, fraxinol, on an MRL / lpr mouse SLE model.

[0073] Animal model and grouping: Forty 12-week-old female MRL / lpr mice (spontaneous SLE model) were randomly divided into 5 groups (n=8): model group, positive control group (prednisone, 10 mg / kg / d), low-dose fraxetine group (25 mg / kg / d), medium-dose fraxetine group (50 mg / kg / d), and high-dose fraxetine group (100 mg / kg / d). Eight age-matched Balb / c mice were used as a normal control group.

[0074] Administration method: Each administration group was administered the corresponding dose by gavage, while the model group and normal control group were given an equal volume of solvent. The administration was continued for 8 weeks.

[0075] Observation indicators:

[0076] 24-hour urinary protein: Urine samples were collected from mice using metabolic cages before administration and 8 weeks after administration, and urinary protein levels were measured using the Coomassie brilliant blue method. Results showed that urinary protein levels were significantly higher in the model group compared to the normal group. All doses of fraxetine significantly reduced urinary protein levels in a dose-dependent manner, with the high-dose group showing effects comparable to the positive control group.

[0077] Serum anti-dsDNA antibody: After the last administration, blood was collected by enucleation, serum was separated, and the level of anti-dsDNA antibody was detected using an ELISA kit. The results showed that all dose groups of fraxetine, especially the medium and high dose groups, significantly reduced the abnormally elevated level of anti-dsDNA antibody in the model mice.

[0078] Kidney histopathological examination: Mouse kidneys were harvested, fixed in formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE) and plasmolysis (PAS). The results showed that in the model group, the glomeruli were enlarged, with mesangial cell proliferation, inflammatory cell infiltration, and thickening of the basement membrane, and immune complex deposition was observed. In the fraxetine treatment group, especially the high-dose group, the pathological changes in the kidneys were significantly alleviated.

[0079] The above experimental results indicate that fraxetine can significantly improve disease activity in BM12 / SLE model mice, reduce proteinuria and autoantibody levels, alleviate kidney pathological damage, and has a clear therapeutic effect on SLE.

[0080] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention. Technical aspects, shapes, and structures not described in detail in this invention are all well-known technologies.

Claims

1. Fraxinus thioglycoside in the preparation of CD4 + Application of drugs that inhibit T-cell activation.

2. The application according to claim 1, characterized in that, Fraxinol reduces CD4+ by inhibiting the T-cell receptor TCR signaling pathway. + T cell activation and proliferation, thereby inhibiting B cell-mediated autoantibody production.

3. The use of fraxetine in the preparation of drugs for the prevention and / or treatment of SLE, wherein the use is achieved by inhibiting CD4. + T-cell activation can prevent and / or treat SLE.

4. The application according to claim 3, characterized in that, Fraxinus luteum prevents and / or treats SLE through one or more of the following pathways: inhibiting the production of autoantibodies, regulating immune cell function, and inhibiting the release of inflammatory factors.

5. A pharmaceutical composition, characterized in that, Containing a therapeutically effective amount of fraxetine, and one or more pharmaceutically acceptable carriers or excipients, for the prevention and / or treatment of systemic lupus erythematosus, or for the inhibition of CD4. + T cell activation.

6. The pharmaceutical composition according to claim 5, characterized in that, The drug is available in oral or injectable form.

7. The pharmaceutical composition according to claim 6, characterized in that, The oral preparation is selected from tablets, capsules, granules, or oral liquids.

8. The pharmaceutical composition according to claim 6, characterized in that, The injection is selected from injection solutions or lyophilized powder injections.

9. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition is a tablet, and by weight, the tablet comprises 40-60 parts of fraxin, 30-50 parts of microcrystalline cellulose, 3-7 parts of sodium carboxymethyl starch, and 0.5-1.5 parts of magnesium stearate.

10. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical composition is an injection solution, and the components of the injection solution, by mass percentage, include 0.05%-0.15% fraxetine, 0.8%-1.1% sodium chloride for injection, and the balance being water for injection.

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