CN116942690B - Application of regulatory factor RFX1 inhibitor in preparation of medicines for preventing and/or treating autoimmune diseases - Google Patents
Application of regulatory factor RFX1 inhibitor in preparation of medicines for preventing and/or treating autoimmune diseases Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
- A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
- A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
- A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
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Abstract
The invention relates to the technical field of medicines, in particular to application of a regulatory factor RFX1 inhibitor in preparation of a medicine for preventing and/or treating autoimmune diseases. The regulatory factor RFX1 inhibitor is adenosine diphosphate, which can inhibit the function of RFX1 in macrophage polarization and inhibit the incidence of mouse colonitis. The research result of the invention reveals that the potential inhibitor ADP of RFX1 provides a potential target for autoimmune inflammation, and as a targeted drug delivery system is developed and matured, targeting different types of immune cells to regulate the expression and activity of RFX1 is an important research direction for treating autoimmune diseases.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to application of a regulatory factor RFX1 inhibitor in preparation of a medicine for preventing and/or treating autoimmune diseases.
Background
Various autoimmune diseases are characterized by unresolved inflammation, systemic Lupus Erythematosus (SLE) is a systemic autoimmune disease, multiple organ inflammation, mainly involving the skin, kidneys, joints and nervous system. Inflammatory Bowel Disease (IBD), including crohn's disease and ulcerative colitis, is an organ-specific autoimmune disease characterized by an unresolved inflammation of the gastrointestinal tract. Macrophages play a vital role in the overall inflammatory process, including initiation, inflammation, regression, and tissue repair. It is distributed in almost all tissues and is involved in the development and progression of autoimmune diseases through phagocytosis, antigen presentation and cytokine secretion. Macrophages exhibit a high degree of plasticity and heterogeneity under environmental signals, and generally tend to differentiate into two major phenotypes, classical activated macrophages (M1) and alternate activated macrophages (M2), known as macrophage polarization. Circulating microparticle-promoted pro-inflammatory M1 macrophages in patients with autoimmune diseases such as SLE can promote T cell activation and induce B cell activation and survival. M1 macrophages can enter the damaged kidneys, promote apoptosis and death of tubular cells, and exacerbate kidney injury. Adoptive grafting of M1 macrophages exacerbates the chloride phosphonate induced increase in SLE severity, while M2 macrophages alleviate this increase. In addition, the M1/M2 ratio of the inflammatory gut lamina propria is also increased in IBD patients, which promotes local macrophage activation, favors gut inflammation and destroys the integrity of the epithelial barrier. Metastasis of M2 macrophages ameliorates chronic inflammation in animal models of IBD. Adoptive grafting of M1 macrophages exacerbates the chloride phosphonate induced increase in SLE severity, while M2 macrophages alleviate this increase. In addition, the M1/M2 ratio of the inflammatory gut lamina propria is also increased in IBD patients, which promotes local macrophage activation, favors gut inflammation and destroys the integrity of the epithelial barrier. Metastasis of M2 macrophages ameliorates chronic inflammation in animal models of IBD. Regulating abnormal activation of macrophage may be used as important target for autoimmune disease treatment.
Various studies have found that some transcription factors are involved in regulating macrophage polarization. For example, kruppel-like factor 5 (KLF 5) directly activates transcription of a variety of pro-inflammatory genes, promotes M1 macrophage polarization, and plays an important role in the pathogenesis of LPS-induced endotoxin shock mice. Inhibiting SOCS3 expression induces polarization of M2 macrophages to inhibit inflammation and promote functional recovery in cerebral hemorrhagic rats. However, the mechanism of aberrant pro-inflammatory macrophage activation in autoimmune diseases is not yet understood. The regulatory factor X (Regulation factor X, RFX) family is an important class of transcription factors, RFX1 being a member of this family, which can bind to the cis-acting element X box, which contains a C-terminal inhibitory domain and an N-terminal activating domain, to obtain dual ability to inhibit and activate transcription of a target gene. Previous studies by the inventors found that down-regulation of RFX1 in peripheral blood cd4+ T cells of SLE patients promoted T cell autoreactive activation and increased Th17 cell differentiation, which was associated with pathogenesis of SLE, and the role of RFX1 and its activity regulatory molecules in macrophages and autoimmune diseases has not been reported.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide an application of a regulatory factor RFX1 inhibitor in preparing medicines for preventing and/or treating autoimmune diseases.
In order to achieve the aim of the invention, the invention adopts the following technical scheme: use of a modulator RFX1 inhibitor, said modulator RFX1 inhibitor being Adenosine Diphosphate (ADP), for the manufacture of a medicament for the prevention and/or treatment of autoimmune diseases.
In some embodiments of the invention, the modulator RFX1 inhibitors of the invention are used to inhibit the function of RFX1 in macrophage polarization.
In some embodiments of the invention, the modulator RFX1 inhibitors of the invention are used to reduce expression of the target gene apodec 3A in classical activated macrophages.
In some embodiments of the invention, the modulator RFX1 inhibitors of the invention are used to reduce LPS-stimulated apodec 3A protein expression in classical activated macrophages.
In some embodiments of the invention, the modulator RFX1 inhibitors of the invention are used to reduce the mRNA and protein expression levels of IL-6, TNF- α in classically activated macrophages.
In some embodiments of the invention, the modulator RFX1 inhibitors of the invention down-regulate CD86 expression of the classical activated macrophage-related molecule and increase CD163 and CD200R expression of the alternative activated macrophage-related molecule in stimulating the classical activated macrophage or alternative activated macrophage.
In some embodiments of the invention, the modulator RFX1 inhibitors of the invention are used to reduce the mRNA expression of the target gene Apobec3 downstream of RFX 1.
In some embodiments of the invention, the modulator RFX1 inhibitors of the invention are used to reduce protein levels of RFX1 and Apobec 3.
The invention also provides a pharmaceutical composition comprising the regulatory factor RFX1 inhibitor.
In some embodiments of the invention, the modulator RFX1 inhibitors of the invention are used to reduce urinary protein or urinary creatinine levels in animals, as well as to reduce the concentration of autoantibodies anti-dsDNA in the serum of animals.
In some embodiments of the invention, the modulator RFX1 inhibitor of the invention reduces the extent of damage to the kidney of an animal and reduces the total immune cell, CD4+ and CD8+ T cell infiltration of CD45+ in the kidney tissue of the animal in the event of damage to the kidney of the animal.
The invention also provides application of the pharmaceutical composition in preparing medicines for preventing and/or treating autoimmune diseases.
The invention has the beneficial effects that: the invention provides an application of a regulatory factor RFX1 inhibitor in preparing a medicament for preventing and/or treating autoimmune diseases, wherein the regulatory factor RFX1 inhibitor is adenosine diphosphate, and can inhibit the function of RFX1 in macrophage polarization and inhibit the incidence of colonitis of mice. The research result of the invention reveals that the potential inhibitor ADP of RFX1 provides a potential target for autoimmune inflammation, and as a targeted drug delivery system is developed and matured, targeting different types of immune cells to regulate the expression and activity of RFX1 is an important research direction for treating autoimmune diseases.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention. In the drawings:
FIG. 1 is a 3D plot of molecular docking results of RFX1 with bound ADP and the pattern of RFX1 and ADP interactions; wherein, the left graph in fig. 1 is a molecular docking result graph of RFX1 and bound ADP; the right panel in fig. 1 is a 3D diagram of RFX1 and ADP interaction pattern.
FIG. 2 shows relative mRNA expression (1 mM) of IL6, IL1B and TNF in the control and ADP treated group M1 hMDM (n=3); wherein the blue bar graph represents control group (LPS), the purple bar graph represents treatment group (LPS+ADP).
FIG. 3 shows the concentrations of IL-6 and TNF-a (1 mM) (n=3) in ELISA assay control and ADP treated group M1 hMDMs culture supernatants; wherein, blue bar graph represents control group (LPS), purple bar graph represents treatment group (LPS+ADP); the left panel in FIG. 3 shows the concentration of IL-6 in ADP-treated group M1 hMDMs culture supernatants; the right panel in FIG. 3 shows the concentrations of IL-6 and TNF-a in the culture supernatants of ADP-treated group M1 hMDMs.
Fig. 4 is a representative flow cytometry peak plot and mean fluorescence intensity MFI statistical histogram (n=4) for CD86, CD64, CD163, and CD200R in control and ADP treated group M1 hMDM; wherein, blue peak and blue bar graphs represent control group (LPS), purple peak and purple bar graphs represent treatment group (lps+adp); panel A in FIG. 4 is a representative flow cytometry peak plot and mean fluorescence intensity MFI statistical histogram of CD86 in ADP treatment group M1 hMDM; panel B in FIG. 4 is a representative flow cytometry peak plot and mean fluorescence intensity MFI statistical histogram of CD64 in ADP treatment group M1 hMDM; panel C of FIG. 4 is a graph of the peak of CD163 representative flow cytometry and the mean fluorescence intensity MFI statistical histogram of ADP-treated group M1 hMDM; panel D in FIG. 4 is a representative flow cytometry peak plot and mean fluorescence intensity MFI statistical histogram of CD200R in ADP treatment group M1 hMDM.
FIG. 5 shows the protein expression of Western Blot for detection of hMDMs of different treatments.
FIG. 6 is a graph showing the results of protein concentration detection of IL-6 and TNF-a in hMDM culture supernatants after LPS and ADP treatment and infection with pLV-NC and pLV-RFX1 (n=3); wherein, blue bar graph represents control group (nc+lps+adp), red bar graph represents treatment group (rfx1+lps+adp); the left panel in FIG. 6 is a graph showing the results of detection of the protein concentration of IL-6 in hMDM culture supernatants after LPS and ADP treatment and infection with pLV-NC and pLV-RFX 1; the right panel in FIG. 6 shows the results of protein concentration detection of TNF-a in hMDM culture supernatants after LPS and ADP treatment and infection with pLV-NC and pLV-RFX 1.
Fig. 7 is the concentration of ADP (n=4) in PMA cells treated with LPS or ADP, respectively, wherein the blue bar graph represents Control (Control), the red bar graph represents lipopolysaccharide treated group (LPS), and the purple bar graph represents ADP treated group (ADP).
Fig. 8 is the daily body weight change level (n=5 per group) of control group and ADP-dosed colitis mice.
Fig. 9 is a representative image of the colon and colon length statistics (n=5 per group) of control group and colitis mice given ADP dosing; wherein, blue bar graph represents negative control group (NC), purple bar graph represents ADP treatment group (ADP); the left plot in fig. 9 is a representative image of each set of colon; the right panel in fig. 9 is a statistical plot of colon length.
FIG. 10 is H of colon tissue of control and ADP treated mice&E staining representative pictures and histological scoring statistics (n=4 per group), scale bar, 500mm; data are mean ± SEM, using two-tailed Student t-test,*p<0.05，**p<0.01，***p<0.001; wherein, blue bar graph represents negative control group (NC), purple bar graph represents ADP treatment group (ADP); the left panel in FIG. 10 shows H of colon tissue of control and ADP treated mice&E staining the representative picture; the right panel in fig. 10 is a statistical plot of histological scores of colon tissue from control and ADP-treated mice.
Fig. 11 shows apodec 3A relative mRNA expression levels (n=4 per group) in M1 hMDM treated with different concentrations of ADP; wherein blue bar graph represents lipopolysaccharide treated group (LPS), orange bar graph represents LPS and 10mM ADP treated results, red bar graph represents LPS and 100mM ADP treated results, and purple bar graph represents LPS and 1mM ADP treated results.
Fig. 12 shows RFX1 relative mRNA expression levels (n=4 per group) in M1 hMDM treated with ADP at different concentrations; wherein blue bar graph represents lipopolysaccharide treated group (LPS), orange bar graph represents LPS and 10mM ADP treated results, red bar graph represents LPS and 100mM ADP treated results, and purple bar graph represents LPS and 1mM ADP treated results.
FIG. 13 shows Western Blot for detecting protein expression levels of APOBEC3A in hMDMs treated differently.
Fig. 14 shows the relative mRNA expression levels of Apobec3 in different concentrations of ADP-treated PMA (n=4 per group); wherein blue bar graph represents lipopolysaccharide treated group (LPS), orange bar graph represents LPS and 10mM ADP treated results, red bar graph represents LPS and 100mM ADP treated results, and purple bar graph represents LPS and 1mM ADP treated results.
Fig. 15 shows relative mRNA expression levels of Rfx1 in different concentrations of ADP-treated PMA (n=4 per group); wherein blue bar graph represents lipopolysaccharide treated group (LPS), orange bar graph represents LPS and 10mM ADP treated results, red bar graph represents LPS and 100mM ADP treated results, and purple bar graph represents LPS and 1mM ADP treated results.
FIG. 16 shows Western Blot for detection of protein expression levels of RFX1 and Apobec3 in different treated PMAs, data expressed as mean.+ -. SEM, using one-way analysis of variance (Dunnett multiple comparison test), p<0.001。
figures 17 and 18 are mouse urinary protein (mg/mL) and urinary protein/urinary creatinine U/C (mg/mL/. Mu.mol/L) levels (n=8 per group) after control and ADP treatment for eight weeks; wherein the blue bar graph represents the negative control group (NC) and the red bar graph represents the ADP-treated group (ADP).
FIG. 19 is an ELISA assay to detect anti-dsDNA IgG concentration in serum of mice at 18 weeks of treatment; wherein the blue bar graph represents the negative control group (NC) and the red bar graph represents the ADP-treated group (ADP).
FIG. 20 is a graph showing the results of H & E and PAS staining for detection of pathological changes in kidney sections of mice (scale bar 50 mm); wherein, the left graph in fig. 20 is a graph of the results of H & E staining for detecting pathological changes in kidney sections of mice; the right panel in fig. 20 is a graph of the results of PAS staining for detecting pathological changes in kidney sections of mice.
FIG. 21 is a flow cytometry measurement of the number of immune cells in the left kidney of mice, expressed as mean.+ -. SEM, using a two-tailed Student t-test, p<0.05，/> p<0.01; wherein, the blue bar graph represents the negative control group (NC), and the red bar graph represents the ADP treatment group (ADP); a, B, C and D in fig. 21 are flow-stained individual immunocyte plots in the control group, respectively; e, F, G and H in fig. 21 are flow-stained individual immunocyte plots in the ADP-dosed group, respectively; FIG. 21 is a chart showing statistics of the number of immune cells in the left kidney of a mouse detected by flow cytometry.
Detailed Description
The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1
This example demonstrates the use of Adenosine Diphosphate (ADP) as an inhibitor of the regulatory factor RFX1 in the preparation of a medicament for the prevention and/or treatment of systemic lupus erythematosus.
1 Experimental method
1.1 Locking interaction technology
(1) Preparation of the Structure
(1) Preparation process of protein structure: the crystalline structure of RFX1 (PDB number: 1DP 7) was first downloaded in the protein crystal structure database (http:// www.rcsb.org/PDB/home. Do). Protein structure preparation module Protein Preparation Wizard in the Schrodinger software package is used for preparing protein structure, and specifically comprises the steps of adjusting the protonation state of amino acid residues according to the set pH condition, complementing hydrogen atoms and possibly missing protein structure, and adopting OPLS-3e to optimize the structure of the protein. And saving the structure after the structure preparation processing as a pdb file for subsequent molecular simulation research.
(2) The collection and structure preparation of inhibitor molecules, namely, preparing the structures of a product library to be screened through a LigPrep module in a Schrodinger software package, generating a 3D conformation, setting the 3D conformation in a protonated state of pH7, and simultaneously optimizing the conformation under an OPLS-3e force field.
(2) Molecular docking
Molecular docking operations were performed using the Glide module in the Schrodinger software package. The prepared protein structure is imported into software, and the prepared inhibitor or the molecular structure in the database is subjected to molecular docking with the protein structure by utilizing the Receptor Grid Generation function in the Glide module, so that the action mode of the existing inhibitor is examined, and the virtual screening research of the existing drug molecules based on molecular docking is realized.
(3) Analysis of receptor-ligand modes of action
The effect of the target protein on the compound molecules was analyzed by interaction visualizer in the Glide module in the Schrodinger software package. In receptor-ligand mutual analysis, the butt joint scores between proteins and small molecules and the corresponding binding modes are mainly analyzed. In the evaluation of the Docking score, a Docking score was mainly considered. In receptor-ligand binding mode assays, the non-binding interactions between proteins and different ligand molecules are mainly analyzed and mainly include: hydrophobic action, hydrogen bonding action, salt bridging action and electrostatic action.
(4) Virtual screening process
The molecular docking process is divided into four rounds for screening:
(1) high throughput coarse screening HTVS coarse screening, reserving 1% of hit structures in the database;
(2) SP standard precision screening, namely reserving 10% hit structure; glide HTVS and SP use a series of hierarchical filters to search for possible positions of ligands in the receptor binding site region. The shape and nature of the receptors are represented on the grid by different field sets, which provide progressively more accurate scoring of ligand postures. An exhaustive count of ligand twist yields a collection of ligand conformations that are examined during docking. In view of these ligand conformations, initial screening is performed deterministically across the entire phase space available for ligands to find promising ligand poses. By initially screening the selected pose, either the ligand (Glide SP & XP) or OPLS2005 (GLIDE HTVS) was refined in the torsion space of the receptor region with a distance dependent dielectric model using OPLS3 4. Finally, a small number of postures are minimized in the region of the receptor, with full ligand flexibility (post-docking minimization or PDM).
(3) XP ultra-high precision screening, 10% hit structure is reserved;
(4) the mode of action and binding energy are analyzed according to the final hit structure.
1.2 Cell culture
Mice were induced by intraperitoneal injection of 4% thioglycolate medium (BD, catalog # 7009838) into mice for four days. After intraperitoneal infusion, macrophages were completely cultured with DMEM containing 10% fbs (Gibco). Isolation of human CD14 from Peripheral Blood Mononuclear Cells (PBMC) by density gradient centrifugation + Monocytes were isolated by CD14 microbeads (Miltenyi Biotec, catalog # 130-05-2021). Induction of CD14 with M-CSF (Absin, catalog #abs 04696) (50 ng/mL) + Monocytes differentiated to hMDM for 8 days. Macrophages were stimulated with Lipopolysaccharide (LPS) (1 mg/mL) (Sigma, catalog No. 82857-67-8) for 24 hours to promote macrophage M1 type polarization
1.3 histological analysis and scoring
The colon and kidney tissues were paraffin embedded and sectioned 5mm thick. Sections were stained with H & E. Histological scoring of colon tissue is assessed by tissue injury and congenital inflammatory cell infiltration. The scoring criteria are as follows: 0 = no tissue injury or inflammatory infiltration; 1 = increased focal epithelial lesions (< 5%) or crypt infiltration of the colon; 2 = submucosa epithelial injury (5% -20%) or inflammatory cell cluster infiltration; 3 = extensive epithelial lesions of the colon (20% -50%) or massive infiltration of mucosal myolayer; 4 = extensive submucosa infiltration or extensive epithelial damage (> 50%) of the colon.
1.4 ADP detection and in vivo administration
The concentration of ADP (Selleckchem, # S9368) in macrophages was measured by ADP assay kit (Sigma, catalog #MAK133) according to the instructions. The procedure is summarized as follows. 10≡4 PMAs were directly cultured in assay 96-well microplates and stimulated with LPS or ADP for 24 hours. Working reagents are used to lyse cells to release ATP and ADP. In the presence of luciferin, ATP reacts immediately with the substrate (D-luciferin) to produce luminescence. ADP is converted to ATP by an enzyme reaction, and then in the former step this newly formed ATP is reacted with D-luciferin. Luminescence (relative light units) is immediately read by a photometer. Standard curves were plotted using values obtained from the appropriate standards and the ADP content of the samples was determined.
The colitis mice in the negative control group (NC) and ADP group were each constructed with 3% dss. DSS (MP Biomedicals, cat No. MFCD 00081551) was added to mice' daily drinking water at a final concentration of 3%. The remaining water was checked daily and replenished in time. The change in body weight of the mice was recorded daily. Daily mouse body weights were normalized to day 0. ADP groups were intraperitoneally injected with ADP (250 mg/kg), and control mice were intraperitoneally injected with an equal amount of physiological saline daily for 7 days.
Female LPR mice in the ADP group were given free drinking water (final concentration 750 mg/L) with ADP added (MCE, catalog number HY-W010918) from week 9. Negative Control (NC) LPR female mice of the same week age were treated with normal drinking water for 18 weeks after 9 weeks.
1.5 SLE mice each index detection and kidney digestion treatment
Kidneys were isolated from sacrificed mice and minced kidneys were digested with collagenase IV (Gibco, cat. No. 17104019). After enrichment and lysis, the cells were incubated with the antibodies in the dark for 30min on ice. Stained cells were analyzed on a Northern Lights flow cytometer (cytok) and data was analyzed using FlowJo software.
The detection in the mouse serum is carried out by adopting the ELISA Kit of the anti-double-stranded DNA antibody/natural DNA antibody (IgG) of the mouse of the Wuhan Huamei bioengineering limited company. The detection of urine protein and creatinine in mouse urine adopts a detection kit of Nanjing to build a bioengineering research institute.
2. Experimental results
RFX1 was identified as a DNA binding protein targeting a specific X box sequence in MHC class II genes. It has an evolutionarily conserved DNA Binding Domain (DBD), which resides in the core of the RFX1 protein and binds to the X box motif present in the targeting DNA. Here, molecular docking techniques are used to explore potential small molecule inhibitors of the RFX1 protein DNA binding region. As shown in the 3D diagram of ADP interfacing with RFX1 in fig. 1, it was found that ADP may bind to the DBD of RFX1 and may affect the function of RFX 1. To verify this prediction, the present invention treats human CD14 type M1 with different concentrations of ADP + Macrophages (hMDMs) of origin, the expression of the target gene APOBEC3A instead of RFX1 was found to be significantly reduced in hmdmms treated with ADP (1 mM) compared to the negative control (fig. 11 and 12). Furthermore, ADP treatment also reduced LPS-stimulated APOBEC3A protein expression in hMDMs (fig. 13). At the same time, the mRNA and protein expression levels of IL-6 and TNF-a in M1 hMDM were also significantly reduced after ADP treatment (FIGS. 2 and 3). Furthermore, the present invention found that in ADP stimulated M1 hMDMs, the expression of the M1-related molecule CD86, but not CD64, was down-regulated, whereas the expression of the M2-related molecules CD163 and CD200R was significantly increased (fig. 4). Overexpression of RFX1 can further counteract ADP-treatment induced inhibition of apodec 3A expression and increase of CD206 expression (fig. 5). Furthermore, ADP treatment counteracted elevated IL-6 and TNF expression in hMDM caused by high RFX1 expression (FIG. 6). The invention further determines the effect of ADP treatment on PMAs, and found that at a concentration of 1mM,ADP reduced mRNA expression of the target gene Apobec3 downstream of RFX1, but did not reduce mRNA expression of Rfx1 (FIGS. 14 and 15). Protein levels of RFX1 and Apobec3 were reduced after ADP treatment as determined by western blot (fig. 16).
In addition, the concentration of ADP in PMA was also examined. It was found that the level of ADP in LPS-induced mouse Peritoneal Macrophages (PMA) was significantly reduced compared to the control group and that ADP incubation increased intracellular ADP levels (fig. 7). The present invention also investigated the effect of ADP administration on DSS-induced inflammatory responses in mice and found that ADP administration reduced weight loss and colonic tissue shortening symptoms in mice with colitis (fig. 8 and 9). ADP-administered colitis mice had reduced colon tissue damage and more complete tissue structure (fig. 10). Finally, the present invention investigated the effect of ADP on the onset of LPR in mice with spontaneous lupus, and found that ADP treatment significantly reduced urine protein, urine protein/urine creatinine levels in mice (fig. 17 and 18). Furthermore, ADP treatment reduced the autoantibody anti-dsDNA concentration in the serum of LPR mice (fig. 19). Renal paraffin section HE and PAS staining showed that the ADP-administered group SLE mice had reduced glomerular and tubular inflammatory immune cell infiltration and reduced renal injury (fig. 20). Cd4+ and cd8+ T cell infiltration was reduced in total immune cells, cd4+ and cd45+ in kidney tissues (fig. 21). ADP administration may therefore be a potential treatment for autoimmune inflammation.
Macrophage phenotype depends on mitochondrial function and ATP/ADP homeostasis. The studies found that M1 macrophages have the lowest ATP content, while M2 macrophages have the highest ATP levels. Furthermore, the metabolic profile detected in macrophages by 1d 1h NMR-based metabolomics suggests a significant increase in oxidative stress, a decrease in mitochondrial respiration, an increase in intracellular ATP in M1 macrophages, and an increase in intercellular ADP and Adenylate (AMP) in M2 macrophages. ADP plays an important role in vascular and cellular responses and is released immediately during inflammation and injury to promote platelet aggregation. The M2 polarization of alveolar macrophages is correlated with the severity of human asthma, and impaired M2 polarization exhibits less eosinophil recruitment and pulmonary inflammation. Bronchoalveolar lavage fluid from asthmatic patients is rich in extracellular ADP, which exacerbates airway inflammation and induces mast cell infiltration in ovalbumin-induced asthma models. However, the role of ADP in autoimmune inflammatory responses is currently unknown, and the present invention finds that ADP can act as a potential inhibitor of RFX1, attenuating LPS-induced macrophage inflammatory responses. Furthermore, continuous ADP administration to DSS-induced colitis mice can inhibit the incidence of colitis mice and reduce intestinal damage.
As a result of the present invention, ADP was found to inhibit the function of RFX 1. Thus, to assess the effect of systemic inhibition of RFX1 on autoimmunity, ADP administration in a SLE mouse model will be used to assess the final effect of RFX1 inhibition on SLE development. In addition, macrophage targeted drug delivery systems, such as macrophage derived extracellular vesicles, have been used to treat autoimmune diseases. The present invention recognizes that targeting different types of immune cells to regulate RFX1 expression and activity is an important research direction for the treatment of autoimmune diseases as targeted drug delivery systems develop and mature.
The present invention allows the use of the modulator RFX1 inhibitor Adenosine Diphosphate (ADP) for the preparation of a pharmaceutical composition for the prevention and/or treatment of autoimmune diseases.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (9)
1. The application of the regulator RFX1 inhibitor in preparing medicines for treating systemic lupus erythematosus and ulcerative colitis is characterized in that the regulator RFX1 inhibitor is adenosine diphosphate.
2. Use of a modulator RFX1 inhibitor according to claim 1 for the preparation of a medicament for the treatment of systemic lupus erythematosus and ulcerative colitis, wherein the modulator RFX1 inhibitor is for inhibiting the function of RFX1 in macrophage polarization.
3. Use of a modulator RFX1 inhibitor according to claim 1 for the preparation of a medicament for the treatment of systemic lupus erythematosus and ulcerative colitis, wherein said modulator RFX1 inhibitor is used to reduce the expression of the target gene apodec 3A in classical activated macrophages;
the regulatory factor RFX1 inhibitor is used for reducing the expression of APOBEC3A protein stimulated by LPS in classical activated macrophages;
the modulator RFX1 inhibitor is used for reducing the mRNA and protein expression level of IL-6 and TNF-alpha in classical activated macrophages.
4. Use of a modulator RFX1 inhibitor according to claim 1 for the preparation of a medicament for the treatment of systemic lupus erythematosus and ulcerative colitis, wherein the modulator RFX1 inhibitor is such that in stimulating classical activated macrophages or alternatively activated macrophages, the expression of CD86, a molecule associated with classical activated macrophages, is down-regulated and the expression of CD163 and CD200R, a molecule associated with alternatively activated macrophages, is increased.
5. Use of a modulator RFX1 inhibitor according to claim 1 for the preparation of a medicament for the treatment of systemic lupus erythematosus and ulcerative colitis, wherein said modulator RFX1 inhibitor is used for reducing mRNA expression of the target gene Apobec3 downstream of RFX 1.
6. Use of a modulator RFX1 inhibitor according to claim 1 for the preparation of a medicament for the treatment of systemic lupus erythematosus and ulcerative colitis, wherein the modulator RFX1 inhibitor is used to reduce the protein levels of RFX1 and Apobec 3.
7. Use of a modulator RFX1 inhibitor according to claim 1 for the preparation of a medicament for the treatment of systemic lupus erythematosus and ulcerative colitis, wherein the modulator RFX1 inhibitor is used for reducing urinary protein or urinary creatinine levels in animals and for reducing the concentration of autoantibodies anti-dsDNA in serum of animals.
8. The use of a modulator RFX1 inhibitor according to claim 1 for the preparation of a medicament for the treatment of systemic lupus erythematosus and ulcerative colitis, wherein the modulator RFX1 inhibitor reduces the extent of damage to the kidney of an animal and reduces cd4+ and cd8+ T cell infiltration in the kidney tissue of an animal in the event of damage to the kidney of an animal.
9. Use of a pharmaceutical composition comprising the modulator RFX1 inhibitor of claim 1 in the manufacture of a medicament for the treatment of systemic lupus erythematosus and ulcerative colitis.
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