Application of isosakuranetin in prevention or treatment of osteoarthritis

Abstract

The invention proves that the isosakuranetin can inhibit the generation of osteoclasts and the functions of the osteoclasts in vitro, protect the bone microstructure of subchondral bones of an OA (osteoarthritis) mouse, relieve the degeneration of articular cartilage of the OA mouse, inhibit the generation of abnormal blood vessels in subchondral marrow cavities in the progress of the OA, and inhibit the osteoclasts in the osteoclasts. The invention proves that the isosakuranetin has a treatment effect on the osteoarthritis for the first time, and discloses an action mechanism of the isosakuranetin.

Description

Technical Field

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

[0002] Osteoarthritis (OA) is a degenerative disease of the entire joint, with cartilage loss, synovial hyperplasia and abnormal subchondral bone remodeling as the main pathological features. The pathogenesis of OA is still unclear, and there is also a lack of effective means to block the progression of OA. Clinically, most OA treatment drugs are mainly for relieving symptoms, often accompanied by a variety of potential side effects, and the long-term efficacy and safety are still 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 the mechanical load 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 excessive activation of subchondral bone OC is a potential measure for targeted OA treatment, and finding a method that can inhibit excessive activation of subchondral bone OC is an urgent problem that needs to be solved. Summary of the invention

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

[0006] The present invention uses a cell membrane chromatography / time-of-flight mass spectrometry (CMC / TOFMS) system to screen out isosakurain, an active ingredient that can effectively inhibit osteoclastogenesis, from Rhizoma Anemarrhenae. Isosakurain is a natural compound with a wide range of biological activities. Previous studies have shown that it has multiple functions such as anti-inflammatory, anti-aging, liver protection, and analgesia. Although there are also studies reporting that flavonoid drugs similar to isosakurain have an anti-osteoporosis effect, there is no report on its role in osteoarthritis and its mechanism of action.

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

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

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

[0010] The present invention also provides application of isosakura vine extract in protecting bone microstructure of osteoarthritis.

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

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

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

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

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

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

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

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

[0019] In one embodiment, the pharmaceutical composition comprises the combined use of isosakuravine 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 an application of a pharmaceutical preparation in the preparation of a drug or reagent for preventing, inhibiting or treating osteoarthritis, wherein the pharmaceutical preparation comprises a therapeutically effective amount of isosakuravine.

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

[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 forms include powders, pills, tablets, and capsules; the semisolid dosage forms include ointments, suppositories, and gels; the liquid dosage forms include lotions, mixtures, solutions, and injections; and the gaseous dosage forms include aerosols and sprays.

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

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows: isosakuratin is a natural compound with a wide range of biological activities, and its anti-inflammatory, anti-aging, liver protection, analgesic and other functions have been preliminarily confirmed, but previous studies have not revealed the relationship between isosakuratin and osteoarthritis, especially its role in osteoarthritis and its mechanism of action have not been reported. The present invention proves that isosakuratin can inhibit the generation and function of osteoclasts in vitro, protect the bone microstructure of the subchondral bone of OA mice, reduce the degeneration of articular cartilage in OA mice, inhibit the generation of abnormal blood vessels in the subchondral bone marrow cavity during the progression of OA, and relieve the joint pain induced by abnormal activation of osteoclasts in OA mice. The present invention proves for the first time that isosakuratin 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 drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. 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 creative work.

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

[0029] Figure 2.ISN inhibits osteoclastogenesis in vitro. a,f Representative TRAP staining (a) and quantitative results (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 quantitative results (g) of bone lacunas induced from 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 subchondral bone of OA mice. a,b Three-dimensional images (upper) and two-dimensional images (lower) of knee joints in sagittal plane 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 gap (Tb.Sp) (f). *P<0.05, **P<0.01 compared with ACLT group. ##P<0.01 compared with sham group. g,h TRAP staining (g) and quantitative results (h) of TRAP+ osteoclasts in tibial subchondral bone marrow at 2 weeks after ACLT. Scale bar: 200 μm (upper). Scale bar: 50 μm (lower). **P<0.01 compared with ACLT group. Sham: sham group treated with PBS. ACLT: ACLT mice treated with PBS. ISN: ACLT mice were treated with ISN.

[0031] Figure 4.ISN improves the degeneration of articular cartilage after ACLT. a, b Representative Safranin O staining at 4 weeks (a) and 8 weeks (b) after ACLT. Scale bar: 200 μm (upper). Scale bar: 50 μm (lower). c Representative immunostaining in articular cartilage at 4 weeks after ACLT, including ACAN (upper), COL II (middle), and MMP-13 (lower). 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. eg Quantitative results 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 subchondral bone of OA mice. a Three-dimensional microangiography images of the sagittal plane of the medial tibial subchondral bone 4 weeks after ACLT. Scale bar = 500 μm. b, c Quantitative results of the number of blood vessels (VN) (b) and the volume of blood vessels (VV) (c). *P < 0.05, **P < 0.01 compared with the ACLT group. d, e Quantitative results of CD31+ (red) and EMCN+ (green) cells in the subchondral bone marrow of the tibia 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 quantitative results (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 quantitative results (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 Quantitative results 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 via the NF-κB / CXCL2 axis. a Heat map of differentially expressed genes (DEGs) between ACLT and ISN samples. b Gene ontology (GO) analysis of biological functions of DEGs. c KEGG analysis of biological functions of DEGs. The red highlighted area is the NF-κB signaling pathway identified and focused. d Western blot analysis of NF-κB (p65) phosphorylation and CXCL2 expression in BMMCs with or without M-CSF and RANKL treated with ISN (15, 30, 60 μg / mL). e Representative immunofluorescence staining of p65 nuclear translocation in osteoclasts induced by BMMCs with or without M-CSF and RANKL treated with ISN (60 μg / mL). 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 by inhibiting subchondral osteoclastogenesis through suppressing NF-κB / CXCL2 axis. DETAILED DESCRIPTION

[0036] In view of the defects of the prior art, the inventor of this case has proposed the technical solution of the present invention after long-term research and extensive practice. The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are 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 entirely.

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

[0040] The term "patient" refers to a subject to which 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 treatment and preventive treatment, wherein the purpose is to inhibit or slow down (mitigate) undesirable physiological changes or conditions, such as the development of osteoarthritis. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, weakening of the extent of the disease, stabilization of the disease state (i.e., no deterioration), 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 and 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, "isosakuravine as the main active ingredient" means that the proportion of isosakuravine 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 contained in the dosage form in addition to the active ingredients when preparing pharmaceutical preparations. These substances are suitable for contact with human or other animal tissues within the scope of reasonable medical judgment and generally have the characteristics of 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., which 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] Dispersant: It can help the active ingredients to be evenly dispersed in the preparation, prevent the active ingredients from aggregating, and improve the stability and uniformity of the drug.

[0047] Wetting agents: such as sodium dodecyl 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, etc.), gelatin slurry, syrup, starch slurry or polyvinyl pyrrolidone, etc., are used to bind drug particles or powders together to form a preparation with a certain shape and strength.

[0049] Regulators: including 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 value of drug preparations to ensure the stability and effectiveness of the drugs, while also being able to adapt to the physiological environment of the human body.

[0050] Solubilizers: such as Tween-80, bile, glycerol, etc., can increase the solubility of the active ingredient 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., which can prevent the active ingredients and other easily oxidized ingredients in drug preparations from being oxidized, thereby extending the shelf life of the drug.

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

[0053] Emulsifiers: such as polysorbate 80, sorbitan monoacrylate, Pluronic F-68, lecithin, soybean lecithin, etc., can make immiscible liquids (such as oil and water) form a uniform emulsion, which is 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 cavity of 4-week-old male C57BL / 6J mice. BMMC cell membranes were coated with activated silica (SiO 2 ) surface and incubated with the Anemarrhena suspension. 2Acting as a carrier and covalently binding to the cell membrane as a stationary phase, the drug suspension is washed with the mobile phase. The affinity relationship between the drug component and the cell membrane protein is judged according to the retention time of the active compound in the drug on the BMMC cell membrane. If the affinity is stronger, the retention time of the compound is longer and the activity potential is greater. Subsequently, the CMC analysis system was used to detect the affinity between Anemarrhena asphodeloides and BMMC membrane receptors and identify potential active ingredients. The 3D contour map of the retention time of the bioactive ingredients was drawn using MATLAB software.

[0058] 2. OA mouse model

[0059] Male C57BL / 6J mice aged 10 weeks were housed in a specific pathogen free (SPF) laboratory with free access to food and water. After inhalation anesthesia with sevoflurane, the mechanical instability OA model was established in the right knee joint by anterior cruciate ligament transection (ACLT). All mice were randomly divided into 3 groups (n=6 in each group): Sham group (sham operation group, mice with only joint capsule transection and treated with phosphate buffered saline (PBS)), ACLT group (modeling group, mice with ACLT modeling and treated with PBS) and ISN group (treatment group, mice with ACLT modeling and treated with ISN). After the mice were euthanized by carbon dioxide inhalation, the knee joint tissues were collected for subsequent experimental detection.

[0060] 3. Drug intervention

[0061] ISN standard solution was dissolved in low concentration of dimethyl sulfoxide (DMSO) (<0.1%). Three days after ACLT surgery, PBS (Sham group and ACLT group) and ISN (2 mg / kg / d) (ISN group) were injected into the peritoneum of mice, respectively, once 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 ingredients for 48 hours. Then, the culture was continued for 2 hours in the presence of 10 μL CCK-8 solution. The absorbance at a wavelength of 450 nm was calculated using a microplate reader.

[0064] 5. Cell induction and intervention

[0065] In a 96-well plate (1 × 10 4BMMCs were cultured with 100 μL of ...

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

[0067] The mouse knee joint was 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 image was loaded into the Data-Viewer software and the data was recorded. The target parameters were quantified using CT-An software: bone volume / total volume (BV / TV) of subchondral bone, total number of trabeculae (Tb.N), trabecular separation (Tb.Sp) and trabecular thickness (Tb.Th). CT-Vol software was used to generate 2D and 3D images.

[0068] 7. Microangiography

[0069] The mice were anesthetized and fixed, and vascular contrast agent (Microphil MV-120, Flow-Tech) was injected through the left ventricular puncture point. After successful angiography, the specimens were stored at 4°C overnight to facilitate the polymerization of the contrast agent. The next day, the mouse knee joints were sampled, immersed in 4% PFA for fixation for 4 days, and then decalcified for 2 weeks. Finally, Micro-CT was used for image scanning and 3D imaging to analyze the relevant vascular parameters in the subchondral bone: including vessel number (Vessel Number, VN) and vessel volume (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 gene.

[0072] 9. Western Blot (WB)

[0073] Proteins were extracted and subjected to WB assay. Primary antibodies included anti-CXCL2, anti-NF-kB p65, and anti-phospho-NF-kBp65. Samples were incubated with secondary antibodies and images were visualized by chemiluminescence.

[0074] 10. Histology and immunohistochemistry

[0075] The knee joints were fixed in 4% PFA for 48 hours, decalcified in 10% EDTA for 2 weeks, and then embedded in paraffin. The 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 evaluated 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 PawWithdrawal 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. The mechanical allodynia of the mice was assessed at the same time of day. Starting with a force of 0.4 grams, acupuncture 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 PlantarAssay

[0079] Heat Plantar was used to measure the 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, the mice 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 evaluations 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] In order to identify potential bioactive monomers in Anemarrhena, we first used a mature high-throughput CMC / TOFMS system to detect the active components in Anemarrhena with high affinity to BMMC cell membranes. The 3D contour results showed that there were three candidate monomers that showed obvious adhesion behavior and relatively stable retention time, namely Broussonin A (BRA), Markogein (MAN) and Isosakuranetin (ISN) ( Figure 1 ac). To further clarify the potential effects of the candidate monomers on OC generation, we first determined the non-cytotoxic concentrations of the three monomers in BMMCs by 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 generation in vitro, while BRA and MAN had no such significant effect ( Figure 2 a and f). In addition, we also detected the expression levels of marker genes associated with OC formation, including cathepsin K (CTSK), matrix metalloproteinase 9 (MMP-9), and TRAcp5B. Consistent with the results of TRAP, 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 lacuna test, and the results also showed that only ISN could significantly inhibit the bone resorption activity of OC induced by RANKL ( Figure 2 e and f). The above results indicate that ISN extracted from Anemarrhena asphodeloides can inhibit the generation and function of OC in vitro.

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

[0088] Given that ISN has a significant inhibitory effect on OC formation in vitro, we then sought to elucidate whether ISN can also show 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 divided them into groups for ISN intervention after modeling.

[0089] We used Micro-CT to detect 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 the bone spur formation and subchondral bone erosion in the modeling group (ACLT) were significantly increased compared with the sham group (Sham). At the same time, the ACLT group showed a trend of enlarged bone marrow cavity and sparse trabeculae ( Figure 3 a, b), the target parameters were 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 bone destruction and bone erosion of subchondral bone was significantly alleviated ( Figure 3 af). We then further evaluated the activation of OCs in the subchondral bone 2 weeks after ACLT surgery by 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 gh). The above results showed that ISN could block the excessive activation of OC and the destruction of bone microstructure in the subchondral bone of mice knee joint induced by ACLT.

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

[0091] Since the subchondral bone and the overlying articular cartilage form a "biomechanical unit", the changes in the subchondral bone microenvironment in OA are closely related to the 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 compared with the Sham group, the ACLT group had more severe proteoglycan loss and a significant increase in OARSI score ( Figure 4 a, b, d). However, ISN intervention significantly alleviated the cartilage degeneration and increased OARSI scores caused by ACLT surgery ( Figure 4 a, 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). Overall, the results showed that ISN alleviated the wear and loss of articular cartilage in the OA mouse model.

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

[0093] Proliferative abnormal angiogenesis in 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 in 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 subchondral bone marrow did not change significantly between the Sham and ISN groups ( Figure 5 d, e). The above results indicate that ISN inhibits the formation of abnormal blood vessels in the subchondral bone marrow cavity during the progression of OA.

[0094] 6. Isocherrin relieves 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, respectively. Immunofluorescence colocalization showed that the number of TRAP+ / Netrin-1+ OC colocalization increased significantly in the ACLT group, but was significantly reduced 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 6 a, b). In addition, immunofluorescence staining of calcitonin gene-related peptide-positive (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 6f). The above 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, we divided them into groups according to whether they had ISN intervention or not. We directly obtained the subchondral bone tissue of the ACLT group and the ISN group, performed transcriptome sequencing to study differentially expressed genes (DEGs), and performed GO and KEGG analysis 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 the common target genes of OC differentiation and bone resorption. To examine the role of ISN in NF-κB pathway activation and CXCL2 expression, we induced OC generation and performed ISN intervention. WB results showed that ISN dose-dependently inhibited the activation of p-P65 during RANKL-induced OC generation. At the same time, the expression of CXCL2 was also significantly downregulated ( Figure 7 d), 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 generation 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 deformation 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 protection scope of the present invention.

Claims

1. The application of isosakuratin in the preparation of drugs or reagents for inhibiting osteoclastogenesis in vitro.

2. The use of isosakuratin in the preparation of drugs or reagents for preventing, inhibiting or treating osteoarthritis.

3. Use of isosakuratin in the preparation of drugs or reagents for preventing, inhibiting or treating joint pain induced by osteoarthritis.

4. Use of a pharmaceutical composition in the preparation of a drug or reagent for preventing, inhibiting or treating osteoarthritis, characterized in that: The pharmaceutical composition comprises a therapeutically effective amount of isosakuramin.

5. Use of a pharmaceutical composition in the preparation of a drug or reagent for preventing, inhibiting or treating joint pain induced by osteoarthritis, characterized in that: The pharmaceutical composition comprises a therapeutically effective amount of isosakuramin.

6. The use according to claim 4 or 5, characterized in that The pharmaceutical composition comprises the combined use of isosakuratin and other drugs for treating osteoarthritis.

7. The use according to claim 4 or 5, characterized in that The pharmaceutical composition also includes pharmaceutically acceptable excipients.

8. Use of a pharmaceutical preparation in the preparation of a drug or reagent for preventing, inhibiting or treating osteoarthritis, characterized in that: The pharmaceutical preparation comprises a therapeutically effective amount of isosakuratin.

9. Use of a pharmaceutical preparation in the preparation of a drug or reagent for preventing, inhibiting or treating joint pain induced by osteoarthritis, characterized in that: The pharmaceutical preparation comprises a therapeutically effective amount of isosakuratin.

10. The use according to claim 8 or 9, characterized in that: The pharmaceutical preparation includes a solid dosage form, a semisolid dosage form, a liquid dosage form or a gas dosage form.

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