Use of CXCR4 antagonists in the preparation of drugs and / or pharmaceutical compositions for the prevention and / or treatment of intestinal fibrosis

Abstract

This invention relates to the field of biomedical technology, and in particular to the use of CXCR4 antagonists in the preparation of drugs and / or pharmaceutical compositions for the prevention and / or treatment of intestinal fibrosis. This invention involves constructing CD19-Cre;CXCR4... flox / flox Conditional gene knockout mice have demonstrated for the first time in vivo that specific knockout of CXCR4 in B cell lineages significantly improves DSS-induced chronic intestinal fibrosis, confirming that CXCR4 is a key functional target driving intestinal fibrosis, rather than merely a related biomarker. In a DSS-induced mouse model of chronic intestinal fibrosis, treatment with the marketed CXCR4 antagonist praxavir significantly reduced intestinal collagen deposition and lowered fibrosis scores, demonstrating excellent anti-fibrotic activity. This research addresses the current lack of effective anti-fibrotic drugs and provides a novel and promising treatment strategy for patients with intestinal fibrosis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the use of CXCR4 antagonists in the preparation of drugs and / or pharmaceutical compositions for the prevention and / or treatment of intestinal fibrosis. Background Technology

[0002] Intestinal fibrosis is a common and serious complication of inflammatory bowel disease (IBD), which can occur in Crohn's disease (CD) and ulcerative colitis (UC). Under the continuous stimulation of long-term chronic inflammation, the extracellular matrix (ECM) in the intestinal wall undergoes a metabolic imbalance, with synthesis far exceeding degradation, leading to excessive deposition of collagen, fibronectin, and other components in various layers of the intestinal wall. This pathological remodeling gradually causes the intestine to lose its normal flexibility and peristaltic function, with the intestinal wall continuously thickening and hardening, eventually leading to intestinal stenosis or even complete obstruction. Clinically, patients often experience recurrent abdominal pain, bloating, nausea, vomiting, and difficulty defecating; in severe cases, endoscopic balloon dilation, stricture incision, or even surgical resection of a segment of the intestine may be necessary. However, even after surgery relieves the mechanical obstruction, postoperative fibrosis is highly prone to recurrence, trapping patients in a vicious cycle of "inflammation-stenosis-surgery-restenosis," severely impairing their quality of life and causing a continuous deterioration in their nutritional status.

[0003] Currently, the standard treatment strategy for IBD primarily focuses on controlling acute inflammation to achieve clinical remission and mucosal healing. Widely used clinical medications include glucocorticoids, immunosuppressants, and biologics, such as anti-tumor necrosis factor-α (TNF-α) antibodies. These treatments effectively inhibit immune cell infiltration and the release of pro-inflammatory factors, reducing mucosal edema, erosion, and ulceration, achieving significant results in inducing and maintaining remission. However, they are largely ineffective against established intestinal fibrosis, failing to degrade excessively deposited ECM or thin the thickened intestinal wall. Existing research suggests that although early, potent anti-inflammatory therapy may delay the progression of fibrosis, once key pro-fibrotic pathways are chronically activated and stable fibrous scar tissue forms, simply controlling inflammation is insufficient to reverse this process. Therefore, no drug currently approved for the prevention or reversal of intestinal fibrosis constitutes a significant and unmet clinical gap.

[0004] To combat fibrosis, researchers have pinned their hopes on targeting key pro-fibrotic pathways, such as transforming growth factor-β (TGF-β) inhibitors. TGF-β is a key cytokine driving the transdifferentiation of fibroblasts into myofibroblasts and promoting the massive synthesis of ECM. While small molecule inhibitors or monoclonal antibodies have shown potential for inhibiting fibrosis in vitro and in animal models, systemic blocking of TGF-β signaling can lead to significant systemic side effects. TGF-β plays a wide range of physiological roles in vivo, including immune regulation, epithelial cell growth control, cardiovascular homeostasis, and tumor suppression. Long-term systemic inhibition of this pathway can lead to serious adverse reactions such as valvular heart disease, keratosis pilaris, bleeding tendencies, and even immune disorders. These safety concerns have greatly limited the clinical translation of TGF-β inhibitors, preventing these drugs from being safely used for the long-term treatment of IBD patients. Similarly, some drugs that have been approved for fibrosis in other organs, such as idiopathic pulmonary fibrosis and nintedanib, are difficult to directly apply to the field of intestinal fibrosis due to their systemic side effects and uncertainties in their local effects on the intestines.

[0005] In summary, the treatment of intestinal fibrosis is facing a dilemma: simple anti-inflammatory treatment cannot overcome existing fibrosis, while systemic anti-fibrotic strategies are difficult to implement due to safety concerns. Given the increasing prevalence of IBD and the accompanying population of patients with fibrotic strictures, developing safe and effective anti-fibrotic drugs has become one of the most pressing clinical needs in the field of gastroenterology. Summary of the Invention

[0006] To fill the technological gap of the current lack of effective anti-intestinal fibrosis drugs, the purpose of this invention is to provide the use of CXCR4 antagonists in the preparation of drugs and / or pharmaceutical compositions for the prevention and / or treatment of intestinal fibrosis.

[0007] CXCR4 is a G protein-coupled receptor with its endogenous ligand SDF-1 (CXCL12). CXCR4 antagonists, such as praxavir (AMD3100), are known to be approved as hematopoietic stem cell mobilizing agents for the treatment of multiple myeloma and lymphoma. While some literature reports on the role of CXCR4 in inflammation and immune regulation, existing technologies do not provide any technical insights or experimental evidence regarding the use of CXCR4 antagonists for the treatment of intestinal fibrosis. This invention is the first to discover that the CXCR4 signaling pathway plays a crucial driving role in the development and progression of intestinal fibrosis. Through gene knockout technology and pharmacological inhibition, it has been demonstrated that inhibiting CXCR4 can significantly improve the symptoms of intestinal fibrosis.

[0008] The objective of this invention can be achieved through the following technical solutions: The first object of the present invention is to provide the use of CXCR4 antagonists in the preparation of medicaments and / or pharmaceutical compositions for the prevention and / or treatment of intestinal fibrosis.

[0009] Preferably, the intestinal fibrosis is intestinal fibrosis associated with inflammatory bowel disease (IBD).

[0010] Preferably, the inflammatory bowel disease is selected from Crohn's disease or ulcerative colitis.

[0011] Preferably, the drug and / or drug composition is a drug and / or drug composition that targets the CXCL12-CXCR4 signaling pathway.

[0012] More preferably, the drug and / or drug composition is for reducing total plasma cells and IgG. + Drugs and / or drug compositions with plasma cell ratios.

[0013] Preferably, the CXCR4 antagonist is Plerixafor (AMD3100).

[0014] A second object of the present invention is to provide a medicament for the prevention and / or treatment of intestinal fibrosis, said medicament containing a CXCR4 antagonist.

[0015] Preferably, the dosage form of the drug includes one or more of the following: suspension, granules, capsules, powders, tablets, emulsions, pills, injections, suppositories, enemas, aerosols, patches, or drops.

[0016] Preferably, the drug contains pharmaceutically acceptable excipients.

[0017] Preferably, the excipients include one or more of the following: diluent, excipient, filler, binder, wetting agent, disintegrant, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

[0018] Preferably, the drug is administered via one of the following methods: intravenous injection, subcutaneous injection, intramuscular injection, oral administration, or transdermal absorption.

[0019] A third object of the present invention is to provide a pharmaceutical composition for the prevention and / or treatment of intestinal fibrosis, wherein the pharmaceutical composition contains the aforementioned pharmaceutical ingredients.

[0020] Compared with the prior art, the present invention has the following beneficial effects: (1) The function of CXCR4 as a therapeutic target for intestinal fibrosis was revealed for the first time: This invention constructs CD19-Cre;CXCR4 flox / floxConditional gene knockout mice have demonstrated for the first time in vivo that specific knockout of CXCR4 in B cell lineages can significantly improve DSS-induced chronic intestinal fibrosis, confirming that CXCR4 is a key functional target driving intestinal fibrosis, rather than just a related biomarker.

[0021] (2) The efficacy of CXCR4 antagonists against intestinal fibrosis was verified for the first time: In a DSS-induced mouse model of chronic intestinal fibrosis, treatment with the marketed CXCR4 antagonist praxaviva significantly reduced intestinal collagen deposition and lowered the fibrosis score, demonstrating excellent anti-fibrotic activity.

[0022] (3) Provided clinical relevance evidence: In intestinal tissue samples from human IBD patients, this invention first discovered that the expression level of CXCR4 in fibrotic sites was significantly higher than that in non-fibrotic sites, further verifying the clinical relevance of this target.

[0023] (4) Filling a technological gap: This invention solves the problem of the lack of effective anti-intestinal fibrosis drugs in the prior art, and provides a new and promising treatment strategy for patients with intestinal fibrosis. Attached Figure Description

[0024] Figure 1 Figure 1 shows the expression of CXCR4 in fibrotic tissue and adjacent non-fibrotic intestinal tissue of patients with IBD intestinal fibrosis in Example 1. Figure A shows the results of Western blot analysis; Figure B shows the results of qPCR quantitative analysis.

[0025] Figure 2 This is a graph showing the qPCR quantitative analysis results of CXCL12 in fibrotic tissue and adjacent non-fibrotic tissue of a patient with intestinal fibrosis in Example 1.

[0026] Figure 3 The graphs show the trends in body weight and disease activity index (DAI) for each group of mice in Example 2.

[0027] Figure 4 The images show representative Masson staining images of mouse colon tissue from each group in Example 2, along with a statistical chart of collagen volume fraction (CVF).

[0028] Figure 5 The image shows the flow cytometry analysis results of the colonic lamina propria lymphocytes in each group of mice in Example 2.

[0029] Figure 6 The graphs show the trends in body weight and disease activity index (DAI) for each group of mice in Example 3.

[0030] Figure 7The images show the pathological evaluation results of the colon tissues of mice in each group in Example 3, including HE staining and Masson staining.

[0031] Figure 8 This is a graph showing the results of Western blot analysis of the expression of Collagen-I and CXCR4 proteins, markers of colonic fibrosis in mice in each group, in Example 3. Detailed Implementation

[0032] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0033] Unless otherwise specified, all reagents used in the following embodiments are commercially available reagents, and all detection methods and techniques used are conventional detection methods and techniques in the art.

[0034] Example 1 This embodiment provides the detection and comparison of CXCL12 and CXCR4 expression levels in fibrotic and adjacent non-fibrotic intestinal tissues of patients with intestinal fibrosis, as detailed below: In this embodiment, the experimental samples were fibrotic and adjacent non-fibrotic intestinal tissue samples from patients with Crohn's disease and intestinal fibrosis. The samples were provided by Shanghai Tenth People's Hospital and were confirmed by pathology. After receiving the samples, they were stored at -80°C for later use.

[0035] (1) Western blot analysis was used to detect the expression level of CXCR4 in fibrotic and adjacent non-fibrotic intestinal tissues of patients with intestinal fibrosis. 1) Take the frozen intestinal tissue sample (already segmented and weighed) from the -80 ℃ freezer and add pre-cooled RIPA lysis buffer (containing 1% protease inhibitor; manufacturer: Thermo, catalog number: 89901) at a ratio of 150 μL per 10 mg sample. Place the sample in a tissue homogenizer for homogenization (4℃, run according to the predetermined program). After homogenization, let stand for 5 min, then centrifuge at 4℃ for 12000 rpm × 5 min. 2) After step 1), aspirate the supernatant and transfer it to a 1.5 mL centrifuge tube. Centrifuge at 4 °C for 14,000 rpm for 5 min. 3) After step 2), aspirate the supernatant and transfer it to a new 1.5 mL centrifuge tube. Then, according to the BCA protein concentration assay kit (Pierce... TM BCA Protein Assay Kit; Manufacturer: Thermo, Catalog No.: 23225) Instructions for use: Determine protein concentration; 4) Prepare loading samples according to protein concentration, ensuring consistent loading amounts for each group of samples; 5) Use Hepes-Tris 8% high-resolution precast gel, and load 10 μg of total protein per well; 6) Set the voltage to 120 V, perform electrophoresis for 60 min, and stop electrophoresis when the bromophenol blue reaches the bottom; 7) Remove the gel plate, gently pry it open to peel off the gel, place the gel in the transfer buffer, and cut the gel according to the size of the protein marker and the target protein. 8) Cut PVDF membranes to the corresponding size according to the adhesive strip, immerse them in anhydrous ethanol for 30 seconds, then soak them in ddH2O for 2 minutes, and then soak them in transfer solution for 5 minutes. 9) Semi-dry transfer at 2.5 A (constant current), maximum voltage 25 V, transfer time 7 min; 10) After the transfer is complete, remove the PVDF membrane and wash it with TBST buffer for 5 min × 3 times; block it by soaking it in 5% skim milk powder at room temperature for 2 h or overnight at 4 ℃; wash it with TBST buffer for 5 min × 3 times. 11) Incubate with primary antibody (Anti-CXCR4 antibody [UMB2], manufacturer: abcam, catalog number: Ab124824; CXCR4 antibody 1:1000 dilution) overnight at 4 ℃. 12) Washing the membrane: Recover the primary antibody and wash the membrane three times with TBST buffer for 5 min each time; 13) Incubation with secondary antibody (Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP; Manufacturer: Thermo, Catalog No.: 31430): Depending on the species of the primary antibody, immerse the PVDF membrane in the secondary antibody diluted 1:10000, place it on a shaker, and incubate at room temperature for 2 h; 14) Wash with TBST buffer for 5 min × 3 times; 15) Development: ECL method was used for development, and Image-Lab was used for image analysis.

[0036] (2) The specific details of real-time quantitative PCR are as follows: 1) Remove the frozen intestinal tissue sample from the -80 ℃ freezer, add 1 mL TRIzol, and homogenize it in a tissue homogenizer (4 ℃, run according to the predetermined program). 2) After homogenization, let stand at room temperature for 5 minutes; 3) Add 200 μL of chloroform, shake vigorously for 15 s, and let stand at room temperature for 3 min; 4) Centrifuge at 12000×g for 15 min at 4 ℃, transfer the upper aqueous phase to a new tube, add 500 μL of isopropanol, and mix well; 5) Centrifuge at 4 ℃, 12000×g for 10 min, discard the supernatant, and wash with 1 mL of 75% ethanol; 6) Centrifuge at 7500×g for 5 min at 4 ℃, discard the ethanol, and dry at room temperature for 20 min; 7) Dissolve in 20-50 μL of RNase-Free water; 8) Use Nanodrop to assess the quality and concentration of the extracted RNA and calculate the RNA volume required for reverse transcription. 9) According to the instructions of the reverse transcription reagent (Hifair® III 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus); manufacturer: Yeasen, catalog number: 11141ES60), reverse transcribe the RNA into cDNA; a. Remove genomic DNA and prepare the reaction mixture in a 200 μL EP tube: Table 1 Summary of Reaction Systems After mixing by blowing, react at 42℃ for 2 min; react at 4℃ for 2 min.

[0037] b. Reverse transcription reaction system: Table 2 Summary of Reverse Transcription Reaction Systems After gently vortexing and centrifuging, the cDNA was synthesized by reacting at 25 °C for 5 min, 55 °C for 15 min, and 85 °C for 5 min. It was then stored at -20 °C for a long time.

[0038] 10) Real-time PCR: Each accessory hole is configured according to the following system: Table 3 Summary of Real-time PCR Systems 11) Computer test.

[0039] The primer sequences involved in Table 3 are shown in Table 4: Table 4 Summary of Primer Sequences The results are as follows: The results are as follows Figure 1 and Figure 2 As shown; via Figure 1 and Figure 2It can be observed that, compared with adjacent non-fibrotic intestinal tissue, the mRNA and protein expression levels of CXCR4 in intestinal fibrosis tissue are significantly higher. Figure 1 A, Figure 1 B) and the mRNA expression level of CXCL12 ( Figure 2 All were significantly increased.

[0040] Example 2 This example provides an investigation into the effects of B / plasma cell-specific CXCR4 gene conditional knockout mice on intestinal fibrosis, as detailed below: (1) Animal model construction: 1) Six 7-week-old male CD19-Cre; CXCR4 mice were subjected to... flox / flox Mice (B cell / plasma cell-specific CXCR4 gene knockout mice, prepared according to: Rickert, RC et al. “B lymphocyte-specific, Cre-mediated mutagenesis in mice.” Nucleic acids research vol. 25,6 (1997): 1317-8. doi:10.1093 / nar / 25.6.1317) were used as the experimental group; another 6 age-matched male CXCR4 knockout mice were used as the experimental group. flox / flox Mice (CXCR4 gene not knocked out, reference: Rickert, RC et al. “B lymphocyte-specific, Cre-mediated mutagenesis in mice.” Nucleic acids research vol. 25,6 (1997):1317-8. doi:10.1093 / nar / 25.6.1317) served as the control group.

[0041] Both groups of mice were induced with DSS to obtain a mouse model of intestinal fibrosis, as detailed below: Each group of mice was given distilled water containing 1.5% sodium dextran sulfate (DSS) freely for 7 days; then, the drinking water was replaced with sterile distilled water, and the mice continued to drink freely for 14 days. The treatment process of 7 days of DSS and 14 days of sterile water was defined as one cycle, and a total of 3 cycles were repeated.

[0042] 2) Observe and record the general condition of the mice daily, and score them using the Disease Activity Index (DAI). The DAI score consists of the following three indicators (Table 4): Table 4 Summary of DAI Scores DAI = (Weight Loss Score + Stool Characteristics Score + Rectal Blood Score) / 3 3) During the endpoint treatment of the mouse model, whole blood was first collected in EDTA anticoagulant tubes by sampling the orbital artery and vein. After centrifugation, the supernatant plasma was separated, aliquoted, and frozen at -80℃ for later use. Subsequently, the complete colon was dissected and separated. After measuring the length, it was processed in segments: the proximal colon was used to prepare a single-cell suspension of the colonic lamina propria; the mid-colon was homogenized on ice in RIPA lysis buffer, centrifuged, and the supernatant was used for ELISA or protein detection; the distal colon was washed with PBS and fixed in 4% paraformaldehyde for subsequent paraffin embedding and pathological staining (as shown in Table 5).

[0043] Table 5. Colonic segmentation treatment (2) Investigation on the assessment of the degree of pathological damage in mouse intestinal tissue using hematoxylin-eosin (HE) staining method: Experimental samples: Colon tissues from mice in the experimental and control groups.

[0044] 1) Sampling and fixation: The excised colon tissue was immediately placed in 4% neutral paraformaldehyde for fixation for 48 h.

[0045] 2) Dehydration and embedding: The fixed tissue blocks were dehydrated in 70% ethanol, 80% ethanol, 95% ethanol and anhydrous ethanol for 10 seconds each. After dehydration, the tissue was removed from the anhydrous ethanol and first placed in a mixture of 50% anhydrous ethanol and 50% xylene for 30 minutes. Then it was immersed in xylene twice for 10 minutes each time. When the tissue blocks became clear (transparent), the tissue was embedded in paraffin to make tissue paraffin blocks.

[0046] 3) Sectioning: Use a paraffin microtome to cut the tissue block into thin sections with a thickness of 4~5 μm, attach them to glass slides, and bake at 60℃ for 2 h for fixation.

[0047] 4) Dewaxing and rehydration: Place the sections in xylene I and II for 10 min each to completely dewax them; then pass them through anhydrous ethanol, 95%, 85%, and 75% ethanol for 5 min each, and finally rinse with distilled water for 2 min.

[0048] 5) Cell nuclear staining: Immerse the sections in hematoxylin staining solution for 10 min (the time depends on the reagent concentration and tissue characteristics), and rinse with tap water to remove excess staining solution.

[0049] 6) Differentiation and Blueing: Immerse in 1% hydrochloric acid ethanol for a few seconds to differentiate until the tissue section turns from dark blue to light red. Immediately rinse with tap water, then place in a weakly alkaline aqueous solution to restore the blue color of the cell nuclei.

[0050] 7) Cytoplasmic staining: Immerse the slide in 0.5% eosin staining solution for 2 min, then rinse quickly with distilled water.

[0051] 8) Dehydration, clearing and sealing: Immerse in 95% ethanol and anhydrous ethanol in sequence for gradient dehydration, 2 min each time, then place in xylene I and II for 5 min each for clearing, add neutral resin, and seal with a coverslip.

[0052] 9) Image Acquisition: Observation is performed using an optical microscope. The cell nucleus should appear as a clear blue-purple color, while the cytoplasm, muscle layer, and connective tissue should appear as varying shades of pink or red.

[0053] (3) Masson's trichrome staining method was used to assess the degree of fibrosis in mouse intestinal tissue (the kit used was the Masson's trichrome staining kit (manufacturer: Beyotime; catalog number: C0189S)): The experimental samples are the same as those in step (2) of this embodiment.

[0054] 1) Sampling and fixation: The excised colon tissue was immediately placed in 4% neutral paraformaldehyde for fixation for 48 h.

[0055] 2) Dehydration and embedding: The fixed tissue blocks were dehydrated in 70% ethanol, 80% ethanol, 95% ethanol and anhydrous ethanol for 10 seconds each. After dehydration, the tissue was removed from the anhydrous ethanol and first placed in a mixture of 50% anhydrous ethanol and 50% xylene for 30 minutes. Then it was immersed in xylene twice for 10 minutes each time. When the tissue blocks became clear (transparent), the tissue was embedded in paraffin to make tissue paraffin blocks.

[0056] 3) Sectioning: Use a paraffin microtome to cut the tissue block into thin sections with a thickness of 4~5 μm, attach them to glass slides, and bake at 60℃ for 2 h for fixation.

[0057] 4) Dewaxing and rehydration: Place the sections in xylene I and II for 10 min each to completely dewax them; then pass them through anhydrous ethanol, 95%, 85%, and 75% ethanol for 5 min each, and finally rinse with distilled water for 2 min.

[0058] 5) Perform staining according to the instructions of the Masson trichrome staining kit.

[0059] 6) Dehydration: After staining, quickly immerse the tissue sections in 70% ethanol, 80% ethanol, 95% ethanol and anhydrous ethanol for 10 seconds each, and clear with xylene 3 times, 2 minutes each time.

[0060] 7) Sealing: Add a drop of neutral resin and cover with a coverslip to seal the slide.

[0061] 8) Image acquisition: Observation is performed using an optical microscope.

[0062] (4) Flow cytometry detection of mouse colonic lamina propria lymphocyte subsets: Experimental sample: proximal colon of mice in the experimental group in step (1); 1) Prepare a single-cell suspension of mouse colonic lamina propria lymphocytes according to the instructions of the Lamina Propria Dissociation Kit (manufacturer: Miltenyi, catalog number: 130-097-410).

[0063] 2) After the single-cell suspension is prepared, Fc Block (anti-CD16 / CD32 antibody) is performed and incubated at 4°C in the dark for 15 min to block non-specific binding.

[0064] 3) After Fc block is completed, add fluorescently labeled antibody. The amount of antibody used per tube should be the dosage recommended in the instructions. Incubate at 4 ℃ in the dark for 30 min.

[0065] 4) After staining, add 1 mL of flow cytometry buffer (a mixture of PBS, FBS (final concentration of 2%) and EDTA (final concentration of 2 mM) to each tube, centrifuge at 300 g for 5 min, discard the supernatant, and wash twice to remove unbound antibodies.

[0066] 5) Add the live and dead dyes and incubate at room temperature in the dark for 15 min to label dead cells.

[0067] 6) After all staining steps are completed, add 1 mL of flow cytometry buffer (a mixture of PBS, FBS (final concentration of 2%) and EDTA (final concentration of 2 mM) to each tube, centrifuge at 300 g for 5 min, discard the supernatant, and wash twice.

[0068] 7) Finally, resuspend the cells in 250 μL of flow cytometry buffer in each tube and perform analysis. Collect data using a flow cytometer and process and analyze the data using FlowJo software.

[0069] The results are as follows: The results are as follows Figures 3-5 As shown, compared with the control group, the experimental group mice had significantly lower Disease Activity Index (DAI) scores, and Masson staining of colon tissue revealed a significant reduction in collagen deposition area and a marked decrease in tissue fibrosis. Furthermore, flow cytometry analysis showed that the experimental group mice had significantly lower levels of total plasma cells and IgG in the colonic lamina propria lymphocytes. + The proportion of plasma cells was significantly lower than that in the control group. These results indicate that the absence of CXCR4 in B cells / plasma cells can effectively alleviate DSS-induced intestinal fibrosis in mice, further confirming the role of CXCR4.+ The key driving role of plasma cells in the progression of intestinal fibrosis.

[0070] Example 3 This embodiment explores the therapeutic effect of AMD-3100 on intestinal fibrosis in mice, as detailed below: (1) Construction of mouse intestinal fibrosis AMD-3100 pharmacological efficacy model 1) After acclimatizing for one week, 24 seven-week-old female C57BL / 6J mice were randomly divided into three groups of eight mice each, according to their body weight: Treatment group (DSS+AMD-3100 group): Mice were given distilled water containing 2% sodium dextran sulfate (DSS) for free drinking for 7 days, and then replaced with sterile distilled water for free drinking for 14 days; the treatment process of 7 days of DSS / 14 days of sterile water was defined as one cycle, and a total of 3 cycles were repeated; AMD-3100 was dissolved in sterile PBS and administered intraperitoneally at a dose of 2.5 mg / kg once daily for 63 consecutive days. Model group (DSS group): Mice were given distilled water containing 2% sodium dextran sulfate (DSS) for free drinking for 7 days, and then replaced with sterile distilled water for free drinking for 14 days. The treatment process of 7 days of DSS / 14 days of sterile water was defined as one cycle, and a total of 3 cycles were repeated. Among them, an equal volume of sterile PBS was injected into the peritoneum daily for 63 consecutive days; Control group: Free access to sterile distilled water for 63 days, and daily intraperitoneal injection of an equal volume of sterile PBS; 2) Observe and record the general condition of the mice daily, and score them using the Disease Activity Index (DAI). The DAI score consists of the following three indicators (as shown in Table 6): Table 6 Summary of DAI Scores DAI = (Weight Loss Score + Stool Characteristics Score + Rectal Blood Score) / 3 3) For the endpoint treatment of mice, whole blood was first collected via the orbital artery and vein into EDTA anticoagulant tubes. After centrifugation, the supernatant plasma was separated, aliquoted, and frozen at -80 °C for later use. Subsequently, the intact colon was dissected and rinsed with PBS to remove intestinal contents. The cecal end was considered the proximal end, and the anal end the distal end. Segmentation treatment was performed according to Table 7.

[0071] Table 7. Segmented treatment of the colon (2) Hematoxylin-eosin (HE) staining method was used to assess the degree of pathological damage in mouse intestinal tissue.

[0072] Experimental samples: colon tissues of mice in the model group, AMD-3100 treatment group, and control group.

[0073] The experimental procedure is the same as step (2) in Example 2.

[0074] (3) Masson's trichrome staining method to assess the degree of fibrosis in mouse intestinal tissue Experimental samples: colon tissues of mice in the model group, AMD-3100 treatment group, and control group.

[0075] The experimental steps are the same as step (3) in Example 2.

[0076] (4) Western blot analysis was used to detect the protein expression levels of Collagen-I and CXCR4, markers of fibrosis, in mouse intestinal tissue. The raw materials used in this embodiment are as follows: ①Collagen I Polyclonal Antibody; Manufacturer: Thermo, Product No.: PA1-26204; ②Anti-CXCR4 antibody [UMB2]; Manufacturer: abcam, Catalog No.: Ab124824; The experimental procedure is the same as step (1) in Example 1.

[0077] The primer sequences involved in this embodiment are specifically shown in Table 8: Table 8 Summary of Primer Sequences The results are as follows: Experimental results are as follows Figures 6-8 As shown, compared with the model group (DSS group), mice in the AMD-3100 treatment group (AMD-3100 group) experienced significantly reduced weight loss, significantly lower Disease Activity Index (DAI) scores, and significantly reduced mucosal structural damage and inflammatory cell infiltration observed in HE staining of colonic tissue. Masson staining also revealed a significant reduction in collagen deposition area. Furthermore, Western blot analysis showed a significant decrease in the protein expression level of the fibrosis marker Collagen-I in the colonic tissue of the treatment group mice, along with a significant downregulation of CXCR4 expression. These results indicate that AMD-3100 can effectively alleviate DSS-induced intestinal fibrosis in mice.

[0078] In summary, the expression of both CXCL12 and CXCR4 was significantly upregulated in patients with CD intestinal fibrosis. Conditional knockout of CXCR4 in B cells / plasma cells significantly alleviated intestinal fibrosis, and treatment with its specific antagonist AMD3100 also effectively reduced intestinal fibrosis. This protective effect is mainly achieved by inhibiting the CXCR4 signaling pathway, suggesting that CXCR4 is a potential intervention target for intestinal fibrosis.

[0079] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the interpretation of the present invention, without departing from the scope of the invention, should be within the protection scope of the present invention.

Claims

1. Use of CXCR4 antagonists in the preparation of drugs and / or pharmaceutical compositions for the prevention and / or treatment of intestinal fibrosis.

2. The application according to claim 1, characterized in that, The intestinal fibrosis mentioned refers to intestinal fibrosis associated with inflammatory bowel disease.

3. The application according to claim 2, characterized in that, The drug and / or drug composition are drugs and / or drug compositions that target the CXCL12-CXCR4 signaling pathway.

4. The application according to claim 1, characterized in that, The CXCR4 antagonist is praxavir.

5. A medicine for the prevention and / or treatment of intestinal fibrosis, characterized in that, The drug contains a CXCR4 antagonist.

6. A medicament for preventing and / or treating intestinal fibrosis according to claim 5, characterized in that, The dosage form of the drug includes one or more of the following: suspension, granules, capsules, powders, tablets, emulsions, pills, injections, suppositories, enemas, aerosols, patches, or drops.

7. A medicament for preventing and / or treating intestinal fibrosis according to claim 5, characterized in that, The drug contains pharmaceutically acceptable excipients.

8. A medicament for preventing and / or treating intestinal fibrosis according to claim 7, characterized in that, The excipients include one or more of the following: diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH adjusters, antioxidants, antibacterial agents, or buffers.

9. A medicament for preventing and / or treating intestinal fibrosis according to claim 5, characterized in that, The drug can be administered via one of the following methods: intravenous injection, subcutaneous injection, intramuscular injection, oral administration, or transdermal absorption.

10. A pharmaceutical composition for the prevention and / or treatment of intestinal fibrosis, characterized in that, The pharmaceutical composition contains the drug as described in claims 5 to 9.

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