Application of namiklast in preparation of medicine for treating intervertebral disc fibrosis through targeted inhibition of macrophage-myofibroblast transformation

Abstract

The invention relates to the technical field of medicines, in particular to a novel medical application of a phosphodiesterase 4B inhibitor namiklast, and particularly relates to an application of namiklast in preparation of a medicine for treating intervertebral disc fibrosis through targeted inhibition of macrophage-myofibroblast transformation.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, specifically to the application of namisitol in the preparation of drugs for treating intervertebral disc fibrosis by targeting and inhibiting macrophage-fibroblast transformation. Background Technology

[0002] Intervertebral disc degeneration is the leading cause of chronic low back pain, affecting over 600 million people worldwide and imposing a significant burden on socioeconomic status and quality of life. The core pathological changes in intervertebral disc degeneration include degradation of the extracellular matrix of the nucleus pulposus cells, rupture of the annulus fibrosus, inflammatory cell infiltration, and pathological fibrosis. Current clinical treatments primarily rely on nonsteroidal anti-inflammatory drugs (NSAIDs), physical therapy, and surgery; however, these methods cannot reverse or effectively slow the pathological progression of intervertebral disc degeneration. Therefore, developing novel treatment strategies that target the core mechanisms of intervertebral disc degeneration is a major challenge that urgently needs to be addressed in this field.

[0003] Recent studies have shown that immune microenvironment dysregulation plays a crucial role in intervertebral disc degeneration. The applicant, through clinical sample analysis, single-cell sequencing, and lineage tracing technology, has for the first time discovered and confirmed the existence of macrophage-myofibroblast transformation in degenerated intervertebral discs. Specifically, infiltrating macrophages, under abnormal mechanical stimulation and an inflammatory microenvironment, can transdifferentiate into myofibroblasts expressing α-smooth muscle actin, directly participating in and driving the fibrotic process of the intervertebral disc, leading to metabolic imbalance and structural damage of the extracellular matrix in the nucleus pulposus. Inhibiting macrophage-myofibroblast transformation has become a potential new strategy for treating intervertebral disc degeneration.

[0004] Namilast is a novel, highly selective phosphodiesterase 4B inhibitor that plays a central role in inflammatory responses by negatively regulating the cAMP / PKA signaling pathway through the hydrolysis of the intracellular second messenger cyclic adenosine monophosphate (cAMP). As the world's first approved phosphodiesterase 4B-specific inhibitor, namilast successfully completed Phase III clinical trials in 2025 and was approved for the treatment of pulmonary fibrosis, with its safety and efficacy clinically validated. However, its role in intervertebral disc degeneration remains completely unknown. Whether it can inhibit intervertebral disc fibrosis or exert its effect by regulating macrophage-myofibroblast transformation has not been reported domestically or internationally. Summary of the Invention

[0005] Application of namisitol in the preparation of drugs for treating intervertebral disc fibrosis by targeting and inhibiting macrophage-fibroblast transformation.

[0006] A drug for treating intervertebral disc fibrosis, wherein the drug is namisitol and a pharmaceutically acceptable carrier or excipient.

[0007] As an optimization, the pharmaceutically acceptable carrier is selected from any one of solvents, emulsifiers, suspending agents, disintegrants, binders, excipients, stabilizers, diluents, gelling agents, preservatives, lubricants, and surfactants.

[0008] As an optimization, the excipients are selected from at least one of hydroxypropyl methylcellulose, hydroxypropyl cellulose, povidone, polyethylene glycol, ethyl cellulose, liposomes, methacrylic acid copolymer, polyvinyl acetate, carboxymethyl ethyl cellulose, carboxymethyl cellulose phthalate, hydroxypropyl methyl cellulose phthalate, hydroxypropyl methyl cellulose acetate succinate, polyacrylic acid resin, polyvinyl carboxylate, alginate, carrageenan, carboxyacetic acid lactone, gum, polyvinyl alcohol, pregelatinized starch, cross-linked starch, sodium carboxymethyl starch, dextrin, polyethylene oxide, chitosan, chitosan, ion exchange resin, and collagen.

[0009] As an optimization, the drug is in a pharmaceutically acceptable dosage form selected from tablets, capsules, injections, granules, suspensions, and solutions.

[0010] Compared with the prior art, the present invention has the following technical effects: A new use for a blockbuster drug has been discovered: for the first time, a novel therapeutic effect of namisitol (a newly launched anti-pulmonary fibrosis drug) on ​​intervertebral disc degeneration has been revealed, expanding its indications to include a new indication with great market potential. The clinical translation pathway is short and low-risk.

[0011] A novel mechanism of action has been revealed: For the first time in IDD, a new signal regulation axis of "PDE4B-cAMP-PKA-QKI (phosphorylation)-STING (m7G modification)-MMT" has been elucidated, which not only provides a solid scientific basis for the therapeutic effect of namisitol, but also provides a series of new targets (such as QKI and STING) for the treatment of IDD, which has important theoretical value. Attached Figure Description

[0012] Figure 1 Combining clinical specimens and animal tissue experiments, this study revealed a positive correlation between macrophage infiltration, fibrosis, and intervertebral disc degeneration (IDD). a) Schematic diagram of the single-cell sequencing workflow for obtaining clinical human specimens; b) Visualized clustering analysis of single-cell sequencing using UMAP; c) Western blotting to detect α-SMA and Col1 expression in intervertebral discs of clinical human specimens; d) IF staining of Col1 and α-SMA in normal and degenerated human nucleus pulposus cells, scale bar = 200 μm; e) Images of intervertebral discs of normal and degenerated mice after Sirius red staining under a polarized light microscope, scale bar = 500 μm; f) IF staining results of CD68, a macrophage marker in intervertebral discs of normal and degenerated mice, scale bar = 500 μm; g) IF staining results of CD68, a macrophage marker in intervertebral discs of normal and degenerated humans, scale bar = 100 μm. Figure 2 MMT plays an important role in IDD. a) Single-cell data analysis of changes in cell composition ratio; b) Tissue staining (HE / Safranin-Fibrin-Green / Sirius Red) and imaging examinations (MRI, X-ray) of mechanically stress-induced mouse IDD, scale bar = 500 μm; c) Intervertebral disc histological score; d) Western blot detection of expression of mouse intervertebral disc fibrosis-related proteins (Col1, α-SMA); ef) Changes in the colocalization of CD68 and α-SMA after in vitro mechanical stress stimulation of Raw 264.7, scale bar = 20 μm; gh) Flow cytometry detection of Cx3cr1-labeled mouse intervertebral disc Td / α-SMA double-positive cells; ij) Immunofluorescence results of Cx3cr1-labeled mouse intervertebral disc Td / α-SMA double-positive cells, scale bar = 20 μm. p < 0.05 indicates statistical significance; Figure 3 PDE4B promotes IDD through MMT. a) Single-cell transcriptome analysis of PDE4 expression levels in different cell types; b) Changes in PDE4 transcription levels in Raw 264.7; c) PDE4B expression levels in human intervertebral disc tissue and human nucleus pulposus cells; d) PDE4B expression levels in Cx3cr1 myeloid-labeled mouse Td+ cells and human macrophage THP-1 cells; e) Statistical graph of PDE4B expression levels; fg) Changes in Td / a-SMA colocalization levels in PDE4B macrophage-specific knockout mice; hi) Changes in the proportion of Td / a-SMA double-positive cells in PDE4B macrophage-specific knockout mice; j) Expression of intervertebral disc fibrosis protein (a-SMA, Col1) in PDE4B macrophage-specific knockout IDD mice; kl) Tissue sections (HE, Safranin-Fibrin Green) showed that the degree of IDD in PDE4B macrophage-specific knockout mice was significantly reduced compared to wild-type mice. p<0.05 indicates a statistically significant difference; Figure 4 Targeted inhibition of PDE4B downregulates STING signaling pathway activation, thereby blocking MMT. a) RNA-Seq flowchart after PDE4B inhibitor Nerandomilast intervention in Raw264.7; b) KEGG analysis of differentially expressed genes; c) Volcano plot of differentially expressed genes; d) GSEA of differentially expressed genes; e) qPCR quantification results of STING; f) Western blot results and quantification analysis of STING / TBK1. p<0.05 indicates statistically significant difference; Figure 5QKI inhibits STING translation via the m7G modification pathway. a) RNA-pull-down / MS experimental flowchart; b) MS protein list fold change analysis; c) QKI protein binding motif; d) RBPsuite 2.0 online prediction of STING mRNA binding to QKI RNA sequence scoring table; e) STING RNA pull-down experimental Western blot results; f) RIP-qPCR showing that Nerandomilast enhances the interaction between QKI and STING RNA; g) Nerandomilast inhibits the translation efficiency of STING mRNA. p < 0.05 indicates statistical significance. Figure 6 Targeted inhibition of PDE4B enhances QKI phosphorylation modification via the classic cAMP / PKA pathway. a) Co-IP / MS flowchart after Nerandomilast intervention in Raw264.7; b) List of proteins analyzed by MS; c) Co-IP validation of interaction with PKA and detection of global phosphorylation levels using QKI; Figure 7 Constructing a macrophage-targeted biomimetic delivery system for the treatment of intervertebral disc degeneration (IDD). a) Flowchart of the nanovesicle delivery system preparation; b) Particle size and potential of drug-loaded nanovesicles; c) Analysis of macrophage membrane and membrane composition of different nano-formulations; d) Transmission electron microscopy morphology of nanovesicles, scale bar = 100 nm; e) In vitro release of the drug-loaded nanosystem; f) In vivo imaging of small animals to detect the sustained-release effect of the drug-loaded nanosystem; g) Quantitative analysis of fluorescence intensity using in vivo imaging; h) In vivo experiment of Nd-PLGA@MacM treatment for intervertebral disc degeneration in rats, results of HE and Safranin-Fix-Green staining of intervertebral disc sections; i) Histological score of intervertebral disc structure. p < 0.05 indicates statistical significance; Figure 8 Cytotoxicity and systemic toxicity evaluation of the biomimetic nano-targeted delivery system. a) CCK8 assay to detect the toxicity of the delivery system to macrophages; b) Blood routine and biochemical tests were performed on venous blood samples from mice treated with the drug delivery system. Detailed Implementation

[0013] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below with reference to specific embodiments and comparisons. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0014] Unless otherwise specified, all equipment used in this embodiment is conventional experimental equipment, and all materials and reagents used are commercially available unless otherwise specified. Experimental methods without special instructions are also conventional experimental methods.

[0015] Example 1: Verification of the role of macrophage infiltration and fibrosis in intervertebral disc degeneration (IDD) 1. Clinical Sample Analysis Sample Acquisition: Clinical surgical samples, approved by the ethics committee and with informed consent from the patients, were acquired, including 5 normal intervertebral disc tissue samples (taken from the non-stress side of scoliosis correction surgery or normal segments from patients with traumatic fractures) and 5 degenerative intervertebral disc tissue samples (taken from the lesion segments of patients with lumbar disc herniation or spinal stenosis). After ex vivo, one portion of the samples was immediately flash-frozen in liquid nitrogen and transferred to a -80°C ultra-low temperature freezer for protein and nucleic acid extraction; the other portion was fixed in 4% paraformaldehyde solution for 24-48 hours for paraffin embedding and sectioning.

[0016] Single-cell transcriptome sequencing: Fresh intervertebral disc tissue was minced and digested with collagenase II (2 mg / mL) and hyaluronidase (1 mg / mL) at 37°C for 2-4 hours to prepare a single-cell suspension. Single-cell capture, library construction, and sequencing were performed using the 10x Genomics Chromium platform. The sequenced data were aligned (reference genome GRCh38), filtered, and UMI counted using Cell Ranger software. Cell clustering analysis, t-SNE / UMAP dimensionality reduction visualization, and cell population annotation were performed using classic cell marker genes (such as macrophage markers: CD68, CD14; myofibroblast markers: ACTA2, COL1A1) to compare the changes in the proportion of various cell types in normal and degenerated tissues.

[0017] Western blotting and tissue sectioning techniques: - Western Blot (WB): Total protein was extracted from tissues and quantified using the BCA method. 30 μg of protein was loaded onto the sample, subjected to SDS-PAGE electrophoresis, transferred to a membrane, blocked, and then incubated overnight at 4°C with anti-CD68 antibody (macrophages), anti-α-SMA antibody (myofibroblasts), anti-Collagen I antibody (fibrosis), and primary antibody GAPDH antibody (internal control), respectively. After washing with TBST, the membrane was incubated at room temperature for 1 hour with HRP-labeled secondary antibody of the corresponding species, developed with ECL chemiluminescence solution, and analyzed for grayscale values ​​using ImageJ software.

[0018] - Immunofluorescence (IF) / Immunohistochemistry (IHC): Paraffin sections were dewaxed, hydrated, and antigen-retrieved before IF or IHC staining. IF staining used primary antibodies against CD68 and α-SMA, incubated overnight at 4°C, followed by incubation for 1 hour the next day with secondary antibodies labeled Cy3 and FITC in the dark. Cell nuclei were counterstained with DAPI, mounted, and observed and photographed under a confocal microscope to analyze colocalization. IHC staining used the HRP-DAB system for development, with hematoxylin counterstaining of cell nuclei. After mounting with neutral resin, the distribution and intensity of positive signals were observed under a light microscope.

[0019] Animal model construction and validation An abnormal mechanical stress (IDD) model was established using 8-week-old C57BL / 6J wild-type mice. After anesthetizing with 1% sodium pentobarbital (50 mg / kg) via intraperitoneal injection, the tail hair was shaved. A customized adjustable mechanical loading device was used to apply an axial static compressive force of 1.2 N to the 8th-9th (Co8-9) and 9th-10th (Co9-10) caudal vertebrae for 4 weeks. No pressure was applied to the control group mice. The tail condition of the mice was observed daily during the period.

[0020] Model evaluation: After the pressure was applied, the mice were euthanized and the compressed caudal intervertebral disc was removed.

[0021] - Micro-CT scans were performed on some samples, and the changes in intervertebral disc height were analyzed after three-dimensional reconstruction.

[0022] - Some samples were paraffin-embedded and sectioned for hematoxylin and eosin (H&E) staining and safranin O-Fast Green staining to assess tissue morphology and proteoglycan loss.

[0023] - The expression levels and spatial distribution of macrophage infiltration (CD68) and fibrosis (α-SMA, Collagen I) indicators in the model were detected by Western blotting and infusion (IF) (methods as above).

[0024] Functional recovery experiment Macrophage clearance: During mechanical compression, clodronate liposomes (2 μL / time, once weekly) were injected locally, while the control group received an equal volume of PBS liposomes. After 8 weeks, samples were collected, and the clearance efficiency of macrophages (CD68+ cells) was verified by inductively coupled plasmapheresis (IF). Changes in fibrosis markers (α-SMA, Collagen I) and nucleus pulposus matrix metabolism markers (Collagen II, Aggrecan) were detected by Western blotting and IF to assess whether macrophage clearance could restore a normal phenotype.

[0025] Fibrosis inhibition: Simultaneously with mechanical compression, the TGF-β receptor inhibitor LY2109761 (10 mg / kg, once weekly) was injected locally, while the control group received an equal volume of solvent. After 8 weeks, tissue samples were collected, and changes in fibrosis markers and nucleus pulposus matrix metabolic markers were detected by Western blotting and infusion (IF) to assess whether fibrosis inhibition could restore a normal phenotype.

[0026] Example 2: Validation of the key role of macrophage-myofibroblast transformation (MMT) in IDD 1. Bioinformatics Analysis Deep mining was performed on the single-cell sequencing data obtained in Example 1. Pseudo-time analysis was performed on macrophage and myofibroblast clusters using software such as Monocle2 or Slingshot to construct cell fate transition trajectories and visualize potential pathways for macrophage transdifferentiation into myofibroblasts. Simultaneously, the percentage of "double-positive" cells expressing both macrophage marker (Cd68) and myofibroblast marker (Acta2) was calculated, and the differences between the normal and degenerated groups were compared.

[0027] Lineage Tracing In Vivo Validation Animal model construction: Cx3cr1-CreERT2 mice were crossed with Rosa26-tdTomato reporter gene mice to obtain offspring mice. Eight-week-old mice were intraperitoneally injected with tamoxifen (75 mg / kg, for 5 consecutive days) to induce macrophage lineage-specific labeling of tdTomato red fluorescent protein. After tamoxifen induction, an abnormal mechanical stress (IDD) model was constructed according to the method in Example 1.

[0028] MMT detection: After model construction, mouse caudal intervertebral discs were harvested and frozen sections were prepared. Immunofluorescence staining was performed using a primary antibody against α-SMA and a secondary antibody labeled with Alexa Fluor 488 (green). Double-positive cells (tdTomato+ (red) and α-SMA+ (green)) were observed and counted under a confocal microscope; these were the cells exhibiting MMT. The proportion of tdTomato+α-SMA+ cells in the single-cell suspension after digestion of the intervertebral disc tissue was quantitatively analyzed by flow cytometry.

[0029] In vitro cell co-culture model Establishment of co-culture system: Mouse macrophage cell line RAW 264.7 and mouse nucleus pulposus cells were obtained. A direct co-culture group (the two cell lines were mixed and seeded at a 1:1 ratio) and an indirect co-culture group (using Transwell chambers, RAW 264.7 cells were seeded in the upper chamber and nucleus pulposus cells were seeded in the lower chamber) were established.

[0030] Mechanical stress stimulation: The co-culture system was placed in a Flexcell FX-5000T cell pressurization system and subjected to dynamic cyclic pressure stimulation of 1.5 MPa and 1.0 Hz for 6 hours per day for 3 consecutive days. The control group was placed in the same incubator without pressure.

[0031] Phenotypic detection: - For the direct co-culture group, after digestion and collection of cells, macrophages and nucleus pulposus cells were sorted by flow cytometry (FACS) based on cell-specific surface markers.

[0032] -WB detection of the expression of MMT markers (α-SMA, Collagen I) in sorted macrophages.

[0033] -qRT-PCR or WB was used to detect the mRNA and protein expression levels of catabolism markers (Mmp13) and anabolism markers (Col2a1, Acan) in nucleus pulposus cells.

[0034] Example 3: Study on the molecular mechanism of namitilast inhibiting MMT and treating IDD 1. Screening of downstream targets for namisitol action RAW 264.7 cells were stimulated with mechanical stress to induce an MMT model, while simultaneously being treated with natriuretic peptide (500 nM) or DMSO (solvent control) for 48 hours. Cells were harvested, and total RNA and total protein were extracted.

[0035] - Transcriptome sequencing (RNA-Seq): Library construction and sequencing were outsourced to a commercial company (Illumina NovaSeq platform). After quality control, alignment, and gene expression quantification of the sequencing data, differentially expressed genes were analyzed using DESeq2 software (screening criteria: |log2FoldChange|>1, adj.p.val<0.05). KEGG and GO enrichment analyses of the differentially expressed genes revealed significant enrichment of the cGAS-STING pathway and significant downregulation of STING gene expression.

[0036] - Proteomics sequencing: TMT labeling quantitative proteomics technology was used to identify differentially expressed proteins, verify RNA-Seq results, and found that STING protein levels were also downregulated.

[0037] STING upstream regulation mechanism verification RNA pull-down and mass spectrometry (MS): Biotin-labeled STING mRNA full-length or 3'UTR fragments were synthesized in vitro via transcription and co-incubated with RAW 264.7 cell lysate. Streptavidin magnetic beads captured the RNA-protein complex. After eluting the bound proteins, SDS-PAGE electrophoresis and silver staining were performed, followed by LC-MS / MS analysis. By comparing with a control group (no RNA or irrelevant RNA), proteins specifically binding to STING mRNA were screened, and the candidate protein QKI was identified using the RBPsuite 2.0 database prediction.

[0038] RNA immunoprecipitation (RIP): Cell lysate was prepared using anti-QKI antibody and control IgG antibody. The co-precipitated RNA was recovered, and the enrichment level of STING mRNA was quantified by qRT-PCR to verify the direct binding of QKI to STING mRNA. This binding was enhanced after namisitol intervention.

[0039] m7G modification analysis: m7G-MeRIP-Seq (m7G methylated RNA immunoprecipitation sequencing) technology was used to enrich m7G-modified RNA fragments in cells from the namitase-treated and control groups. Sequencing analysis was then performed to analyze changes in m7G modification sites and levels on STING mRNA. miCLIP (miRNA binding site identification technology) was used to further pinpoint the binding and modification sites of QKI on STING mRNA.

[0040] Upstream mechanism of QKI / STING signal axis Co-IP / MS screening of interacting proteins: After treating RAW 264.7 cells with namitase or DMSO, immunoprecipitation was performed using an anti-QKI antibody. After eluting the complex, LC-MS / MS analysis was performed to screen for differentially expressed proteins that interact with QKI under namitase regulation. The classic PDE4B-cAMP-PKA pathway was then used to screen for the kinase PKA.

[0041] Interaction verification: - Co-IP: Co-IP was performed using anti-PKA antibody and anti-QKI antibody respectively, and then the presence of the counterpart protein was detected by Western blotting to verify the interaction between endogenous PKA and QKI.

[0042] -GST Pull-down: Prokaryotic expression and purification of GST-QKI fusion protein and His-PKA protein. GST-QKI was immobilized on glutathione magnetic beads, incubated with His-PKA protein, and after elution, the His tag was detected by Western blotting to verify whether the two directly interacted.

[0043] Phosphorylation regulation verification: - After cell treatment with namitase, proteins were extracted, and changes in QKI protein phosphorylation levels were detected using Phos-tag™ SDS-PAGE.

[0044] - Perform phosphorylated proteomics sequencing to screen differential phosphorylation sites regulated by namisitol, and combine with prediction software to identify potential phosphorylation sites of PKA on QKI (such as Ser / Thr sites).

[0046] Example 4: Construction and efficacy evaluation of a macrophage membrane biomimetic delivery system (Nera@MM-NP) 1. Fabrication and characterization of the delivery system Macrophage membrane extraction: RAW 264.7 cells were collected, and macrophage membrane fragments were extracted using hypotonic lysis, differential centrifugation, and sucrose gradient ultracentrifugation. Membrane protein concentrations were determined using the BCA method.

[0047] Preparation of drug-loaded nanoparticles (Nd-PLGA): A nanoprecipitation method was used. 2 mg of natamycin and 20 mg of PLGA (50:50 lactoglycolic acid copolymer) were dissolved in 1 mL of acetonitrile to form the organic phase. The organic phase was slowly injected into 4 mL of pure water under high-speed magnetic stirring (800 rpm) using a syringe pump, and stirred for 4 hours to completely evaporate the organic solvent. The solution was then transferred to a dialysis bag (MWCO 3500) and dialyzed against pure water for 24 hours to remove unencapsulated drug and organic solvent. The resulting suspension was filtered through a 0.22 μm microporous membrane for sterilization.

[0048] Membrane coating (Nd-PLGA@MacM): The extracted macrophage membrane and Nd-NP were mixed at a mass ratio of membrane protein to PLGA of 1:5. Using a mini extruder (Avanti, USA), the mixture was extruded 11 times each through a 400 nm and a 200 nm polycarbonate membrane, so that the cell membrane fused on the surface of the nanoparticles to form a coating layer.

[0049] Characterization: - Particle size and Zeta potential: The hydrated particle size of Nd-PLGA@MacM was 115.52 ± 5.8 nm, the PDI was 0.124, and the Zeta potential was -6.14 ± 3.5 mV, as determined by dynamic light scattering.

[0050] - Morphology: Transmission electron microscopy revealed that the particles were spherical with a distinct core-shell structure.

[0051] - Encapsulation efficiency and drug loading: A small amount of Nd-PLGA@MacM suspension was centrifuged at high speed, and the supernatant was collected. The free drug content was determined by HPLC. The encapsulation efficiency was calculated to be (62.5±3.1)%, and the drug loading was (8.2±0.4)%.

[0052] - In vitro release: Nd-PLGA@MacM was placed in PBS (pH 7.4) and incubated with shaking at 37°C. The supernatant was collected by centrifugation at predetermined time points, and the amount of drug released was determined by HPLC to plot the drug release curve.

[0053] In vivo pharmacodynamics and safety evaluation Animal Model and Grouping: A mouse IDD model was constructed according to the method in Example 1. The mice were randomly divided into four groups (n=8): PBS control group, free natriuretic peptide group (2 mg / kg), Nera-NP group (2 mg / kg), and Nera@MM-NP group (2 mg / kg). Administration was via tail vein injection twice weekly for four weeks.

[0054] Therapeutic effect evaluation: -MRI: After the last administration, T2-weighted MRI scans of the lumbar spine of small animals were performed to measure the intervertebral disc height index and perform Pfirrmann classification.

[0055] - Histology: The intervertebral discs were removed from the euthanized animals, and after paraffin sectioning, H&E staining and Safranin O-Fixed Green staining were performed for histological scoring.

[0056] - Molecular biology detection: The expression of MMT markers (α-SMA), fibrosis markers (Collagen I), and matrix metabolism markers (Collagen II) in intervertebral disc tissue was detected by Western blotting and infusion (WB) and infusion (IF).

[0057] Safety assessment: -In vivo toxicity: The toxicity of different drug formulations to nucleus pulposus cells was detected using a CCK8 assay kit; -In vivo toxicity: After treatment, blood samples were collected from mice for routine blood tests.

[0058] Experimental conclusion: To obtain the cellular composition characteristics of clinically degenerated intervertebral discs and normal intervertebral disc tissues, single-cell transcriptome sequencing was first performed on 5 clinically degenerated samples and 5 normal control samples. Figure 1 a). UMAP visualization analysis showed significant infiltration of myofibroblasts and macrophages in the degenerated intervertebral disc tissue. Figure 1b). Further analysis using Western blotting (WB), immunofluorescence (IF), immunohistochemistry (IHC), and Sirius red staining consistently revealed significantly increased expression of fibrosis markers α-SMA and Collagen I in both clinical and animal tissue samples, along with a marked increase in type I / III collagen deposition. Figure 1 (cg) confirmed that the degree of intervertebral disc fibrosis and the level of macrophage infiltration both increased during the IDD process, and the two were significantly positively correlated.

[0059] In-depth analysis of single-cell sequencing data revealed that the number of infiltrating macrophages and myofibroblasts significantly increased with the progression of degeneration. Figure 2 a). To simulate this process in vivo, a mouse model of IDD induced by abnormal mechanical stress was established. This model successfully reproduced the core pathological features of IDD and exhibited a significant fibrotic phenotype. Figure 2 bd). In in vitro experiments, abnormal mechanical stress stimulation can lead to a significant increase in the colocalization of CD68 and α-SMA in the macrophage cell line Raw 264.7 (bd). Figure 2 Using a Cx3cr1-CreERT2; Rosa26-tdTomato lineage-traced mouse model, flow cytometry analysis revealed a significant increase in the proportion of tdTomato+α-SMA+ double-positive cells in the intervertebral discs of IDD model mice. Figure 2 This result was further validated at the tissue immunofluorescence level (gh). Figure 2 The above evidence clearly indicates that macrophage-myofibroblast transformation (MMT) occurs during the IDD process, and that MMT plays a crucial role in the development of IDD.

[0060] To investigate the upstream regulatory mechanism of MMT, the expression of PDE4 family members in single-cell data was analyzed, and it was found that PDE4B was most significantly upregulated in macrophages. Figure 3 ab). Validated by clinical samples and animal models, PDE4B expression was significantly increased in degenerated intervertebral disc tissue, but showed no significant change in nucleus pulposus cells. Figure 3 c, e). Flow cytometry sorting of Cx3cr1 lineage cells in IDD mice and Western blot analysis revealed significantly increased PDE4B expression in macrophages of the IDD group. Raw 264.7 cell line also showed the same trend after mechanical stress stimulation. Figure 3 d, e). To clarify the function of PDE4B, a macrophage-specific PDE4B knockout mouse model (PDE4BmacKO) was constructed. Experimental results showed that in the IDD model, PDE4B deficiency significantly reduced the co-localization level of tdTomato and α-SMA. Figure 3 fg) and the proportion of double-positive cells ( Figure 3 hi), and effectively inhibited the expression of fibrosis markers ( Figure 3 j) and delaying IDD progress ( Figure 3 These results demonstrate that PDE4B promotes IDD by regulating the MMT process.

[0061] Nerandomilast is a highly selective PDE4B inhibitor that has completed Phase III clinical trials and been approved for marketing in the field of pulmonary fibrosis, but its mechanism of action in pulmonary fibrosis (IDD) remains unclear. This study simulated the pathological state of IDD by abnormal stress stimulation of Raw264.7 cells, followed by transcriptome sequencing (RNA-Seq) after nerandomilast intervention. The analysis showed that differentially expressed genes were significantly enriched in the cGAS-STING signaling pathway (…). Figure 4 (b, d), among which STING expression was downregulated most significantly ( Figure 4 c). This result was validated at both the protein and mRNA levels. Figure 4 Previous studies have reported that the cGAS-STING pathway can promote MMT. The results of this study show that inhibiting PDE4B can block MMT and alleviate IDD by downregulating the activation of the STING pathway.

[0062] In the transcriptome of cells treated with namisitol, in addition to the STING pathway, the RNA degradation pathway was also significantly enriched. To elucidate its downstream mechanisms, interacting proteins were screened using STING mRNA pull-down combined with mass spectrometry analysis. Figure 5 a) found that the binding difference of QKI was the largest ( Figure 5 b). Bioinformatics analysis predicts that QKI can recognize STING mRNA via the ACUAAC motif ( Figure 5 cd), and RNA pull-down experiments confirmed that QKI and STING mRNA binding was enhanced after namiturite intervention (c ... Figure 5 e). RIP-qPCR experiments further verified the direct interaction between QKI protein and STING mRNA ( Figure 5 f). As an m7G modification recognition protein, QKI can regulate mRNA stability and translation efficiency. Dual-luciferase reporter gene assays confirmed that namisitol treatment can inhibit the translation efficiency of STING (f). Figure 5 g). The above results indicate that QKI participates in the regulation of MMT by inhibiting the translation of STING mRNA through an m7G-dependent pathway.

[0063] Based on this, we further explored the upstream regulatory mechanism of QKI. Co-IP / MS experiments using namitase-treated cells and QKI as a decoy were conducted. Figure 6 a) It was found that PKA was one of the most significantly different interacting proteins. Figure 6 b). Co-IP experiments confirmed that namisulant can enhance the interaction between PKA and QKI and significantly increase the global serine / threonine phosphorylation level ( Figure 6 c). Given that PDE4B negatively regulates PKA activity through the hydrolysis of cAMP, the results of this study indicate that inhibiting PDE4B can activate PKA, thereby promoting QKI phosphorylation, enhancing its function of inhibiting STING translation in an m7G-dependent manner, and ultimately blocking MMT and intervertebral disc fibrosis.

[0064] Based on the above mechanism, a biomimetic nanodelivery system coated with a macrophage membrane was constructed for targeted delivery of namitase to macrophages. The fabrication process of this system is as follows: Figure 7 As shown in figure a, its particle size was characterized as 115.52 ± 5.8 nm, its potential as -6.14 ± 3.5 mV, and its PDI as 0.124 ( ). Figure 7 b) Membrane protein composition results indicate that Nd-PLGA@MacM effectively retains macrophage membrane surface antigen components. Figure 7 c) Transmission electron microscopy results show that Nd-PLGA@MacM exhibits a near-spherical shape. Figure 7 (d) In vitro drug release results showed that Nd-PLGA@MacM maintained 80% drug release on day 15, demonstrating a significant sustained-release effect in vitro. In vivo imaging in small animals also showed that by week 4, some fluorescent material remained at the intervertebral disc acupuncture site in the Nd-PLGA@MacM group, while the fluorescent material in the drug-only group disappeared by week 2. These experimental results indicate that this nanodelivery method has good sustained-release capability in vivo. Figure 7 e.g., through in vivo animal experiments, we found that this biomimetic nanodelivery system can significantly inhibit IDD (…). Figure 7 The nanomaterial (Nd-PLGA@MacM) exhibited good in vivo therapeutic effects. Further investigation into the safety of this nanomaterial revealed that the CCK8 assay showed extremely low toxicity to macrophages, and mouse blood routine tests demonstrated good biocompatibility. These results collectively indicate that the Nd-PLGA@MacM targeted delivery system provides an effective tool for precise intervention in intradiscal micro-thrombotic manipulation (MMT).

[0065] Finally, it should be noted that the above embodiments and experimental examples are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. The application of namisulant in the preparation of drugs for treating intervertebral disc fibrosis by targeting and inhibiting macrophage-fibroblast transformation is characterized by, The drug uses namisitol as the active ingredient and is formulated into a pharmaceutically acceptable dosage form using pharmaceutically acceptable excipients.

2. A drug for treating intervertebral disc fibrosis, characterized in that, The drug is namisitol and a pharmaceutically acceptable carrier or excipient.

3. The drug according to claim 2, characterized in that, The pharmaceutically acceptable carrier is selected from any one of solvents, emulsifiers, suspending agents, disintegrants, binders, excipients, stabilizers, diluents, gelling agents, preservatives, lubricants, and surfactants.

4. The drug according to claim 2, characterized in that, The excipients are selected from at least one of hydroxypropyl methylcellulose, hydroxypropyl cellulose, povidone, polyethylene glycol, ethyl cellulose, liposomes, methacrylic acid copolymer, polyvinyl acetate, carboxymethyl ethyl cellulose, carboxymethyl cellulose phthalate, hydroxypropyl methyl cellulose phthalate, hydroxypropyl methyl cellulose acetate succinate, polyacrylic acid resin, polyvinyl carboxylate, alginate, carrageenan, carboxyacetic acid lactone, gum, polyvinyl alcohol, pregelatinized starch, cross-linked starch, sodium carboxymethyl starch, dextrin, polyethylene oxide, chitosan, chitosan, ion exchange resin, and collagen.

5. The drug according to claim 2, characterized in that, The drug is in a pharmaceutically acceptable dosage form, selected from any one of tablets, capsules, injections, granules, suspensions, and solutions.

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