Application of prussian blue in preparation of medicine for relieving or treating pain

Abstract

The invention discloses application of Prussian blue in preparation of a medicine for relieving or treating pain. The invention provides an application of Prussian blue or a combination of Prussian blue and an Smoc2 agonist and / or a P2X7R inhibitor in preparation of a medicine for relieving or treating pain. According to the invention, the ability of PBNP to up-regulate back root ganglion fibroblasts to secrete SMOC2 is determined through biological experiments, and mechanical pain perception can be regulated and postoperative pain can be relieved; the strategy not only promotes in-situ secretion of natural analgesic substances through single administration to provide lasting pain relief, but also effectively avoids complex biological effects of drugs on proteins / ions, contributes to reduction of adverse reactions, and overcomes the bottlenecks of drug addiction, tolerance and the like. The invention provides a strategy for activating endogenous analgesic substances based on bioactive nanoparticles of Prussian blue nanoparticles for pain treatment for the first time, and has wide application prospects and high clinical application value.

Description

Technical Field

[0001] The invention relates to application of Prussian blue in preparing medicine for relieving or treating pain, and belongs to the technical field of biomedicine. Background Art

[0002] Postoperative pain is an inevitable consequence for surgical patients, and about 20%-60% of patients still face the problem of inadequate postoperative pain control. This poor control is a significant risk factor for the development of chronic pain, thus slowing down recovery from surgery. This situation greatly reduces the quality of life of patients and increases the burden on the medical system. At present, opioid analgesics (such as morphine, pethidine, etc.) widely used in clinical practice achieve analgesic effects by inhibiting the pain area of ​​the cerebral cortex of the central nervous system, rather than blocking pain signals from the source of pain. They are prone to cause plastic changes in neural circuits and have disadvantages such as controllability, easy off-target, strong drug tolerance, strong addiction, and short analgesia time. Therefore, based on the basic mechanism of pain, it is urgent to develop new strategies and technologies for non-drug-resistant, non-addictive, and long-acting analgesia, which has important scientific significance and clinical value.

[0003] Nanotechnology offers the potential for precise targeting and controlled drug release in medicine, which is expected to improve pain management and bring new ideas to the development of new analgesics. By adjusting the structure, size and surface properties of nanoparticles, the pharmacokinetics and biodistribution of existing drug ingredients can be changed, thereby improving drug delivery and therapeutic effects (such as nerve growth inhibitors and functional substances). For example, for metastatic bone cancer pain caused by tumor-nerve interaction, researchers designed and constructed magnesium aluminum double hydroxide supramolecular materials (LDHs) loaded with nerve growth inhibitor AZ-23, and modified the bone targeting molecule sodium propidronate (ALD) on its surface to obtain LDH / AZ-ALD therapeutic agents. LDH / AZ-ALD therapeutic agents cut off the signal communication between tumors and nerves, successfully inhibited bone cancer pain, and prevented the malignant progression of tumors, providing new ideas for the clinical treatment of bone tumors and bone cancer pain. In order to achieve effective ionic analgesia, researchers constructed MgB2 nanomaterials, which can dynamically generate two-dimensional BH nanosheets with boron-like structures in situ in a physiological environment. By utilizing the interaction between BH delocalized π electrons and sodium and potassium cations, the distribution and transmembrane diffusion of ions around nerves are regulated, and the conductivity of membrane ion channels is changed, thereby significantly increasing the threshold of nerve action potentials, inhibiting nerve excitability, and ultimately achieving long-term, high-intensity relief of intractable pain. However, current nanomedicine analgesia focuses on precise targeted drug delivery, which is essentially a treatment of existing drugs and does not fundamentally solve the problem of analgesics. Bioactive nanomaterials can be phagocytosed by cells, indicating that they have the potential to stay in the target site for a long enough time. If bioactive nanomaterials can act as catalysts to upregulate the secretion of natural analgesics and provide long-term pain relief at the molecular level, this will be a breakthrough in the development of new non-opioid analgesic candidates.

[0004] Peripheral dorsal root ganglia (DRG) are key components of pain signaling and pain management and have great potential in the treatment of pain. In addition to DRG neurons, non-neuronal cells such as satellite glial cells and immune cells also significantly affect the regulation of pain signals. The role of DRG fibroblasts in the development and progression of pain is becoming increasingly clear. New therapeutic approaches targeting DRG and peripheral systems remain a hot topic for pain management. Secreted modular calcium-binding protein 2 (Smoc2) is a member of the extracellular calcium-binding protein family and is classified as a modular extracellular protein known for secreting acidic and cysteine-rich molecules. This protein is widely expressed in multiple adult mouse tissues, such as ovaries, heart, muscles, and spleen. Recent studies have shown that Smoc2 is mainly expressed in fibroblasts within DRG. Single-cell transcriptome analysis of DRGs demonstrated the importance of Smoc2 in the development of pain. Functionally, Smoc2 inhibits the coupled activation of adjacent DRG neurons induced by noxious mechanical stimulation by blocking the function of satellite glial P2X7 receptors (P2X7R). This finding provides the possibility that Smoc2 agonists or P2X7R inhibitors could be potential targets for pain relief.

[0005] Prussian blue (PB) is an antidote approved by the U.S. Food and Drug Administration for the clinical treatment of thallium and other radioactive poisoning. When its size reaches the nanoscale, PBNP not only exhibits the unique properties of nanomaterials, but also enhances its intrinsic bioactivity. In previous studies, we found that Prussian blue nanoparticles (PBNP) can be phagocytosed / adhered to cells to maintain normal microenvironmental homeostasis or correct neurological dysfunction, thereby achieving the prevention and repair of tissue and organ damage. PBNP has the advantages of long-term retention, homeostasis maintenance / disorder correction, rich intrinsic bioactivity and controllable structure. However, the role and mechanism of PBNP in pain are still unclear. Summary of the invention

[0006] The purpose of the present invention is to address the shortcomings of existing drugs for relieving or treating pain, and for the first time propose a strategy based on Prussian blue nanoparticles (PBNP) to activate endogenous analgesics for pain treatment.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The first aspect of the present invention provides the use of Prussian blue in preparing a medicament for relieving or treating pain.

[0009] Furthermore, the pain includes mechanical pain.

[0010] Furthermore, the mechanical pain includes postoperative pain.

[0011] Furthermore, the medicine comprises an active ingredient and a pharmaceutically acceptable carrier and / or excipient, and the active ingredient is Prussian blue or its nanoparticles.

[0012] In some embodiments of the present invention, the Prussian blue nanoparticles are prepared by a hydrothermal method under acidic conditions using potassium ferrocyanide and polyvinyl pyrrolidone as raw materials.

[0013] Furthermore, the dosage form of the drug includes tablets, powders, suspensions, granules, capsules, injections, sprays, solutions, enemas, emulsions, films, suppositories, patches, nasal drops or pills.

[0014] Furthermore, the drug is administered orally or parenterally.

[0015] The second aspect of the present invention provides the use of Prussian blue in preparing a reagent for promoting the secretion of SMOC2 in fibroblasts.

[0016] The third aspect of the present invention provides the use of Prussian blue combined with a Smoc2 agonist and / or a P2X7R inhibitor in the preparation of a medicament for relieving or treating pain.

[0017] In a fourth aspect of the present invention, a pharmaceutical composition is provided, comprising a first active ingredient and a second active ingredient, wherein the first active ingredient is Prussian blue or its nanoparticles, and the second active ingredient is a Smoc2 agonist and / or a P2X7R inhibitor. Since the present invention has shown through biological experiments that PBNP regulates mechanical pain perception and relieves postoperative pain by upregulating the ability of dorsal root ganglion fibroblasts to secrete SMOC2, and Smoc2 agonists or P2X7R inhibitors are potential targets for relieving pain, the combined drug strategy is expected to provide a new pain treatment solution.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention first determined the ability of PBNP to upregulate the secretion of SMOC2 by dorsal root ganglion fibroblasts, which helps to regulate mechanical pain perception and relieve postoperative pain ( Figure 1 ); This strategy not only provides lasting pain relief by promoting the in situ secretion of natural analgesics through a single administration, but also effectively avoids the complex biological effects of drugs on proteins / ions, helps to reduce the occurrence of adverse reactions, and overcomes bottlenecks such as drug addiction and tolerance; This research strategy shows broad application prospects in pain management and even neuromodulation, and provides a new solution for the treatment of pain-related diseases; It is expected to achieve more effective pain control and improve the quality of life of patients with pain. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The principle of the bioactive nanoparticles (PBNP) provided by the present invention for upregulating the secretion strategy of natural analgesics for pain treatment: Taking Prussian blue nanoparticles (PBNP) as an example, the present invention proves through behavioral, histopathological, transcriptomics and viral intervention methods that PBNP can affect the activity of the extracellular matrix region, upregulate the secretion of SMOC2 in dorsal root ganglion fibroblasts, regulate mechanical pain perception, and relieve postoperative pain.

[0021] Figure 2 Preparation and characterization of PBNPs: (A) Schematic diagram of PBNP synthesis; (B) SEM, (C) TEM images, (D) elemental mapping, (E) TG-DSC curves, (F) Raman spectrum of PBNP, (G) FTIR spectrum, (H) XPS analysis, (I) Mössbauer spectrum, and (J) X-ray diffraction spectrum of PBNP.

[0022] Figure 3 Intraplantar injection of 75 μg / mL PBNP relieved postoperative mechanical pain: (A) Behavioral test design; After 3 days of acclimatization, the baseline mechanical and thermal pain of mice was measured; The incision pain model was established and PBNP was injected into the plantar area. The paw withdrawal frequency of mice was measured at 2 hours, 5 hours and 1-6 days after surgery. Three concentrations of PBNP were tested (7.5, 75 and 750 μg / mL); (B) Paw withdrawal frequency of 0.07 g von Frey filaments, n = 10 per group, two-way ANOVA with Bonferroni post hoc test, *p < 0.05, incision group vs. incision + 75 μg / mL PBNP group; (C) Area under the curve of B, one-way ANOVA with Bonferroni post hoc test, *p < 0.05, incision group vs. incision + 75 μg / mL PBNP group; (D) Paw withdrawal frequency of 0.4 g von Frey filaments, n=10 per group, two-way ANOVA followed by Bonferroni post hoc test, *p<0.05, incision group vs. incision + 75 μg / mL PBNP group; (E) Area under the curve of D, one-way ANOVA followed by Bonferroni post hoc test, **p<0.01, incision group vs. incision + 75 μg / mL PBNP group; Hargreaves test paw withdrawal latency on the ipsilateral (F) or contralateral (G) side, n=10 per group, two-way ANOVA followed by Bonferroni post hoc test; (H) Eosin staining of DRG and PBNP on day 6 after incision and injection, scale bar: 25 μm.

[0023] Figure 4Intraplantar injection of 75 μg / mL PBNP reduces pain-like movement types in the 3D motion capture system: (A) Workflow of the 3D motion capture and learning framework. Two groups of mice (incision or incision + 75 μg / mL PBNP) were tested 1 day after incision surgery and PBNP injection. 3D motion was captured for each mouse and the videos were then sent for reconstruction and computation for further analysis; (B) Diagram of the 3D motion system. Two cameras recorded the top and side views of the mouse; (C) Coordinate diagram of 16 body parts of the mouse tracked in each camera view; (D) Z-score clustering of 40 identified movement types in the incision or incision + 75 μg / mL PBNP groups, n = 10 per group, blue arrows indicate pain-like behaviors; (E) Comparison of movement type scores in the incision (black) or incision + 75 μg / mL PBNP groups (red). A total of 12 movement types were significantly different between the two groups. Blue indicates incision + 75 μg / mL compared to the incision group. The scores of the PBNP group were decreased, while those of the incision + 75 μg / mL PBCP group were increased in red; M1: firing; M11: wiping face; M12: freezing; M20: wiping face; M21: firing; M22: standing and sniffing; M23: looking good; M24: freezing; M27: climbing; M28: hunching; M34: looking up; M38: sniffing; Two-way ANOVA followed by Bonferroni post hoc test *p<0.05, **p<0.01, ***p<0.001 for the incision group compared with the incision + 75 μg / mL PBNP group; (F) Top and side view reconstructions of pain-related movements (wiping face: M11 and M20; freezing: M12 and M24); (G) Dimensional representation of the two groups (black: incision group; red: incision + 75 μg / mL PBNP group); points in 3D space decreased in size from 40 motion scores; (H) x-axis projection values ​​of G; Student's t-test, t=3.506, **p<0.01, incision group vs. incision+75 μg / mL PBNP group.

[0024] Figure 5DRG RNA sequencing showed that affecting DRG extracellular matrix activity was the reason for PBNP analgesia: (A) DRG RNA sequencing process, 4-5 lumbar DRGs from the incision or incision + 75μg / mL PBNP group were collected 1d after incision and injection; 8 DRGs from 4 mice were collected as one sample; three samples from each group were sent for further sequencing and analysis; (B); Sample correlation and clustering of each sample; (C); Principal component analysis (PCA) of each sample; Red dots represent the incision group; Blue dots represent the incision + 75μg / mL PBNP group; Red dots and blue dots are separate; (D) Volcano plot of differentially expressed genes (DEGs); Red dots represent upregulated genes; Blue dots represent downregulated genes; Gray dots represent genes with no difference; (E) Counts of DEGs; Compared with the incision group, incision + 75μg / mL In the PBNP group, 278 were upregulated and 14 were downregulated; (F) Cluster diagram of DEGs; (G) Gene Ontology (GO) enrichment of DEGs; a total of five terms were related to the extracellular matrix (ECM) across biological processes, cellular components, and molecular functions (red dashed boxes); (H) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis; ECM receptor interactions were the top 2 pathways in the enriched terms (red dashed boxes).

[0025] Figure 6DRG Smoc2 enhancement after intraplantar injection of 75 μg / mL PBNP: (A) Workflow for potential target validation; (B) RT-qPCR of ECM-related genes; n = 3 per group; Student's t test, *Smoc2 (t = 4.186), Col6a1 (t = 0.3612), Col6a2 (t = 1.121), Col8a2 (t = 1.190), Col14a1 (t = 0.4849), Apod (t = 0.8170), Ogn (t = 0.0543), Ccl 11(t=0.5729), Mmp2(t=0.6127), *p<0.05, incision group vs. incision+75μg / mL PBNP group; (C) Western blot of Smoc2 (53kDa) in lumbar DRG 1d after injection of different concentrations of PBNP; (D) Relative value in C; n=3 for each group; one-way ANOVA followed by Bonferroni post hoc test *p<0.05, incision group vs. incision+75μg / mL PBNP group. (E) Immunofluorescence of Smoc2 (green) in lumbar DRGs 1 day after injection of different concentrations of PBNP; (F) Relative percentage of fluorescence intensity in E; n = 5 per group; one-way ANOVA followed by Bonferroni post hoc test *p < 0.05, ***p < 0.001, incision group compared with incision + 75 μg / mL PBNP group; scale bar: 50 μm; (G) Colocalization of fibroblast markers PDGFRα (green) and Smoc2 (red); scale bar: 25 μm.

[0026] Figure 7Figure PBNP enhances Smoc2 secretion in L929 and DRG fibroblasts in vitro: (A) Immunofluorescence of L929 fibroblast cell line; L929 was stimulated by adeno-associated virus-mediated fibroblast-specific protein 1 with a Flag tag; blue: DAPI labeled nuclei; green: anti-Flag labeled fibroblasts; (B) Concentration of Smoc2 in the culture medium after incubation with solution alone, 7.5 or 75 μg / mL PBNP for 5 h or 24 h; n=4 per group per time point; one-way ANOVA followed by Bonferroni post hoc test *p<0.05 for solution alone group vs. 75 μg / mL PBNP group; (C) Relative mRNA expression of L929 after incubation with 7.5 or 75 μg / mL PBNP solution alone for 24 h; n=3 per group; one-way ANOVA followed by Bonferroni post hoc test **p<0.05 for solution alone group vs. 75 μg / mL Compared with the PBNP group, p<0.01; (D-E) CCK-8 assay for the survival rate (D) and proliferation rate (E) of L929 cells incubated with PBNP; n=4 per group; (F) Immunofluorescence of DRG primary fibroblasts; DRG primary fibroblasts were affected by adeno-associated virus promoted by fibroblast-specific protein 1 with a Flag tag; blue: DAPI labeled nuclei; green: anti-Flag labeled fibroblasts. (G) Concentration of Smoc2 in the culture medium after incubation with solution alone, 7.5 or 75 μG / mL PBNP for 5 or 24 hours; n=4 per group at each time point; one-way ANOVA followed by Bonferroni post hoc test **p<0.01,

[0027] ***p<0.001, difference between the solution group and the 7.5 or 75 μg / mL PBNP group at each time point; (H) Relative mRNA expression of DRG primary fibroblasts after incubation with 7.5 or 75 μg / mL PBNP solution for 24 hours; n=3 per group; one-way ANOVA followed by Bonferroni post hoc test **p<0.01 between the solution group and the 75 μg / mL PBNP group; (IJ) CCK-8 assay to detect the survival rate (I) and proliferation rate (J) of DRG primary fibroblasts incubated with PBNP; n=4 per group.

[0028] Figure 8Knockdown of DRG Smoc2 abolished the analgesic effect of PBNP: (A) Workflow of DRG microinjection and behavioral testing; mice were acclimated for 3 days and baseline of mechanical pain was measured; then L4-5 was microinjected with Smoc2 shRNA or scrambled RNA; after 3 weeks of stable viral expression, incision surgery was performed and 75 μg / mL PBNP was injected; von Frey test and 3D motion capture were measured; (B, E) Validation of the effect of Smoc2 shRNA, respectively (B) Immunofluorescence of DRG Smoc2 after microinjection; (C) Relative percentage of fluorescence intensity in B; scrambled RNA group n=6, shRNA group n=5; Student's t-test, t=3.090, *p<0.05, scrambled RNA group vs. shRNA group; (D) DRG after microinjection Western blot analysis of Smoc2; (E) relative values ​​in D; n = 4 per group; Student's t-test, t = 3.543, *p < 0.05, scrambled RNA group vs. shRNA group; (F) claw pulling frequency was 0.07 g von Frey filaments; scrambled RNA group n = 10, shRNA group n = 11; two-way ANOVA followed by Bonferroni post hoc test, *p < 0.05, from 5 hours to 6 days, scrambled RNA group vs. shRNA group; (G) area under the curve of F one-way ANOVA and Bonferroni post hoc test, **p < 0.01, scrambled RNA group vs. shRNA group; (H) claw pulling frequency was 0.4 g von Frey filaments; n is the same as F; two-way ANOVA followed by Bonferroni post hoc test, *p<0.05, from 5h to 6d, scramble RNA group compared with shRNA group; (I) H area under the curve of one-way ANOVA and Bonferroni post hoc test, **p<0.01, scramble RNA group compared with shRNA group; (J) Comparison of movement type scores between scramble RNA group and shRNA group; scramble RNA group n=12, shRNA group n=11; There were 4 movement types with significant differences between the two groups; compared with the incision group, blue indicates a decrease in the score of the incision + 75μg / mL PBNP group, while red indicates an increase in the score of the incision + 75μg / mL PBCP group; M7: face wiping; M9: freezing; M13: sniffing; M18: face wiping; two-way ANOVA followed by Bonferroni post hoc test *p<0.01, scramble RNA group compared with shRNA group.05; (K) Z-score clustering of 40 identified movement types in the scramble RNA group or shRNA group; n is the same as in J; blue arrows indicate pain-like behaviors; (L) Top and side view reconstructions of pain-related movements (wiping face: M7 and M18; freezing: M9); (M) Dimensional representation of the two groups (black: scramble RNA group; red: shRNA group); points in 3D space reduced in size from the 40 movement scores; (N) x-axis projection values ​​of M; Student's t-test, t = 3.506, *p < 0.05, scramble RNA group vs. shRNA group. .

[0029] Fig. 9 The dynamic light scattering (DLS) particle size analysis results of PBNP in normal saline.

[0030] Fig.10 is the Zeta potential of PBNP in physiological saline.

[0031] Fig.11 The absorption spectrum of PBNP in salt water detected by UV and visible spectrophotometer;

[0032] Fig.12 is the Brunauer-Emmett-Teller measurement result of PBNP;

[0033] Fig.13 Figure 2 Contralateral paw withdrawal threshold after incision and PBNP injection: (A) Paw withdrawal frequency was 0.07 g von Frey filament; n=10 per group; (B) Paw withdrawal frequency was 0.4 g von Frey filament; n=10 per group.

[0034] Fig.14 Eosin staining images of (A) skin and (B) sciatic nerve on day 6 after incision and injection (scale bar: 50 μm);

[0035] Fig.15 Figure 2 shows the contralateral paw withdrawal threshold after shRNA or scRNA microinjection and incision + PBNP injection: (A) the paw withdrawal frequency is 0.07 g von Frey filament; n = 10 for the scrambled RNA group and n = 11 for the shRNA group; (B) the paw withdrawal frequency is 0.4 g von Frey filament; n is the same as in A.

[0036] Fig.16 Uncropped Western blot staining: (A) relative expression of DRG Smoc2 after incision and PBNP injection; (B) relative expression of DRG-Smoc2 after scRNA or shRNA microinjection. DETAILED DESCRIPTION

[0037] In order to make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0038] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0039] Some of the materials, instruments and methods designed in the following embodiments are as follows:

[0040] Raw materials for the preparation of PBNPs:

[0041] Analytical grade potassium ferricyanide (K3[Fe(CN)6]), polyvinylpyrrolidone (PVP, K30) and hydrochloric acid (HCl, 36.0%) were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0042] Preparation of PBNP:

[0043] K3[Fe(CN)6] (396 mg) and PVP (5 g) were dissolved and mixed in 1 M HCl (40 mL) at room temperature. After stirring until clear, the solution was transferred to an oven and heated at 80 °C for 20 h. The final product was collected by centrifugation, washed three times with deionized water, and then dispersed in saline or lyophilized and stored at 4 °C.

[0044] Characterization of PBNPs:

[0045] The synthesized PBNPs were freeze-dried to obtain PBNP powders. After being dispersed in anhydrous ethanol, the PBNP dispersion was evenly dripped on the support film and loaded on a copper mesh for TEM analysis. Transmission electron microscopy (TEM) and high-resolution TEM images were obtained using a JEOL-2100FTEM (Tokyo, Japan). SEM requires that the PBNP powders be fixed on the sample stage using a conductive adhesive before the sample morphology is evaluated using a Zeiss Gemini 300 scanning electron microscope (SEM, Oberkochen, Germany). Elemental mapping was analyzed by Berlin Bruker Nano Co., Ltd. (Esprit 2.x), Germany. The hydrodynamic diameter and Zeta potential were determined by dynamic light scattering (DLS, Malvern Zetasizer Nano-Z, Malvern, UK). Ultraviolet-visible (UV-vis) spectra were recorded using a PerkinElmer Lambda 750 spectrophotometer (Waltham, Massachusetts, USA). Thermogravimetric differential scanning calorimetry (TG-DSC) was performed using a Hitachi STA200 to simultaneously measure thermogravimetric change (TG), thermal effect information (DSC), and differential thermogravimetric (DTG) parameters by temperature control in various gas environments. Raman spectroscopy was performed using a Renishaw Raman system (Renishaw inVia, London, UK). PBNP powder was directly mixed and ground with potassium bromide powder at a ratio of 1 mg / 100 mg, and the mixture was then pressed into a transparent sheet for Fourier transform infrared spectroscopy (FTIR). FTIR spectra were characterized using a Fourier transform infrared spectrometer (Nicolet iS20, Thermo Fisher Scientific). The surface valence state of iron was evaluated by X-ray photoelectron spectroscopy (XPS, Thermo Scientific K-Alpha, New York, NY, USA). The Mössbauer spectra of PBNP powder measured by a Mössbauer spectrometer (MFD-500AV-02, Topologic Systems) provide detailed information on its microstructure, electronics, and magnetic properties. The PBNP powders were arranged as randomly as possible to obtain X-ray diffraction (XRD) patterns on an X-ray diffractometer (Bruker D8 Advance, Karlsruhe, Germany) to ensure that the X-rays can penetrate and interact with the target crystal structure. The BET test determined the specific surface area and pore structure of the material by calculating the relationship between the amount of gas adsorbed and the sample mass on a Gemini 2360 instrument (Micromeritics, Norcross, GA).

[0046] Construction of postoperative pain model:

[0047] All animal surgeries were performed in strict accordance with international ethical guidelines and the guidelines of the Shanghai Sixth People's Hospital on the care and use of laboratory animals. The experimental protocol followed the principles and recommendations of the International Association for the Study of Pain (IASP) on the ethical use of laboratory animals. C57BL / 6 mice weighing between 22 and 25 g were selected for the study. They were housed with ad libitum access to water and standard laboratory food pellets. Before the start of the experiment, the mice were acclimated to their environment, which was maintained at a temperature of 24 °C, 60% humidity, and a 12-h light / dark cycle for 3 days. The construction procedure of the postoperative pain model (plantar incision surgery) was as follows: Mice were anesthetized using isoflurane and received oxygen at a rate of 0.8–1.0 liters per minute via nasal cannula. After disinfecting the left hind plantar area with iodine, a 0.5-cm longitudinal incision was carefully made using a surgical blade starting from the edge of the heel and extending 0.2 cm toward the toes. During the incision, special attention was paid to maintaining the integrity of the muscle when incising the plantar skin and fascia. Gentle pressure was applied to control any bleeding, and the skin was closed using 4-0 sutures. The wound was covered with bacitracin ointment. After the surgery, the mice were allowed to wake up and then returned to their cages to rest. Usually, the wound heals well within 5 to 7 days. In all experiments, the contralateral paw was used as a control. In the case of the control group mice, they were anesthetized but no surgical intervention was performed.

[0048] DRG microinjection:

[0049] Direct dorsal root ganglion (DRG) microinjections were performed using recombinant adeno-associated virus (AAV) containing Smoc2 shRNA to downregulate Smoc2 expression within the L4 / 5 DRG (scrambled RNA was used as a control). Specifically, a midline incision was made in the lower lumbar region to access the L4 and L5 articular processes, which were subsequently resected using bone forceps. Once the DRGs were exposed, the viral solution was injected into two sites within the L4 and L5 DRGs using a glass micropipette connected to a Hamilton syringe. Ten minutes after the injection, the micropipette was carefully removed. The surgical area was rinsed with sterile saline and the skin incision was closed with sutures. Once the mice were fully awake and showed no signs of abnormal behavior, they were returned to their cages.

[0050] Mouse pain behavior test:

[0051] Mechanical pain test (von Frey filaments for frequency testing): Mechanical pain testing was performed daily, starting from the day before surgery and continuing for 2 hours, 5 hours, and 1-6 days. Before each test, each mouse was placed in a small transparent plastic cage of 7.5×15×15 cm for at least 30 minutes to ensure that they reached a calm and stable state, which indicated that they had been adapted. The plantar surface of the left and right hind paws of the mice was stimulated with 0.07g and 0.4g von Frey filaments. Each filament was applied 10 times. There was at least one minute interval between each adjacent test. When the mouse showed a pain response, including foot withdrawal or foot licking, the positive effect of the foot withdrawal response could be determined. The frequency of withdrawal reactions for each mouse was recorded (positive reactions / 10×100%).

[0052] Thermal stimulation (laser heat pain test):

[0053] Before each test, mice were placed in a small transparent plastic cage of 7.5 × 15 × 15 cm for at least 30 minutes to allow them to reach a calm state and adapt to the surrounding environment. The heat source was directed to the plantar surface of the left and right soles of the mice. When the mice showed pain responses such as foot withdrawal or foot licking, the heat source was immediately turned off. The time from the start of stimulation to the cessation of the heat source was recorded for each trial, which was repeated three times. The average of these values ​​was calculated to determine the heat pain foot withdrawal latency. To ensure safety and prevent plantar burns, a 20-second observation cutoff time was set, and the minimum interval between adjacent tests was at least 10 minutes.

[0054] 3D Motion Capture and Learning Framework:

[0055] The mice were acclimated to the recording box for 10 min, and then four cameras (two on the upper side and two on the lower side) were used to record 16 anchor points of each mouse within 30 min. The 16 anchor points included nose, left ear, right ear, neck, left forelimb, right forelimb, left hindlimb, right hindlimb, left forefoot, right forefoot, left hind paw, right hind paw, back, base of tail, middle of tail, and end of tail ( Figure 4 C). After acquiring the 16 body part data, the pre-trained DeepLabCut model was used to track the 2D coordinates of each camera view. Then, the 3D skeleton of the mouse was reconstructed by fusing the 2D coordinates of the four views. BehaviorAtlas was used to decompose and segment the mouse motion. The motion segments of all mice were merged to construct a similarity matrix. Subsequently, a dimensionality reduction algorithm combining manifold approximation projection and hierarchical clustering was used for motion clustering. The motion types were then defined for further analysis.

[0056] Western blot analysis:

[0057] Bilateral L4-5 DRGs were collected, snap frozen, and homogenized in chilled SDS lysis buffer (P0013G, Beyotime). The crude homogenate was centrifuged at 12000g for 15 minutes at 4°C. The resulting supernatant was collected and any pellets containing nuclei and debris were discarded. The protein concentration was quantified, and the samples were subsequently heated at 100°C for 15 minutes and loaded onto SDS-PAGE gels. After electrophoresis, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes (IPVH00010, Immobilon-P). The PVDF membrane was blocked overnight at 4°C with 1% bovine serum albumin (BSA) solution and then incubated with mouse anti-Smoc2 (1:500, sc-376104, Santa Cruz Biotechnology) antibody at 4°C overnight under gentle agitation. GAPDH (1:3000, ZB15004-HRP-100, Servicebio) or Actin (1:50000, AB49900, Abcam) was used as a loading control. After washing the membrane, it was further incubated with horseradish peroxidase-conjugated goat anti-mouse secondary antibody (1:20007076S, Cell Signaling Technology) for 1 h at room temperature. The blot was visualized using the ECL Plus detection system. The band density was quantified using Image J software. Supplementary Figure 8 Uncropped staining is listed in . Immunofluorescence. Mice were perfused with a 4% paraformaldehyde (PFA) solution in phosphate-buffered saline (PBS) and then fixed overnight in a 4% PFA solution in PBS. Afterwards, DRG tissues were harvested and initially dehydrated in a 20% sucrose solution. Once the tissue had completely settled at the bottom of the container, it was then transferred to a 30% sucrose solution for further dehydration until it was equally settled. The dissected tissues were processed to generate sections with a thickness of 20 μm for immunofluorescence staining. The sections were carefully washed with PBS and subsequently treated with immunostaining blocking buffer for 1 hour at room temperature before the staining process began. Double staining was performed using mouse anti-Smoc2 antibody (1:200, sc-376104, Santa Cruz Biotechnology) and rabbit anti-PDGFRα antibody (1:1000, 3174S, Cell Signaling Technology). Images were captured using a fluorescence microscope (Olympus, DP80) and analyzed using Image J software. To ensure the reliability of the data, five to six sections were counted from each DRG per mouse. Each treatment group consisted of 3 to 4 mice.

[0058] RNA transcriptome sequencing:

[0059] 24 hours after surgery, 4-5 dorsal root ganglia (DRG) from the lumbar spine were collected from the incision alone group or the incision + Prussian blue group. For each sample, eight DRGs from four mice were pooled together to ensure sufficient RNA for sequencing. Each experimental group consisted of three biological replicates. Total RNA purity and concentration were assessed using Nanodrop 2000, while the integrity of total RNA was assessed using Agilent2100 / LabChip GX. After successful sample testing (grade > B+), the library construction process was as follows: (1) enrichment of eukaryotic mRNA using magnetic beads containing Oligo(dT); (2) random mRNA fragmentation by adding fragmentation buffer; (3) synthesis of the first and second cDNA chains using mRNA as a template, followed by purification of the cDNA; (4) processing of the purified double-stranded cDNA, including end repair, A-tail addition, ligation with sequencing adapters, and fragment size selection using AMPure XP beads; (5) the last step involved PCR enrichment to generate a cDNA library. After the library was constructed, a Qubit 3.0 fluorescence quantitative analyzer was used for preliminary quantification to ensure a minimum concentration of 1 ng / μL. The insert fragments of the library were verified using the Qsep400 high-throughput analysis system. Once the insert fragments met the expected standards, the effective concentration of the library was accurately quantified using the Q-PCR method (effective concentration>2nM) to ensure the quality of the library. After successfully passing the quality check of the library, the Illumina NovaSeq6000 sequencing platform was used in the PE150 mode for the sequencing process. All subsequent analyses, including differential expression, hierarchical clustering, and functional annotation, were performed using the BMKCloud platform (www.biocloud.net).

[0060] RT-qPCR:

[0061] DRGs from four mice were pooled together to obtain sufficient RNA. Total RNA was extracted from the samples using the EZBioscience Tissue RNA Purification Kit PLUS (EZBioscience, USA) according to the manufacturer's instructions. RNA was then reverse transcribed into cDNA using HiScript II qRT SuperMix (Vazyme, China). TM SYBRColor PCR Master Mix (Vazyme, China) was used to perform RT-PCR on a LightCycler 480 instrument II. The relative expression level of the target gene was calculated using the 2-ΔΔCt method and normalized to the expression of gapdh according to the threshold cycle (Ct). The specific primers are listed in Table 1 below:

[0062] Table 1. RT-PCR primers

[0063]

[0064]

[0065] Cell culture:

[0066] L929 cell line: L929 cells (Fuheng Bio) were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco) containing 10% fetal bovine serum (Sigma) and 1% penicillin-streptomycin (Gibco) in a humidified incubator at 37°C and 5% CO2. Cells were seeded at a density of (2.5×104) cells / mL. For cell thawing, frozen L929 cells were quickly thawed in a 37°C water bath, transferred to a 15mL centrifuge tube containing 10mL preheated DMEM, centrifuged at 200g for 5 minutes, resuspended in fresh medium, and incubated at 37°C.5% CO2. Cells were passaged at a confluence rate of 80-90% by washing with 1×PBS (Servicebio), detaching with 0.25% trypsin-EDTA (Gibco), centrifuging at 200g for 5 minutes, and re-seeding in fresh medium.

[0067] Primary DRG fibroblasts: Mouse L4-5 DRGs were collected in cold DMEM / F12 medium (Gibco) containing 10% fetal bovine serum (Sigma), then transferred and treated with an enzyme solution containing 5 mg / mL dispase and 1 mg / mL collagenase type I in HBSS. DRGs were dissociated and resuspended in DMEM / F12, simmered for 10 minutes and the suspension was collected. The suspension was then plated in a 5 cm diameter culture vessel for future experiments. Fibroblasts were incubated at 37°C, 95% oxygen and 5% carbon dioxide.

[0068] Statistical analysis:

[0069] All data are presented as mean ± standard error. Data were analyzed in Prism 8.0. Two-tailed, unpaired Student's t-test, one-way or two-way ANOVA were used. When ANOVA showed significant differences, relative comparisons between means were tested by Bonferroni post hoc test. p < 0.05 was considered significant.

[0070] Example 1

[0071] Preparation and characterization of PBNPs:

[0072] PBNP (PBNP) was synthesized by an improved hydrothermal method using potassium ferrocyanide, polyvinyl pyrrolidone and hydrochloric acid as raw materials. Figure 2A). Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analyses showed that PBNPs were spherical, uniform in size, and had excellent monodispersity, indicating a highly consistent morphology ( Figure 2 B, C). Dynamic light scattering (DLS) measurements showed that the hydrodynamic diameter of PBNPs was approximately 120.6 nm ( Fig. 9 ). The zeta potential of PBNP is -7.78 V, indicating that the surface of PBNP has a negative charge ( Fig.10 PBNPs exhibited strong absorbance in the first biological window, which was attributed to the electronic transition of the Fe-C≡N-Fe part of Prussian blue, with a distinct peak at 700 nm ( Fig.11 ). Elemental mapping showed that iron, potassium, carbon, nitrogen, and oxygen were uniformly distributed in PBNPs ( Figure 2 D). The elemental composition of the solid PBNPs was further verified by thermal stability measurements performed in thermogravimetric analysis (TG-DSC) curves from room temperature to 1000 °C under oxidizing, reducing, and inert atmospheres. Figure 2 E). In the Raman spectrum, the Fe(II)-CN-Fe(III) group of PBNP is at 2154 cm -1 There is a strong and sharp single band at 534cm -1 There is an obvious Fe-C vibration peak ( Figure 2 F), which is considered to be the hallmark of Prussian blue. Fourier transform infrared spectroscopy (FTIR) also showed a peak at 2088 cm 1 The absorption peak at , corresponds to the stretching vibration of -CN- in the Fe-CN-Fe bond of PBNP ( Figure 2 G). To investigate the material preparation, high-resolution XPS spectra were recorded and curve fitting revealed a peak at 707.7 eV in the Fe2p spectrum, indicating the presence of Fe(II) ( Figure 2 H). Mössbauer spectroscopy further revealed the structure of Fe4[Fe(CN)6]3 through electronic configuration and coordination ( Figure 2 I). like Figure 2 As shown in Figure 1, the comparison of X-ray diffraction with the standard card (JCPDS No. 73-0687) confirmed the single crystal structure of PBNP. The diffraction angles of 2θ = 20.52°, 29.04°, 41.42°, 46.54° and 51.20° correspond to the diffraction planes of (200), (220), (400), (420) and (422), respectively, indicating that PBNP has good crystallinity. In addition, the N2 absorption-desorption results show that the BET surface area of ​​PBNP is 98 m 2 g -1 , with an average pore size of 13.36 nm, confirming the mesoporous nature of PBNPs ( Fig.12 ).

[0073] Example 2

[0074] PBNP relieves postoperative pain caused by incisions:

[0075] This example evaluates whether intraplantar injection of PBNP can relieve postoperative pain. Three days before surgical incision, mice were acclimated to the experimental environment and baseline measurements of mechanical and thermal pain responses were obtained. Figure 3 As shown in A. Three different doses of PBNP were tested, namely 7.5μg / mL, 75μg / mL and 750μg / mL. The simple incision group was injected with 40μL PBS as a control. In the 0.07g von Frey filament frequency test, the 75μg / mL PBNP group had a significant decrease in the paw withdrawal threshold at 5 hours and 1 day after injection ( Figure 3 BC), tested at 0.4g von Free filament frequency ( Figure 3 DE), the withdrawal threshold was significantly reduced 1-3 days after injection. The response to mechanical pain remained unchanged in the incision-only group and the groups given 7.5 μg / mL and 750 μg / mL PBNP. However, the injection of PBNP did not show any improvement in thermal pain ( Figure 3 F). Contralateral behavior remained consistent across all groups ( Figure 3 G, Fig.13 AB). Eosin staining confirmed that PBNP can be transported from the skin to the DRG via the sciatic nerve ( Figure 3 G, Fig.14 AB). These results indicate that administration of 75 μg / mL PBNP can alleviate mechanical pain after incision. It should be noted that mice recovered relatively quickly from incision pain within 6 days. Therefore, the difference was mainly observed in the initial period after injection. The phenomenon of blocking only mechanical nociception but not thermal pain should be considered in further molecular target exploration. Based on the above evidence, we chose 75 μg / mL PBNP as our working concentration and day 1 as the observation time point for further experiments.

[0076] Example 3

[0077] PBNP alleviates pain-like behaviors in a 3D motion capture and learning framework:

[0078] To further confirm the analgesic effect of PBNP, this example applied a 3D motion capture and learning framework system, which has made progress in providing comprehensive animal movement in drug discovery. The activity of the incision alone or incision + 75 μg / mL PBNP group was recorded one day after surgery and injection, and then analyzed ( Figure 4 A). Four cameras ( Figure 4B), two for the upper side and two for the lower side, 16 anchor points were recorded for each mouse over a 30-min period ( Figure 4 C). Reconstruction and clustering of the recordings identified 40 movement types (Table S1) and their scores for each group ( Figure 4 Compared with the simple incision group, the scores of the incision + 75 μg / mL PBNP group in 9 types of exercise were significantly reduced, and the scores in 3 types of exercise were increased ( Figure 4 E). Among them, the two types of painful movements, face rubbing (M11, M20) and freezing (M12, M24), were significantly reduced in the PBNP injection group ( Figure 4 F). Further analysis of dimensionality reduction ( Figure 4 G) and the associated projection values ​​on the x-axis ( Figure 4 H) also showed that the movement type of the PBNP injection group was different from that of the simple incision group. 3D motion capture and learning framework systems are now hot topics and research trends in the field of behavioral detection of various disease models and species. In addition to direct pain-like movement types, it can be found that the scores of several anxiety-like behaviors such as firing (M1 and M21), climbing (M27), and hunching (M28) were reduced in the incision + 75μg / mL PBNP group. Anxiety is a strong pain-related behavior. The reduction in anxiety-like behavior suggests that PBNP injection can relieve pain. It should be mentioned that each movement type was reconstructed from video recordings. Based on these results, we believe that 3D motion capture provides another piece of evidence that the administration of 75μg / mL PBNP can relieve postoperative pain caused by incision.

[0079] Example 4

[0080] PBNP affects the activity of DRG extracellular matrix:

[0081] Since DRG is crucial for pain transmission, in order to clarify the mechanism of PBNP analgesia, this example performed a large number of RNA sequence analyses on lumbar DRG in the single incision group or the incision + 75 μg / mL PBNP group ( Figure 5 A). Both sample correlation and principal component analysis (PCA) revealed differences between the two groups ( Figure 5 BC). When the fold change threshold (FC) was ≥1.5 and the false discovery rate (FDR) was <0.05, a total of 292 genes in the incision + 75 μg / mL PBNP group were defined as differentially expressed genes (DEGs), 278 genes were upregulated, and 14 genes were downregulated compared with the simple incision group ( Figure 5DF). We then performed Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis on these genes. Notably, GO analysis highlighted five terms related to the extracellular matrix (ECM) across biological process (BP), cellular component (CC), and molecular function (MF) ( Figure 5 G). KEGG enrichment also identified ECM interactions ( Figure 5 F). The above eosin staining evidence showed that PBNPs were located around DRG neurons after injection ( Figure 3 G). Considering this collective evidence, we focused our attention on the role of the ECM of DRG in mediating the analgesic effect of PBNP. PBNP upregulated the expression of Smoc2 in DRG. Next, we validated the ECM-related DEGs and phenotypes derived from the sequence data ( Figure 6 A). Compared with the simple incision group, the Smoc2 mRNA expression in DRG of the incision + 75 μg / mL PBNP group was significantly upregulated by about 1.5 times ( Figure 6 B). Western blotting further confirmed that the Smoc2 protein level was significantly increased in the 75 μg / mL PBNP-injected group, but there was no significant change in the 7.5 μg / mL or 750 μg / mL PBNP-injected groups ( Figure 6 CD). The relative percentage of fluorescence intensity in the 75μg / mL PBNP injection group increased by about 3 times compared with the single incision group. No significant changes were observed in the 7.5μg / mL or 750μg / mL PBNP injection groups ( Figure 6 EF). Given that Smoc2 has been reported to be expressed in DRG fibroblasts21, colocalization of the fibroblast marker PDGFRα with Smoc2 was also confirmed ( Figure 6 G). A previous report by Zhang et al. (Fibroblastic SMOC2Suppresses Mechanical Nociception byInhibiting Coupled Activation of Primary Sensory Neurons) showed that fibroblast Smoc2 can suppress mechanical pain but does not affect thermal pain. Here, we observed that injection of 75 μg / mL PBNP only affected the mechanical behavior of postoperative pain but not the thermal test. The combination of RT-qPCR, western blot and immunofluorescence data suggested that PBNP may exert analgesic effects through DRG fibroblast Smoc2. The molecular and nociceptive phenotypes are consistent with the above reports. Collectively, these findings suggest that DRG fibroblast Smoc2 is a potential key molecular target for PBNP to mediate its analgesic effects.

[0082] Example 5

[0083] PBNP increases the secretion of Smoc2 in fibroblasts:

[0084] Since Smoc2 is a secreted protein, this example examined whether PBNPs can enhance Smoc2 secretion in fibroblasts in vitro. Figure 7 AE) and DRG primary fibroblasts ( Figure 7 FJ). In the L929 cell line, it was observed that fibroblast-specific protein (fsp)-promoted AAV labeled the majority of cells ( Figure 7 A). Notably, incubation with 75 μg / mL PBNP for 5 h resulted in a significant upregulation of Smoc2 concentration in the culture medium. However, no significant changes were observed after 24 h of incubation ( Figure 7 B). The relative mRNA expression level of Smoc2 in the 75 μg / mL PBNP group also increased ( Figure 7 C). Similar results were observed in the 7.5 μg / mL PBNP incubation group ( Figure 7 BC). CCK-8 assay showed that 75 μg / mL PBNP did not affect the cell viability of L929 ( Figure 7 D) and proliferation ( Figure 7 E). Further experiments were performed using primary DRG fibroblast cultures as described previously and incubated with PBNPs. These primary fibroblasts can also be labeled with fsp ( Figure 7 F). Interestingly, incubation with 7.5 μg / mL PBNPs resulted in increased Smoc2 concentrations in the culture medium both after 5 and 24 h, whereas 75 μg / mL PBNP-incubation induced higher concentrations of Smoc2 only after 5 h, but not after 24 h ( Figure 7 G). However, the relative mRNA expression level of Smoc2 was only upregulated in the 75 μg / mL PBNP group ( Figure 7 H). According to CCK-8 assay, 75 μg / mL PBNP did not affect the cell viability of DRG primary fibroblasts ( Figure 7 I) and proliferation ( Figure 7J). In vitro data suggest that PBNPs can enhance Smoc2 secretion from fibroblasts without affecting cell growth. The observed changes in absolute Smoc2 concentrations between the L929 cell line and primary DRG fibroblasts may be due to differences in their density and physiological state. Degradation of Smoc2 in the culture medium may be the reason why the differences observed at the 24-hour time point were less pronounced. In addition, the in vitro environment of fibroblasts may not fully replicate natural conditions. However, these experiments provide convincing evidence that PBNPs can stimulate Smoc2 secretion from fibroblasts, potentially contributing to their analgesic effects.

[0085] Example 6

[0086] Knockdown of DRG Smoc2 abolished the analgesic effect of PBNP:

[0087] To further investigate whether Smoc2 is an important gene for the analgesic effect of PBNP on postoperative pain, we microinjected Smoc2 shRNA and control virus into DRG to knock down the expression of DRG-Smoc2. Once the viral expression was stable, we established the model and subsequently injected 75 μg / mL PBNP into the plantar, followed by a series of behavioral tests ( Figure 8 A). Immunofluorescence results showed that the relative percentage of fluorescence intensity of DRG fibroblasts injected with Smoc2 shRNA decreased by nearly 10% compared with the control virus injection group ( Figure 8 BC). Western blotting showed that Smoc2 shRNA virus reduced the Smoc2 protein content in DRG by about 50% ( Figure 8 DE), uncropped Western blot staining analysis results as shown Fig.16 Compared with mice given control virus on the same side, mice treated with Smoc2 shRNA virus showed a decrease in the number of skeletal muscle cells at low ( Figure 8 FG) and high ( Figure 8 The paw withdrawal frequency at the mechanical threshold of HI increased significantly. The paw withdrawal frequency of the contralateral side remained stable ( Fig.15 AB). One day after model construction, 3D motion analysis showed significant differences in four specific motion scores between mice injected with control virus and mice injected with Smoc2 shRNA ( Figure 8 J). The Smoc2 shRNA-injected group showed an increase in the types of movements when wiping the face (M7 and M18) and freezing (M19), which are associated with pain-like behaviors ( Figure 8 KL). Further inspection of dimensionality reduction ( Figure 8 M) and its corresponding projection on the x-axis ( Figure 8N) also highlighted the differences in exercise type between the groups. These results suggest that inhibition of Smoc2 expression in DRG can reverse the analgesic effect produced by intraplantar injection of 75 μg / mL PBNP. Based on the combined evidence from our in vitro and in vivo experiments, we infer that the analgesic effect of PBNP may originate from the increase in Smoc2 secretion by fibroblasts in DRG. In DRG, Smoc2 is mainly expressed in fibroblasts. It inhibits the coupled activation of adjacent DRG neurons triggered by noxious mechanical stimulation by inhibiting satellite glial P2X7 receptors 21. Our experimental results suggest that PBNP can induce fibroblasts in DRG to release more Smoc2, thereby alleviating postoperative pain.

[0088] The above description is only a preferred embodiment of the present invention and is not any formal or substantial limitation of the present invention. It should be pointed out that a person skilled in the art can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. Use of Prussian blue in the preparation of medicines for relieving or treating pain.

2. The use according to claim 1, characterized in that The pain includes mechanical pain.

3. The use according to claim 1, characterized in that The medicine comprises an active ingredient and a pharmaceutically acceptable carrier and / or auxiliary material, and the active ingredient is Prussian blue or its nanoparticles.

4. The use according to claim 1, characterized in that The dosage form of the drug includes tablets, powders, suspensions, granules, capsules, injections, sprays, solutions, enemas, emulsions, films, suppositories, patches, nasal drops or pills.

5. The use according to claim 1, characterized in that The drug is administered orally or parenterally.

6. Use of Prussian blue in the preparation of reagents for promoting SMOC2 secretion in fibroblasts.

7. Use of Prussian blue combined with Smoc2 agonists and / or P2X7R inhibitors in the preparation of drugs for relieving or treating pain.

8. A pharmaceutical composition, characterized in that The invention comprises a first active ingredient and a second active ingredient, wherein the first active ingredient is Prussian blue or its nanoparticles, and the second active ingredient is a Smoc2 agonist and / or a P2X7R inhibitor.

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