Application of sargassum fusiforme nanovesicles in preparation of drugs for treating shoulder stiffness

Abstract

The application discloses application of Sargassum fusiforme nano vesicles in preparation of drugs for treating shoulder joint stiffness, and belongs to the field of biological medicine. In the application, the large particle impurities of Sargassum fusiforme are removed through a differential centrifugation method, and then the Sargassum fusiforme nano vesicles are separated and extracted by using a hollow fiber column. Through shoulder joint stiffness animal model experiments, it is found that the Sargassum fusiforme nano vesicles can inhibit the formation of type I and type III collagen of joint capsules and inhibit the activation of myofibroblasts, thereby playing a role in treating joint stiffness; and the treatment effect of the Sargassum fusiforme nano vesicles on shoulder joint stiffness is also confirmed through analysis of the shoulder joint activity after treatment. The application provides a new drug treatment strategy for clinical diseases such as frozen shoulder and postoperative joint stiffness.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the application of Sargassum fusiforme nanovesicles in the preparation of drugs for treating shoulder joint stiffness. Background Technology

[0002] Shoulder stiffness is a common shoulder condition characterized by capsular fibrosis, limited range of motion, and pain. It can occur primary (diagnosed as "frozen shoulder" or "adhesive capsulitis") or secondary to shoulder trauma, surgery, or prolonged immobilization. Pathological features include chronic inflammation of the joint capsule, abnormal activation of fibroblasts, and excessive deposition of collagen fibers (especially type I and type III collagen), leading to capsular thickening, contracture, and decreased compliance.

[0003] Currently, clinical treatment for shoulder stiffness mainly includes nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroid injections, physical therapy, and manual release techniques. For refractory or severe cases of stiffness, arthroscopic release surgery is often necessary. However, existing drug treatments often lack targeting, and long-term use can lead to side effects or limited efficacy. While surgical intervention can improve range of motion in the short term, the risk of postoperative inflammation and refibrosis remains high, and the effects are not lasting for some patients. Therefore, developing a novel drug that can regulate joint capsule fibrosis, inhibit fibroblast activation, and reduce collagen deposition at the pathological mechanism level has significant clinical importance and application prospects.

[0004] In recent years, plant-derived nanovesicles have gradually become a hot topic in drug research due to their natural origin, good biocompatibility, and potential bioactivity. Sargassum fusiforme, a medicinal seaweed, is rich in polysaccharides, polyphenols, terpenes, and other active ingredients, possessing anti-inflammatory, antioxidant, and immunomodulatory pharmacological effects. However, there are currently no research reports on the use of Sargassum fusiforme nanovesicles in the treatment of shoulder joint stiffness. Summary of the Invention

[0005] The purpose of this invention is to provide the application of Sargassum fusiforme nanovesicles in the preparation of drugs for treating shoulder joint stiffness, in order to solve the problems existing in the prior art. Sargassum fusiforme nanovesicles are extracted and separated from Sargassum fusiforme by differential centrifugation combined with hollow fiber column filtration. These Sargassum fusiforme nanovesicles provide a new drug treatment strategy for clinical diseases such as frozen shoulder and postoperative joint stiffness.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides the application of Sargassum fusiforme nanovesicles in the preparation of drugs for treating or relieving shoulder joint stiffness, wherein the Sargassum fusiforme nanovesicles are isolated from Sargassum fusiforme.

[0007] Preferably, the separation method includes the following steps: Sargassum fusiforme was mixed with PBS solution, then crushed and ground to obtain a grinding solution; The grinding slurry was centrifuged at differential speed to obtain the supernatant. The supernatant is filtered using a hollow fiber column, and the retained concentrate is collected to obtain the product.

[0008] Preferably, the mass-to-volume ratio of the Sargassum fusiforme to the PBS solution is 1:(3-5).

[0009] Preferably, the grinding time is 1 minute each time, for a total of 3-5 times; after each grinding is completed, the mixture is placed on ice for 3-10 minutes before the next grinding is performed.

[0010] Preferably, the differential centrifugation is performed as follows: the grinding liquid is centrifuged at 1000 g for 10 min, the precipitate is discarded after centrifugation, the supernatant is centrifuged at 3000 g for 20 min, the precipitate is discarded after centrifugation, and the supernatant is centrifuged at 10000 g for 40 min.

[0011] Preferably, the hollow fiber column has a pore size of 200 nm.

[0012] The present invention also provides a medicament for treating or relieving shoulder stiffness, the medicament comprising the Sargassum fusiforme nanovesicles.

[0013] Preferably, the drug is used to regulate the formation of collagen fibers in the joint capsule, the activation of fibroblasts, and the range of motion of the shoulder joint.

[0014] The present invention discloses the following technical effects: This invention extracts nanoparticles from Sargassum fusiforme using differential centrifugation combined with hollow fiber column filtration. Characterization confirmed that the extract contains nanovesicles, demonstrating that this invention successfully extracts Sargassum fusiforme nanovesicles. Furthermore, using an animal model of shoulder joint stiffness, it was found that Sargassum fusiforme nanovesicles inhibit the formation of type I and type III collagen in the joint capsule and suppress myofibroblast activation, indicating that Sargassum fusiforme nanovesicles affect joint capsule collagen fiber formation and fibroblast activation. Post-treatment shoulder joint range of motion analysis also confirmed the therapeutic effect of Sargassum fusiforme nanovesicles on shoulder joint stiffness. This invention provides a new drug treatment strategy for clinical diseases such as frozen shoulder and postoperative joint stiffness. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 Extraction and characterization of a concentrated solution rich in Sargassum fusiforme nanovesicle particles; A: Extraction flow chart; B: Transmission electron microscopy observation results; C: Particle size; D: Potential; Figure 2 The pathological effects of Sargassum fusiforme-derived nanovesicles on the joint capsule tissue in the axillary bursa (HE staining). Figure 3 The pathological effects of Sargassum fusiforme-derived nanovesicles on the joint capsule tissue in the axillary bursa (Masson staining). Figure 4 The pathological effects of Sargassum fusiforme-derived nanovesicles on type I collagen fibers in the axillary bursa joint capsule tissue; A: Staining results; B: Statistical results of type I collagen staining area; Figure 5 The pathological effects of Sargassum fusiforme-derived nanovesicles on type III collagen fibers in the axillary bursa joint capsule tissue; A: Staining results; B: Statistical results of type III collagen staining area; Figure 6 The pathological effects of Sargassum fusiforme-derived nanovesicles on αSMA in the axillary bursa joint capsule tissue; A: Staining results; B: Statistical results of αSMA protein staining area; Figure 7 The effect of Sargassum fusiforme nanovesicles on shoulder joint range of motion; A: Schematic diagram of shoulder joint range of motion test; B: Statistical results of shoulder joint range of motion data in different groups. Detailed Implementation

[0017] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0018] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0019] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0020] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0021] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0022] Example 1 I. Experiment on the extraction of Sargassum fusiforme nanovesicles The specific flowchart of the extraction is as follows: Figure 1 As shown in Figure A, the operation steps are as follows: (1) Take 50 g of frozen Sargassum fusiforme from the -30℃ freezer, wash it, and accurately weigh an appropriate amount of Sargassum fusiforme using an electronic balance. Put the Sargassum fusiforme mass and 1×PBS volume into a blender at a ratio of 1:5 (g:v) and grind it at the maximum speed (45000 rpm / min) for 1 min. Place it on ice for 5 min and repeat this operation 5 times to prevent overheating from destroying the components. Finally, pour the juice-like grinding liquid into several 50 mL centrifuge tubes. (2) Centrifuge the grinding liquid at 1000 g for 10 min. After centrifugation, discard the precipitate and take the supernatant into a 50 mL centrifuge tube. Centrifuge at 3000 g for 20 min. After centrifugation, discard the precipitate and take the supernatant into a 50 mL centrifuge tube. Centrifuge the supernatant at 10000 g for 40 min. (3) After the differential centrifugation, the supernatant was first rinsed with PBS. Then, the supernatant obtained by centrifugation was filtered through a 200 nm hollow fiber column. The retained product was the concentrate, and the product flowing directly out of the hollow fiber column was the filtrate. The concentrate was rich in Sargassum fusiforme nanovesicle particles. The concentrate was used for further research. The hollow fiber column was rinsed with PBS to remove residual particles.

[0023] II. Characterization Experiment of Sargassum fusiforme Nanovesicles 1. Transmission electron microscopy (TEM) (1) Take out 10 μL of the concentrated solution rich in Sargassum nanovesicle particles and drop it onto a copper grid with a pore size of 2 nm and let it stand at room temperature for 5 min.

[0024] (2) Use filter paper to absorb the liquid on the side of the filter screen, add 2% phosphotungstic acid solution, and negative stain for 2 min at room temperature.

[0025] (3) Blot the negative stain with filter paper, add 10 μL of double-distilled water to wash three times, and then let it air dry at room temperature. Then it can be observed using a transmission electron microscope (TEM).

[0026] TEM image Figure 1 As shown in Figure B, the results indicate that the Sargassum fusiforme nanovesicle particles in the concentrate have a nanoscale vesicle-like structure.

[0027] 2. Nanoparticle size tracking instrument for particle size analysis Take 50 μL of the concentrated solution rich in Sargassum nanovesicle particles, dilute it 10,000 times with ultrapure water, and then take 1 mL of the diluted solution for measurement. The particle size and concentration of the concentrated solution are measured using a Malvern Panak Nanoparticle Size Tracker (NTA).

[0028] The results are as follows Figure 1 As shown in Figure C, the size of the Sargassum fusiforme nanovesicles in the concentrated solution is 122.5 ± 51.8 nm, and the concentration after dilution is 6.04 × 10⁻⁶. 10 Particle count / mL.

[0029] 3. Zeta potentiometer detects vesicle potentials. Take 200 μL of the concentrated solution rich in Sargassum nanovesicle particles, dilute it 100 times with PBS, and measure the zeta potential. First, vortex the diluted solution for 10 s to mix it, avoiding violent shaking. Then, filter it with a 0.45 μm filter membrane to remove large particle aggregates. Add the filtrate to the quartz sample cell to measure the potential.

[0030] The results are as follows Figure 1 As shown in Figure D, the potential of Sargassum nanovesicle particles in the concentrate is -39.33mV.

[0031] III. Effects of Sargassum fusiforme nanovesicles on joint stiffness in SD rats (1) Animal model: Female SD rats (weighing 250-280g) were selected, and a shoulder joint stiffness model was constructed using unilateral shoulder joint plaster fixation. The following groups were set up, with 12 rats in each group: Control group: No model established + intraperitoneal injection of normal saline; Model group: Modeling + intraperitoneal injection of physiological saline; Treatment group: Modeling + intraperitoneal injection of Sargassum nanovesicles.

[0032] (2) The drug was administered on the first day after modeling and continued for 8 weeks. The injection dose was 0.5 mL / 100 g, twice a week. After 8 weeks of treatment, the animals were sacrificed, and blood was collected from the hearts of 6 animals in each group to separate and extract serum. The serum was centrifuged at 3000×g for 15 min, and the supernatant was stored at -80℃.

[0033] (3) Serum TNF-α and IL-6 levels were detected using ELISA. Following the ELISA kit instructions, standards or serum samples and biotinylated detection antibodies were added to pre-coated antibody-containing 96-well plates and incubated at 37°C for 2 h. After washing, HRP-labeled streptavidin was added and incubated at 37°C for 30 min. After washing, TMB chromogenic substrate was added and incubated in the dark for 15 min. The reaction was terminated by adding stop solution. The absorbance at 450 nm was measured using a microplate reader, and the sample concentration was calculated using a standard curve.

[0034] The results are shown in Table 1: Compared with the control group, the serum levels of TNF-α and IL-6 in the model group were significantly increased, while after treatment with Sargassum fusiforme nanovesicles, the serum levels of inflammatory factors TNF-α and IL-6 were significantly lower than those in the model group. P <0.05), suggesting that Sargassum nanovesicles have anti-inflammatory effects.

[0035] Table 1. Effects of concentrated solution rich in Sargassum fusiforme nanovesicle particles on serum inflammatory factors in rats with shoulder joint stiffness. IV. Shoulder Joint Pathological Assessment Eight weeks after administration, shoulder joint tissues from different treatment groups were collected, fixed in 4% paraformaldehyde, embedded in paraffin, stained for pathological sections, and mounted with neutral resin.

[0036] 1. HE staining Dewaxed 5 μm thick paraffin sections to water. The sections were then immersed in hematoxylin staining solution for 5 min, rinsed with running water to remove excess stain, differentiated with hydrochloric acid-ethanol for a few seconds, and immediately rinsed back to blue. The sections were then immersed in eosin staining solution for 2 min. After staining, the sections were dehydrated with graded ethanol solutions, cleared with xylene, and finally mounted with neutral resin.

[0037] 2. Masson staining Dewaxed 5 μm thick paraffin sections to water. Stained with Weigert iron hematoxylin solution for 5 min, differentiated with hydrochloric acid-ethanol, and then blued in running water. Subsequently, stained with Ponceau S-acid fuchsin solution for 5 min, and treated with 1% phosphomolybdic acid solution for 2 min. Finally, counterstained with aniline blue solution for 2 min, rapidly differentiated with glacial acetic acid solution, dehydrated and cleared, and then mounted.

[0038] 3. Immunohistochemical staining Immunohistochemical staining was used to detect the expression levels of type I collagen (ColI), type III collagen (ColIII), and α-smooth muscle actin (α-SMA) in tissue sections. The specific procedures are as follows: (1) Dewaxing and hydration of sections The paraffin-embedded tissue sections were heated in a 65°C oven for 2 hours to fully melt the wax. The sections were then dewaxed three times in xylene for 10 minutes each time; then rehydrated in anhydrous ethanol, 95% ethanol, 85% ethanol and 75% ethanol for 5 minutes each time; finally, they were rinsed three times in phosphate-buffered saline (PBS) for 5 minutes each time.

[0039] (2) Antigen retrieval The slides were placed in sodium citrate buffer (pH 6.0) and antigen retrieval was performed using either microwave or autoclaving. Specifically, the slides were placed in an antigen retrieval chamber containing the retrieval solution and heated in a microwave oven on high until boiling, then the microwave was turned off. This process was repeated three times, with a 5-minute interval between each heating. Alternatively, the slides were heated in an autoclave at 121°C for 3 minutes. After retrieval, the slides were allowed to cool naturally to room temperature and then rinsed three times with PBS, 5 minutes each time.

[0040] (3) Endogenous peroxidase blocking To eliminate endogenous peroxidase activity, the slices were immersed in a 3% hydrogen peroxide-methanol solution and incubated at room temperature in the dark for 15 min. After incubation, the slices were rinsed three times with PBS for 5 min each time.

[0041] (4) Serum blocking Add 5% normal goat serum (homogeneous with the secondary antibody) blocking solution and block at room temperature for 30 min to reduce nonspecific background staining. After blocking, discard the blocking solution without washing.

[0042] (5) Primary antibody incubation Add appropriately diluted primary antibody working solution (anti-ColI, ColIII, and α-SMA antibodies, dilution ratio according to kit instructions), place the slides in a humidified chamber, and incubate overnight (12-16 h) at 4°C. After incubation, wash three times with PBS for 5 min each time.

[0043] (6) Secondary antibody incubation Add HRP-labeled goat anti-rabbit IgG secondary antibody working solution (dilution ratio according to kit instructions) and incubate at room temperature for 1 hour. After incubation, wash three times with PBS for 5 minutes each time.

[0044] (7) Color reaction Add DAB chromogenic solution (prepared fresh) and incubate at room temperature in the dark for 3-10 minutes. Control the chromogenic time under a microscope. Terminate the reaction when the positive signal turns brownish-yellow and the background remains unstained. Rinse three times with distilled water after termination.

[0045] (8) Nuclear counterstaining Counterstain cell nuclei with hematoxylin solution and incubate at room temperature for 3-5 min. Then rapidly differentiate with 0.5% hydrochloric acid-ethanol differentiation solution (about 5-10 s), and rinse with running tap water for 15 min to return to blue.

[0046] (9) Sealing and observation The sections were dehydrated by sequentially immersing them in 75% ethanol, 85% ethanol, 95% ethanol, and anhydrous ethanol for 5 minutes each, followed by clearing in xylene three times for 5 minutes each time. Finally, the sections were mounted with neutral resin and observed and images were acquired under an optical microscope or a fluorescence microscope.

[0047] 4. Results and Analysis like Figure 2 As shown, HE staining results indicated that: in the control group, the axillary bursa tissue (mainly collagen fibers) was arranged in an orderly manner, with less lymphocyte infiltration on the joint side; in the model group, the joint capsule tissue was arranged in a disordered manner, varying in size, with some areas clumped together, and more lymphocyte infiltration on the joint side (red arrow); in the treatment group, the joint capsule tissue was arranged in an orderly manner along the longitudinal direction of the joint capsule, with uniform layering, and less lymphocyte infiltration on the joint side (red arrow).

[0048] like Figure 3 As shown, Masson staining results indicated that the axillary bursa joint capsule tissue in the control group was relatively thin, with collagen fibers arranged in an orderly layered manner; the collagen fibers in the axillary bursa joint capsule tissue of the model group were arranged in a disordered manner and showed obvious densification, indicating a high degree of fibrosis; the collagen fibers in the joint capsule of the treatment group were wavy and relatively loosely arranged, with no obvious dense fibrous clusters.

[0049] like Figure 4 As shown, the immunohistochemical staining results of type I collagen showed that, compared with the control group, the area of ​​positive type I collagen staining in the axillary sac of the model group was significantly increased. P <0.01), while after treatment with Sargassum nanovesicles, the area of ​​positive staining for type I collagen in the axillary fossa of the treatment group was significantly smaller than that of the model group ( P <0.01). Statistical results of type I collagen positive staining area (nominal signal value): control group: 23503.755±6826.590 μm. 2 The model set is 171854.795±25722.733μm. 2 The treatment group had a concentration of 86302.355 ± 15792.583 μm.2 .

[0050] like Figure 5 As shown, the immunohistochemical staining results of type III collagen showed that, compared with the control group, the area of ​​positive staining for type III collagen in the axillary sac was significantly increased in the model group. P <0.01), while after treatment with Sargassum nanovesicles, the staining area of ​​type III collagen in the axillary fossa of the shoulder joint in the treatment group was significantly less than that in the model group ( P <0.01), the control group had a thicker layer of type III collagen on the surface of the joint capsule, indicating significant joint capsule fibrosis, while the treatment group had a thinner layer of type III collagen. Statistical results of the positive staining area (nominal signal value) of type III collagen: Control group: 22856.362±5836.733 μm 2 The model set was 161937.994±23532.978 μm. 2 The treatment group had a concentration of 98940.381 ± 10790.583 μm. 2 .

[0051] like Figure 6 As shown, the immunohistochemical staining results of αSMA protein showed that, compared with the control group, the area of ​​αSMA protein-positive staining in the axillary sac of the model group was significantly increased. P <0.01, after treatment with Sargassum fusiforme nanovesicles, the expression of αSMA in the axillary bursa of the shoulder joint in the treatment group was significantly lower than that in the model group ( P <0.01 indicates lower myofibroblast activation in the treatment group. Statistical results of αSMA protein positive staining area (nominal signal value): Control group: 3114.409±753.711 μm 2 The model group was 15659.511±5358.396 μm. 2 The treatment group had a concentration of 9814.240 ± 3150.762 μm. 2 .

[0052] The above results indicate that Sargassum nanovesicles can inhibit the formation of type I and type III collagen in joint capsules and inhibit the activation of myofibroblasts, thereby playing a role in treating joint stiffness and providing a new drug treatment strategy for clinical diseases such as frozen shoulder and postoperative joint stiffness.

[0053] V. Shoulder joint range of motion measurement Six rats were taken from each group, anesthetized and euthanized, and the rats' upper limbs were removed intact. The scapula was fixed to a flat plate, and a 0.4 N tension was applied to the distal end of the upper limb. A straight line was drawn along the scapular spine, and a straight line was drawn between the distal end of the scapular spine and the olecranon of the ulna. The angle between the two lines was recorded as the range of motion of the shoulder joint.

[0054] like Figure 7As shown, the results indicated that the shoulder joint range of motion was 175.308±7.022 degrees in the control group, 103.255±10.376 degrees in the model group, and 123.790±15.523 degrees in the treatment group. Compared with the control group, the shoulder joint range of motion was significantly reduced in the model group. P <0.01%, after treatment with Sargassum fusiforme nanovesicles, the shoulder joint range of motion in the treatment group was significantly increased ( P <0.05), shoulder joint stiffness was significantly improved, indicating that Sargassum nanovesicles have a therapeutic effect on shoulder joint stiffness.

[0055] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of Sargassum fusiforme nanovesicles in the preparation of drugs for treating shoulder joint stiffness, characterized in that, The Sargassum nanovesicles were isolated from Sargassum.

2. The application as described in claim 1, characterized in that, The separation method includes the following steps: Sargassum fusiforme was mixed with PBS solution, then crushed and ground to obtain a grinding solution; The grinding slurry was centrifuged at differential speed to obtain the supernatant. The supernatant is filtered using a hollow fiber column, and the retained concentrate is collected to obtain the product.

3. The application as described in claim 2, characterized in that, The mass-to-volume ratio of Sargassum fusiforme to the PBS solution is 1:(3-5).

4. The application as described in claim 2, characterized in that, Each grinding session lasts 1 minute, for a total of 3-5 times. After each grinding session, place the mixture on ice for 3-10 minutes before proceeding with the next grinding session.

5. The application as described in claim 2, characterized in that, The differential centrifugation is performed as follows: the grinding liquid is centrifuged at 1000 g for 10 min. After centrifugation, the precipitate is discarded, and the supernatant is centrifuged at 3000 g for 20 min. After centrifugation, the precipitate is discarded, and the supernatant is centrifuged at 10000 g for 40 min.

6. The application as described in claim 2, characterized in that, The hollow fiber column has a pore size of 200 nm.

7. A medicine for treating or relieving shoulder stiffness, characterized in that, The drug comprises Sargassum nanovesicles as described in any one of claims 1-6.

8. The drug as described in claim 7, characterized in that, The drug is used to regulate the formation of collagen fibers in the joint capsule, the activation of fibroblasts, and the range of motion of the shoulder joint.

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