Application of miR-130b-5p in preparation of medicine for treating osteoarthritis as well as sustained release system and preparation method of miR-130b-5p

Abstract

The invention discloses application of miR-130b-5p in preparation of a medicine for treating osteoarthritis, a sustained release system and a preparation method, and belongs to the technical field of medicines, and the sustained release system is ZIF-8 (at) miR-130b-5p constructed by ZIF-8 loaded miR-130b-5p. The miR-130b-5p provided by the invention can be used for remarkably relieving cartilage cell injury in an inflammation environment and improving a local inflammation state. In an OA mouse model, the ZIF-8 (at) miR-130b-5p sustained-release system disclosed by the invention can be used for remarkably improving the inflammatory response and cartilage injury of a joint focus area and remarkably improving the motor function.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to an osteoarthritis drug, particularly to the application of miR-130b-5p in the preparation of a drug for treating osteoarthritis, as well as its sustained-release system and preparation method. Background Technology

[0002] Osteoarthritis (OA) is a common chronic degenerative joint disease, mainly characterized by degeneration of articular cartilage, chronic inflammation of the synovium, subchondral bone remodeling, and osteophyte formation. Clinical symptoms include joint pain, functional impairment, and limited mobility. With the increasing aging of the global population, the prevalence of OA is rising, placing a significant economic burden on patients and societal healthcare resources. Currently, there are no effective treatments to slow or reverse the progression of OA; therefore, a deeper understanding of the pathogenesis of OA and the development of effective treatments have significant clinical and socioeconomic value.

[0003] In recent years, mesenchymal stem cells (MSCs) have become a hot research topic in the field of OA biotherapy due to their multiple roles in immunomodulation and tissue repair promotion. In particular, exosomes secreted by MSCs have shown great potential in anti-inflammatory and tissue regeneration aspects. However, existing studies have found that the efficacy of MSC exosomes in treating OA is inconsistent. Summary of the Invention

[0004] This invention provides the application of miR-130b-5p in the preparation of drugs for treating osteoarthritis, as well as a sustained-release system and preparation method, to overcome the shortcomings of the prior art.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides the use of miR-130b-5p in the preparation of a medicament for treating osteoarthritis.

[0007] In a second aspect, the present invention provides a medicament for treating osteoarthritis: the active ingredient of the medicament is miR-130b-5p.

[0008] Thirdly, the present invention provides a sustained-release system for treating osteoarthritis: the sustained-release system is ZIF-8@miR-130b-5p constructed by loading miR-130b-5p onto ZIF-8.

[0009] Fourthly, the present invention provides a method for preparing the above-mentioned sustained-release system: adding miR-130b-5p solution to zinc nitrate solution, stirring rapidly, then rapidly adding 2-methylimidazole solution, stirring rapidly, to obtain ZIF-8@miR-130b-5p.

[0010] Furthermore, the miR-130b-5p solution is a DMSO solution of miR-130b-5p with a concentration of 40–60 μg / mL, preferably 50 μg / mL.

[0011] Further, the zinc nitrate is zinc nitrate hexahydrate; the mass ratio of miR-130b-5p, zinc nitrate hexahydrate, and 2-methylimidazole is 0.040–0.060:110–125:2000–3000, preferably 0.050:117:2500.

[0012] Furthermore, after adding the 2-methylimidazole solution, the rapid stirring time is 3 to 8 minutes, preferably 5 minutes.

[0013] Further, after adding the 2-methylimidazole solution and stirring rapidly, the precipitate was collected by centrifugation, and the solid was collected by freeze-drying, which is the ZIF-8@miR-130b-5p.

[0014] Fifthly, the present invention provides the application of the above-described sustained-release system in the preparation of a drug for treating osteoarthritis.

[0015] Furthermore, the sustained-release system is used in the preparation of drugs that improve the inflammatory microenvironment, cartilage damage, and synovial lesions in osteoarthritis.

[0016] The beneficial effects of this invention are as follows:

[0017] Firstly, this invention addresses the issues of unstable efficacy and unclear mechanisms of action in current stem cell therapy for osteoarthritis by providing an effective drug component for osteoarthritis, miR-130b-5p. miR-130b-5p is a miRNA enriched in the exosomes of young MSCs, which can effectively improve the inflammatory microenvironment, cartilage damage, and synovial lesions in osteoarthritis. It exhibits anti-inflammatory and cartilage repair-promoting effects in the treatment of osteoarthritis, enhancing the targeting and controllability of biotherapy. The proposed active ingredient breaks away from treatment models dependent on whole-cell or exosome components, providing a new research direction for RNA-targeted therapy.

[0018] Secondly, this invention also provides a ZIF-8-based micro / nano sustained-release system. By loading miR-130b-5p onto ZIF-8 material, stable encapsulation and precise release of miR-130b-5p at the lesion site of osteoarthritis are achieved. This sustained-release system exhibits excellent sustained-release performance and biocompatibility, significantly prolonging the duration of local drug efficacy, reducing the frequency of administration, and enhancing the stability and compliance of treatment effects. Furthermore, this cell-free therapeutic approach possesses advantages such as ease of operation, high controllability, and high translational potential, providing a novel approach and technological platform for precision therapy using sustained-release systems. Attached Figure Description

[0019] Figure 1 This is a flowchart of the extraction process for exosomes derived from young MSCs;

[0020] Figure 2 This is a TEM image of exosomes derived from young MSCs;

[0021] Figure 3 This is a Western blot result of Alix and Tsg101 exosomes derived from young MSCs;

[0022] Figure 4 This is a particle size distribution map of exosomes derived from young MSCs;

[0023] Figure 5 These are TEM images of ZIF-8 and ZIF-8@miR-130b-5p;

[0024] Figure 6 This is a graph showing the miRNA release performance of ZIF-8@miR-130b-5p under different pH conditions;

[0025] Figure 7 These are the XRD patterns of stimulated ZIF-8, ZIF-8, and ZIF-8@miR-130b-5p;

[0026] Figure 8 These are hydrodynamic particle size distribution diagrams for ZIF-8 and ZIF-8@miR-130b-5p.

[0027] Figure 9 This is a graph showing the qRT-PCR results of inflammatory factors and factors promoting anti-inflammation and repair in various groups of young MSC-derived exosome cells experiments;

[0028] Figure 10 This is a Western blot result of inflammatory factors and factors promoting anti-inflammation and repair in each group of experiments using young MSC-derived exosome cells;

[0029] Figure 11These are immunofluorescence staining images of various groups in experiments using exosomes derived from young MSCs.

[0030] Figure 12 These are flow cytometry structural diagrams of each group in experiments using exosomes derived from young MSCs.

[0031] Figure 13 These are H&E staining images from various groups in animal experiments using young MSC-derived exosomes.

[0032] Figure 14 These are immunofluorescence staining images of various groups in animal experiments using exosomes derived from young MSCs;

[0033] Figure 15 These are toluidine blue and H&E staining images from various groups in experiments on young MSC-derived exosomes.

[0034] Figure 16 These are immunofluorescence and immunohistochemical staining images of various groups in animal experiments with exosomes derived from young MSCs.

[0035] Figure 17 These are immunofluorescence and immunohistochemical staining images of various groups in animal experiments with exosomes derived from young MSCs.

[0036] Figure 18 This is a graph showing the transcriptomic sequencing results of exosomes derived from young and aged MSCs;

[0037] Figure 19 This is a graph showing the qRT-PCR results of inflammatory factors and factors promoting anti-inflammatory and repair-related factors in each group of miR-130b-5p cell experiments;

[0038] Figure 20 This is a Western blot result of inflammatory factors and factors promoting anti-inflammation and repair in each group of miR-130b-5p cell experiments;

[0039] Figure 21 The image shows the qRT-PCR results of inflammatory factors and anti-inflammatory and repair-related factors in each group of miR-130b-5p and ZIF-8@miR-130b-5p cell experiments.

[0040] Figure 22 This is a Western blot result of inflammatory factors and anti-inflammatory and repair-related factors in each group of miR-130b-5p and ZIF-8@miR-130b-5p cell experiments.

[0041] Figure 23 The images show the results of immunofluorescence staining and flow cytometry analysis of each group in the miR-130b-5p and ZIF-8@miR-130b-5p cell experiments.

[0042] Figure 24 These are the H&E and immunofluorescence staining images of each group in animal experiments with miR-130b-5p and ZIF-8@miR-130b-5p.

[0043] Figure 25 These are toluidine blue and H&E staining images of each group in animal experiments with miR-130b-5p and ZIF-8@miR-130b-5p;

[0044] Figure 26 These are immunofluorescence and immunohistochemical staining images of various groups in animal experiments with miR-130b-5p and ZIF-8@miR-130b-5p.

[0045] Figure 27 These are immunohistochemical and immunofluorescence staining images of various groups in animal experiments with miR-130b-5p and ZIF-8@miR-130b-5p.

[0046] Figure 28 These are MicroCT images of various groups in animal experiments using miR-130b-5p and ZIF-8@miR-130b-5p.

[0047] Figure 29 These are open field experiment results for each group in animal experiments with miR-130b-5p and ZIF-8@miR-130b-5p. Detailed Implementation

[0048] The present invention will be further described below with reference to specific embodiments.

[0049] This invention provides the application of miR-130b-5p and the ZIF-8@miR-130b-5p sustained-release system constructed by loading miR-130b-5p onto ZIF-8 in the preparation of drugs for treating osteoarthritis.

[0050] The preparation method of ZIF-8@miR-130b-5p is as follows: First, dissolve 117 mg of zinc nitrate hexahydrate in 1 mL of ultrapure water. Second, weigh 2.5 g of 2-methylimidazole and dissolve it in 8 mL of water. Add 1 mL of miR-130b-5p solution (50 μg / mL, DMSO) to the zinc nitrate solution and stir rapidly. Quickly add the 2-methylimidazole solution to the above mixture and stir rapidly for 5 minutes. Finally, centrifuge to collect the precipitate (6500 rpm, 30 min), freeze-dry to collect the solid, and obtain ZIF-8@miR-130b-5p.

[0051] Characterization of miR-130b-5p and ZIF-8@miR-130b-5p and related substances: such as Figure 1 As shown, EVs were isolated from MSC conditioned medium using differential / ultracentrifugation coupled with membrane filtration. Typical vesicle morphology was observed using transmission electron microscopy, as shown... Figure 2 As shown. Figure 3 As shown, Western blot detected Alix and TSG101 in the EV fraction but not in the cell lysate, indicating reliable separation purity and identity. The particle size distribution of exosomes was determined by particle tracking / dynamic light scattering, and the results are as follows... Figure 4 As shown, the main peak is located in the exosome-scale range, with particle sizes mainly ranging from 150 to 300 nm. The morphology of ZIF-8 before and after loading was examined using transmission electron microscopy, as shown below. Figure 5 As shown in the figure, electron microscopy revealed that ZIF-8 and ZIF-8@miR-130b-5p exhibited uniform morphology and good dispersibility. The miRNA release performance of the ZIF-8@miR-130b-5p sustained-release system was assessed at pH 6.5 and pH 7.4, respectively, and the results are as follows. Figure 6 As shown, the results indicate that release is significantly accelerated at pH 6.5 and slower at pH 7.4, consistent with acid-triggered characteristics. The XRD patterns of stimulated ZIF-8, ZIF-8, and ZIF-8@miR-130b-5p are shown below. Figure 7 As shown in the figure, ZIF-8@miR-130b-5p retains the characteristic diffraction peaks of ZIF-8, indicating that the crystal structure is maintained after loading. The hydrodynamic particle size distributions of ZIF-8 and ZIF-8@miR-130b-5p are shown in the figure. Figure 8 As shown in the figure, the hydrodynamic particle size shifts to the right after loading, and the surface miR-130b-5p is successfully bonded.

[0052] The above results confirm the successful preparation and characterization of MSC-EVs and verify the structure and acid-responsive release behavior of ZIF-8@miR-130b-5p.

[0053] The efficacy of exosomes derived from young MSCs containing miR-130b-5p in the treatment of osteoarthritis is as follows:

[0054] Cellular experiments: Cultured macrophages (RAW264.7 cells) were divided into three groups: a control group, an LPS group supplemented with LPS, and an LPS+Exo group supplemented with both LPS and exosomes (Exo) derived from young MSCs. After 24 hours, the expression of inflammatory factors and factors promoting anti-inflammatory and repair-related processes were detected in each group. The results are as follows: Figure 9 and 10As shown, the results indicated that Exo significantly inhibited the expression of LPS-induced inflammatory factors (TNF-α, iNOS, IL-6, COX2, CD86) and promoted the expression of anti-inflammatory and repair-related factors (CD206, Arg1, IL-10, CD163). Immunofluorescence staining was performed on each group, and the results are shown below. Figure 11 As shown, the results indicate that Exo can effectively clear the accumulation of reactive oxygen species in LPS-induced macrophages; flow cytometry analysis of each group yielded the following results. Figure 12 As shown, the results indicate that Exo can effectively reduce reactive oxygen species in LPS-induced macrophages and inhibit inflammatory responses in macrophages.

[0055] Animal experiments: Mice were divided into three groups: a sham-operated group (injecting saline into the joint cavity of sham-operated mice), a DMM group (injecting saline into the joint cavity of mice with medial meniscus instability (DMM)-induced osteoarthritis model), and a DMM+Exo group (injecting exosomes into the joint cavity of DMM-induced model mice). Intra-articular injections were performed every two weeks during the animal experiments. After 12 weeks of treatment, the experimental animals were sacrificed, and knee joint specimens were obtained.

[0056] The method for establishing the DMM-induced OA model in mice was as follows: Ten-week-old male C57BL / 6 mice were selected for the experiment. After isoflurane inhalation anesthesia, routine skin preparation was performed, the right knee joint was disinfected, and a medial patellar incision was made to expose the knee joint. The joint capsule was opened to expose the joint cavity. The patella was dislocated laterally, the knee joint was flexed, and under direct vision, the connection between the medial meniscus and the intermediate ridge of the tibial plateau was severed using a #23 scalpel blade, causing medial meniscus instability. The cartilage surface was not damaged during the operation, hemostasis was achieved thoroughly, the patella was repositioned, and the joint capsule and skin incision were sutured sequentially. In the sham surgery group, the medial meniscus was not severed, and the remaining procedures were the same as in the model group. No fixation measures were taken postoperatively, and penicillin was administered intramuscularly daily for three consecutive days. After three days, the knee joint of the mice was observed for infection. Mice without infection continued to the next stage of the experiment.

[0057] H&E staining was performed on the specimens from each group, and the results are as follows: Figure 13 As shown, the results indicated that Exo effectively alleviated synovial inflammation in DMM mice and reduced inflammation scores; immunofluorescence staining of specimens from each group yielded the following results. Figure 14 As shown, the results indicated that Exo could downregulate the expression of inflammation-related markers CD14 and CD86, upregulate the protective marker CD206, and significantly improve OARSI scores; toluidine blue and H&E staining were performed on the specimens of each group, and the results are as follows. Figure 15 As shown, the results indicated that Exo could increase the thickness of the uncalcified cartilage layer and the number of chondrocytes, thus improving cartilage structure; immunofluorescence and immunohistochemical staining were performed on the specimens of each group, and the results are as follows. Figure 16 and 17As shown, the results indicate that Exo can significantly reduce the expression of inflammatory factors COX2, TNF-α, and IL-6 in the synovium, and Exo can upregulate cartilage matrix components Col II and Aggrecan, inhibit the degradation enzymes MMP-13 and ROS, and improve cartilage matrix degradation.

[0058] The above results confirm the anti-inflammatory and chondrogenic protective effects of exosomes derived from young MSCs both in vitro and in vivo. qRT-PCR, Western blot, and immunological analyses showed that exosomes effectively inhibited LPS-induced inflammatory cytokine expression, promoted the upregulation of anti-inflammatory factors, and significantly reduced ROS levels. Results in the DMM mouse model indicated that exosomes alleviated synovial inflammation, improved cartilage thickness and cell number, restored Col II and Aggrecan expression, and inhibited MMP-13. Preliminary conclusions suggest that young MSC exosomes can regulate the inflammatory microenvironment, inhibit oxidative stress, and protect cartilage tissue.

[0059] Transcriptomic sequencing was used to compare exosomes from young and senescent MSCs, and the results were as follows: Figure 18 As shown, the results indicated that the content of miR-130b-5p in exosomes from young MSCs was significantly higher than that in exosomes from senescent MSCs. Preliminary conclusions suggest that miR-130b-5p is a key functional molecule in the anti-inflammatory and cartilage repair-promoting effects of exosomes from young MSCs.

[0060] The efficacy trials of miR-130b-5p and ZIF-8@miR-130b-5p in treating osteoarthritis are as follows:

[0061] Cellular experiments: Cultured macrophages were divided into three groups: a control group, an LPS group (containing LPS), and a miR-130b-5p group (containing both LPS and miR-130b-5p). After 24 hours, the expression of inflammatory factors and factors promoting anti-inflammatory and repair-related processes were detected in each group. The results are as follows: Figure 19 and 20 As shown, the results indicated that miR-130b-5p could inhibit the expression of inflammatory factors (TNF-α, iNOS, IL-6, COX2, CD86) and promote the expression of anti-inflammatory factors (CD206, Arg1, IL-10, CD163). Cultured macrophages were divided into five groups: a control group, an LPS-treated group, a miR-130b-5p group treated with both LPS and miR-130b-5p, a ZIF-8 group treated with both LPS and ZIF-8, and a ZIF-8@miR-130b-5p group treated with both LPS and ZIF-8@miR-130b-5p. After 24 hours, the expression of inflammatory factors and factors promoting anti-inflammatory and repair-related processes in each group was detected, and the results are shown below. Figure 21 and 22As shown, the results indicated that ZIF-8@miR-130b-5p also significantly inhibited the expression of inflammatory factors and promoted the transcription of protective factors; immunofluorescence staining and flow cytometry analysis were performed on each group, and the results are as follows. Figure 23 As shown, the results indicate that ZIF-8@miR-130b-5p can effectively clear the accumulation of reactive oxygen species in LPS-induced macrophages and inhibit the formation of macrophage inflammatory factors.

[0062] The above results confirm the in vitro function of miR-130b-5p and its ZIF-8 sustained-release delivery system. The results showed that both free miR-130b-5p and the ZIF-8@miR-130b-5p complex significantly inhibited LPS-induced expression of inflammatory factors (TNF-α, iNOS, IL-6, etc.), promoted the transcription of anti-inflammatory factors (CD206, Arg1, IL-10, etc.), and effectively scavenged intracellular ROS. Preliminary conclusions indicate that ZIF-8@miR-130b-5p not only retains the anti-inflammatory and antioxidant effects of miR-130b-5p but also achieves stable and controllable drug release.

[0063] Animal experiments: Mice were divided into 5 groups: a sham-operated group (injected with 8 μl of saline solution into the joint cavity), a control group (injected with 8 μl of saline solution into the joint cavity), a miR-130b-5p group (injected with 8 μl of miR-130b-5p into the joint cavity), a ZIF-8 group (injected with 8 μl of ZIF-8 into the joint cavity), and a ZIF-8@miR-130b-5p group (injected with 8 μl of ZIF-8@miR-130b-5p into the joint cavity). Intra-articular injections were performed every two weeks during the experiments. After 12 weeks of treatment, the animals were sacrificed, and knee joint specimens were obtained.

[0064] H&E and immunofluorescence staining were performed on the specimens of each group, and the results are as follows: Figure 24 As shown, H&E staining results indicated that ZIF-8@miR-130b-5p effectively alleviated synovial inflammation in an OA mouse model. Immunofluorescence staining showed that ZIF-8@miR-130b-5p effectively improved the proportion of inflammatory macrophages (CD14, CD86, CD106) in the synovium of the OA mouse model. Toluidine blue and H&E staining were performed on samples from each group, and the results are as follows: Figure 25 As shown in the figure, the results indicated that ZIF-8@miR-130b-5p effectively improved cartilage wear in the OA mouse model, significantly increasing the number of chondrocytes and the thickness of the cartilage layer. Immunofluorescence and immunohistochemical staining were performed on specimens from each group, and the results are as follows: Figure 26As shown, the results indicated that ZIF-8@miR-130b-5p effectively reduced the levels of COX2, TNF-α, and IL-6 in the synovium of an OA mouse model, alleviating inflammatory cartilage damage in the OA mouse model. Immunohistochemistry and immunofluorescence staining were performed on specimens from each group, and the results are attached. Figure 27 As shown, the results indicate that ZIF-8@miR-130b-5p can effectively increase Col II and Aggrecan levels in cartilage tissue of OA mouse models, reduce Mmp13, promote matrix repair, and treat osteoarthritis. MicroCT scans were performed on samples from each group, and the results are attached. Figure 28 As shown in the figure, the results indicate that ZIF-8@miR-130b-5p can effectively alleviate pathological manifestations such as osteophyte formation and subchondral bone sclerosis in the knee joint of OA model mice; open field experiments were performed on specimens from each group, and the results are attached. Figure 29 As shown in the figure, the results indicate that the motor function of OA model mice treated with ZIF-8@miR-130b-5p was significantly improved.

[0065] The above results confirm the in vivo efficacy of ZIF-8@miR-130b-5p in a DMM mouse model of osteoarthritis. Histological and immunological results showed that this sustained-release system effectively alleviated synovitis, improved cartilage wear, increased chondrocyte number and thickness, and reduced inflammatory factor levels. Immunohistochemistry further confirmed that it could increase cartilage matrix components Col II and Aggrecan, and reduce MMP-13 expression. Micro-CT scans showed a significant reduction in osteophyte formation and subchondral bone sclerosis; behavioral experiments showed significant improvement in motor function. Preliminary conclusions indicate that the ZIF-8@miR-130b-5p sustained-release system possesses significant anti-inflammatory, chondrogenic, and functional recovery effects in vivo.

[0066] In this invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the reagents, materials, and procedures used herein are all widely used in the relevant fields.

[0067] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of miR-130b-5p in the preparation of drugs for treating osteoarthritis.

2. A medicament for treating osteoarthritis, characterized in that: The active ingredient of the drug is miR-130b-5p.

3. A sustained-release system for treating osteoarthritis, characterized in that: The sustained-release system is ZIF-8@miR-130b-5p constructed by loading miR-130b-5p onto ZIF-8.

4. The method for preparing the sustained-release system as described in claim 3, characterized in that: The miR-130b-5p solution was added to the zinc nitrate solution and stirred rapidly. Then, the 2-methylimidazole solution was added rapidly and stirred rapidly to obtain the ZIF-8@miR-130b-5p.

5. The method for preparing the sustained-release system according to claim 4, characterized in that: The miR-130b-5p solution is a DMSO solution of miR-130b-5p with a concentration of 40–60 μg / mL.

6. The method for preparing the sustained-release system according to claim 4, characterized in that: The zinc nitrate is zinc nitrate hexahydrate; The mass ratio of miR-130b-5p, zinc nitrate hexahydrate, and 2-methylimidazole is 0.040–0.060:110–125:2000–3000.

7. The method for preparing the sustained-release system according to claim 4, characterized in that: After adding the 2-methylimidazole solution, the stirring time is 3 to 8 minutes.

8. The method for preparing the sustained-release system according to claim 4, characterized in that: After adding the 2-methylimidazole solution and stirring rapidly, the precipitate was collected by centrifugation, and the solid was collected by freeze-drying, which is the ZIF-8@miR-130b-5p.

9. The use of the sustained-release system as described in claim 3 in the preparation of a medicament for treating osteoarthritis.

10. The application according to claim 9, characterized in that: The sustained-release system is used in the preparation of drugs that improve the inflammatory microenvironment, cartilage damage, and synovial lesions in osteoarthritis.

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