Use of luteolin in preparation of a medicine for preventing and / or treating systemic lupus erythematosus

Abstract

This invention discloses the application of luteolin in the preparation of drugs for the prevention and / or treatment of systemic lupus erythematosus (SLE), belonging to the field of natural product chemistry. This invention discovers the pathological changes caused by immune imbalance and the excessive production of autoantibodies due to the overactivation of Tfh cells in SLE patients, and studies the molecular mechanism by which luteolin inhibits Tfh differentiation, providing a new approach for the application of single-component traditional Chinese medicine in SLE treatment. This contributes to a deeper understanding of the disease mechanism and provides theoretical support for new drug development. Compared with existing technologies, the single-component luteolin of this invention can effectively inhibit Tfh cell differentiation. Compared with compound traditional Chinese medicine, the use of a single component has advantages in efficacy control and efficacy evaluation. Furthermore, luteolin is widely available, inexpensive, and has no toxic side effects, making it suitable for long-term SLE treatment.

Description

Technical Field

[0001] This invention relates to the field of natural medicinal chemistry, and in particular to the use of luteolin in the preparation of medicaments for the prevention and / or treatment of systemic lupus erythematosus. Background Technology

[0002] Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by the production of autoantibodies, the deposition of immune complexes, and chronic inflammation. It commonly affects women of childbearing age and can involve multiple organs and systems, including the skin, joints, kidneys, and central nervous system. Currently, the main treatments for SLE include glucocorticoids, antimalarial drugs (such as hydroxychloroquine), immunosuppressants (such as mycophenolate mofetil, cyclophosphamide, and tacrolimus), and biologics (such as belimumab). However, these drugs have significant side effects, poor efficacy in some patients, and a high relapse rate after discontinuation. Therefore, developing novel SLE treatments that combine good efficacy with enhanced safety is a crucial issue that urgently needs to be addressed in this field.

[0003] The pathogenesis of SLE is closely related to the abnormal activation of the adaptive immune response, among which the central role of follicular helper T cells (Tfh) in driving the disease progression of SLE is becoming increasingly prominent. Tfh cells are CD4+ cells. + A specific subset of T cells, primarily located in lymphoid follicles and germinal centers, provides crucial auxiliary signals for B cell differentiation, affinity maturation, and antibody class switching by secreting cytokines such as IL-21 and CXCR5. Recent studies have shown that the abnormal proliferation of Tfh cells and the resulting abnormal increase in autoantibody-secreting plasma cells are important pathogenic mechanisms driving SLE. Tfh cells are significantly increased in the peripheral blood and lymphoid tissues of SLE patients, and their number is positively correlated with disease activity scores and anti-dsDNA antibody titers, making them an important potential target for SLE treatment.

[0004] The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor belonging to the bHLH-PAS family. After binding to its ligand, the AHR dissociates from the cytoplasmic complex and translocates into the nucleus, forming a heterodimer with the aryl hydrocarbon receptor nuclear transporter (ARNT). This heterodimer binds to heteroreceptor response elements (XREs) in the promoter regions of target genes, regulating the expression of various genes, including Cyp1a1 and Cyp1b1. The AHR signaling pathway plays a crucial role in immune regulation, participating in the regulation of CD4+. + The direction of T cell differentiation.

[0005] Cynaroside is a naturally occurring flavonoid glycoside widely distributed in medicinal plants such as chrysanthemum and honeysuckle. Existing pharmacological studies have confirmed that cynaroside possesses various pharmacological activities, including anti-inflammatory, antioxidant, neuroprotective, and antitumor effects, primarily exerting its effects through the regulation of Nrf2 / Keap1, NF-κB, and MAPK signaling pathways. However, whether cynaroside affects the progression of SLE by regulating Tfh cell differentiation through the AHR-Cyp1a1 axis remains unreported. Summary of the Invention

[0006] The purpose of this invention is to provide the application of luteolin in the preparation of drugs for the prevention and / or treatment of systemic lupus erythematosus (SLE), thereby addressing the problems existing in the prior art. This invention reveals for the first time a novel mechanism by which luteolin alleviates SLE by activating the AhR signaling pathway, upregulating Cyp1a1 expression, and inhibiting Tfh cell differentiation, providing a theoretical basis and experimental support for the treatment of SLE with luteolin.

[0007] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of this invention is the use of luteolin or its pharmaceutically acceptable salts, esters, or solvates in the preparation of a medicament for the prevention and / or treatment of systemic lupus erythematosus.

[0008] The second technical solution of the present invention is a medicament for the prevention and / or treatment of systemic lupus erythematosus, comprising a therapeutically effective amount of luteolin or its pharmaceutically acceptable salt, ester, solvate, and a pharmaceutically acceptable carrier.

[0009] The third technical solution of the present invention is the application of luteolin or its pharmaceutically acceptable salts, esters, or solvates in the preparation of reagents for reducing the production of autoantibodies.

[0010] Based on the above technical solution, the present invention has the following technical effects: This invention reveals the pathological changes caused by excessive activation of Tfh cells in SLE patients, leading to immune imbalance and the production of large amounts of autoantibodies. It investigates the molecular mechanism by which luteolin inhibits Tfh differentiation, providing new insights into the application of traditional Chinese medicine monomers in SLE treatment. This contributes to a deeper understanding of the disease's mechanisms and provides theoretical support for new drug development.

[0011] Compared with existing technologies, the single herbal ingredient luteolin in this invention can effectively inhibit Tfh cell differentiation. Compared with compound herbal formulas, the use of a single ingredient has advantages in efficacy control and treatment evaluation. In addition, luteolin is widely available, inexpensive, and has no toxic side effects, making it suitable for long-term treatment of SLE. Attached Figure Description

[0012] Figure 1 This is a diagram illustrating the mechanism of action of luteolin in this invention.

[0013] Figure 2 Example 1 shows that luteolin can alleviate lupus-like symptoms in vivo. In the figures, A represents a comparison of spleen size among groups; B represents statistical analysis of spleen index; C represents a comparison of end-stage urinary protein levels among groups; D represents the levels of inflammatory cytokines and autoantibodies in mouse serum; E represents representative images of hematoxylin-eosin (HE) and periodic acid-Schiff (PAS) staining of kidney tissue; F represents quantitative analysis of kidney pathological scores; G represents representative immunohistochemical staining images of complement C3b deposition in kidney tissue; H represents representative immunohistochemical staining images of immunoglobulin G (IgG) deposition in kidney tissue; I represents quantitative analysis results of C3b deposition in the kidney; and J represents quantitative analysis results of IgG deposition in the kidney.

[0014] Figure 3 Example 2 shows that luteolin can reduce the proportion of Tfh and plasma cells in the spleen of lupus erythematosus mice. Wherein, A represents TfH (CXCR5) in spleen tissue. + CD4 + A) Flow cytometry results of TfH in spleen tissue; B) Quantitative analysis of TfH in spleen tissue by flow cytometry; C) Plasma cells (CD138) in spleen tissue. + CD19 + The flow cytometry results of ) are shown in Figure 1, where D represents the quantitative analysis of flow cytometry results of plasma cells in spleen tissue.

[0015] Figure 4 Example 3 shows that luteolin can directly inhibit Tfh cell differentiation in vitro. In this example, A represents the initial CD4+ levels detected by flow cytometry under Tfh polarization model culture conditions (Tfh polarization model culture system: anti-IL-4 antibody, anti-IFN-γ antibody, IL-6, IL-21, and TGF-β1). + Tfh (CXCR5) in T cells + PD-1 + The percentage of A is given by A. B represents the quantitative statistical analysis result of A. C represents the effect of luteolin on Tfh cell differentiation detected by flow cytometry. D represents the quantitative statistical analysis result of C.

[0016] Figure 5Example 4 illustrates the mechanism of action of luteolin in inhibiting Tfh cell differentiation. In the diagram, A is a Venn diagram showing the intersection of differentially expressed genes among the groups; B is a volcano plot showing the expression characteristics of the top 50 differentially expressed genes between the model group and the luteolin-treated group; C is a cluster heatmap showing the expression characteristics of the top 50 differentially expressed genes between the model group and the luteolin-treated group; D shows KEGG signaling pathway enrichment analysis of differentially expressed genes obtained from the comparison between the luteolin-treated group and the model group; E shows AHR-related signaling pathway enrichment analysis of differentially expressed genes obtained from the comparison between the luteolin-treated group and the model group; and F shows the verification of key differentially expressed genes using real-time quantitative PCR. Cyp1a1 , Cyp1b1 , Ovol1 , Mgll , Rslcan18 , Wdr81 G represents the relative mRNA expression level, G represents the expression level of Cyp1a1 protein in spleen tissue of MRL / Lpr mice detected and quantitatively analyzed by Western blotting, and H represents the expression level of Cyp1a1 protein in Tfh cells detected and quantitatively analyzed by Western blotting.

[0017] Figure 6 Example 4 illustrates the mechanism of action of luteolin in inhibiting Tfh cell differentiation. In this diagram, A represents the binding mode and binding energy of luteolin to the AHR receptor through molecular docking analysis; B shows the root mean square deviation of the luteolin-AHR complex backbone over simulation time; C shows the root mean square fluctuation curve of the AHR protein amino acid residues; D is a three-dimensional free energy landscape analysis diagram of the luteolin-AHR complex; E shows the radius of gyration of the luteolin-AHR complex over simulation time; F shows the number of hydrogen bonds between luteolin and AHR over simulation time; G is a two-dimensional free energy landscape analysis diagram of the luteolin-AHR complex; and H is an AHR luciferase reporter gene assay to quantitatively detect AHR luciferase activity under the influence of luteolin. Detailed Implementation

[0018] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0019] This invention provides the use of luteolin or its pharmaceutically acceptable salts, esters, and solvates in the preparation of medicaments for the prevention and / or treatment of systemic lupus erythematosus.

[0020] In some specific implementations, the luteolin glycoside inhibits the differentiation of follicular helper T cells by activating the aryl hydrocarbon receptor signaling pathway and upregulating the expression of its downstream target gene cytochrome P450 1A1.

[0021] In some specific implementations, the luteolin reduces the production of autoantibodies by inhibiting the differentiation of follicular helper T cells and reducing the generation of downstream plasma cells.

[0022] In some specific implementations, the systemic lupus erythematosus includes lupus nephritis.

[0023] This invention also provides a medicament for the prevention and / or treatment of systemic lupus erythematosus, comprising a therapeutically effective amount of luteolin or a pharmaceutically acceptable salt, ester, or solvate thereof, and a pharmaceutically acceptable carrier.

[0024] The present invention also provides the use of luteolin or its pharmaceutically acceptable salts, esters, and solvates in the preparation of reagents for reducing the production of autoantibodies.

[0025] The molecular formula of luteolin is C 21 H 20 O 11 The structural formula is: .

[0026] This invention is the first to evaluate the therapeutic effect of luteolin on SLE using MRL / Lpr mice in animal experiments. Combined with in vitro cell experiments, the experimental data revealed that luteolin inhibits Tfh differentiation, reduces autoantibody production, and alleviates the progression of SLE by activating the AHR signaling pathway and upregulating the expression of the downstream AHR target gene Cyp1a1. Its mechanism of action is as follows: Figure 1 As shown.

[0027] Example 1 Luteolin can alleviate lupus symptoms in the body. 1. Experimental methods and procedures: 1) Animal grouping and intervention: 7-8 week old female MRL / Lpr mice (SLE spontaneous model mice, purchased from Shanghai Slack Laboratory Animal Co., Ltd.) were randomly divided into three groups (6 mice in each group): (1) model mouse group, (2) model mouse + high concentration of luteolin treatment group, and (3) model mouse + low concentration of luteolin treatment group. Starting from 12 weeks of age, mice in the high and low concentration groups of luteolin were administered luteolin by gavage daily at doses of 50 mg / kg / day and 20 mg / kg / day, respectively, while the model group was given an equal volume of solvent.

[0028] 2) Disease activity assessment: Urinary protein levels in mice were measured weekly during the administration period. After administration, serum was collected to detect anti-dsDNA antibody titers, ANA titers, and inflammatory factors. Kidney tissue was taken for HE staining and PAS staining to observe renal pathological changes, and glomerulonephritis scoring and immune complex (IgG, C3) deposition were detected.

[0029] 2. Experimental Results: This study investigated the in vivo intervention effect of luteolin on lupus-like symptoms using an MRL / Lpr spontaneous SLE mouse model. Results showed that compared with the model control group, the high-concentration luteolin group significantly alleviated splenomegaly and significantly reduced the spleen index. Figure 2 In mice of concentrations A and B, the terminal urinary protein levels in both the high and low concentration luteolin groups were significantly lower than those in the model group. Figure 2 In the treatment of luteolin (C), renal function impairment was effectively improved; at the same time, luteolin intervention significantly reduced the levels of autoantibodies such as anti-dsDNA antibody and anti-ANA antibody in the serum of model mice, as well as the levels of pro-inflammatory cytokines such as IL-1β, TNF-α, IL-4, MCP-1, and G-CSF, thus regulating immune imbalance (C). Figure 2 (D); Renal histopathological examination showed that luteolin significantly reduced the pathological changes of lupus nephritis in the kidneys of model mice, such as inflammatory cell infiltration, glomerular structural disorder, and mesangial proliferation, and the renal pathological score was significantly reduced (D). Figure 2 Immunohistochemical results further confirmed that luteolin can significantly inhibit the deposition of IgG and C3 immune complexes in kidney tissue (E, F); Figure 2 (China GJ). The above results indicate that luteolin can effectively alleviate lupus-like symptoms in SLE model mice in vivo, exert significant renal protective and immunomodulatory effects, and provide in vivo experimental evidence for its use in the treatment of autoimmune diseases such as SLE.

[0030] Example 2 Luteolin can reduce the proportion of Tfh and plasma cells in the spleen of lupus mice. 1. Experimental methods and procedures: After drug administration, the spleens of MRL / Lpr mice were isolated, and single-cell suspensions were prepared by grinding. After red blood cell lysis and washing, the cell concentration was adjusted. Flow cytometry was used to label and stain the cells with CD4, CXCR5, and PD-1 antibodies to detect the proportion of Tfh cells in the spleen. The proportion of plasma cells in the spleen was detected by labeling and staining the cells with CD19 and CD138 antibodies. The cell proportions were then detected and analyzed by flow cytometry.

[0031] 2. Experimental Results: This embodiment further explores how luteolin affects Tfh and plasma cell differentiation. Using an MRL / Lpr spontaneous SLE mouse model, the regulatory effect of luteolin on immune cell subsets in the spleen of SLE mice was investigated. Results showed that compared with the model control group, the proportion of Tfh cells in the spleen of mice in both high and low concentration luteolin groups was significantly lower than that in the model group, with the high concentration group showing a more pronounced inhibitory effect. Figure 3(A, B); Meanwhile, the proportion of plasma cells in the spleen of mice in the high-concentration luteolin group was significantly lower than that in the model group, and the proportion of plasma cells in the low-concentration group also showed a decreasing trend. Figure 3 (C, D). The above results indicate that luteolin can effectively inhibit the abnormal activation and proliferation of Tfh cells in the spleen of SLE model mice, reduce the production of downstream plasma cells, and thus inhibit the production of autoantibodies.

[0032] Example 3 Luteolin can directly inhibit the differentiation of Tfh cells in vitro. 1. Experimental methods and procedures: Using Mouse CD4 + The T Cell Isolation Kit obtains initial CD4 from the spleen of MRL / Lpr mice through negative selection. + T cells were divided into a control group (cultured in vitro with only 10 μg / mL anti-IL-4 antibody and 10 μg / mL anti-IFN-γ antibody), a model group (Tfh-induced differentiation group, with 10 μg / mL anti-IL-4 antibody, 10 μg / mL anti-IFN-γ antibody, 100 ng / mL IL-6, 100 ng / mL IL-21 and 10 ng / mL TGF-β1), and a luteolin intervention group (co-cultured in the Tfh cell polarization induction system with luteolin at a final concentration of 3.125 μg / mL).

[0033] Both the model group and the luteolin-treated group were cultured in vitro using a Tfh cell induction system (containing IL-6, IL-21, TGF-β1, and anti-IFN-γ and anti-IL-4 antibodies). The control group received anti-IFN-γ and anti-IL-4 antibodies, while the luteolin-treated group received luteolin-treated intervention during induction culture. The culture period was 5 days. After culture, CD4 counts in each group were measured by flow cytometry. + T cells Tfh cells (CXCR5) + PD-1 + )Proportion.

[0034] 2. Experimental Results: To further investigate the direct effects of luteolin on Tfh cell differentiation, this example uses CD4... + A Tfh differentiation model induced by naïve T cells in vitro was established to investigate the direct regulatory effect of luteolin on Tfh cell differentiation. Results showed that, compared with the control group, the Tfh-induced model group had significantly higher CD4 counts. + The proportion of Tfh cells in T cells was significantly increased, and an in vitro Tfh differentiation model was successfully constructed. Figure 4 (A, B) Compared with the model group, the proportion of Tfh cells in the luteolin intervention group was significantly reduced, indicating that luteolin can directly inhibit naïve CD4+.+ T cell differentiation into Tfh cells ( Figure 4 (C, D)

[0035] Example 4 Mechanism of action of luteolin in inhibiting Tfh cell differentiation 1. Experimental methods and procedures: 1) Transcriptome sequencing and enrichment analysis: Tfh cells induced to differentiate in vitro from the luteolin-treated group and the model group in Example 3 were used to extract total RNA for transcriptome sequencing, and differentially expressed genes (DEGs) were analyzed. Key signaling pathways regulated by luteolin were screened by KEGG pathway enrichment analysis.

[0036] 2) Molecular docking and molecular dynamics simulation: Molecular docking technology was used to analyze the binding ability and binding sites of luteolin and AHR protein, and the binding energy (BE) was calculated; the stability of the luteolin-AHR complex was verified by molecular dynamics simulation (RMSD, RMSF, Rg, hydrogen bond and other parameters).

[0037] 3) Dual-luciferase reporter gene experiment: Construct a dual-luciferase reporter gene vector containing wild-type (WT) and mutant (MUT) AhRE sequences to verify the activation effect of luteolin on AHR transcriptional activity.

[0038] 2. Experimental Results: This embodiment systematically elucidates the molecular mechanism by which luteolin inhibits Tfh cell differentiation through transcriptome sequencing, molecular docking, molecular dynamics simulation, and dual-luciferase reporter gene assays. Transcriptome sequencing results showed that a large number of differentially expressed genes existed in Tfh cells after luteolin intervention. KEGG pathway enrichment analysis identified the AHR signaling pathway as the core pathway regulated by luteolin, with significant enrichment of AHR-related pathways (…). Figure 5 qPCR and Western blot results confirmed that luteolin significantly upregulated the expression of downstream target genes Cyp1a1 and Cyp1b1 of AHR, effectively activating the AHR-Cyp1a1 axis both in vivo and in vitro. Figure 5 (FH). Molecular docking results showed that luteolin can stably bind to AHR protein with a binding energy of -6.1097 kcal / mol, exhibiting good binding activity. Figure 6 (A); Molecular dynamics simulations further verified the structural stability of the luteolin-AHR complex. Figure 6 (BG). Dual-luciferase reporter gene assays confirmed that luteolin significantly enhanced the luciferase activity of wild-type AhRE, but had no significant effect on mutant AhRE, clarifying that luteolin can directly activate the transcriptional activity of AHR (AH). Figure 6(H). The above results indicate that luteolin can directly bind to and activate AHR, upregulate downstream target molecules of Cyp1a1, and thus inhibit the abnormal differentiation of Tfh cells, providing a clear molecular mechanism basis for its treatment of SLE.

[0039] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. The use of luteolin or its pharmaceutically acceptable salts, esters, or solvates in the preparation of medicaments for the prevention and / or treatment of systemic lupus erythematosus.

2. The application according to claim 1, characterized in that, The luteolin glycoside inhibits the differentiation of follicular helper T cells by activating the aryl hydrocarbon receptor signaling pathway and upregulating the expression of its downstream target gene cytochrome P450 1A1.

3. The application according to claim 1, characterized in that, The luteolin glycoside reduces the production of autoantibodies by inhibiting the differentiation of follicular helper T cells and reducing the generation of downstream plasma cells.

4. The application according to claim 1, characterized in that, The systemic lupus erythematosus includes lupus nephritis.

5. A medicament for the prevention and / or treatment of systemic lupus erythematosus, characterized in that, It contains a therapeutically effective amount of luteolin or its pharmaceutically acceptable salt, ester, solvate, and a pharmaceutically acceptable carrier.

6. The use of luteolin or its pharmaceutically acceptable salts, esters or solvates in the preparation of reagents for reducing the production of autoantibodies.

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