Use of polygonatum exosome in preparation of medicine for preventing and treating osteoporosis

Abstract

The application discloses application of a polygonatum exosome in preparation of a medicine for preventing and treating osteoporosis, and the polygonatum exosome has good biocompatibility, can significantly inhibit differentiation of osteoclasts, promote differentiation of osteocytes, effectively target bone tissues, significantly improve osteoporosis, and has potential in preparation of a medicine product for preventing and treating osteoporosis. The polygonatum exosome is rich in sources, has potential active natural ingredients, and is low in cost, and is suitable for large-scale batch production.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the application of Polygonatum exosomes, specifically to the application of Polygonatum exosomes in the preparation of drugs for the prevention and treatment of osteoporosis. Background Technology

[0002] Osteoporosis (OP) is a common chronic bone metabolic disease characterized by decreased bone density and mass, deterioration of bone microstructure, increased bone fragility, and bone pain, significantly increasing the risk of fractures. Epidemiological data shows that more than 200 million people worldwide currently suffer from osteoporosis. With the increasing aging population and changes in lifestyle, osteoporosis has become one of the most common skeletal diseases globally, leading to a severe socioeconomic and medical burden.

[0003] Currently, there are many types of drugs used in clinical practice to treat osteoporosis, such as bisphosphonates and estrogen-like hormones. Although these drugs have certain therapeutic effects, long-term use is often accompanied by side effects, such as gastrointestinal discomfort, increased risk of cardiovascular disease, and even complications such as atypical fractures. Therefore, there is an urgent need to explore treatment drugs that are safer, more effective, and have fewer side effects.

[0004] As the unique advantages of traditional Chinese medicine (TCM) in disease prevention and treatment become increasingly apparent, numerous studies on TCM preparations for treating osteoporosis have been reported. TCM herbs, due to their characteristics of "multiple components, multiple targets, and holistic regulation," possess significant advantages in preventing and treating osteoporosis. However, traditional TCM herbs suffer from problems such as poor water solubility of active ingredients in the body, low bioavailability, and poor targeting, which limit their clinical efficacy.

[0005] Polygonatum is a traditional Chinese tonic herb, and historical herbal texts have recorded its effects of "nourishing the kidneys and replenishing essence, strengthening tendons and bones," making it suitable for adjunctive treatment of osteoporosis and other bone-related diseases. Existing studies have shown that Polygonatum polysaccharides exhibit significant effects in free radical scavenging, phagocytosis by immune cells, and the recovery of immune function in immunosuppressed mice. However, systematic research on the role of Polygonatum exosomes in the prevention and treatment of osteoporosis is currently lacking, and the application of Polygonatum exosomes in the preparation of drugs for the prevention and treatment of osteoporosis has not been reported. Summary of the Invention

[0006] Therefore, in order to solve the technical problems existing in the prior art, the present invention proposes the application of Polygonatum exosomes in the preparation of drugs for the prevention and treatment of osteoporosis.

[0007] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: This invention provides the application of Polygonatum exosomes in the preparation of drugs for the prevention and treatment of osteoporosis.

[0008] Specifically, the application is the use of Polygonatum exosomes in the preparation of drugs that inhibit osteoclast differentiation.

[0009] Specifically, the application is the use of Polygonatum exosomes in the preparation of drugs that promote osteogenic differentiation of bone marrow mesenchymal stem cells.

[0010] Specifically, the application is the use of Polygonatum exosomes in the preparation of drugs that improve the microstructure of cancellous bone.

[0011] Specifically, the Huangjing exosomes are Huangjing exosome-like nanovesicles.

[0012] Specifically, the Polygonatum exosomes are prepared by the following method: S1. Processing Polygonatum sibiricum; S2. Prepare Polygonatum filtrate: Crush and filter the processed Polygonatum to obtain Polygonatum filtrate; S3. Purify the Polygonatum exosomes by centrifuging and filtering the Polygonatum filtrate, and take the supernatant after filtration to obtain the Polygonatum exosomes.

[0013] Specifically, in step S1, the processing of Polygonatum includes at least one step of steaming Polygonatum at 100-110℃ for 2-3 hours per steaming.

[0014] Specifically, step S2 includes: mixing the processed Polygonatum with a buffer solution, grinding and pulverizing it, collecting the grinding liquid, and filtering the grinding liquid to obtain Polygonatum filtrate.

[0015] Specifically, step S3 includes: centrifuging the Polygonatum filtrate, collecting the supernatant, centrifuging the supernatant at least once, taking the centrifuged precipitate and resuspending it with a buffer solution, filtering the obtained resuspended liquid, and collecting the supernatant to obtain the Polygonatum exosomes.

[0016] Specifically, the drug also includes pharmaceutically acceptable excipients.

[0017] The technical solution of the present invention has the following advantages compared with the prior art: The application of Polygonatum exosomes provided by this invention in the preparation of drugs for the prevention and treatment of osteoporosis. Polygonatum exosomes have good biocompatibility, can significantly inhibit osteoclast differentiation and promote osteoblast differentiation, effectively target bone tissue, and significantly improve osteoporosis. They have the potential to prepare drugs for the prevention and treatment of osteoporosis. Polygonatum exosomes are abundant, are natural components with potential activity, and are inexpensive, making them suitable for large-scale mass production. Attached Figure Description

[0018] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein... Figure 1 This is a transmission electron microscope image of the exosomes of Polygonatum sibiricum; Figure 2 This is a particle size distribution diagram of Polygonatum exosomes; Figure 3 This is a Zeta potential distribution diagram of Polygonatum exosomes; Figure 4 This is a fluorescence image of exosomes from Polygonatum sibiricum staining cells for cell viability; Figure 5 The bar chart shows cell viability on the first and fourth days after treatment with different concentrations of Polygonatum exosomes. Figure 6 These are TRAP staining images of macrophages after 6 days of treatment with different concentrations of Polygonatum exosomes. Figure 7 This corresponds to a quantitative PCR graph of TRAP enzyme activity. Figure 8 This corresponds to the Ctsk immunofluorescence staining pattern; Figure 9 These are ALP and Alizarin Red staining images after processing bone marrow mesenchymal stem cells with Polygonatum exosomes; Figure 10 This corresponds to a quantitative graph of ALP enzyme activity; Figure 11 This corresponds to a quantitative assay of intracellular calcium deposition. Figure 12 This is a Western blot (WB) assay for the expression of osteogenic-related proteins. Figure 13 These are fluorescence images of mouse heart, liver, spleen, lung, kidney and bone tissues at different time points after intravenous injection of PBS, DiD and DiR labeled Polygonatum exosomes. Figure 14 This is a quantitative fluorescence image of each organ in the DiD injection group; Figure 15 This is a quantitative fluorescence image of each organ in the DiD-PELNs-injected group; Figure 16 This is a timeline diagram of OVX modeling and drug administration in mice; Figure 17 This is a Micro-CT scan of the femur of a mouse after drug treatment; Figure 18 This is an H&E staining image of a bone tissue section. Detailed Implementation

[0019] 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.

[0020] 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.

[0021] 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 to or equivalent to those described herein may be used in the implementation or testing of this invention.

[0022] Unless otherwise specified, all materials used in the following embodiments are commercially available. Unless otherwise specified, all methods described are conventional methods.

[0023] Example 1. Experimental materials and reagents Experimental reagents: fresh Polygonatum sibiricum, PBS buffer solution (phosphate buffer solution) (1X, pH=7.4).

[0024] 2. Experimental instruments and materials: stirrer; ultracentrifuge (Beckman, OptimaXE-90), microporous filter membrane (0.22μm).

[0025] This embodiment provides the application of Polygonatum exosomes in the preparation of a drug for the prevention and treatment of osteoporosis, wherein the Polygonatum exosomes are Polygonatum exosome-like nanovesicles, and the drug also includes pharmaceutically acceptable excipients.

[0026] Polygonatum exosomes were prepared through the following steps: S1. Processing Polygonatum: Steam fresh Polygonatum at 100-110℃ at least once, with each steaming time being 2-3 hours. In this embodiment, preferably, the number of steaming times is 9. After each steaming, place the Polygonatum in a ventilated place to obtain processed Polygonatum.

[0027] S2. To prepare the Polygonatum filtrate, cut the processed Polygonatum into small pieces, mix it with PBS buffer solution, grind it in a mixer, and then filter it coarsely with gauze to obtain the Polygonatum filtrate.

[0028] S3. Purify Polygonatum exosomes: Centrifuge the Polygonatum filtrate at 10000g for 1 hour at 4℃, collect the supernatant, and centrifuge the supernatant twice at 12000g for 90 minutes each time. Take the precipitate each time and resuspend it in pre-cooled PBS buffer solution. Filter the obtained resuspended solution through a 0.22μm microporous membrane one by one, and take the supernatant to obtain high-purity Polygonatum exosomes.

[0029] The application of Polygonatum exosomes provided in this embodiment in the preparation of drugs for the prevention and treatment of osteoporosis. Polygonatum exosomes have good biocompatibility, can significantly inhibit osteoclast differentiation and promote osteoblast differentiation, effectively target bone tissue, and significantly improve osteoporosis. They have the potential to prepare drugs for the prevention and treatment of osteoporosis. Polygonatum exosomes are abundant, are natural components with potential activity, and are inexpensive, making them suitable for large-scale mass production.

[0030] Experimental Example 1. Characterization by transmission electron microscopy (TEM) Take 10 μL of purified Polygonatum exosomes prepared according to the steps provided in the example, drop it onto a carbon film copper grid, let it stand for 1 min to allow sedimentation, remove excess liquid, then drop 10 μL of uranium acetate solution onto the copper grid, stain for 1 min, and blot away the floating liquid again with filter paper. After the sample is naturally dried at room temperature for 5 min, it is observed and imaged using a transmission electron microscope (TEM, 80 kV). The TEM image is shown in Figure 1. Figure 1 As can be seen, the prepared Polygonatum exosomes are intact, uniform in size, and exhibit a typical cup-shaped vesicle structure, which is complete and stable.

[0031] 2. Particle size distribution and concentration determination Take 1 μL of purified Polygonatum sibiricum exosomes prepared according to the steps provided in the example, and dilute to 1000 μL with PBS buffer. Load the diluted exosome sample into a clean syringe and place it into a PMX120 nanoparticle tracking analysis (NTA) system for detection, recording the particle size distribution and particle concentration. The results are as follows: Figure 2 As shown, the test results indicate that the prepared Polygonatum exosomes have a concentrated particle size distribution and uniform size, with an average particle size of approximately 141.9 nm.

[0032] 3. Zeta potential measurement 20 μL of purified Polygonatum exosomes prepared according to the steps provided in the example was diluted to 1 mL with PBS buffer and mixed well, then transferred to a disposable electrophoresis cuvette. The Zeta potential was measured at 25 °C using a Zetasizer Nano ZS dynamic light scattering (DLS) analyzer (Malvern). Each sample was measured three times to obtain the average value. The experimental results are shown in Figure 3. The test results indicate that the prepared Polygonatum exosomes have a negatively charged surface, a Zeta potential of approximately -10 mV, and good colloidal stability and other physicochemical properties.

[0033] 4. Detection of cytotoxicity of Polygonatum exosomes Rat bone marrow mesenchymal stem cells (BMSCs) in the logarithmic growth phase were collected and prepared into 5 × 10⁻⁶ cells. 4 Cell suspensions of 100 μL / mL were seeded into each well of a 96-well plate and cultured overnight at 37 °C with 5% carbon dioxide (CO2) to allow cell adhesion and growth. The next day, different concentrations of exosome solutions (0, 0.2, 1, 10, 50 μg / mL) prepared with complete culture medium were added to each well. After 24 h of treatment, the cells were stained for viability using a Live / Dead cell staining kit (Beyotime, C2015), and cell viability was observed under a confocal microscope. The results are shown below. Figure 4 As shown in Figure 5. Simultaneously, cell viability was assessed according to the CCK-8 reagent kit (Beyotime, C0037) instructions: 10 μL of CCK-8 reagent and 100 μL of complete culture medium were added to each well, and the mixture was incubated at room temperature in the dark for 1 h. The absorbance of each well was measured at 450 nm. The results are shown in Figure 5. Within the concentration range of 0–50 μg / mL, the Polygonatum exosomes prepared according to the steps provided in the examples did not significantly inhibit the activity of BMSCs, indicating that they have good biocompatibility and low cytotoxicity. In the figure, ns indicates no significant difference.

[0034] 5. Test on the inhibitory effect of Polygonatum exosomes on osteoclast differentiation To investigate the inhibitory effect of Polygonatum exosomes on osteoclast differentiation, macrophages were prepared at a ratio of 2 × 10⁻⁶. 4 cells / cm 2Cells were seeded in 48-well plates and induced to undergo osteoclastogenesis induction using induction medium containing M-CSF (30 ng / mL) and RANKL (50 ng / mL). Simultaneously, different concentrations (0, 2, 10, and 50 μg / mL) of Polygonatum exosomes were added for 6 days. After induction, cells were stained using a tartrate acid phosphatase (TRAP) staining kit (Solepro, G1492), and the formation of multinucleated TRAP-positive cells was observed under a microscope (e.g., ...). Figure 6 As shown), the TRAP enzyme activity level in cell secretions was detected and quantitatively analyzed using a TRAP enzyme activity assay kit (Yuanye Biotechnology, R22113). Figure 7 (As shown). Furthermore, to further evaluate the expression of osteoclast-related markers, the expression of Catepsin K (Ctsk) was detected using immunofluorescence staining. The specific procedure was as follows: Macrophages after induced differentiation were fixed on coverslips in 4% paraformaldehyde for 15 minutes, then washed three times with PBS buffer, 5 minutes each time. Subsequently, they were incubated with 0.5% Triton X-100 at room temperature for 10 minutes to permeate the cell membrane, and non-specific binding sites were blocked for 30 minutes with PBS blocking solution containing 5% bovine serum albumin (BSA). After blocking, Ctsk primary antibody (SantaCruz, sc-48353, 1:200) was added and incubated overnight at 4°C. The next day, unbound primary antibody was washed away three times with PBS buffer, 5 minutes each time, and then fluorescently labeled secondary antibody (Proteintech, SA00013-1, 1:200) was added and incubated at room temperature in the dark for 1 hour. After incubation, the cells were washed three times with PBS buffer, followed by DAPI staining of the nuclei for 5 minutes. Finally, residual dye was washed away with PBS buffer. After mounting, Ctsk fluorescence signals were observed and captured under a laser confocal microscope to assess Ctsk expression in osteoclasts (e.g., Figure 8 (As shown in the image). The above tests indicate that Polygonatum exosomes can effectively inhibit osteoclast differentiation of macrophages.

[0035] 6. Test on the promoting effect of Polygonatum exosomes on osteogenic differentiation of bone marrow mesenchymal stem cells Bone marrow mesenchymal stem cells (BMSCs) were distributed at a density of 5000 cells / cm³. 2 Cells were seeded in 48-well plates and co-cultured with exosomes of Polygonatum sibiricum (10 μg / mL) in osteogenic induction medium. After 7 days of induction culture, cells were stained for alkaline phosphatase (ALP), and enzyme activity levels were measured using an ALP activity assay kit (Beyotime, P0321) to assess early osteogenic differentiation (e.g., ...). Figure 9-10(As shown). After 21 days of induction culture, the formation of intracellular calcium nodules was observed using Alizarin Red staining, and the calcium ion content in the staining solution was measured using a calcium ion quantitative detection kit (Beyotime, S1063) to reflect the degree of late osteogenic differentiation (e.g., ...). Figure 9 , Figure 11 (As shown). Total protein from BMSCs was then extracted for Western blot analysis. First, cells were thoroughly lysed using RIPA lysis buffer. After lysis for 30 minutes, the cells were centrifuged at 12000 g for 10 minutes at 4°C, and the supernatant was collected as the total protein sample. Protein concentration was determined using the BCA method, and an equal volume of protein (30 μg) was added to loading buffer and denatured at 95°C for 5 minutes. The sample was loaded onto a 12% SDS-PAGE gel for electrophoresis. After electrophoresis, the protein was transferred to a PVDF membrane. After transfer, the membrane was blocked in TBST with 5% skim milk at room temperature for 1 hour to block non-specific binding sites. Then, the membrane was incubated overnight at 4°C with primary antibodies against ALP (Abclonal, A12396), RUNX2 (Proteintech, 20700-1-AP), and Col1 (Proteintech, 14695-1-AP). The following day, the membrane was washed three times with TBST for 10 minutes each time, and then incubated with the corresponding HRP-labeled secondary antibody at room temperature for 1 hour. After washing away excess secondary antibody, the sample was visualized using chemiluminescent (ECL) reagent, and the signal was recorded using an imaging system. Finally, GAPDH was used as an internal control for semi-quantitative analysis to compare the expression levels of bone-related proteins in each component (e.g., ...). Figure 12 The test results showed that Polygonatum exosomes could significantly promote osteogenic differentiation of bone marrow mesenchymal stem cells.

[0036] 7. Biodistribution test of Polygonatum exosomes in vivo To evaluate the distribution characteristics of exosomes (PELNs) in vivo and their bone-targeting ability, the Polygonatum sibiricum exosomes prepared according to the preparation steps provided in the examples were used at a concentration of 1×10⁻⁶. 11Particles were labeled with fluorescent dyes. The specific method is as follows: Polygonatum exosomes were added to a pre-prepared DiD working solution, gently mixed at room temperature in the dark, and incubated for 30 minutes to allow the fluorescent dye to fully bind to the exosome membrane surface. After incubation, the mixture was centrifuged at 120,000 × g at 4 °C for 90 minutes. The supernatant was discarded, and the precipitate was resuspended in sterile PBS to completely remove unbound free dye, ultimately obtaining DiD-labeled PELNs (DiD-PELNs). For animal experiments, 8-week-old female mice were used. DiD-PELNs were slowly injected into the body via the tail vein; the control group was injected with an equal volume of PBS or DiD solution. On days 1, 2, 3, 4, 5, and 6 after injection, mice were sacrificed, and their major organs (heart, liver, spleen, lungs, kidneys) and bone tissue such as the femur were removed. After rinsing off any remaining blood with PBS, the tissues were placed in a small animal in vivo imaging system for imaging analysis. Imaging conditions were set at an excitation wavelength of 644 nm and an emission wavelength of 665 nm. Fluorescence signal images were acquired under uniform exposure parameters, and the fluorescence intensity of each tissue site was quantitatively measured using accompanying analysis software to assess the distribution pattern of PELNs in vivo and their bone-targeting enrichment ability (e.g., Figure 13-15 As shown in the figure, the test results indicate that Polygonatum exosomes can effectively target bone tissue and remain in bone tissue for more than 6 days.

[0037] 8. Therapeutic effect of Polygonatum exosomes on a mouse model of osteoporosis. To evaluate the therapeutic effect of Polygonatum sibiricum exosomes (PELNs) in a mouse model, this study used 8-week-old female mice to establish an osteoporosis model through bilateral ovariectomy (OVX). Three groups were established: a Sham group (sham-operated control), a PBS group (OVX+PBS control), and an exosome group (OVX+PELNs) (n = 6). After surgery, the mice were routinely fed for 6 weeks to ensure model stability, followed by drug administration: the exosome group received slow intravenous injection of PELNs via the tail vein at a dose of 5 × 10⁻⁶. 10 Particles / 100 μL, once weekly for 6 consecutive weeks; the PBS group received an equal volume of PBS as a control during the same period. Animals were sacrificed after administration, and femurs and tibias were collected for subsequent analysis (e.g., ...). Figure 16 (As shown). First, the femoral sample was scanned using a Micro-CT system. Three-dimensional reconstruction was then used to analyze the structural parameters of the trabecular bone to evaluate the recovery of bone mass and bone microstructure (e.g., Figure 17 (As shown). Subsequently, femoral samples were decalcified with 10% EDTA, embedded in paraffin, and sectioned for H&E staining. The morphology of bone trabeculae and the repair of bone tissue structure were observed under an optical microscope to further verify the promoting effect of PELNs on bone tissue remodeling at the histological level (e.g. Figure 18(As shown). Micro-CT analysis results showed that in the ovariectomy (OVX) mouse osteoporosis model, Polygonatum exosomes significantly improved the microstructure of cancellous bone and increased key bone parameters such as bone volume fraction and trabecular thickness.

[0038] Based on the above test results, the plant exosomes derived from Polygonatum provided in this embodiment of the invention can be used to prepare drugs for the prevention and treatment of osteoporosis, especially drugs that inhibit osteoclast differentiation of macrophages, promote osteogenic differentiation of bone marrow mesenchymal stem cells, and improve the microstructure of cancellous bone.

[0039] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. The application of Polygonatum exosomes in the preparation of drugs for the prevention and treatment of osteoporosis, characterized in that, The Polygonatum exosomes were prepared by the following method: S1. Processing Polygonatum: Steam fresh Polygonatum at 100-110℃ at least once, each time for 2-3 hours. After each steaming, place the Polygonatum in a ventilated place to air dry, and you will get processed Polygonatum. S2. To prepare the Polygonatum filtrate, cut the processed Polygonatum into small pieces, mix it with PBS buffer solution, grind it in a mixer, and then filter it coarsely with gauze to obtain the Polygonatum filtrate. S3. Purify Polygonatum exosomes. Centrifuge the Polygonatum filtrate at 10000g for 1 hour at 4℃. Collect the supernatant and centrifuge the supernatant twice at 12000g for 90 minutes each time. Take the precipitate each time and resuspend it in pre-cooled PBS buffer solution. Filter the obtained resuspended solution through a 0.22μm microporous membrane. Take the supernatant to obtain Polygonatum exosomes.

2. The application according to claim 1, characterized in that, The steaming process is repeated 9 times.

3. The application according to claim 1 or 2, characterized in that, The exosomes of Polygonatum are exosome-like nanovesicles.

4. The application according to claim 3, characterized in that, The drug also includes pharmaceutically acceptable excipients.

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