Use of calycosin in preparation of drugs for preventing and treating osteoarthritis and drug combination

Abstract

The application relates to the field of biological medicine, and particularly relates to the application of calycosin in the preparation of a medicine for preventing and treating osteoarthritis and a medicine combination. The calycosin can inhibit abnormal activation of osteoclasts induced by RANKL by regulating the coupling process of osteoclast metabolism and oxidative stress mediated by PGAM2, and can improve abnormal remodeling of subchondral bone, thereby delaying or improving the progression of osteoarthritis. The application first proposes that the calycosin can regulate the coupling process of osteoclast metabolism and oxidative stress by acting on the metabolic enzyme PGAM2, and then intervene in the abnormal activation of osteoclasts induced by RANKL, improve the abnormal remodeling of subchondral bone, and realize the effect of delaying or improving the progression of osteoarthritis. Compared with the existing classic signal pathway, the application provides a disease intervention mechanism from the perspective of "cell metabolism and redox homeostasis regulation", and provides an action target and a theoretical basis for the modified treatment of osteoarthritis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application and drug combination of calycosin (Cal) in the preparation of drugs for the prevention and treatment of osteoarthritis. Background Technology

[0002] Osteoarthritis (OA) is a degenerative joint disease characterized by degeneration of articular cartilage, abnormal subchondral bone remodeling, chronic low-grade inflammation, and joint dysfunction. Knee osteoarthritis is the most common type. Currently, clinical treatment for osteoarthritis primarily focuses on symptomatic relief, including nonsteroidal anti-inflammatory drugs (NSAIDs), analgesics, intra-articular injections, physical therapy, and end-stage joint replacement. While these methods can temporarily relieve pain, they are insufficient to effectively halt the early structural degenerative process of the disease, and particularly difficult to target key pathological aspects such as abnormal subchondral bone remodeling and osteochondral unit imbalance.

[0003] Recent studies have shown that subchondral bone plays a crucial role in the development and progression of osteoarthritis. In the early stages of osteoarthritis, subchondral bone can exhibit a high-turnover remodeling state, characterized by abnormal osteoclast activation, increased bone resorption, and disordered trabecular microstructure. Abnormally activated osteoclasts not only disrupt bone homeostasis but can also further accelerate cartilage degeneration by altering the local biomechanical environment, promoting the release of inflammatory factors, and facilitating neurovascular invasion. Therefore, inhibiting excessive osteoclast activation and regulating subchondral bone remodeling is considered a potential intervention strategy.

[0004] In existing technologies, studies have explored interventions for bone metabolism-related diseases by inhibiting osteoclast differentiation and function. For example, patent application CN202411775397.1, based on a RANKL (Receptor Activator of Nuclear Factor-κB Ligand)-induced osteoclast differentiation model, found that TOFA (5-(tetradecyloxy)-2-furoic acid) can inhibit osteoclast formation and bone resorption by reducing intracellular reactive oxygen species (ROS) levels and downregulating the expression of osteoclast-related genes such as NFATc1 (activated T cell nuclear factor c1) and Ctsk (cathepsin K). Other studies have reported that various natural products or small molecule compounds can inhibit osteoclast differentiation by regulating classic signaling pathways such as NF-κB (nuclear factor κB), MAPK (mitogen-activated protein kinase), NFATc1, or ROS, for the intervention of osteoporosis and osteolytic diseases, as illustrated in patent application CN201710811286.5. However, most of these studies focus on simple bone metabolism disorders such as osteoporosis, and interventions for the complex pathological network of interactions between cartilage, subchondral bone, and the inflammatory microenvironment in osteoarthritis are still very limited. Effective disease modification strategies for early subchondral bone remodeling abnormalities are still insufficient.

[0005] Verbena isoflavones are isoflavone active ingredients derived from traditional Chinese medicinal herbs such as Astragalus membranaceus, and have been shown to possess anti-inflammatory, antioxidant, immunomodulatory, and tissue-protective effects. However, there is currently a lack of reports on their ability to slow the progression of osteoarthritis by regulating osteoclast metabolism and subchondral bone remodeling. Therefore, developing disease-modifying intervention strategies for osteoarthritis based on verbena isoflavones has significant clinical implications. Summary of the Invention

[0006] The purpose of this invention is to provide the application of verbascoside in the preparation of drugs for the prevention and treatment of osteoarthritis. Verascoside inhibits RANKL-induced abnormal activation of osteoclasts and improves abnormal subchondral bone remodeling, thereby delaying or improving the progression of osteoarthritis.

[0007] To achieve the above objectives, the present invention provides the following technical solution: the application of verbascoside in the preparation of drugs for the prevention and treatment of osteoarthritis, wherein verbascoside inhibits RANKL-induced abnormal osteoclast activation by regulating the PGAM2-mediated osteoclast metabolism and oxidative stress coupling process, and at the same time improves abnormal subchondral bone remodeling, thereby delaying or improving the progression of osteoarthritis.

[0008] Furthermore, PGAM2 is the direct target of the verbascoside isoflavone; the verbascoside isoflavone exerts its effect by binding to PGAM2 and enhancing its protein stability.

[0009] Furthermore, PGAM2 is involved in regulating osteoclast glycolysis and mitochondrial redox balance.

[0010] Furthermore, the osteoclast abnormal activation phenotype includes at least one of the following phenotypes: RANKL-induced enhanced osteoclast differentiation, increased formation of TRAP-positive multinucleated osteoclasts, enhanced microfilament ring formation, and enhanced bone resorption-related structures or functions. Furthermore, the oxidative stress includes an increase in intracellular total reactive oxygen species (ROS) levels and / or an increase in the accumulation of mitochondrial-derived ROS.

[0011] Furthermore, the verrucous isoflavones can improve RANKL-induced mitochondrial dysfunction, which includes a decrease in mitochondrial membrane potential.

[0012] Furthermore, the effective in vitro concentration of the verrucous isoflavone is any value between 1.25 μM and 20 μM; and the effective in vivo dose is any value between 5 mg / kg and 100 mg / kg.

[0013] Furthermore, the type of osteoarthritis includes any one of knee osteoarthritis, post-traumatic osteoarthritis, degenerative osteoarthritis, and senile osteoarthritis.

[0014] This application provides a pharmaceutical composition for the prevention and treatment of osteoarthritis, comprising verbascoside and pharmaceutically acceptable excipients.

[0015] Furthermore, the dosage form of the pharmaceutical composition includes any one of tablets, capsules, granules, oral liquids, injections, lyophilized powder injections, sustained-release formulations, controlled-release formulations, nanoformulations, liposome formulations, intra-articular injection formulations, targeted subchondral bone delivery formulations, and topical formulations.

[0016] The beneficial effects of this invention are as follows: The application of verbascoside in the preparation of drugs for the prevention and treatment of osteoarthritis, as provided in this application, is the first to propose that verbascoside can regulate the coupling process of osteoclast metabolism and oxidative stress by acting on the metabolic enzyme PGAM2, thereby intervening in RANKL-induced abnormal osteoclast activation and improving abnormal subchondral bone remodeling, ultimately achieving the effect of delaying or improving the progression of osteoarthritis. Compared with existing technologies that mainly focus on classic signaling pathways such as NF-κB, MAPK, or NFATC1, this application, from the perspective of "cellular metabolism and redox homeostasis regulation," provides a new disease intervention mechanism, namely, a metabolic regulation mechanism based on PGAM2. This provides a new target and theoretical basis for the modification therapy of osteoarthritis and expands the regulatory pathway of abnormal subchondral bone remodeling.

[0017] The application of verbascoside in the preparation of drugs for the prevention and treatment of osteoarthritis, as provided in this application, involves reducing the content of total intracellular ROS and the accumulation of mitochondrial-derived ROS by regulating PGAM2-related metabolic processes, and improving abnormal mitochondrial membrane potential. This corrects the RANKL-induced abnormal activation state at the metabolic level, thereby inhibiting excessive differentiation and hyperfunction of osteoclasts. In this process, verbascoside does not directly inhibit osteoclast activity, but rather regulates abnormal osteoclast activation by restoring cellular metabolic homeostasis and redox balance; therefore, verbascoside exerts its effects without affecting normal bone metabolism.

[0018] This application demonstrates that isoflavones not only inhibit osteoclast differentiation but also simultaneously inhibit the formation of TRAP-positive multinucleated osteoclasts and the establishment of bone resorption structures such as microfilament rings. It achieves synergistic intervention at multiple key stages, including cell differentiation, scaffold remodeling, and the formation of bone resorption function, thereby systematically blocking the abnormal activation process of osteoclasts. It also demonstrates that isoflavones can act on both cartilage and subchondral bone, improving the structural integrity of the cartilage layer and the degree of matrix degradation, reducing OARSI scores, and significantly alleviating articular cartilage degeneration, achieving overall protection of key pathological structures of the joint.

[0019] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0020] Figure 1 The image shows the TRAP (Tartrate-Resistant Acid Phosphatase) staining results of the effect of verbascoside on RANKL-induced osteoclast differentiation in Example 1 of this invention. Figure 2 This is an immunofluorescence observation result of the effect of verbascoside on RANKL-induced osteoclast microfilament ring formation in Example 2 of the present invention; Figure 3 The image shows the Western blot (WB) results of the effect of verbascoside on the expression of osteoclast differentiation-related proteins in Example 3 of this invention. Figure 4 This is a Micro-CT three-dimensional reconstruction and quantitative analysis result of the effect of verbascoside on the subchondral bone microstructure of mice with medial meniscus instability (DMM)-induced osteoarthritis in Example 4 of the present invention. Figure 5The figure shows the histological staining results and OARSI (Osteoarthritis Research Society International) score analysis results of the effect of verbascoside on articular cartilage degeneration in DMM-induced osteoarthritis mice in Example 5 of this invention. Figure 6 This is a diagram showing the experimental results of the use of the Cellular Thermal Shift Assay (CETSA) method in Example 6 of the present invention to verify the binding stability of verbascoside isoflavones with PGAM2 (phosphoglycerate mutase 2). Figure 7 The figure shows the detection results of the effect of PGAM2 silencing on the expression of osteoclast differentiation-related proteins in Example 7 of the present invention; Figure 8 The figure shows the results of detecting the effect of verbascoside isoflavone on the level of total reactive oxygen species (ROS) in osteoclasts using the DCFH-DA fluorescent probe in Example 8 of this invention. Figure 9 This is a graph showing the results of detecting the effect of salamidoflavonoids on the mitochondrial ROS level of osteoclasts using the MitoSOX Red fluorescent probe in Example 8 of this invention. Figure 10 This is a graph showing the results of JC-1 staining to detect the effect of verbascoside isoflavones on the mitochondrial membrane potential of osteoclasts in Example 9 of the present invention. Detailed Implementation

[0021] The technical solutions of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0022] It should be noted that the fibrous actin ring (F-actin ring, or microfilament ring for short) is a closed ring-shaped cytoskeleton structure formed by the polymerization and assembly of fibrous actin (F-actin), located in the "sealing zone" at the cell edge.

[0023] The preferred embodiment of this application illustrates the use of verbascoside in the preparation of drugs for the prevention and treatment of osteoarthritis. This verbascoside can inhibit RANKL-induced abnormal osteoclast activation and improve subchondral bone remodeling by regulating the PGAM2 (phosphoglycerate mutase 2)-mediated coupling process between osteoclast metabolism and oxidative stress, thereby delaying or improving the progression of osteoarthritis. Traditionally, PGAM2 is primarily considered a key metabolic enzyme in the glycolysis pathway, participating in cellular energy metabolism, particularly related to the metabolic activities of high-energy-demand tissues such as muscle, and is not a conventional regulator of bone metabolism or a target associated with osteoarthritis.

[0024] This application, through experimental research, discovered that verbascoside can interact with PGAM2 and enhance its thermal stability, indicating a binding relationship between verbascoside and PGAM2. Based on this result, PGAM2 was selected as a candidate target protein in the action of verbascoside to further analyze its functional role in osteoclast differentiation. Further experimental studies revealed that intervening in PGAM2 expression levels can affect the regulatory effect of verbascoside on RANKL-induced osteoclast differentiation-related phenotypes. This indicates that PGAM2 participates in the regulation of abnormal osteoclast activation. Therefore, it can be concluded that verbascoside may regulate abnormal osteoclast activation and subchondral bone remodeling by acting on PGAM2-related metabolic regulatory nodes and intervening in the coupled process of osteoclast metabolism and oxidative stress. This "metabolic enzyme-oxidative stress coupled regulation" mechanism was confirmed in subsequent experiments, providing a new molecular mechanism basis for the intervention of abnormal subchondral bone remodeling in osteoarthritis.

[0025] In one embodiment, PGAM2 is the direct target of verbascoside. Verascoside binds to PGAM2 and enhances its stability, thereby affecting PGAM2-related metabolic regulatory functions and thus regulating the activity of signaling pathways during osteoclast differentiation, leading to a decrease in the expression levels of osteoclast differentiation-related marker molecules. In other words, verbascoside regulates osteoclast generation and differentiation upstream by acting on PGAM2, a metabolically relevant node, thereby inhibiting abnormal osteoclast activation.

[0026] In one embodiment, PGAM2 participates in regulating osteoclast glycolysis and mitochondrial redox balance. Specifically, PGAM2 regulates osteoclast glycolysis levels, thereby reducing reactive oxygen species (ROS) generation during metabolism, while simultaneously regulating mitochondrial oxidative stress and improving mitochondrial redox imbalance, thus modulating osteoclast metabolism and influencing its differentiation process. This regulatory effect can inhibit the positive feedback amplification effect between metabolic processes and oxidative stress, reducing metabolically driven ROS accumulation and its sustained enhancing effect on mitochondrial function. Furthermore, it can weaken the energy metabolism support and oxidative stress amplification mechanisms upon which abnormal osteoclast activation depends at the metabolic level, inhibiting excessive osteoclast differentiation and enhanced bone resorption.

[0027] In one embodiment, the abnormal osteoclast activation phenotype includes at least one of the following: RANKL-induced enhanced osteoclast differentiation, increased formation of TRAP-positive multinucleated osteoclasts, enhanced microfilament ring (F-actin ring) formation, and enhanced bone resorption-related structures or functions.

[0028] In one embodiment, oxidative stress includes elevated intracellular total reactive oxygen species (ROS) levels and / or increased accumulation from mitochondria. Versicolor isoflavones can significantly reduce intracellular total ROS levels and mitochondrial accumulation, alleviating RANKL-induced oxidative stress load, thereby inhibiting oxidative stress-mediated osteoclast activation signaling amplification.

[0029] In one embodiment, verbascoside isoflavones can ameliorate RANKL-induced mitochondrial dysfunction, including a decrease in mitochondrial membrane potential. Verascoside isoflavones can maintain mitochondrial functional stability by improving mitochondrial membrane potential, thereby enhancing mitochondrial energy metabolism homeostasis and reducing osteoclast activation induced by mitochondrial dysfunction.

[0030] In one embodiment, the preferred in vitro effective concentration of verbascoside is any value between 1.25 μM and 20 μM. Within this effective concentration range, verbascoside can stably inhibit osteoclast differentiation in vitro. In other embodiments, the preferred in vivo effective dose of verbascoside is any value between 5 mg / kg and 100 mg / kg. Within this effective dose range, verbascoside can exert a protective effect on subchondral bone structure in vivo. This also allows for the determination of a reproducible efficacy window and a safe and effective dose range, providing a dosage basis for the translation and application of in vitro and in vivo efficacy results.

[0031] In one embodiment, the types of osteoarthritis include at least one of knee osteoarthritis, post-traumatic osteoarthritis, degenerative osteoarthritis, and senile osteoarthritis. Experimental verification revealed that isoflavones exhibited an inhibitory effect on disease progression in different types of osteoarthritis models, and demonstrated a cartilage structure-protective effect in both traumatic and age-related osteoarthritis models. This result also suggests that the mechanism of action of isoflavones may be related to the underlying abnormal bone remodeling process common in osteoarthritis, rather than relying on a single etiology-specific pathway.

[0032] One embodiment provides a pharmaceutical composition for the prevention and treatment of osteoarthritis. The pharmaceutical composition includes verbascoside and pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients include, but are not limited to: fillers or diluents for improving the formability and compressibility of tablets or capsules, such as lactose, microcrystalline cellulose (MCC), corn starch, pregelatinized starch, mannitol, and sorbitol; binders for improving granule / tablet strength, such as polyvinylpyrrolidone, hydroxypropyl methylcellulose, and starch paste; disintegrants for promoting release and improving bioavailability, such as croscarmellose sodium and carboxymethyl starch sodium; solubilizers for improving the solubility and bioavailability of verbascoside, such as polyethylene glycol, Tween 80 cyclodextrin, and its derivatives; and sustained-release materials for improving the duration of action, such as waxy matrices of carbomer + lipid systems and Eudragit® acrylic resins. By introducing excipients, the formulation compatibility and administration properties of verbascoside can be improved, its in vivo stability and bioavailability can be enhanced, and its delivery capability in different routes of administration can be strengthened. Introducing excipients also improves the feasibility of processing and application of the drug composition, making verbascoside suitable for multiple forms of administration, thereby increasing its application flexibility and overall efficacy in the treatment of osteoarthritis to a certain extent.

[0033] In one embodiment, the dosage form of the pharmaceutical composition includes one or more of the following: tablets, capsules, granules, oral liquids, injections, lyophilized powder for injection, sustained-release formulations, controlled-release formulations, nanoformulations, liposome formulations, intra-articular injection formulations, subchondral bone targeted delivery formulations, and topical formulations. Nanoformulations include any one of polymer nanoparticles, lipid nanoparticles, exosome-loaded drug systems, and hydrogel sustained-release systems. By designing different formulation forms, the distribution characteristics of the drug at the target site can be improved to a certain extent, such as increasing the local drug concentration in the joint cavity and subchondral bone, prolonging the drug's duration of action (e.g., sustained-release or controlled-release formulations), and improving targeted delivery efficiency (e.g., nanoformulations or liposome formulations).

[0034] One embodiment also provides a method for screening candidate drugs for the prevention and treatment of osteoarthritis. The method uses PGAM2 as the drug target and evaluates the regulatory effects of candidate substances on osteoclast metabolism and oxidative stress state, thereby screening candidate substances with anti-osteoclast activity.

[0035] Example 1

[0036] To investigate the effect of verbascoside on RANKL-induced osteoclast differentiation, an in vitro osteoclast differentiation model was established using RAW264.7 cells. Specifically, RAW264.7 cells were seeded in multi-well culture plates and cultured. After reaching adherence, macrophage colony-stimulating factor (M-CSF) was added for pretreatment to maintain cell viability, followed by the addition of RANKL to induce osteoclast differentiation. This study included a control group, a RANKL-induced group (RANKL group), a RANKL + 10 μM verbascoside group (RANKL + 10 μM Cal group), and a RANKL + 20 μM verbascoside group (RANKL + 20 μM Cal group). The control group consisted of a blank group pretreated with M-CSF but without RANKL induction. The RANKL group consisted of experimental groups pretreated with RANKL followed by RANKL induction. The RANKL+10μM Cal group consisted of experimental groups inducing RANKL followed by intervention with 10μM verbascoside. The RANKL+20μM Cal group consisted of experimental groups inducing RANKL followed by intervention with 20μM verbascoside. All groups were cultured under the same conditions for 4-6 days, with the drug-containing medium being changed periodically to maintain a stable induction environment. After induction, the medium was discarded, and the cells were fixed with fixative and subjected to TRAP staining. Multiple fields of view were randomly selected for microscopic observation and photographic recording after staining. Cells that were TRAP-positive, significantly enlarged, and contained three or more nuclei were defined as mature osteoclasts and were counted. The results are as follows: Figure 1 As shown, the scale bars in the figures are all 500 μM. In other embodiments, mouse bone marrow-derived macrophages (BMMs) can also be used to establish an in vitro osteoclast differentiation model.

[0037] Depend on Figure 1It was found that the cell count in the control group was normal, and no obvious TRAP-positive multinucleated cells were observed. In the RANKL group, a large number of strongly TRAP-positive multinucleated osteoclasts were observed, with significantly enhanced cell fusion, exhibiting typical bone resorption-related giant cell morphology. Compared with the RANKL group, the number of TRAP-positive multinucleated cells and the degree of cell fusion were significantly reduced in the RANKL+10μM Cal group and the RANKL+20μM Cal group, showing a clear dose-dependent effect. The inhibitory effect was most significant in the RANKL+20μM Cal group, with only a small number of weakly positive or immature mononuclear cells observed. Furthermore, compared with the RANKL group, the density of TRAP-positive areas was significantly reduced in the RANKL+10μM Cal group and the RANKL+20μM Cal group treated with isoflavones. This indicates that isoflavones have an inhibitory effect on osteoclast fusion and differentiation, exhibiting a certain degree of concentration dependence. This result indicates that RANKL significantly promotes osteoclast differentiation and multinucleated giant cell formation, resulting in a large number of TRAP-positive multinucleated osteoclasts; while verbascoside effectively inhibits RANKL-induced osteoclast differentiation and reduces the formation of TRAP-positive multinucleated osteoclasts. This also confirms that verbascoside mainly blocks osteoclast generation at the cellular morphology level by intervening in the early fusion and maturation stages of osteoclasts.

[0038] Example 2

[0039] To further explore the effects of verbascoside on the bone resorption structure of osteoclasts, a RANKL-induced osteoclast model was established using mouse bone marrow-derived macrophages (BMMs). Specifically, BMMs were seeded in multi-well culture plates and cultured. After reaching adherence, M-CSF was added for pretreatment to maintain cell viability, followed by the addition of RANKL to induce osteoclast differentiation. Similar to Example 1, this example also included a RANKL induction group, a RANKL + 10 μM verbascoside group, and a RANKL + 20 μM verbascoside group. All groups were induced and cultured under the same conditions for 4-6 days, with the drug-containing medium being replaced periodically to maintain a stable induction environment. After induction, cells were fixed with paraformaldehyde to maintain cytoskeleton stability. Subsequently, the cell membranes in each group were permeabilized with a permeabilizing agent to allow the fluorescent probe to enter the cells. Then, microfilaments were stained with FITC-labeled phalloidin-FITC to observe cytoskeleton structure; simultaneously, DAPI was used to label cell nuclei to assess cell fusion and multinucleation. The cytoskeleton and fusion were observed using confocal or fluorescence microscopy. Results are as follows: Figure 2 As shown, the scale bars in the figure are all 200 μm.

[0040] Depend on Figure 2 It was observed that the RANKL group showed distinct closed or semi-closed microfilament ring structures, significantly increased cell volume, and marked multinucleated fusion, exhibiting characteristics typical of mature osteoclasts. This indicates that RANKL can significantly induce osteoclasts to form typical microfilament ring structures and multinucleated fused cells, constructing the cytoskeleton basis required for bone resorption function. Compared to the RANKL group, the RANKL+10μM Cal group showed a significant reduction in the number of microfilament ring structures, decreased structural integrity, and discontinuous or partially broken states. Simultaneously, the number of multinucleated cells decreased, and the degree of nuclear fusion was significantly reduced, indicating a certain degree of inhibition of bone resorption structure formation. This suggests that low-dose isoflavones can inhibit osteoclast cytoskeleton remodeling, but still retain the ability to form structures. In the RANKL+20μM Cal group, the number of microfilament ring structures was significantly reduced, even almost disappearing, the cytoskeleton was diffusely distributed, multinucleated fused cells were significantly reduced, and the nuclei were dispersed with no obvious fusion trend; bone resorption structures were also essentially blocked. This indicates that the inhibitory effect of verbascoside on RANKL-induced microfilament ring formation and cell fusion increases with increasing concentration. This further demonstrates that verbascoside can inhibit RANKL-induced microfilament ring formation and cell fusion, i.e., by intervening in osteoclast cytoskeleton remodeling, it blocks bone resorption at the structural level in a dose-dependent manner, providing a basis for its ability to improve abnormal subchondral bone remodeling in osteoarthritis.

[0041] Example 3

[0042] To further explore the regulatory role of verbascoside on osteoclast differentiation at the molecular level, a RANKL-induced RAW264.7 cell model was used for Western blot analysis. Specifically, RAW264.7 cells were seeded in multi-well culture plates and cultured. After reaching the adherent state, M-CSF was added for pretreatment to maintain cell viability, followed by the addition of RANKL to induce osteoclast differentiation. Similar to Example 1, this example also included a control group, a RANKL-induced group, a RANKL + 10 μM verbascoside group, and a RANKL + 20 μM verbascoside group. After induction, cells were collected, lysed, and total protein was extracted. After centrifugation to remove impurities, Western blotting was used for detection. The detection indicators included key osteoclast differentiation markers such as NFATC1, MMP9 (matrix metalloproteinase-9, a matrix degradation-related protein), and ACP5 (tartrate-resistant acid phosphatase 5, a maturation marker protein), with GAPDH used as an internal control. ImageJ was used for grayscale analysis to calculate the relative expression levels. The results are as follows: Figure 3 As shown.

[0043] Depend on Figure 3It was found that, compared with the control group, the expression level of NFATC1 in the RANKL group was significantly upregulated, and MMP9 and ACP5 were also significantly increased, indicating that RANKL induction enhanced bone matrix degradation capacity and osteoclast maturation markers. This further demonstrates that RANKL induction can successfully activate the osteoclast differentiation program. Compared with the RANKL group, the expression level of NFATC1 in the RANKL+10μM Cal group was significantly reduced, but did not return to the baseline level, i.e., the expression level in the control group; the expression levels of MMP9 and ACP5 were also significantly reduced, but still higher than the expression levels of MMP9 and ACP5 in the control group. This indicates that, after intervention with this dose of isoflavone, differentiation signals can be blocked to a certain extent. When the dose of isoflavone is increased to about 20μM, such as in the RANKL+20μM Cal group, the expression level of NFATC1 was close to the baseline level, and the expression levels of MMP9 and ACP5 were also significantly reduced, indicating that osteoclast differentiation and function were significantly inhibited, and this showed a clear dose-dependent effect. This result demonstrates that isoflavones can simultaneously inhibit the expression levels of key regulatory nodes (NFATc1) and functional executive proteins (MMP9, ACP5) of osteoclast differentiation at the molecular level, thereby effectively blocking RANKL-induced osteoclast differentiation and maturation.

[0044] Example 4

[0045] To investigate the effects of verbascoside on subchondral bone structure in osteoarthritis induced by medial meniscus instability (DMM), a knee osteoarthritis model was established using C57BL / 6 mice through DMM to simulate abnormal subchondral bone remodeling and osteochondral interface degeneration. In this study, 24 C57BL / 6 mice were randomly and equally divided into a control group, a DMM model group, a DMM combined with verbascoside treatment group, and a verbascoside alone group, and knee osteoarthritis animal models were established in each group. After model establishment, each group of mice underwent a drug intervention phase. The DMM combined with verbascoside treatment group and the verbascoside alone group received intraperitoneal injection (ip) of 40 mg / kg verbascoside every other day to maintain a relatively stable in vivo exposure level for 4 consecutive weeks. Both the control group and the DMM model group received an equal volume of physiological saline intraperitoneally during the same period, with the same administration frequency as the experimental group, to eliminate the influence of injection operation factors on the experimental results. After the intervention, knee joint specimens of mice in each group were collected, fixed with 4% paraformaldehyde, and the surrounding soft tissue was removed, leaving only the bony structure for use in Micro-CT scanning analysis. High-resolution Micro-CT was used for three-dimensional reconstruction, and the microstructure of the subchondral bone region of the proximal tibia was quantitatively analyzed. The main evaluation indicators included trabecular volume fraction (BV / TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp) to comprehensively reflect the subchondral bone remodeling status and changes in bone microstructure integrity. The results are as follows: Figure 4 As shown.

[0046] Depend on Figure 4 It was found that the subchondral bone structure of mice in the control group was intact, with regular and continuous trabecular arrangement. In contrast, the subchondral bone of mice in the DMM model group showed significant structural disorder, mainly characterized by sparse and coarse trabeculae, irregular arrangement, abnormal remodeling of the subchondral bone plate, and the formation of osteophyte-like structures. This indicates that the osteoarthritis model successfully induced abnormal subchondral bone remodeling. Compared with the control group, no significant structural abnormalities were observed in the isoflavone-only treatment group, indicating that isoflavone has no significant adverse effects on bone structure under normal physiological conditions. Compared with the DMM model group, the subchondral bone structure of mice in the DMM combined with isoflavone treatment group was significantly improved, with more regular trabecular arrangement and enhanced structural continuity, significantly reduced abnormal thickening of the subchondral bone plate and osteophyte formation, showing an overall trend towards restoration to normal structure. This demonstrates that isoflavone can significantly improve DMM-induced abnormal subchondral bone remodeling, inhibit degenerative changes in bone structure, and delay the progression of osteoarthritis at the tissue structure level.

[0047] Example 5

[0048] To further investigate the effects of isoflavone on cartilage degeneration in osteoarthritis, an osteoarthritis model was constructed using the same method as in Example 4 to simulate the process of articular cartilage degeneration and bone-cartilage interface structure destruction. Similar to Example 4, 24 C57BL / 6 mice were randomly and equally divided into a control group, a DMM model group, and a DMM combined with isoflavone intervention group. The DMM combined with isoflavone treatment group and the isoflavone alone group received isoflavone intervention at a dose of 40 mg / kg, administered every other day for 4 consecutive weeks. The control group and the DMM model group received an equal volume of physiological saline concurrently, with the same frequency as the experimental group. After the intervention, knee joint tissue was collected, fixed, decalcified, and embedded in paraffin before sectioning. The articular cartilage structure was observed using conventional cartilage histological staining methods (such as Safranin O / Fast Green or HE staining), and semi-quantitative analysis was performed according to the internationally recognized OARSI (Osteoarthritis Research Society International) scoring system. The results are as follows: Figure 5 As shown.

[0049] Depend on Figure 5 It was found that the articular cartilage structure of mice in the control group was intact, with clear cartilage layers, smooth surface, and uniform matrix staining, indicating that the cartilage tissue was in a normal homeostatic state. In contrast, the articular cartilage of mice in the DMM model group showed significant degenerative changes, manifested as irregular cartilage surface, thinning of the cartilage layer, weakened matrix staining, and disordered or even absent chondrocyte arrangement, with a corresponding significant increase in OARSI scores. This indicates that the degree of cartilage damage in mice in the DMM model group was significantly aggravated, and the pathological process of osteoarthritis was significantly accelerated. Compared with the control group, no significant structural abnormalities were observed in the isoflavone-only treatment group. This indicates that isoflavone has no significant adverse effect on articular cartilage structure under normal physiological conditions. Compared with the articular cartilage of mice in the DMM model group, the cartilage degeneration phenomenon in mice treated with DMM combined with isoflavone was significantly improved. The cartilage surface was relatively intact, the degree of structural disorder was reduced, the cartilage layer thickness was restored compared with the DMM model group, the weakening trend of matrix staining was suppressed, and the OARSI score decreased significantly. This indicates that verbascoside can effectively alleviate DMM-induced articular cartilage degeneration, improve cartilage tissue structural integrity, and reduce OARSI scores, thereby delaying the progression of osteoarthritis at the histopathological level. This result also confirms that verbascoside has good chondroprotective effects and potential disease-modifying value.

[0050] To investigate the mechanism by which verbascoside inhibits RANKL-induced abnormal osteoclast activation and improves subchondral bone remodeling, this application conducted a systematic experimental study. The results indicate that verbascoside may exert its effect of delaying or improving the progression of osteoarthritis by regulating the PGAM2-mediated coupling process between osteoclast metabolism and oxidative stress, inhibiting RANKL-induced abnormal osteoclast activation, and improving subchondral bone microstructural disorder.

[0051] Example 6

[0052] To further verify whether there is a direct interaction between verbascoside and PGAM2, a Cellular Thermal Shift Assay (CETSA) was used to assess its intracellular targeting binding ability and its impact on protein stability. Osteoclast precursor cells or RAW264.7 cells were used in the experiment. After RANKL induction, they were treated with verbascoside or DMSO (control), respectively, and multiple temperature gradients were set to thermally denature the cellular proteins. Specifically, cell lysates or intact cells were heated at different temperature gradients, causing unbound or weakly bound proteins to denature and precipitate, while proteins stable with small molecules remained soluble at higher temperatures. Subsequently, the supernatant proteins were obtained by centrifugation, and the residual levels of PGAM2 under different temperature conditions were detected by Western blot, with GAPDH used as an internal control for calibration. Results are as follows: Figure 6 As shown.

[0053] Depend on Figure 6 It was found that, compared with the DMSO-treated control group, under the same temperature gradient conditions, the PGAM2 in the verbascoside-treated group still exhibited a higher level of soluble residue at higher temperatures, indicating a direct binding between verbascoside and PGAM2. Thermal stability curve analysis showed that after verbascoside treatment, the melting temperature (Tm value) of PGAM2 shifted upwards, with the overall curve shifting towards higher temperatures, indicating that the structural stability of PGAM2 was significantly enhanced in the presence of verbascoside. This result demonstrates that verbascoside can directly interact with PGAM2 intracellularly and significantly improve the thermal stability of PGAM2, thus indicating that PGAM2 is a direct binding target of verbascoside. This confirms the mechanism by which "verascoside regulates PGAM2-related functions, influencing the coupling process of osteoclast metabolism and oxidative stress, thereby inhibiting abnormal osteoclast activation and improving subchondral bone remodeling in osteoarthritis."

[0054] Example 7

[0055] To verify the role of PGAM2 in the inhibition of osteoclast differentiation by verbascoside, a PGAM2 deficiency model was constructed by specifically silencing PGAM2 using small interfering RNA (siRNA). Specifically, based on RAW264.7 cells, four groups were established: a control group, a RANKL-induced group (RANKL group), a RANKL + verbascoside group (RANKL + Cal group), and a PGAM2 siRNA transfection combined with verbascoside treatment group (PGAM2 siRNA + RANKL + Cal group). In the control group, no induction or intervention was performed on RAW264.7 cells. In the RANKL group, RAW264.7 cells were stimulated with RANKL to induce differentiation. In the RANKL + verbascoside group, verbascoside was administered to RANKL-stimulated RAW264.7 cells as an intervention. In the PGAM2 siRNA+RANKL+Cal group, RANKL was administered to siRNA-transfected RAW264.7 cells for stimulation, followed by intervention with isoflavones. The expression of PGAM2 in siRNA-transfected RAW264.7 cells was as follows: Figure 7 As shown in Figure A. After induction culture, the expression levels of osteoclast differentiation-related proteins (such as NFATc1, MMP9, and ACP5) were detected by Western blot. Simultaneously, osteoclast morphological examination (such as TRAP staining or observation of related cell fusion / bone resorption structures) was used to comprehensively evaluate the degree of osteoclast differentiation. Results are as follows: Figure 7 As shown.

[0056] Depend on Figure 7 As shown in Figure B, compared with the RANKL group, the expression of osteoclast differentiation-related proteins such as NFATc1, MMP9, and ACP5 was significantly downregulated in the RANKL+Cal group, indicating that isoflavones have a significant inhibitory effect on RANKL-induced osteoclast differentiation. However, compared with the RANKL+Cal group, in the PGAM2 siRNA+RANKL+Cal group, under PGAM2 silencing conditions, the expression of differentiation-related proteins rebounded, and the number of multinucleated osteoclasts increased, indicating that the inhibitory effect induced by isoflavones was reversed. Figure 7The results showed that osteoclast formation was significantly reduced in the RANKL+Cal group; however, in the PGAM2 siRNA+RANKL+Cal group, the number of multinucleated cells increased under PGAM2 silencing conditions, indicating that the inhibitory effect of verbascoside on osteoclast formation was weakened and the osteoclast differentiation trend was restored. This result demonstrates that the expression status of PGAM2 directly affects the inhibitory effect of verbascoside on osteoclast differentiation, and PGAM2 silencing weakens the anti-osteoclast effect of verbascoside. This further confirms that PGAM2 participates in regulating the process of osteoclast differentiation inhibition mediated by verbascoside. As a key target protein of the coupling regulation axis between verbascoside and osteoclast metabolism and oxidative stress, PGAM2 has important biological significance in the regulation of abnormal subchondral bone remodeling in osteoarthritis.

[0057] Example 8

[0058] A RANKL-induced osteoclast differentiation model was established using mouse bone marrow-derived macrophages or RAW264.7 cells to evaluate the effect of verbascoside on osteoclast oxidative stress levels. Specifically, RAW264.7 cells were seeded and cultured in multi-well plates. After reaching adherence, M-CSF was added for pretreatment to maintain cell viability. Similar to Example 1, this example also included a control group, a RANKL-induced group, a RANKL + 10 μM verbascoside group, and a RANKL + 20 μM verbascoside group. After induction, cells were collected and incubated with a DCFH-DA fluorescent probe, which allowed the probe to enter the cells and be oxidized to generate fluorescent substances, reflecting the total intracellular ROS level. Flow cytometry or fluorescence microscopy was then used to quantify the ROS level, and the results are shown below. Figure 8 As shown in the figure. Simultaneously, mitochondrial superoxide anions were labeled using the MitoSOX Red specific fluorescent probe to reflect mitochondrial oxidative stress levels, and the results are shown in the figure. Figure 9 As shown.

[0059] Depend on Figure 8 It was found that in the RANKL group, under RANKL-induced conditions, intracellular fluorescence signals were significantly enhanced, indicating a significant increase in intracellular total reactive oxygen species (ROS) levels, and osteoclasts were in a state of enhanced oxidative stress. In the RANKL + 10 μM and RANKL + 20 μM isoflavone groups, intracellular fluorescence signals were significantly weakened after isoflavone intervention. Furthermore, compared to the RANKL + 10 μM isoflavone group, the RANKL + 20 μM isoflavone group showed a more significant decrease in intracellular total ROS levels, indicating that isoflavone can reduce RANKL-induced intracellular total ROS levels in a dose-dependent manner. Figure 9It was found that in the RANKL group, mitochondrial ROS levels were significantly increased under RANKL-induced conditions, indicating that mitochondrial oxidative stress was significantly activated. In the RANKL + 10 μM verbascoside and RANKL + 20 μM verbascoside groups, mitochondrial ROS levels were significantly reduced after verbascoside treatment. Among them, the inhibitory effect was more significant in the RANKL + 20 μM verbascoside group, and its mitochondrial ROS level was close to that in the control group.

[0060] This result indicates that RANKL induction significantly enhances oxidative stress in osteoclasts, manifested by a simultaneous increase in both total ROS and mitochondrial ROS levels; while verbascoside effectively reduces these oxidative stress levels, exhibiting a clear dose-dependent inhibitory trend. This suggests that verbascoside can intervene in the amplification of oxidative stress in osteoclasts by inhibiting mitochondrial ROS production and improving cellular redox homeostasis, thereby inhibiting the biological effects related to abnormal osteoclast activation.

[0061] Example 9

[0062] To evaluate the regulatory effect of verbascoside on RANKL-induced mitochondrial dysfunction, changes in mitochondrial membrane potential in osteoclasts were detected using the JC-1 fluorescent probe. Specifically, RAW264.7 cells were seeded and cultured in multi-well plates. After reaching adherence, M-CSF was added for pretreatment to maintain cell viability. Similar to Example 1, this example also included a control group, a RANKL-induced group, a RANKL + 10 μM verbascoside group, and a RANKL + 20 μM verbascoside group. After induction, cells were collected, and JC-1 staining working solution was added. Cells were incubated at 37°C in the dark for 20–30 min to allow the JC-1 probe to fully penetrate the cells and reflect the mitochondrial membrane potential. After incubation, cells were washed 2–3 times with JC-1 staining buffer or serum-free medium to remove unbound probes. Subsequently, fluorescence microscopy or confocal microscopy were used for imaging observation. Fluorescence signals of JC-1 aggregates (red channel, reflecting high membrane potential) and JC-1 monomers (green channel, reflecting low membrane potential) were acquired, and cell nuclei were counterstained with DAPI for localization and counting analysis. Image acquisition conditions were kept consistent across groups to ensure comparability of results. Results are as follows: Figure 10 As shown.

[0063] Depend on Figure 10It was found that cells in the control group mainly exhibited strong JC-1 aggregate fluorescence signals, indicating that the mitochondrial membrane potential was maintained at a high level. In the RANKL group, under RANKL induction conditions, the fluorescence signal of JC-1 aggregates was significantly weakened, while the signal of JC-1 monomers was significantly enhanced, indicating a decrease in mitochondrial membrane potential and significant damage to mitochondrial function. Compared with the RANKL group, in the RANKL+10μM and RANKL+20μM isoflavone groups, the fluorescence signal of JC-1 aggregates was significantly restored after isoflavone treatment, while the signal of JC-1 monomers was correspondingly weakened. Furthermore, the recovery of JC-1 aggregate fluorescence signal in the RANKL+20μM isoflavone group was more significant, indicating a significant recovery of mitochondrial membrane potential compared to the RANKL group, showing a certain concentration-dependent improvement trend.

[0064] This result indicates that RANKL can induce a decrease in mitochondrial membrane potential in osteoclasts, leading to mitochondrial dysfunction; while verbascoside can effectively improve this abnormal change and maintain mitochondrial membrane potential stability. This suggests that verbascoside can improve cellular energy metabolism and inhibit abnormal osteoclast activation by improving RANKL-induced mitochondrial dysfunction.

[0065] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0066] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. The application of verbascoside in the preparation of drugs for the prevention and treatment of osteoarthritis, characterized in that, The isoflavones described herein inhibit RANKL-induced abnormal osteoclast activation by regulating the PGAM2-mediated coupling process between osteoclast metabolism and oxidative stress, while simultaneously improving abnormal subchondral bone remodeling, thereby delaying or improving the progression of osteoarthritis.

2. The application as described in claim 1, characterized in that, PGAM2 is the direct target of the verbascoside isoflavone; the verbascoside isoflavone exerts its effect by binding to PGAM2 and enhancing its protein stability.

3. The application as described in claim 2, characterized in that, The PGAM2 is involved in regulating osteoclast glycolysis and mitochondrial redox balance.

4. The application as described in claim 1, characterized in that, The osteoclast abnormal activation phenotypes include at least one of the following: RANKL-induced enhanced osteoclast differentiation, increased formation of TRAP-positive multinucleated osteoclasts, enhanced microfilament ring formation, and enhanced bone resorption-related structures or functions.

5. The application as described in claim 1, characterized in that, The oxidative stress includes an increase in the level of total intracellular reactive oxygen species (ROS) and / or an increase in the accumulation of mitochondrial-derived ROS.

6. The application as described in claim 1, characterized in that, The isoflavone can improve RANKL-induced mitochondrial dysfunction, which includes a decrease in mitochondrial membrane potential.

7. The application as described in claim 1, characterized in that, The effective in vitro concentration of the verrucous isoflavone is any value between 1.25 μM and 20 μM; the effective in vivo dose is any value between 5 mg / kg and 100 mg / kg.

8. The application as described in claim 1, characterized in that, The types of osteoarthritis mentioned include any one of knee osteoarthritis, post-traumatic osteoarthritis, degenerative osteoarthritis, and senile osteoarthritis.

9. A pharmaceutical composition for the prevention and treatment of osteoarthritis, characterized in that, This includes verbascoflavonoids and pharmaceutically acceptable excipients.

10. The pharmaceutical composition according to claim 9, characterized in that, The dosage form of the pharmaceutical composition includes any one of tablets, capsules, granules, oral liquids, injections, lyophilized powder injections, sustained-release formulations, controlled-release formulations, nanoformulations, liposome formulations, intra-articular injection formulations, targeted subchondral bone delivery formulations, and topical formulations.

Images