Application of isosakuratin in preventing or treating osteoarthritis

Abstract

The present invention demonstrates that isosakurabin can inhibit the formation and function of osteoclasts in vitro, protect the bone microstructure of the subchondral bone in mice with osteoarthritis (OA), reduce articular cartilage degeneration in OA mice, inhibit the formation of abnormal blood vessels in the subchondral bone marrow cavity during OA progression, and alleviate joint pain induced by abnormal osteoclast activation in OA mice. This invention demonstrates for the first time that isosakurabin has a therapeutic effect on osteoarthritis and reveals its mechanism of action.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and particularly relates to the use of isosakurain in preparing drugs for preventing, inhibiting or treating osteoarthritis. Background Art

[0002] Osteoarthritis (OA) is a degenerative disease affecting all joints, characterized by cartilage loss, synovial hyperplasia, and abnormal subchondral bone remodeling. The pathogenesis of OA remains unclear, and effective interventions to halt its progression are lacking. Clinically, OA treatments primarily focus on symptom relief, often with potential side effects, and their long-term efficacy and safety remain poor.

[0003] For a long time, targeting cartilage matrix degradation has been an inherent concept in the treatment of OA, but in recent years, the key role of subchondral bone in OA has gradually attracted attention. Articular cartilage and subchondral bone form a structural and functional unit. Subchondral bone not only provides mechanical support for the overlying articular cartilage, but also can respond quickly to mechanical loads applied to the joint surface. During the initiation of OA, mechanical instability causes abnormal activation of osteoclasts (OC) in the subchondral bone, leading to accelerated early subchondral bone turnover and increased bone loss, which in turn exacerbates OA progression. In addition, the neovascularization mediated by OC can cause abnormal vascularization of the subchondral bone and joint pain.

[0004] Therefore, inhibiting the excessive activation of OC in subchondral bone is a potential measure for targeted OA treatment, and finding a method that can inhibit the excessive activation of OC in subchondral bone is an urgent problem that needs to be solved. Summary of the Invention

[0005] The main purpose of the present invention is to provide research on the therapeutic effect and molecular mechanism of isosakuranetin (ISN) on osteoarthritis, so as to overcome the shortcomings of the existing technology.

[0006] The present invention utilizes a cell membrane chromatography / time-of-flight mass spectrometry (CMC / TOFMS) system to screen isosakurain, an active ingredient that effectively inhibits osteoclastogenesis, from Anemarrhena Rhizoma. Isosakurain is a natural compound with a wide range of biological activities. Previous studies have demonstrated its multiple benefits, including anti-inflammatory, anti-aging, liver protection, and analgesia. While studies have reported that flavonoids similar to isosakurain have anti-osteoporosis effects, their role in osteoarthritis and their mechanism of action have not been reported.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0008] The present invention provides the use of isosakuratin in preparing a drug for inhibiting osteoclastogenesis in vitro.

[0009] The present invention also provides the use of isosakuratin in preparing an agent for inhibiting osteoclastogenesis in vitro.

[0010] The present invention also provides the application of isosakuratin in protecting the bone microstructure of osteoarthritis.

[0011] The present invention also provides the use of isosakuratin in alleviating articular cartilage degeneration in osteoarthritis.

[0012] The present invention also provides the use of isosakuratin in inhibiting abnormal angiogenesis in the subchondral bone marrow cavity during the progression of osteoarthritis.

[0013] The present invention also provides the use of isosakuratin in preparing a drug for preventing, inhibiting or treating osteoarthritis.

[0014] The present invention also provides the use of isosakuratin in preparing a reagent for preventing, inhibiting or treating osteoarthritis.

[0015] The present invention also provides the use of isosakuratin in preparing a drug for preventing, inhibiting or treating joint pain induced by osteoarthritis.

[0016] The present invention also provides the use of isosakuratin in preparing a reagent for preventing, inhibiting or treating joint pain induced by osteoarthritis.

[0017] The present invention also provides a use of a pharmaceutical composition in preparing a drug or reagent for preventing, inhibiting or treating osteoarthritis, wherein the pharmaceutical composition comprises a therapeutically effective amount of isosakuratin.

[0018] The present invention also provides a use of a pharmaceutical composition in preparing a drug or reagent for preventing, inhibiting or treating joint pain induced by osteoarthritis, wherein the pharmaceutical composition comprises a therapeutically effective amount of isosakuratin.

[0019] In one embodiment, the pharmaceutical composition comprises the combined use of isosakuratin and other drugs for treating osteoarthritis.

[0020] In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0021] The present invention also provides a use of a pharmaceutical preparation in preparing a drug or reagent for preventing, inhibiting or treating osteoarthritis, wherein the pharmaceutical preparation comprises a therapeutically effective amount of isosakuratin.

[0022] The present invention also provides a use of a pharmaceutical preparation in preparing a drug or reagent for preventing, inhibiting or treating joint pain induced by osteoarthritis, wherein the pharmaceutical preparation comprises a therapeutically effective amount of isosakuratin.

[0023] In one embodiment, the pharmaceutical formulation comprises a solid dosage form, a semisolid dosage form, a liquid dosage form, or a gaseous dosage form.

[0024] In one embodiment, the solid dosage form includes powders, pills, tablets, and capsules; the semisolid dosage form includes ointments, suppositories, and gels; the liquid dosage form includes lotions, mixtures, solutions, and injections; and the gaseous dosage form includes aerosols and sprays.

[0025] In one aspect, the present invention is directed to the prevention, inhibition or treatment of osteoarthritis, using isosakuratin as the main active ingredient.

[0026] Compared with the prior art, the present invention has the following advantages: isosakurabin is a natural compound with a wide range of biological activities. Its anti-inflammatory, anti-aging, hepatoprotective, and analgesic functions have been preliminarily confirmed. However, previous studies have not revealed the relationship between isosakurabin and osteoarthritis, and in particular, its role in osteoarthritis and its mechanism of action have not been reported. The present invention demonstrates that isosakurabin can inhibit the formation and function of osteoclasts in vitro, protect the bone microstructure of the subchondral bone in OA mice, reduce articular cartilage degeneration in OA mice, inhibit the formation of abnormal blood vessels in the subchondral bone marrow cavity during OA progression, and alleviate joint pain induced by abnormal osteoclast activation in OA mice. The present invention demonstrates for the first time that isosakurabin has a therapeutic effect on osteoarthritis and reveals its mechanism of action. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0028] Figure 1 BRA, MAN, and ISN are three bioactive components identified from Rhizoma Anemarrhenae. ac Representative three-dimensional images of BRA (a), MAN (b), and ISN (c) obtained using a CMC / TOFMS system. df CCK-8 assays of the cytotoxicity of BRA (d), MAN (e), and ISN (f) in bone marrow-derived mononuclear cells (BMMCs).

[0029] Figure 2ISN inhibits osteoclastogenesis in vitro. a, f Representative TRAP staining (a) and quantification (f) of osteoclasts induced from BMMCs. BMMCs were cultured with or without M-CSF and RANKL and treated with BRA (120 μg / mL), MAN (60 μg / mL), or ISN (60 μg / mL). Scale bar: 100 μm. **P < 0.01, compared with the RANKL group. bd mRNA expression levels of CTSK (b), MMP-9 (c), and TRAcp5B (d). *P < 0.05, **P < 0.01, compared with the RANKL group. e, g Images (e) and quantification (g) of bone lacunas in osteoclasts induced from BMMCs treated with BRA (120 μg / mL), MAN (60 μg / mL), or ISN (60 μg / mL). Scale bar: 100 μm. **P<0.01 compared with the RANKL group.

[0030] Figure 3 ISN restores knee bone microarchitecture in the subchondral bone of OA mice. a, b Three-dimensional images (top) and two-dimensional images (bottom) of the sagittal plane of the knee joint at 4 weeks (a) and 8 weeks (b) after ACLT. cf Quantitative microCT results of tibial subchondral bone, including bone volume / total volume (BV / TV) (c), bone thickness (Tb.Th) (d), bone number (Tb.N) (e), and bone space (Tb.Sp) (f). *P < 0.05, **P < 0.01 compared with the ACLT group. ##P < 0.01 compared with the sham group. g, h TRAP staining (g) and quantification (h) of TRAP+ osteoclasts in the tibial subchondral bone marrow at 2 weeks after ACLT. Scale bar: 200 μm (top). Scale bar: 50 μm (bottom). **P < 0.01 compared with the ACLT group. Sham: sham group treated with PBS. ACLT: ACLT mice treated with PBS. ISN: ACLT mice were treated with ISN.

[0031] Figure 4ISN improves articular cartilage degradation after ACLT. a, b Representative Safranin O staining at 4 weeks (a) and 8 weeks (b) after ACLT. Scale bar: 200 μm (top). Scale bar: 50 μm (bottom). c Representative immunostaining of ACAN (top), COL II (middle), and MMP-13 (bottom) in articular cartilage at 4 weeks after ACLT. Scale bar: 20 μm. d OARSI histological grading of articular cartilage at 4 weeks and 8 weeks after ACLT. **P < 0.01 compared with the ACLT group. e.g. Quantification of ACAN (e), COL II (f), and MMP-13 (g) in articular cartilage at 4 weeks after ACLT. *P < 0.05, **P < 0.01 compared with the ACLT group.

[0032] Figure 5 ISN protects against abnormal angiogenesis in the subchondral bone of OA mice. a Three-dimensional microangiography image of the sagittal section of the medial tibial subchondral bone 4 weeks after ACLT. Scale bar = 500 μm. b, c Quantification of vessel number (VN) (b) and vessel volume (VV) (c). *P < 0.05, **P < 0.01 compared with the ACLT group. d, e Quantification of CD31+ (red) and EMCN+ (green) cells in the tibial subchondral bone marrow 4 weeks after ACLT (d) and representative immunofluorescence double staining (e). Scale bar: 50 μm. **P < 0.01 compared with the ACLT group.

[0033] Figure 6 ISN alleviates joint pain in OA mice. a, b Representative immunofluorescence double staining (a) and quantification (b) of TRAP+ (red) and Netrin-1+ (green) cells in the subchondral bone marrow 2 weeks after ACLT. Scale bar: 50 μm. **P < 0.01 compared with the ACLT group. c, d Representative immunofluorescence double staining (c) and quantification (d) of CGRP+ cells in the subchondral bone marrow 2 weeks after ACLT. Scale bar: 50 μm. **P < 0.01 compared with the ACLT group. e, f Quantification of pain-related behaviors and 50% PWT (e) and PWL (f) from 0 to 8 weeks after ACLT. *P < 0.05, **P < 0.01 compared with the ACLT group.

[0034] Figure 7ISN regulates osteoclastogenesis through the NF-κB / CXCL2 axis. a Heatmap of differentially expressed genes (DEGs) between ACLT and ISN samples. b Gene Ontology (GO) analysis of the biological functions of DEGs. c KEGG analysis of the biological functions of DEGs. Red highlighted regions indicate the NF-κB signaling pathway identified and focused. d Western blot analysis of NF-κB (p65) phosphorylation and CXCL2 expression in BMMCs treated with ISN (15, 30, and 60 μg / mL) with or without M-CSF and RANKL. e Representative immunofluorescence staining of p65 nuclear translocation in osteoclasts induced by ISN (60 μg / mL) in BMMCs treated with or without M-CSF and RANKL. BMMCs were treated with ISN (60 μg / mL) in the presence or absence of CU-T12-9. Scale bar: 20 μm.

[0035] Figure 8 The mechanism by which ISN alleviates osteoarthritis is by inhibiting the NF-κB / CXCL2 axis to suppress subchondral osteoclastogenesis. DETAILED DESCRIPTION

[0036] In view of the shortcomings of the prior art, the inventors of this case, after long-term research and extensive practice, have proposed the technical solution of the present invention. The technical solution of the present invention will be clearly and completely described below. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making any creative effort shall fall within the scope of protection of the present invention.

[0037] definition:

[0038] The term "inhibit" means to reduce by a measurable amount or to prevent completely.

[0039] The term "therapeutically effective amount" refers to an amount of isosakuramin that is effective in treating an osteoarthritis disease or condition in a patient such as a mammal.

[0040] The term "patient" refers to a subject to whom the isosakuratin of the present invention is administered. Patients include, but are not limited to, humans, rats, mice, guinea pigs, non-human primates, pigs, goats, cattle, horses, dogs, cats, birds, and poultry. Typically, the patient is a rat, mouse, dog, human, or non-human primate, more typically a human.

[0041] The term "treat or treatment" refers to therapeutic and prophylactic treatments, wherein the purpose is to suppress or slow down (mitigate) undesirable physiological changes or conditions, such as the development of osteoarthritis. For purposes of the present invention, useful or desired clinical outcomes include, but are not limited to, alleviation of symptoms, weakening of the extent of the disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial or complete), whether detectable or undetectable. "Treatment" may also mean an extension of survival, an improvement in quality of life, compared to the expected survival without treatment. Those in need of treatment include those already suffering from the disease or condition and those susceptible to the disease or condition.

[0042] As used herein, "isosakurabin as the main active ingredient" means that the proportion of isosakurabin in the active ingredients is more than 10%; preferably, more than 20%; preferably, more than 30%; preferably, more than 40%; preferably, more than 50%; preferably, more than 60%; preferably, more than 65%; preferably, more than 70%; preferably, more than 75%; preferably, more than 80%; preferably, more than 85%; preferably, more than 90%; preferably, more than 95%.

[0043] The term "pharmaceutically acceptable excipients" refers to substances, other than the active ingredient, included in the dosage form when preparing a pharmaceutical preparation. These substances are suitable for contact with human or other animal tissues within the scope of reasonable medical judgment and generally have characteristics such as safety, non-toxicity, and no adverse biological reactions. Specifically, they include but are not limited to the following categories:

[0044] Fillers (diluents): such as lactose, powdered sugar, dextrin, starch and its derivatives, cellulose and its derivatives, inorganic calcium salts (such as calcium sulfate, calcium phosphate, calcium hydrogen phosphate, precipitated calcium carbonate, etc.), sorbitol, glycine, etc., are used to increase the volume of the drug preparation so that the active ingredients can be evenly dispersed.

[0045] Lubricants (glidants or anti-adhesives): including micro-powdered silica gel, magnesium stearate, talc, aluminum hydroxide, boric acid, hydrogenated vegetable oil, polyethylene glycol, etc., can reduce the friction between drug particles and between drugs and production equipment, ensure the smooth production process of drug preparations, and also help swallowing and release of drugs.

[0046] Dispersants: They can help the active ingredients disperse evenly in the preparation, prevent the active ingredients from aggregating, and improve the stability and uniformity of the drug.

[0047] Wetting agents: such as sodium lauryl sulfate, water or alcohol, can increase the contact area between drug particles and liquid medium, making the drug easier to be wetted and facilitating the preparation and use of the preparation.

[0048] Binders: such as syrup, gum arabic, gelatin, sorbitol, tragacanth, cellulose and its derivatives (such as microcrystalline cellulose, sodium carboxymethyl cellulose, ethyl cellulose or hydroxypropyl methyl cellulose), gelatin paste, syrup, starch paste or polyvinyl pyrrolidone, etc., used to bind drug particles or powders together to form a preparation with a certain shape and strength.

[0049] Regulators: include hydrochloric acid, citric acid, potassium hydroxide (sodium), sodium citrate and buffers (including sodium dihydrogen phosphate and disodium hydrogen phosphate), etc., which can adjust the pH of drug preparations to ensure the stability and effectiveness of the drugs, while also adapting to the physiological environment of the human body.

[0050] Solubilizers: such as Tween-80, bile, glycerol, etc., can increase the solubility of active ingredients in the solvent, allowing the drug to form a uniform solution or suspension.

[0051] Antioxidants: including sodium sulfite, sodium bisulfite, sodium metabisulfite, dibutyl benzoic acid, etc., can prevent the active ingredients and other easily oxidized ingredients in pharmaceutical preparations from being oxidized, thereby extending the shelf life of the drugs.

[0052] Antibacterial agents: such as 0.5% phenol, 0.3% cresol, 0.5% chlorobutanol, etc., are used to inhibit the growth of microorganisms in pharmaceutical preparations to ensure the quality and safety of drugs.

[0053] Emulsifiers: such as polysorbate 80, sorbitan monophosphate, Pluronic F-68, lecithin, soy lecithin, etc., can make immiscible liquids (such as oil and water) form uniform emulsions, which are suitable for the preparation of emulsion drug preparations.

[0054] Disintegrants: including starch and its derivatives (such as sodium carboxymethyl starch, sodium starch glycolate, pregelatinized starch, modified starch, hydroxypropyl starch, corn starch, etc.), polyvinyl pyrrolidone or microcrystalline cellulose, etc., which can promote the rapid disintegration of drug preparations in the body and accelerate the release and absorption of active ingredients.

[0055] 1. Experimental steps

[0056] 1. Identification of bioactive ingredients

[0057] BMMCs were collected from the bilateral femoral bone marrow cavities of 4-week-old male C57BL / 6J mice. BMMC cell membranes were coated on an activated silica (SiO2) surface and incubated with a suspension of Anemarrhena Rhizoma. The SiO2 served as a carrier, covalently binding to the cell membrane as a stationary phase, while the drug suspension was flushed with a mobile phase. The retention time of the active compound on the BMMC cell membrane was used to determine the affinity between the drug component and the cell membrane protein. Stronger affinity, longer retention time, and greater potential activity were associated with the compound. A CMC analysis system was then used to determine the affinity between Anemarrhena Rhizoma and BMMC membrane receptors and identify potential active ingredients. A 3D contour map of the retention time of the bioactive ingredients was plotted using MATLAB software.

[0058] OA mouse model

[0059] Ten-week-old male C57BL / 6J mice were housed in a specific pathogen-free (SPF) laboratory with free access to food and water. After inhalation anesthesia with sevoflurane, a mechanically unstable OA model was established in the right knee joint using anterior cruciate ligament transaction (ACLT). All mice were randomly divided into three groups (n = 6 per group): the sham group (sham group, mice with only the joint capsule removed and treated with phosphate-buffered saline (PBS)), the ACLT group (model group, mice with ACLT modeling and treated with PBS), and the ISN group (treatment group, mice with ACLT modeling and treated with ISN). Mice were euthanized by carbon dioxide inhalation, and knee joint tissues were collected for subsequent experimental examinations.

[0060] 3. Drug intervention

[0061] ISN standard solution was dissolved in low concentration dimethyl sulfoxide (DMSO) (<0.1%). Three days after ACLT surgery, mice were intraperitoneally injected with PBS (Sham group and ACLT group) and ISN (2 mg / kg / d) (ISN group), respectively, every other day for a total of 8 weeks.

[0062] 4. Cytotoxicity test

[0063] CCK-8 assay was performed according to standard methods. BMMCs were seeded on 96-well plates (1×10 4 Cells were co-cultured with different concentrations of the identified active ingredient for 48 hours. The culture was then continued for 2 hours in the presence of 10 μL of CCK-8 solution. The absorbance at 450 nm was calculated using a microplate reader.

[0064] 5. Cell induction and intervention

[0065] In a 96-well plate (1 × 10 4 BMMCs were cultured in a 100 μL (100 μL) PBS solution. OCs were induced with M-CSF (50 ng / mL) and RANKL (50 ng / mL), along with intervention with non-toxic doses of identified components. TRAP staining was performed after 7 days of cell culture. OCs were then seeded onto biomimetic synthetic plates for 7 days before bone resorption assays. Results were observed using light microscopy, and positive cells were counted using Image J software.

[0066] 6. Micro-computed tomography (μMicro-CT)

[0067] Mouse knee joints were scanned using μMicro-CT (8 μm resolution, 50 kV voltage, and 60 A current). The sagittal plane at the center of the tibial plateau was selected as the target area, and the scanned images were loaded into Data-Viewer software and the data were recorded. CT-An software was used to quantify the target parameters: bone volume / total volume (BV / TV) of subchondral bone, total number of trabeculae (Tb.N), trabecular separation (Tb.Sp), and trabecular thickness (Tb.Th). 2D and 3D images were generated using CT-Vol software.

[0068] 7. Microangiography

[0069] Mice were anesthetized and immobilized, and an angiographic contrast agent (Microphil MV-120, Flow-Tech) was injected via a left ventricular puncture site. After successful angiography, the specimens were stored at 4°C overnight to allow for polymerization of the contrast agent. The following day, the knee joints were sampled and fixed in 4% PFA for four days. Following decalcification for two weeks, micro-CT scanning and 3D imaging were performed to analyze vascular parameters in the subchondral bone, including vessel number (VN) and vessel volume (VV).

[0070] 8. Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR)

[0071] cDNA was extracted and analyzed by RT-qPCR using 2 -ΔΔCT method to detect the expression level of target genes.

[0072] 9. Western Blot (WB)

[0073] Proteins were extracted and subjected to Western blotting. Primary antibodies included anti-CXCL2, anti-NF-kB p65, and anti-phospho-NF-kB p65. Samples were incubated with secondary antibodies, and images were visualized by chemiluminescence.

[0074] 10. Histological and immunohistochemical examinations

[0075] Knee joints were fixed in 4% PFA for 48 hours, decalcified in 10% EDTA for 2 weeks, and then embedded in paraffin. Specimens were prepared into 4-μm sagittal sections and stained with TRAP, Safranin O / Fast Green, and immunohistochemistry. The severity of the cartilage histological phenotype was assessed using the Osteoarthritis Research Society International (OARSI) scoring system, and the results were quantified using Image J software.

[0076] 11. Von Frey Test

[0077] Mechanical hyperalgesia was assessed using Von Frey filaments, and the hind paw withdrawal threshold (PWT) was recorded. To acclimate the mice to the test environment, they were first placed in a cage with a steel mesh bottom for 20 minutes. The force of the filaments ranged from 0.04 to 2.0 grams. Mechanical allodynia was assessed in mice at the same time each day. Starting with a force of 0.4 grams, a needle prick was performed in the mid-plantar area of ​​the right hind paw of the mouse, and 50% PWT was recorded using the Dixon method.

[0078] 12. Heat Plantar Assay

[0079] Heat Plantar was used to measure the hind paw withdrawal latency (Paw Withdrawal Latency, PWL). Before the start of the experiment, the device was calibrated to provide a standard PWL of approximately 10 seconds, and the cutoff latency was set to 20 seconds to avoid damage to the plantar tissue of the mice. In order to allow the mice to adapt to the test environment, they were first placed in a cage with a steel mesh bottom for 20 minutes. A 40-watt infrared heat source was used to stimulate the middle plantar area of ​​the right hind paw of the mouse. The time from the start of the heat stimulation to the withdrawal of the hind paw was recorded, and a total of 3 assessments were made within 5 minutes, and the average value was recorded.

[0080] 13. GO and KEGG analysis

[0081] The differentially expressed genes were uploaded to the DAVID database (https: / / david.ncifcrf.gov / summary.jsp) for GO and KEGG analysis.

[0082] 2. Experimental Results

[0083] 1. Isocherrin is the active ingredient extracted from Anemarrhena asphodeloides

[0084] To identify potential bioactive monomers in Anemarrhena Rhizoma, we first used a well-established high-throughput CMC / TOFMS system to detect active components in Anemarrhena Rhizoma that have high affinity for BMMC cell membranes. 3D contour analysis results showed that three candidate monomers exhibited significant adhesion behavior and relatively stable retention times: Broussonin A (BRA), Markogein (MAN), and Isosakuranetin (ISN). Figure 1 ac). To further clarify the potential effects of candidate monomers on OC generation, we first determined the non-cytotoxic concentrations of the three monomers in BMMCs using the CCK-8 assay (BRA < 256 μg / mL, MAN < 128 μg / mL, ISN < 128 μg / mL), and then selected the non-cytotoxic concentrations (BRA = 120 μg / mL, MAN = 60 μg / mL, ISN = 60 μg / mL) for subsequent experiments ( Figure 1 df).

[0085] 2. Isocherrin can inhibit osteoclastogenesis in vitro

[0086] We treated BMMC with these three monomers and then performed TRAP staining. The results showed that among the three candidate monomers, only ISN could significantly inhibit RANKL-induced OC formation in vitro, while BRA and MAN had no such significant effect ( Figure 2 a and f). In addition, we also examined the expression levels of marker genes associated with OC formation, including cathepsin K (CTSK), matrix metalloproteinase-9 (MMP-9), and TRAcp5B. Consistent with the TRAP results, only ISN was able to significantly downregulate the expression of these genes ( Figure 2 To further explore whether these three candidate drugs affect the bone resorption function of OC, we subsequently performed a bone pit test, and the results also showed that only ISN could significantly inhibit the RANKL-induced bone resorption activity of OC ( Figure 2 e and f). The above results indicate that ISN extracted from Anemarrhena asphodeloides can inhibit the formation and function of OC in vitro.

[0087] 3. Isocherrin can protect the bone microstructure of subchondral bone in OA mouse model

[0088] Given that ISNs significantly inhibit OC formation in vitro, we next sought to elucidate whether ISNs could exhibit the same inhibitory effect in vivo and stabilize the bone microstructure of subchondral bone during OA progression. We established an OA model in 10-week-old male C57BL / 6J mice by ACLT surgery and then administered ISN intervention to each group.

[0089] We used Micro-CT to examine the changes in the bone structure of the knee joints in different groups. Four to eight weeks after modeling, Micro-CT of the knee joints showed that compared with the sham group (Sham), the model group (ACLT) had significantly increased bone spur formation and subchondral bone erosion. At the same time, the ACLT group showed a trend of enlarged bone marrow cavity and sparse trabeculae ( Figure 3 a, b), target parameters showed decreased bone volume / total volume (BV / TV), trabecular number (Tb.N), trabecular thickness (Tb.Th) and increased trabecular separation (Tb.Sp) ( Figure 3 cf). However, these pathological morphologies and parameter changes were normalized after ISN intervention, that is, the degree of subchondral bone destruction and bone erosion was significantly alleviated ( Figure 3 We then further evaluated the activation of OCs in the subchondral bone 2 weeks after ACLT surgery using TRAP staining. The results showed that the increase in the number of TRAP-positive OCs in the ACLT group was significantly inhibited by ISN, reaching a level comparable to that of the OCs in the Sham group ( Figure 3 These results indicate that ISN can block ACLT-induced excessive activation of OC in subchondral bone and destruction of bone microstructure in the mouse knee joint.

[0090] 4. Isocherrin can reduce articular cartilage degeneration in OA mouse models

[0091] Since the subchondral bone and the overlying articular cartilage form a "biomechanical unit", changes in the subchondral bone microenvironment in OA are closely related to cartilage degeneration. To further verify the effect of ISN on articular cartilage in OA progression, we performed histological analysis of the knee joints at 4 and 8 weeks under different intervention conditions. Safranin O / FastGreen staining showed that the ACLT group had more severe proteoglycan loss compared with the Sham group, and the OARSI score was significantly increased ( Figure 4 a, b, d). However, ISN intervention significantly alleviated the cartilage degeneration and increased OARSI score caused by ACLT surgery ( Figure 4a, b, d). In immunohistochemistry and fluorescence staining, the expression level of cartilage degradation marker (MMP-13) increased in the ACLT group, but normalized after ISN treatment. In contrast, the levels of cartilage synthesis markers (ACAN and COL II) decreased after ACLT surgery compared with the sham control group, and these changes were significantly improved by ISN intervention, further suggesting the protective effect of ISN on articular cartilage ( Figure 4 c, eg). Taken together, these results indicate that ISN alleviates articular cartilage wear and loss in an OA mouse model.

[0092] 5. Inhibitory effect of isosakuratin on abnormal angiogenesis in subchondral bone of OA mice

[0093] Proliferative abnormal angiogenesis in the subchondral bone plays a key role in the occurrence and development of OA. Therefore, in addition to studying the changes in subchondral structure and cartilage morphology, we also performed microangiography and Micro-CT to detect the vascularization of the subchondral bone marrow. We found that 4 weeks after ACLT surgery, the degree of angiogenesis in the subchondral bone marrow cavity was significantly increased, as manifested by increased vessel number (VN) and vessel volume (VV), and ISN intervention significantly improved these changes, restoring the degree of vascularization to a level comparable to that of the Sham group ( Figure 5 ac). In addition, double immunofluorescence staining of H-type vessels showed that the co-localization expression level of CD31+ / EMCN+ cells was abnormally enhanced in ACLT mice, but was significantly reduced after ISN treatment. However, the vascularization phenotype of the subchondral bone marrow did not change significantly between the Sham and ISN groups ( Figure 5 d, e). These results indicate that ISN inhibits the formation of abnormal blood vessels in the subchondral bone marrow cavity during OA progression.

[0094] 6. Isocherrin alleviates joint pain induced by abnormal osteoclast activation in OA mice

[0095] Increased OCs and sensory nerve invasion in the subchondral bone marrow are another pathological feature of OA progression, which is associated with joint pain. To explore whether ISN has an inhibitory effect on pain caused by overactivated OCs in ACLT mice, we performed immunofluorescence colocalization and behavioral tests. Immunofluorescence colocalization showed that the number of TRAP+ / Netrin-1+ OC colocalization increased significantly in the ACLT group, but decreased significantly after ISN intervention, indicating that Netrin-1-positive OCs gradually increased in the subchondral bone marrow during OA progression, but were significantly inhibited by ISN ( Figure 6a, b). In addition, immunofluorescence staining of calcitonin gene-related peptide (CGRP+) sensory nerve fibers also showed similar results ( Figure 6 c, d). Consistent with the histological changes, the Von Frey behavioral test showed that the pain threshold (PWT) of the ACLT group was significantly reduced from 0 to 8 weeks after surgery compared with the Sham group. The intervention of ISN can increase the pain threshold of mice, suggesting that ISN can improve knee pain in OA mice ( Figure 6 e). In addition, the hot plate test also showed a similar pain response trend in the three groups as in the von Frey test ( Figure 6 f). These results indicate that ISN can reduce the secretion of Netrin-1 by OC and alleviate mechanical and thermal hyperalgesia in OA mice.

[0096] 7. Isocherrin inhibits the formation of subchondral osteoclasts by inhibiting the RANKL-induced NF-κB / CXCL2 signaling pathway

[0097] After OA mice were modeled, they were divided into groups according to whether they had received ISN intervention. Subchondral bone tissues of the ACLT and ISN groups were directly obtained for transcriptome sequencing to investigate differentially expressed genes (DEGs). GO and KEGG analyses were performed to determine the biological functions and signaling pathways ( Figure 7 ac). The results showed that C–X–C motif chemokine ligand 2 (CXCL2) was significantly downregulated in the ISN-treated group ( Figure 7 a). CXCL2 is a chemokine involved in inflammatory and immune responses and promotes the recruitment of polymorphonuclear cells during OC maturation. We also found significant changes in the NF-κB signaling pathway ( Figure 7 c). The NF-κB signaling pathway plays a key role in regulating common target genes for OC differentiation and bone resorption. To examine the role of ISN in NF-κB pathway activation and CXCL2 expression, we induced OC formation and administered ISN intervention. Western blot results showed that ISN dose-dependently inhibited p-P65 activation during RANKL-induced OC formation. Simultaneously, CXCL2 expression was significantly downregulated ( Figure 7d), indicating that ISN can affect the expression level of CXCL2 by regulating NF-κB activity. CU-T12-9 is an agonist that activates the NF-κB-dependent signaling pathway. By immunofluorescence staining, we further revealed that RANKL-induced P65 nuclear translocation was significantly blocked by ISN, suggesting that ISN inhibits the NF-κB pathway during OC generation. However, this effect of ISN on P65 nuclear translocation was partially reversed after CU-T12-9 intervention ( Figure 7 e). Taken together, these results suggest that ISN inhibits OC formation by blocking RANKL-induced NF-κB / CXCL2 axis.

[0098] It should be understood that the technical solution of the present invention is not limited to the above-mentioned specific implementation cases. Any technical variations made according to the technical solution of the present invention without departing from the scope of protection of the purpose of the present invention and the claims shall fall within the scope of protection of the present invention.

Claims

1. Use of isosakuratin as the sole active ingredient in the preparation of drugs for preventing or treating osteoarthritis.

2. Use of a pharmaceutical composition in the preparation of a drug for preventing or treating osteoarthritis, characterized in that: The pharmaceutical composition comprises a therapeutically effective amount of isosakurabin, wherein the isosakurabin serves as the sole active ingredient in the pharmaceutical composition.

3. The use according to claim 2, characterized in that The pharmaceutical composition also includes pharmaceutically acceptable excipients.

4. The use according to claim 2, characterized in that The pharmaceutical composition is used in combination with other drugs for treating osteoarthritis.

5. Use of a pharmaceutical preparation in the preparation of a drug for preventing or treating osteoarthritis, characterized in that: The pharmaceutical preparation comprises a therapeutically effective amount of isosakurabin, wherein the isosakurabin serves as the sole active ingredient.

6. The use according to claim 5, characterized in that The pharmaceutical preparation includes a solid dosage form, a semisolid dosage form, a liquid dosage form or a gaseous dosage form.

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