Composition for treating arthritis and its preparation method and application

Abstract

This invention relates to the field of biomedicine, and in particular to a composition with therapeutic effects on arthritis, its preparation method, and its application. The invention provides a composition with therapeutic effects on arthritis, which synergistically enhances the therapeutic effect of shikonin by actively targeting M1 subtype macrophages and regulating their metabolic pathways. Simultaneously, shikonin, as an AMPK activator, further enhances the cytotoxic effect of macrophages, forming a positive feedback loop and enhancing the remodeling of the immune microenvironment. Therefore, it can effectively delay and control the progression of rheumatoid arthritis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a composition capable of treating arthritis, a preparation method thereof and an application thereof. Background Art

[0002] Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by erosive polyarthritis. The pathological manifestation of RA is diffuse and invasive hyperplasia of the synovial membrane in joints throughout the body, and it is known as a tumor-like disease. RA can occur in joints throughout the body, especially in small joints, and is characterized by persistence and symmetry. Under the influence of persistent chronic inflammatory stimulation and "tumor-like" synovial hyperplasia and infiltration, cartilage tissue is invaded and degraded, and bone tissue is absorbed and destroyed. Therefore, RA patients develop joint deformities and even disability in the late stage of the disease. In addition, RA also affects other organs, such as the respiratory system, digestive system, circulatory system, nervous system, and blood system, bringing mental, physical, and economic burdens to patients. At present, the pathogenesis of RA is still unclear, and the clinical treatment goal is mainly to control inflammation, inhibit inflammatory factors, and thus delay the progression of RA disease. Treatment options primarily include systemic anti-rheumatic drugs (DMARDs), nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids (Glu), and biologics. However, conventional drug treatments often face challenges such as poor drug selectivity, low drug utilization, and side effects from long-term use, as well as ineffective therapeutic effects and the development of drug tolerance.

[0003] Macrophages play a crucial role in the innate immune response in rheumatoid arthritis. Macrophages produce a large and diverse array of proinflammatory cytokines in inflamed joints. These cytokines not only regulate and activate the adaptive immune response but also promote synovial proliferation and infiltration, as well as bone destruction through the RANKL pathway, which activates osteoclasts. In chronic inflammation, proinflammatory cytokines increase vascular permeability. Upon arrival at inflamed tissues, immune cells such as macrophages infiltrate the inflamed vessels through a process of aggregation, rolling, and adhesion, ultimately entering the inflammatory microenvironment. Most macrophages are classified into two subtypes: the proinflammatory M1 and the immunomodulatory M2, each with distinct metabolic preferences. M1 macrophages rapidly generate large amounts of ATP through aerobic glycolysis to meet the demands of proliferation and differentiation, while M2 macrophages generate energy through oxidative phosphorylation (OXPHOS). By regulating the macrophage metabolic pathway, the remodeling of M1 macrophages into M2 macrophages is regulated, thereby promoting the expression of cytokines with immune regulatory functions. Therefore, the immune microenvironment homeostasis of the inflamed joint can be rebuilt, thereby effectively delaying or inhibiting the progression of rheumatoid arthritis.

[0004] Efferocytosis is a term for the daily removal of dead cells in healthy organisms, and it plays a key role in maintaining the homeostasis of the immune microenvironment. Macrophages, as a classic phagocyte, remove dead cells through three steps: "eat me", "find me" and digestion. At the same time, macrophages produce a large number of immunomodulatory cytokines in this process and reshape towards the M2 type. Because dead cells can be regarded as a source of metabolic raw materials, such as fatty acids and amino acids. Therefore, efferocytosis can regulate macrophage metabolic pathways by promoting fatty acid oxidation (FAO) and OXPHOS, thereby promoting the production of immunomodulatory cytokines.

[0005] In response to the problems faced by DMARDs, such as poor drug selectivity, low drug utilization, various side effects caused by long-term drug use, ineffective therapeutic effects of drugs, or the body's tolerance to drugs, the current treatment strategy mainly focuses on the development of multifunctional active targeted drug delivery platforms, such as chemically synthesized nanoparticles, cell carriers, and cell membrane-encapsulated drug carriers.

[0006] Chemically synthesized nanoparticles are a drug delivery platform based on chemically synthesized nanoparticles. Bringing nanoparticles to the vicinity of target antigens presents numerous challenges and challenges, including complex nanoparticle structure, ligand binding chemistry, ligand-target interactions, host immune responses, difficulty in target selection, and drug resistance due to target-negative cell populations. When using cells as drug delivery vehicles, considerations must be given to phagocytosis and self-digestion of the drug, the ability of living cells to function in vivo, and whether they can evade host immune attack. For example, the hydrophobicity and toxicity of substances like shikonin limit their anti-inflammatory therapeutic effects in vivo. The process of encapsulating drug delivery vehicles with cell membranes results in significant loss of raw materials, and the cell membrane itself has limited anti-inflammatory therapeutic effects. Therefore, drugs prepared using these methods have limited anti-inflammatory effects. Therefore, it is necessary to find an effective treatment for rheumatoid arthritis. Summary of the Invention

[0007] The present invention aims to provide a composition having the effect of treating arthritis, and its preparation method and application, so as to solve the problems existing in the above-mentioned prior art. The composition of the present invention can effectively treat rheumatoid arthritis.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] The present invention provides a composition with the effect of treating arthritis. The composition is prepared from dead macrophages (DM) and shikonin (SHK).

[0010] The present invention provides a method for preparing the above-mentioned composition, which is characterized by comprising the following steps:

[0011] The macrophages were treated with liquid nitrogen to obtain dead macrophages;

[0012] After the dead macrophages are thawed, they are mixed with a PBS aqueous solution containing shikonin and incubated to obtain a composition.

[0013] Preferably, the volume ratio of the dead macrophages to the PBS aqueous solution containing shikonin is 3×10 7 Pieces: 1mL.

[0014] Preferably, the concentration of polyvinyl pyrrolidone in the PBS aqueous solution containing shikonin is 2 mg / mL, and the concentration of shikonin is 0.5 mg / mL.

[0015] The present invention provides use of the above composition or the composition prepared by the above preparation method in preparing anti-rheumatoid arthritis drugs.

[0016] The present invention provides an anti-rheumatoid arthritis drug, the active ingredient of which includes the above-mentioned composition or the composition prepared by the above-mentioned preparation method.

[0017] Preferably, the drug further comprises medically acceptable excipients.

[0018] Preferably, the dosage form of the drug includes injection or gel.

[0019] Preferably, the injection includes an intravenous injection or an intra-articular injection.

[0020] The present invention discloses the following technical effects:

[0021] The present invention discloses for the first time that in the targeted treatment of inflammation, DM prepared by liquid nitrogen treatment can target M1 subtype macrophages and activate efferocytosis, and DM itself can play a good anti-inflammatory role by regulating the metabolic pathway of macrophages. SHK, an effective ingredient extract of the traditional Chinese medicine Lithospermum officinale, is loaded into DM. SHK has a good anti-inflammatory effect, and SHK can inhibit the M2 subtype of pyruvate kinase (PK), inhibit the glycolysis metabolic pathway of the cell, activate the AMPK pathway, thereby promoting the OXPHOS pathway, and remodeling the M1 macrophages to the M2 subtype. The combination of DM / SHK synergistically enhances the therapeutic effect of SHK by actively targeting the M1 subtype macrophages and regulating their metabolic pathways. At the same time, SHK, as an activator of AMPK, further enhances the efferocytosis of macrophages, forming a positive feedback loop, and enhancing the effect on the remodeling of the immune microenvironment homeostasis, thereby effectively delaying and controlling the disease progression of rheumatoid arthritis.

[0022] Moreover, DM has the advantages of active targeting, convenient preparation, excellent biocompatibility, long circulation and ideal drug encapsulation rate. Compared with the traditional mobile communication system with cells as carriers (TDD system), DM has the advantages of simple preparation, low cost and easy preservation, and tissue-specific cells can be selected to prepare carriers according to the target tissue. DM loses cell activity, so there is no need to worry about the tolerance of carrier cells to loaded drugs and the physiological or pathological effects of carrier cells in the body when living cells are used as carriers. Compared with cell membrane derivatives, DM has a complete cell structure and a more complete protein composition structure, which provides an ideal basis for DM to exert its own therapeutic effect. At the same time, the combination of DM and SHK further enhances the targeting and therapeutic effect on M1 subtype macrophages. Therefore, the combination of DM and SHK can better regulate the phenotype of macrophages, thereby reshaping the homeostasis of the immune microenvironment and playing a good role in delaying and controlling the progression of rheumatoid arthritis.

[0023] During the preparation of the composition of the present invention, live macrophages are placed in liquid nitrogen and then resuspended in a serum-free cell cryopreservation solution. Under normal circumstances, the cell cryopreservation solution can protect the cells from damage caused by ice crystal formation during the cryopreservation process, thus ensuring that most cells survive when the cells are revived. The purpose of treating the cells with liquid nitrogen is to exceed the protective capacity of the cell cryopreservation solution, so that the ice crystals formed in the cells during the rapid drop in temperature will pierce the cell membrane, causing cell death. The pierced cell membrane permeability is increased, allowing small molecule drugs to freely enter and exit the cell, but the ice crystals formed must not destroy the cell morphology. Traditional cell cryopreservation solution is serum + DMSO. In experiments, it was found that this configuration of cell cryopreservation solution does not preserve the intact morphology of cells, that is, many cells appear broken. Therefore, we chose a commercial serum-free cell cryopreservation solution, which can better protect cells in normal use. This also helps to cause cell death during ice crystal formation and increase cell membrane permeability, but the cell structure remains intact.

[0024] Polyvinylpyrrolidone (PVP) is a solubilizing agent. Shikonin is highly hydrophobic and readily soluble in DMSO, but it precipitates immediately upon exposure to water. We have found that PVP can help dissolve shikonin in water, forming a shikonin aqueous solution at a concentration of 2 mg / mL. PVP is not a therapeutically active ingredient. Therefore, in our treatment of arthritic mice, we injected the shikonin aqueous solution into the tail vein, producing a corresponding therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 Schematic diagram of DM / SHK preparation and treatment of CIA mice;

[0027] Figure 2Preparation and characterization of DM / SHK, where a is a schematic diagram of the preparation of DM by the LNT-based method, created using www.biorender.com, b is a volcano plot showing the expression changes between DM and live macrophages, and DEPs (sample size 3) are defined as log2|FC|>1.5 and P<0.05, c is a GO component enrichment bubble plot classifying up-regulated and down-regulated DEPs (sample size 3), and red text indicates up-regulated components, d is the preparation process of DM / SHK, e is a scanning electron microscopy image of live macrophages (Live M), DM and DM / SHK, with a sample size of 3, and the scale bars from left to right are 10μm, 10μm, 5μm and 1μm, and f is a live macrophage (Live M), DM and DM / SHK transmission electron microscopy images, sample size is 3, the first row is low-power images, scale bar is 2 μm, the second row is high-power images, scale bar is 1 μm, gh live macrophages (LiveM), DM and DM / SHK diameter (g) and zeta potential (h), sample size is 6, data were analyzed by ANOVA and presented as mean ± SEM;

[0028] Figure 3Targeting of DM / SHK, a is a Western blot showing the expression of adhesion proteins CD44 and CD18 on Live M, DM, and DM / SHK, b is a representative fluorescence image of DM or DM / SHK binding to non-activated or activated EA.hy926, the first three columns, the scale is 100 μm, the last column, the scale is 50 μm, c is a quantitative assessment of the brightness level of DM or DM / SHK attachment to non-stimulated or stimulated EA.hy926 using fluorescence intensity analysis, the sample size is 3, statistical analysis was performed using t-test and shown as mean ± SEM, d is an example of fluorescence images of the distribution of free-Cy5.5, Cy5.5-labeled DM, and Cy5.5 / SHK-labeled DM in collagen-induced arthritis mice at different time points after injection, e is an in vitro fluorescence image showing free-Cy5.5 , DM labeled with Cy5.5 and DM labeled with Cy5.5 / SHK in major organs, f is a fluorescence image showing the distribution of free-Cy5.5, DM labeled with Cy5.5 and DM / SHK labeled with Cy5.5 in the synovium of joints, the nuclei were stained with DAPI (blue), while cy5.5, DM or DM / SHK were labeled with Cy5.5 (red), the measurement scale is 100 μm, g is the fluorescence intensity of DM or DM / SHK in major organs was analyzed using a quantitative method, the sample size was 3, the statistical analysis was performed using ANOVA and shown as the mean ± SEM, h is a quantitative assessment of the brightness level of DM or DM / SHK dispersion in the synovium of joints (n = 3), the statistical analysis was performed using ANOVA and shown as the mean ± SEM.

[0029] Figure 4DM / SHK triggers and enhances the phagocytic function of M1 subtype macrophages. a is a flow cytometric image showing DM stained with Annexin V-FITC / PI at different time intervals in liquid nitrogen. PtdSer is labeled by Annexin V. The sample size is 3. b is the “find” in non-activated or LPS (lipopolysaccharide) activated macrophages. Heat map of the relative mRNA expression levels of me" signals CD36, ELMO3, Mfge8 and MerTK, with a sample size of 3. c is a representative fluorescence image of DM or DM / SHK engulfed by non-activated or LPS-stimulated macrophages, with DAPI (blue) staining for nuclei, SF488-Phalloidin (green) staining for cytoskeleton structure, and pHrodo (red) staining for DM or DM / SHK. The measurement scale is 10 μm. d is a quantitative analysis of the fluorescence intensity of DM or DM / SHK engulfed by macrophages, with a sample size of 3. Statistical analysis was performed using an unpaired Student's t-test and is shown as the mean ± SEM. e is a Western blot showing the expression of AMPK phosphorylation in M1 subtype macrophages in each treatment group, with normal macrophages serving as a negative control. f is the figure The process of triggering double phagocytosis in macrophages is illustrated. This figure was created by www.biorender.com. g is a heat map of CD36, ELMO3, Mfge8, and MerTK mRNA expression levels in M1 subtype macrophages that undergo double phagocytosis. The sample size is 3. h is a representative fluorescence image of DM or DM / SHK double phagocytosis by macrophages stimulated with LPS. The nuclei are stained with DAPI (blue), the cytoskeleton structure is stained with SF488-Phalloidin (green), and DM or DM / SHK is stained with pHrodo (red). The measurement scale is 10 μm. i is the quantitative analysis of the fluorescence intensity of DM or DM / SHK double phagocytosis in macrophages. The sample size is 3. Statistical analysis was performed using an unpaired Student's t-test and shown as the mean ± SEM.

[0030] Figure 5Figure 3 In vivo effectiveness of DM / SHK in treating collagen-induced arthritis in mice. a This study demonstrated the therapeutic effects of intravenous injection of PBS, DM, SHK, and DM / SHK on collagen-induced arthritis mice, with healthy mice serving as the control group. b The arthritis scores of each group were assessed every three days throughout the treatment of collagen-induced arthritis mice, with a sample size of 5. c Data were analyzed using ANOVA and shown as mean ± SEM. On the 45th day after injection, the thickness of the paw of mice in each group was evaluated, with a sample size of 5. The data were analyzed using ANOVA and are shown as the mean ± SEM. d is an image of the arthritic paw of arthritic mice in each group. e is a representative H&E and Masson-stained image of the synovium of each group. The measurement scale is 100 μm. fh are the levels of proinflammatory cytokines IL-1β (f), IL-6 (g), and TNF-α (h) in the serum of mice in each group. The sample size is 3. The data were analyzed using ANOVA and are shown as the mean ± SEM. im are the mRNA levels of proinflammatory cytokines IL-1β (i), IL-6 (j), TNF-α (k) and anti-inflammatory mediators IL-10 (l) and Arg-1 (m) measured in the synovium of collagen-induced arthritis mice in each group. The sample size is 3. The data were analyzed using ANOVA and are shown as the mean ± SEM.

[0031] Figure 6 This is the therapeutic effect of different treatments on CIA mice. DETAILED DESCRIPTION

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0034] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0035] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0036] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0037] Example 1 Preparation of DM / SHK (composition with therapeutic effect on arthritis)

[0038] The preparation process of DM / SHK is as follows Figure 1 and Figure 2 As shown in d:

[0039] RAW 264.7 cells were cultured in DMEM high glucose medium containing 10% fetal bovine serum (FBS) in an incubator at 37°C and 5% CO2. 7 When the cells were isolated, they were washed three times with 1×PBS, and then collected using a cell scraper. The cells were centrifuged at 300×g for 5 minutes at room temperature, and then the supernatant was removed. Serum-free cell freezing solution (from New Saimei Company, product number C40100, for specific steps, please refer to the official website manual) was added to resuspend the cells and transferred to a cryopreservation tube. The cryopreservation tube was then directly placed in liquid nitrogen for storage to obtain DM. The preparation process is as follows: Figure 2 As shown in a in .

[0040] After freezing in liquid nitrogen for 24 h, DM (cell number is about 3×10 7DM (DM) was quickly thawed in a 37°C water bath. The cells were placed in a centrifuge tube and centrifuged at 700×g for 5 minutes at room temperature. The supernatant was discarded to obtain ready-to-use DM (if not used immediately, it can be temporarily stored in a 4°C refrigerator). The thawed DM was resuspended in 1× PBS and centrifuged at 700×g for 5 minutes at room temperature. This washing process was repeated three times. After the final centrifugation (only cell pellets remain), 1 mL of PBS containing 0.5 mg / mL SHK and 2 mg / mL polyvinyl pyrrolidone (PVP) was added. The cells were resuspended in PBS and incubated in a 37°C water bath for 2 hours. The cells were then centrifuged at 700×g for 5 minutes at room temperature. The supernatant was discarded to obtain DM / SHK. The cells were then resuspended in 1× PBS and stored in a 4°C refrigerator.

[0041] Example 2 Related Characterization

[0042] RAW 264.7 cells were collected and centrifuged to obtain live macrophages (Live M). Peptides were obtained by digesting the total protein extracted from Live M and DM prepared in Example 1 with Trypsin enzyme. Peptides were labeled using iTRAQ kit according to the manufacturer's instructions, and then the changes in DM total protein were determined by bioinformatics analysis. Figure 2 As shown in Figure b, a total of 6997 proteins were detected in Live M and DM, of which 2745 proteins showed significant expression changes in the DM group (fold change > 1.5, P < 0.05). Among the various proteins showing differential expression (DEPs), 850 were upregulated, while 1895 were downregulated. Figure 2 As shown in the c, GO component enrichment revealed that the majority of upregulated differentially expressed proteins (DEPs) were associated with cell and organelle membrane proteins, while the majority of downregulated DEPs were expressed in the cytoplasm and cytoplasm. Proteomic analysis of DM revealed that a large amount of protein was lost from the cell slurry because the cell membrane was punctured by ice crystals, increasing permeability. During the DM and DM / SHK preparation processes, proteins flowed out of the cell, while proteins on the cell membrane system remained. Because the cell morphology was well preserved, proteins fixed to the membrane did not lose, or the loss was minimal.

[0043] During the preparation of DM / SHK in Example 1, cells and debris were discarded by differential centrifugation, and the supernatant containing only SHK was collected. The SHK content was determined using high-performance liquid chromatography (HPLC), and the encapsulation efficiency (EE) was calculated using the formula: EE = (total SHK - SHK in supernatant) / total SHK × 100%. The calculated result showed that the drug loading efficiency of DM for SHK was 46±4%.

[0044] Live M, DM prepared in Example 1, and DM / SHK prepared in Example 1 were mixed with 2.5% glutaraldehyde and treated at 4°C for 4 hours. After three washes with PBS, the cells were fixed with 1% potassium permanganate at 4°C for 1 hour and dehydrated with varying concentrations of ethanol. The cells were then resuspended in anhydrous ethanol, placed on silicon, and dried. Finally, the silicon surface was coated with gold. After two rounds of anhydrous ethanol dehydration, the cells were then immersed in a mixture of epoxy resin and acetone with a volume ratio of 1:1, a mixture of epoxy resin and acetone with a volume ratio of 2:1, and a mixture of epoxy resin and acetone with a volume ratio of 3:1 at 37°C.

[0045] After preparing semi-thin sections, the desired position was determined using toluene blue staining. Subsequently, 70 nm ultrathin sections were prepared. They were soaked in 1% uracil hydrochloride for 10 min and then washed with deionized water for 10 min. After that, they were soaked in 1% lead citrate hydrochloride for 10 min and then washed with deionized water for 10 min. Transmission electron microscopy and scanning electron microscopy were used to determine that DM / SHK maintained an intact cell structure, and dynamic light scattering (DLS) was used to analyze the particle size and potential changes of Live M, DM, and DM / SHK. The results are shown in Figure 2. Figure 2 As shown in eh, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) imaging showed the fluffy cell structure of DM, and in the case of SHK ligation, DM / SHK exhibited a contracted cell structure (indicated by white arrows), with SHK attached to the surface and nucleus of DM.

[0046] Example 3 Targeting of DM / SHK

[0047] Preparation process of DM / SHK:

[0048] RAW 264.7 cells were cultured in DMEM high glucose medium containing 10% fetal bovine serum (FBS) in an incubator at 37°C and 5% CO2. 7 When the cells were isolated, they were washed three times with 1×PBS, and then collected using a cell scraper. The cells were centrifuged at 300×g for 5 min at room temperature, and the supernatant was removed. Serum-free cell freezing solution (from New Saimei Company, product number C40100, for specific steps, please refer to the official website manual) was added to resuspend the cells and transferred to a cryopreservation tube. The cryopreservation tube was then directly placed in liquid nitrogen for storage to obtain DM. The preparation process is as follows: Figure 2 As shown in a in .

[0049] After freezing in liquid nitrogen for 2, 4, 24 or 24 h, DM (cell number is about 3×10 7DM (DM) was quickly thawed in a 37°C water bath. The cells were placed in a centrifuge tube and centrifuged at 700×g for 5 minutes at room temperature. The supernatant was discarded to obtain ready-to-use DM (if not used immediately, it can be temporarily stored in a 4°C refrigerator). The thawed DM was resuspended in 1× PBS and centrifuged at 700×g for 5 minutes at room temperature. This washing process was repeated three times. After the final centrifugation (only cell pellets remain), 1 mL of PBS containing 0.5 mg / mL SHK and 2 mg / mL polyvinyl pyrrolidone (PVP) was added. The cells were resuspended in PBS and incubated in a 37°C water bath for 2 hours. The cells were then centrifuged at 700×g for 5 minutes at room temperature. The supernatant was discarded to obtain DM / SHK. The cells were then resuspended in 1× PBS and stored in a 4°C refrigerator. Live M, prepared DM, and prepared DM / SHK were evaluated for surface efferocytosis-associated phosphatidylserine (PtdSer) expression on DM at different time intervals using the Annexin V-FITC / PI apoptosis kit according to the manufacturer's instructions. The results are shown in Figure 5. Figure 4 As shown in (a), we documented evidence of PtdSer exposure on the external surface of DMs, with PtdSer exposure detected in over 90% of DMs after 24 h of liquid nitrogen treatment.

[0050] Live M, DM, DM / SHK cell membrane proteins were obtained using a membrane and cytoplasmic protein extraction kit according to the manufacturer's guidelines. Live M, DM, DM / SHK cell membrane proteins were diluted to a concentration of 2 mg / mL, and then the amount of protein in the diluted membrane protein solution was measured using a BCA protein assay kit according to the manufacturer's guidelines. After separation on 8% SDS-PAGE, the proteins were electronically transferred to a PVDF membrane and blocked with protein blotting blocking buffer at room temperature for 10 minutes. Then, they were incubated with CD44 and CD18 antibodies (CD44 antibodies were purchased from abcam, and CD18 antibodies were purchased from abc am) at 4°C overnight. Subsequently, they were treated with HRP-labeled secondary antibodies (purchased from proteintech) for 1 hour at room temperature. Finally, the bands were identified using ECL reagents, and the results are shown in Figure 2. Figure 3 As shown in a, Live M, DM, and DM / SHK can express adhesion proteins CD44 and CD18.

[0051] After EA.hy926 cells were cultured, they were incubated for 24 hours in a culture medium containing 50 ng / mL TNF-α (the basic culture medium of the culture medium was DMEM high glucose medium containing 10% fetal bovine serum). EA.hy926 cultured in a normal culture medium (normal culture medium was DMEM high glucose medium containing 10% fetal bovine serum) was used as a control group. Subsequently, DM and DM / SHK labeled with DID were added, respectively, and cultured at 37°C and 120 rpm on a shaker for 4 hours. Afterwards, the cells were fixed with 4% formaldehyde for 15 minutes. After incubation with SF488-Phalloidin for 15 minutes, the cells were observed using a fluorescence microscope, and the fluorescence intensity was quantitatively analyzed using ImageJ software to determine the targeted adhesion effect of DM / SHK on inflammatory vascular endothelial cells in vitro. The results are shown in Figure 2. Figure 3 As shown in b and c, the adhesion rate of DM or DM / SHK to activated EA.hy926 cells was significantly higher than that of untreated cells.

[0052] Example 4 Study on the effect of DM / SHK triggering and enhancing M1 subtype macrophage efferocytosis

[0053] RAW264.7 cells incubated in conventional culture medium (conventional culture medium is DMEM high glucose medium containing 10% fetal bovine serum) were recorded as the control group, and RAW 264.7 cells cultured and treated for 24 hours in a culture medium containing 1 μg / mL LPS (lipopolysaccharide) (the basic culture medium of this culture medium is DMEM high glucose medium containing 10% fetal bovine serum) were recorded as the experimental group. Subsequently, pHrodo red-labeled DM and DM / SHK were added to the experimental group, and the cells were incubated at 37°C for 24 hours. Subsequently, the cells were incubated with SF488-Phalloidin for 5 minutes and DAPI stained for 5 minutes, and then the burial effect of DM / SHK in M1 subtype macrophages was observed by CLSM, and the fluorescence intensity was quantitatively analyzed using ImageJ software. The results are shown in Figure 2. Figure 4 As shown in Figures c and d, pHrodo dye emits red fluorescence when exposed to an acidic environment. Therefore, LPS-stimulated macrophages exhibited higher fluorescence signal intensity compared to normal macrophages, demonstrating that M1 macrophages phagocytize and digest more DM and DM / SHK in the same timeframe. Furthermore, M1 macrophages phagocytize and digest DM / SHK, followed by DM and then digestion, produced higher fluorescence signal intensity than M1 macrophages phagocytize and then digest DM, demonstrating that DM / SHK can promote cellular internalization with the help of SHK and increase the number of phagocytic cells.

[0054] RAW264.7 cells incubated in conventional culture medium (conventional culture medium is DMEM high glucose medium containing 10% fetal bovine serum) were designated as the control group (A, Normal LPS(-)), and RAW264.7 cells cultured and treated in culture medium containing 1 μg / mL LPS (basal culture medium of this culture medium is DMEM high glucose medium containing 10% fetal bovine serum) for 24 h were designated as the experimental group (B, PBS LPS(+));

[0055] Subsequently, DM (C, DM LPS(-)) and DM / SHK (F, DM / SHK LPS(-)) were added to the control group, and DM (D, DM LPS(+)) and DM / SHK (G, DM / SHK LPS(+)) were added to the experimental group;

[0056] In the experimental group, the MerTK inhibitor UNC 2881 was added at a concentration of 28 nM, followed by the addition of DM (E, DM + UNC 2881 LPS (+)) and DM / SHK (H, DM / SHK + UNC 2881 LPS (+));

[0057] The above treatments were incubated at 37°C for 24 h, and then the mRNA expression levels of CD36, ELMO3, Mfge8, and MerTK were detected by qPCR and then normalized with GAPDH as a reference. The results are shown in Figure 4 As shown in b, the above four genes are related genes in the "eat me" process. Compared with normal macrophages, M1 macrophages showed higher expression levels of related genes in the process of phagocytosis of DM and DM / SHK. The expression levels of efferocytosis-related genes in the process of M1 macrophages phagocytosis of DM / SHK were significantly higher than those in the process of M1 macrophages phagocytosis of DM. This shows that DM / SHK synergistically can activate efferocytosis.

[0058] RAW264.7 cells incubated in conventional culture medium (conventional culture medium is DMEM high glucose medium containing 10% fetal bovine serum) were recorded as the control group, and RAW 264.7 cells cultured and treated for 24 hours in a culture medium containing 1 μg / mL LPS (the basal culture medium of this culture medium is DMEM high glucose medium containing 10% fetal bovine serum) were recorded as the experimental group. Subsequently, DM and DM / SHK were added, and after incubation for 24 hours, pHrodo red-labeled DM and DM / SHK were added, respectively, and incubated for another 24 hours at 37°C. Subsequently, the cells were incubated with SF488-Phalloidin for 5 minutes, stained with DAPI for 5 minutes, and then observed by CLSM, and the fluorescence intensity was quantitatively analyzed using ImageJ software. Figure 4As shown in Figures f, h, and i, the secondary phagocytosis of DM and DM / SHK by M1 macrophages can produce a higher phagocytic rate compared with the primary phagocytosis, and the phagocytic rate of DM / SHK is higher than that of DM, proving the enhancing effect of DM / SHK on endocytosis.

[0059] RAW264.7 cells incubated in conventional culture medium (conventional culture medium is DMEM high glucose medium containing 10% fetal bovine serum) were recorded as the control group, and RAW 264.7 cells cultured and treated for 24 hours in a medium containing 1 μg / mL LPS (the base medium of this culture medium is DMEM high glucose medium containing 10% fetal bovine serum) were used as the experimental group. Subsequently, AMPK inhibitor Compound C was added at a concentration of 110nM, and DM and DM / SHK were added respectively. After incubation for 24 hours, the cells were washed twice with PBS, and then DM and DM / SH K were added again, and incubated for another 24 hours at 37°C. After that, the mRNA expression levels of CD36, ELMO3, Mfge8 and MerT K were detected by qPCR, and then normalized to GAPDH as a reference. Figure 4 In Figures f and g, M1 macrophages phagocytized DM and DM / SHK for the second time produced higher levels of efferocytosis-related gene expression compared to the first phagocytosis, and phagocytized DM / SHK further produced higher levels of efferocytosis-related gene expression compared to the phagocytosis of DM, thus proving that DM / SHK synergistically enhances efferocytosis.

[0060] RAW 264.7 cells were cultured and treated with a solution containing 1 μg / mL LPS for 24 hours. Subsequently, PBS, DM (the amount of DM was 5 times that of RAW 264.7 cells), SHK (the final concentration of SHK was 1 μM / mL), and DM / SHK (the amount of DM was 5 times that of RAW 264.7 cells and the final concentration of SHK was 1 μM / mL) were added, and incubated at 37°C for 24 hours. The negative control group was conventional macrophages (RAW 264.7 cells), which were recorded as Normal. Using ripa solution, a total protein solution was obtained, and then Western blot analysis was performed to detect p-AMPK and AMPK-α, while β-actin was used as an internal reference gene. The results are shown in Figure 2. Figure 4 As shown in Figure e, DM itself can activate AMPK protein to phosphorylate it. SHK is known to promote AMPK protein phosphorylation. The DM / SHK group showed higher levels of phosphorylated AMPK protein expression. This shows that DM / SHK can synergistically activate the AMPK pathway and play a role in regulating inflammation.

[0061] Example 5

[0062] 1. Distribution of DM / SHK in CIA mice

[0063] 1.1. Establishment of CIA mouse model (mouse arthritis model)

[0064] Type II bovine collagen (CⅡ, 2 mg / mL, Chondrex) and complete Freund's adjuvant (CFA, Chondrex) were mixed at a volume ratio of 1:1, and the mixture contained 2 mg / mL of inactivated Mycobacterium tuberculosis.

[0065] Eighteen eight-week-old female DBA / 1J mice were injected at the base of the tail with 100 μL of a mixture containing inactivated Mycobacterium tuberculosis to induce CIA. Twenty-one days after the initial immunization, a second immunization was performed with 100 μL of a mixture of CⅡ and incomplete Freund's adjuvant (IFA, Chondrex) (CⅡ:IFA, 1:1, volume ratio) to increase the likelihood of arthritis.

[0066] 1.2 Distribution of DM / SHK in CIA mice

[0067] On the 60th day after the initial injection, the average arthritis score of CIA mice reached 14, with a scoring scale of 0 indicating no signs of redness or swelling; 1 indicating mild redness and swelling limited to the tarsal or ankle joints; 2 indicating mild redness and swelling spreading from the ankle to the tarsal bones; 3 indicating moderate redness and swelling spreading from the ankle to the metatarsal joints; and 4 indicating severe redness and swelling affecting the ankle, foot, and toe joints, or limb stiffness. The arthritis score was recorded for each mouse, and the total score of the four paws was calculated. Subsequently, CIA mice were intravenously injected with a solution containing 10 μg of Cy5.5 (single agent), DM labeled with Cy5.5, and DM / SHK labeled with Cy5.5, respectively. Six CIA mice were injected for each treatment, and fluorescence images were obtained using an in vivo imaging system (IVIS) at 0.5, 1, 3, 6, 12, 24, and 48 hours after injection. Images of CIA mice before injection were used as bright fields, as shown in Figure 2. Figure 3 As shown in Figure d, cy5.5, as a simulation of the in vivo circulation of SHK alone, was basically metabolized in the body at 24h and 48h. In contrast, DM and DM / SHK still showed high fluorescence signals enriched in various inflamed joints at 48h after injection. 48h after injection, CIA mice were euthanized, and the IVIS system was used to detect fluorescence in major organs and inflamed joints during in vitro imaging, as shown in Figure d. Figure 3As shown in Figures e and g, DM and DM / SHK basically converge in the inflamed joints and do not stay in other organs. In vitro, inflamed knee joint tissues of mice were obtained, fixed with 4% formaldehyde, and decalcified with 20% (w / v) ethylenediaminetetraacetic acid (EDTA) solution, which was replaced once a day for 30 days. Afterwards, the inflamed knee joints were sectioned, stained with DAPI, and observed under a fluorescence microscope. The fluorescence intensity was quantitatively analyzed using ImageJ software. The results are shown in Figure 3. Figure 3 In Figures f and h, cy5.5 was not enriched in the inflamed synovium, while DM and DM / SHK were significantly enriched in the inflamed synovium of inflamed joints. This demonstrates the in vivo targeted therapeutic effect of DM / SHK on multiple inflamed joints.

[0068] 2. Treatment plan for CIA mice (see the schematic diagram of treatment of CIA mice for details) Figure 1 )

[0069] 2.1. Establishment of CIA mouse model (mouse arthritis model)

[0070] Type II bovine collagen (CⅡ, 2 mg / mL, Chondrex) and complete Freund's adjuvant (CFA, Chondrex) were mixed at a volume ratio of 1:1, and CFA contained 2 mg / mL inactivated Mycobacterium tuberculosis.

[0071] Twenty-five eight-week-old female DBA / 1J mice were injected at the base of the tail with 100 μL of a mixture containing inactivated Mycobacterium tuberculosis to induce CIA. Twenty-one days after the initial immunization, a second immunization was performed with 100 μL of a mixture of CⅡ and incomplete Freund's adjuvant (IFA, Chondrex) (CⅡ:IFA, 1:1 volume ratio) to increase the likelihood of arthritis.

[0072] After the second immunization, mice were evaluated every two days. The evaluation criteria were: 0 means no signs of redness or swelling; 1 means mild redness or swelling limited to the tarsal or ankle joints; 2 means mild redness or swelling spreading from the ankle to the tarsal joints; 3 means moderate redness or swelling spreading from the ankle to the metatarsal joints; 4 means severe redness or swelling affecting the ankle, foot and toe joints, or limb stiffness. The arthritis score was recorded for each mouse, and the total score of the four paws was calculated. The evaluation results are as follows: Figure 5 As shown in bd, the joint scores of the healthy group (Normal), PBS group, DM group, SHK group, and DM / SHK group were 0, 14, 9.2, 7.8, and 4.2, respectively, and the foot thickness measurements were 2.016 cm, 3.362 cm, 2.812 cm, 2.774 cm, and 2.382 cm, respectively.

[0073] CIA mice with an average clinical score higher than 1 were randomly divided into four groups, with 5 mice in each group, and injected with PBS, DM (5×10 6 / mouse), SHK (final concentration was 5 mg / kg) or DM / SHK (DM was 5×10 6 Each mouse was treated with DM / SHK at a final concentration of 5 mg / kg. Healthy 8-week-old female DBA / 1J mice were used as negative controls. Starting 28 days after initial immunization, CIA mice were intravenously injected every other day. DM / SHK targeted the inflamed joints through the homing effect of macrophages on inflamed tissues. 48 hours after injection, the drug still showed good enrichment in the inflamed joints. Figure 3 dh in). 45 days after the initial immunization, CIA mice were euthanized. The specific procedures are detailed in Figure 5 a in .

[0074] 2.2 Histological Analysis

[0075] After CIA mice were euthanized, the knee joints were fixed and decalcified for 30 days, and the joints and fixed organs were embedded and sectioned for histopathological and immunohistochemical analysis. To examine the morphology of the synovium and cartilage, tissue sections were stained with H&E and Masson's trichrome. Figure 5 As shown in Figure e, DM / SHK has an inhibitory effect on the synovium and a protective effect on bones and cartilage, which shows that DM / SHK delays the progression of arthritis in CIA mice.

[0076] 2.3 Cytokine Assays in CIA Mice

[0077] The concentrations of IL-1β, IL-6, and TNF-α in the serum of each group of CIA mice were detected using ELISA kits according to the manufacturer's instructions. Afterwards, the samples were detected using a microplate reader at wavelengths of 450 nm and 630 nm. After grinding the CIA mice in liquid nitrogen, total RNA was extracted from the hind knee joints using the Trizol method to obtain a solution containing total DNA. GAPDH was used as a reference gene to normalize the relative mRNA expression levels of IL-1β, IL-6, TNF-α, Arg-1, and IL-10. The results are shown in Figure 2. Figure 5 As shown in the figure, the levels of proinflammatory cytokines in CIA mice treated with DM / SHK were close to normal levels, the immunomodulatory cytokines were statistically significantly increased, the proliferation of synovial tissue was inhibited, and the cartilage and bone tissue were successfully protected, approaching the normal joint ( Figure 5 The reduced expression levels of systemic inflammatory factors and inflammatory factors in local inflammatory joints can prove that the immune homeostasis in the joints has been remodeled, which can effectively delay the progression of RA ( Figure 5 im).

[0078] Example 6

[0079] According to the method used by TING-CHAO CHOU in a 1984 article, the synergistic effect (combination index, CI) was calculated using the following formula:

[0080]

[0081] Among them, D1 and D2 are the IC of the two drugs acting alone. 50 Dx(1,2) is the dose of each drug corresponding to the IC50 of the two drugs when used together. CI < 1 indicates synergistic effect; CI = 1 indicates additive effect; CI > 1 indicates antagonistic effect.

[0082] We used the Bio-Tech Nitric Oxide (NO) assay kit to test the inhibitory effects of different concentrations or cell numbers on LPS-activated macrophage NO production in vitro. NO is produced by M1 macrophages, and reduced production can demonstrate an inhibitory effect on macrophage inflammation. RAW 264.7 cells incubated in conventional culture medium (conventional culture medium is DMEM high glucose medium containing 10% fetal bovine serum) were recorded as the control group. RAW 264.7 cells were cultured and treated with culture medium containing 1 μg / mL LPS (the basic culture medium of this culture medium is DMEM high glucose medium containing 10% fetal bovine serum) for 24 hours. SHK was treated at concentrations of 0.5 μM, 1 μM, 2 μM or 4 μM for 24 hours. DM and LPS-activated macrophages in culture dishes were treated at a ratio of 1:1, 1:2, 1:5 or 1:10 for 24 hours. DM / SHK (dose corresponding to single drug) was used for drug screening. Finally, we found that when the DM dose was 1:5 and SHK was 2 μM, the CI value was 0.628, and when the DM dose was 1:10 and SHK was 2 μM, the CI value was 0.516, which proved that DM and SHK had a synergistic anti-inflammatory effect.

[0083] Example 7 The therapeutic effects of MTX, DM / MTX, and DM / SHK on CIA mice

[0084] Methotrexate (MTX) is the first-line drug for the treatment of RA. Therefore, DM / MTX was prepared to study its therapeutic effect on CIA mice.

[0085] 1. Preparation of DM / MTX

[0086] RAW 264.7 cells were cultured in DMEM high glucose medium containing 10% fetal bovine serum (FBS) in an incubator at 37°C and 5% CO2.7 When the cells were isolated, they were washed three times with 1×PBS, and then collected using a cell scraper. The cells were centrifuged at 300×g for 5 min at room temperature, and the supernatant was removed. Serum-free cell freezing solution (from New Saimei Company, product number C40100, for specific steps, please refer to the official website manual) was added to resuspend the cells and transferred to a cryopreservation tube. The cryopreservation tube was then directly placed in liquid nitrogen for storage to obtain DM. The preparation process is as follows: Figure 2 As shown in a in .

[0087] After freezing in liquid nitrogen for 24 h, DM (cell number is about 3×10 7 ) and thaw quickly in a 37°C water bath, place in a centrifuge tube and centrifuge at 700g for 5 minutes at room temperature. After discarding the supernatant, you will get the usable DM (if not used in time, you can temporarily store it in a 4°C refrigerator). The thawed DM is resuspended by pipetting with 1×PBS and centrifuged at 700×g for 5 minutes at room temperature in a centrifuge. The above washing process is repeated 3 times. After the last washing and centrifugation (only cell clumps are left at this time), add 1mL of PBS aqueous solution containing SHK. The concentration of SHK in the PBS solution is 0.5mg / mL. After pipetting and resuspending, place in a 37°C water bath and incubate for 2h. Then place in a centrifuge and centrifuge at 700g for 5 minutes at room temperature. After discarding the supernatant, you will get DM / MTX. After that, resuspend with 1×PBS and store in a 4°C refrigerator.

[0088] During the DM / MTX preparation process, cells and debris were discarded by differential centrifugation, and the supernatant containing only MTX was collected. The SHK content was determined using high-performance liquid chromatography (HPLC). The encapsulation efficiency (EE) was calculated using the formula: EE = (total MTX - MTX in supernatant) / total MTX × 100%. The calculated result showed that the drug loading efficiency of DM for MTX was 23%.

[0089] 2. Treatment options for CIA mice

[0090] Type II bovine collagen (CⅡ, 2 mg / mL, Chondrex) and complete Freund's adjuvant (CFA, Chondrex) were mixed at a volume ratio of 1:1, and CFA contained 2 mg / mL inactivated Mycobacterium tuberculosis.

[0091] Thirty-five eight-week-old female DBA / 1J mice were injected at the base of the tail with 100 μL of a mixture containing inactivated Mycobacterium tuberculosis to induce CIA. Twenty-one days after the initial immunization, a second immunization was performed with 100 μL of a mixture of CⅡ and incomplete Freund's adjuvant (IFA, Chondrex) (CⅡ:IFA, 1:1 volume ratio) to increase the likelihood of arthritis.

[0092] After the second immunization, mice were evaluated every two days on a scale of 0 (no redness or swelling); 1 (mild redness or swelling limited to the tarsal or ankle joints); 2 (mild redness or swelling spreading from the ankle to the tarsal joints); 3 (moderate redness or swelling spreading from the ankle to the metatarsal joints); and 4 (severe redness or swelling affecting the ankle, foot, and toe joints, or limb stiffness). The arthritis score was recorded for each mouse, and the total score for all four paws was calculated.

[0093] CIA mice with an average clinical score of 1 were randomly divided into 6 groups, with 5 mice in each group, and injected with PBS, DM (5×10 6 / ), SHK (final concentration 5 mg / kg), DM / SHK (DM 5×10 6 / mouse, SHK final concentration was 5mg / kg), MTX (final concentration was 5mg / kg) or DM / MTX (DM was 5×10 6 The CIA mice were euthanized 45 days after the initial immunization and treated with MTX at a final concentration of 5 mg / kg. At the same time, healthy 8-week-old female DBA / 1J mice were used as negative controls. Starting from 28 days after the initial immunization, the CIA mice were intravenously injected every other day. The results are shown in the figure below. Figure 6 As shown. Figure 6 It can be seen that the inhibitory effect of DM / MTX on arthritis is greater than that of MTX, and the inhibitory effect of DM / SHK on arthritis is greater than that of DM / MTX.

[0094] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A composition for treating arthritis, characterized in that: The composition is prepared from dead macrophages and shikonin; The preparation method of the composition comprises the following steps: The macrophages were treated with liquid nitrogen to obtain dead macrophages; After the dead macrophages are thawed, they are mixed and incubated with a PBS aqueous solution containing shikonin to obtain the composition.

2. The method for preparing the composition according to claim 1, characterized in that: The following steps are involved: The macrophages were treated with liquid nitrogen to obtain dead macrophages; The dead macrophages are thawed, mixed with a PBS aqueous solution containing shikonin, and incubated to obtain a composition.

3. The preparation method according to claim 2, characterized in that: The number volume ratio of the dead macrophages to the PBS aqueous solution containing shikonin is 3×10 7 Piece: 1mL.

4. The preparation method according to claim 3, characterized in that: The concentration of polyvinyl pyrrolidone in the PBS aqueous solution containing shikonin is 2 mg / mL, and the concentration of shikonin is 0.5 mg / mL.

5. Use of the composition according to claim 1 or the composition prepared by the preparation method according to claims 2 to 4 in the preparation of anti-rheumatoid arthritis drugs.

6. An anti-rheumatoid arthritis drug, characterized in that: The active ingredient comprises the composition according to claim 1 or a composition prepared by the preparation method according to claims 2-4.

7. The drug according to claim 6, characterized in that The medicament also includes medically acceptable excipients.

8. The drug according to claim 7, characterized in that The dosage form of the drug includes injection or gel.

9. The drug according to claim 8, characterized in that The injection includes intravenous injection or intra-articular injection.

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