Application of amygdalin in inhibition of cartilage cell death and fibrosis and composition

Abstract

The invention discloses application of amygdalin in inhibition of cartilage cell death and fibrosis and a composition. The invention discloses and confirms that cartilage cell ferroptosis is a key pathological mechanism for driving cartilage fibrosis and scar formation. The invention proves that the natural plant extract amygdalin can effectively inhibit cartilage cell ferroptosis induced by IL-17A and other factors, and the mechanism at least partially relates to regulation of IL-17A / GPX4 signal axis, especially by up-regulating expression of key anti-ferroptosis protein GPX4. The inhibition effect on ferroptosis causes the expression of a fibrocartilage marker, and meanwhile, the expression of a hyaline cartilage anabolism marker is improved, which indicates that AMD can be effectively used for preparing the anti-cartilage fibrosis medicine. The present invention discloses a pharmaceutical composition comprising AMD and Mg < 2 + >, AMD provides a synergistic or protective effect for the cartilage protection effect of Mg < 2 + > by inhibiting ferroptosis and exerting an antioxidant effect. The invention provides a new conservative treatment strategy which targets ferroptosis and has a good application prospect for the treatment of cartilage fibrosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a method and pharmaceutical composition for treating joint diseases by regulating cell death pathways. Specifically, it relates to a new use of amygdalin in preparing drugs for preventing or treating cartilage fibrosis-related diseases, as well as related pharmaceutical compositions containing amygdalin. Background Art

[0002] Cartilage fibrosis, also known as hyaline cartilage fibrosis, is a complex pathological process involving the formation of ciliary tears, ulcers, cartilage defects, and eventual replacement by fibrous tissue due to persistent wear and tear of the hyaline cartilage. Traditionally, knee fibrosis is primarily thought to result from the abnormal proliferation of scar tissue caused by repeated injury or prolonged immobilization after surgery. However, recent studies have revealed the central role of fibrochondrocytes in degenerative osteoarthritis and have increasingly highlighted the crucial role of cell death, particularly programmed cell death, in driving the fibrotic process. Given the extremely limited regenerative capacity of hyaline cartilage, inhibiting cartilage fibrosis and promoting its regeneration are key goals in the treatment of joint diseases, particularly with the crucial importance of early intervention. While existing therapies, such as novel biomaterials, hold promise, widespread clinical application faces challenges such as high cost, long-term biocompatibility, immunogenicity, and a lack of sufficient clinical validation. Therefore, the search for safer, more cost-effective, and effective conservative treatment strategies, particularly drugs targeting key molecular mechanisms of fibrosis, has become a research hotspot. Amygdalin (AMD), also known as vitamin B17, is a natural cyanogenic glycoside extracted from various plants and exhibits multiple biological activities. However, the potential toxicity of hydrocyanic acid produced by its metabolism after oral administration limits its systemic application. On the other hand, magnesium ions (Mg2+) have been shown to have chondroprotective effects, but these effects are easily attenuated by oxidative stress.

[0003] The core pathological feature of fibrosis is the abnormal deposition and remodeling of the extracellular matrix, which leads to tissue structural disorder and loss of function. In this process, the fate of chondrocytes is crucial. When chondrocytes die in an unfavorable microenvironment, especially when the dead cells are not effectively cleared, the signaling molecules released will strongly activate the fibrotic response. Different cell death modes have different effects on tissue repair and the outcome of fibrosis. Ferroptosis is an iron-dependent form of programmed cell death characterized by lipid peroxidation. In recent years, it has been found to be closely related to a variety of fibrotic diseases. Interleukin-17A (IL-17A) is not only an important fibrosis inducer, but may also trigger ferroptosis in chondrocytes through specific signaling pathways. Glutathione peroxidase 4 (GPX4) is a key enzyme in cells that resists lipid peroxidation and inhibits ferroptosis. Its dysfunction or downregulation may make chondrocytes more susceptible to ferroptosis, thereby exacerbating fibrosis. Therefore, the development of natural drugs that can simultaneously target the IL-17A signaling pathway, regulate GPX4 activity, and inhibit ferroptosis provides a promising new strategy for combating cartilage fibrosis.

[0004] In response to these issues and new insights into the mechanisms of cartilage fibrosis, this paper proposes the innovative application of AMD in the preparation of drugs that target the inhibition of chondrocyte ferroptosis and anti-cartilage fibrosis. The drug is administered via intra-articular injection, aiming to: 1) allow AMD to act directly on diseased cartilage, increasing local drug concentration and enhancing the efficacy of inhibiting ferroptosis and fibrosis; 2) bypass digestive tract and liver metabolism, significantly reducing the risk of systemic toxicity caused by hydrocyanic acid; and 3) AMD's anti-inflammatory and antioxidant properties may directly counteract factors that induce ferroptosis and provide protection for Mg2+, synergistically enhancing cartilage protection.

[0005] It is worth noting that although amygdalin has been used to prepare drugs for the treatment of various visceral organ diseases, it is the first time that it has been clearly applied to inhibit cartilage fibrosis, especially by regulating the ferroptosis mechanism mediated by the IL-17A / GPX4 axis. Considering the heavy burden that the high cost of surgery brings to society and individuals, people have been seeking more economical and effective conservative treatment methods. Therefore, the intra-articular injection treatment method of amygdalin combined with magnesium ions proposed in the present invention aims to provide an economical and effective conservative treatment option, which effectively inhibits fibrocartilage formation and promotes hyaline cartilage regeneration by precisely intervening in the core links of cell death and fibrosis. Summary of the Invention

[0006] The purpose of the present invention is to address the common problem of cartilage fibrosis in joint diseases (covering degenerative, traumatic, inflammatory joint diseases and postoperative cartilage damage repair, etc.), especially based on the new discovery of the mechanism of action of IL-17A-induced chondrocyte ferroptosis in fibrosis, to provide a new use of the natural drug amygdalin for targeted inhibition of ferroptosis and regulation of GPX4 to combat cartilage fibrosis and its pharmaceutical composition.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solutions: As a first aspect, the present invention provides the use of amygdalin in the preparation of a medicament for inhibiting articular cartilage fibrosis, wherein the inhibitory effect involves regulating chondrocyte ferroptosis. The amygdalin, also known as vitamin B17, has a molecular formula of C20H27NO11.

[0008] The amygdalin inhibits cartilage fibrosis by inhibiting ferroptosis of articular chondrocytes, and the inhibition of ferroptosis is achieved by upregulating the expression or activity of glutathione peroxidase 4 in chondrocytes.

[0009] Furthermore, the drug is formulated into a dosage form for intra-articular injection, wherein the concentration of amygdalin in the solution for injection is 1-10 mmol / L, and the solvent includes PBS buffer, physiological saline (or other commonly used solvents).

[0010] Preferably, the concentration of the AMD in the solution for injection is 10 mmol / L.

[0011] As a second aspect, the present invention provides an anti-cartilage fibrosis pharmaceutical composition comprising a therapeutically effective amount of amygdalin and a pharmaceutically acceptable carrier, wherein the composition is used to inhibit articular cartilage fibrosis in mammals, wherein the inhibitory effect is achieved by inhibiting ferroptosis of articular chondrocytes.

[0012] Preferably, the pharmaceutical composition comprises a mixed solution of the active ingredient amygdalin (AMD) and magnesium ions (Mg2+), which can synergistically inhibit chondrocyte ferroptosis and resist fibrosis. The magnesium ions are preferably magnesium chloride, and the ratio of MgCl to AMD is 1:96-100 by weight.

[0013] Furthermore, the pharmaceutical composition is used by intra-articular injection, and when administered by intra-articular injection, the concentration of amygdalin is 1-10 mmol / L.

[0014] Preferably, the amygdalin and magnesium chloride are dissolved in a solvent, the concentration of AMD in the solution is 10 mmol / L, the concentration of MgCl is 0.5 mmol / L, and the solvent includes PBS buffer, physiological saline (or other commonly used solvents).

[0015] The core mechanism of the present invention is that AMD or its combination with Mg2+, administered by intra-articular injection, can effectively regulate the intra-articular microenvironment, particularly the IL-17A / GPX4 signaling axis. AMD can inhibit the IL-17A signaling pathway (downregulating IL-17RA and TRAF6 expression) and, crucially, inhibit chondrocyte ferroptosis by promoting the expression of the anti-ferroptosis core protein GPX4. This inhibitory effect on ferroptosis is one of the important mechanisms of its anti-cartilage fibrosis effect.

[0016] Through this mechanism, the drug of the present invention can inhibit the formation of fibrocartilage and promote the regeneration of hyaline cartilage. When AMD is used in combination with Mg2+, AMD can also reduce oxidative stress and protect the cartilage-protective effects of Mg2+, achieving synergistic effects that are significantly better than using Mg2+ alone.

[0017] As a third aspect, the present invention also provides the use of amygdalin in inhibiting ferroptosis and / or preparing a ferroptosis inhibitor.

[0018] When the drug of the present invention is used, it is in an injectable dosage form and needs to be dissolved in physiological saline or other commonly used solvents (such as PBS buffer) to prepare a drug solution. In addition, the drug preparation solution can be mixed with other pharmaceutically acceptable excipients (such as sodium hyaluronate) to lubricate the tissues within the joint after injection into the joint cavity, reducing friction and impact between tissues, thereby synergistically inhibiting the occurrence of cartilage fibrosis and promoting the repair of hyaline cartilage.

[0019] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0020] 1) The present invention conducted experiments on human and mouse articular cartilage samples, confirming that IL-17A plays an extremely important role in the process of cartilage fibrosis.

[0021] 2) This invention reveals and confirms the key driving role of ferroptosis in the process of cartilage fibrosis. In vitro experiments show that the ferroptosis inducer Erastin can promote the cartilage fibrosis phenotype, while the ferroptosis inhibitor Fer-1 can reverse this effect, directly proving that targeted inhibition of ferroptosis is an effective strategy for combating cartilage fibrosis. 3) This invention discovers and elucidates for the first time a new mechanism by which the natural plant extract amygdalin inhibits cartilage fibrosis by regulating specific molecular pathways: AMD can inhibit chondrocyte ferroptosis by inhibiting the IL-17A signaling pathway and, crucially, by upregulating GPX4 expression, thereby reducing the expression of the fibrocartilage marker COL1 and increasing the expression of the hyaline cartilage marker COL2, achieving the dual effects of inhibiting fibrosis and promoting hyaline cartilage regeneration.

[0022] 4) The anti-chondrogenic drug combination proposed in the present invention has shown good cartilage repair and regeneration effects in in vivo models, and the repaired cartilage morphology is closer to natural hyaline cartilage.

[0023] 5) Administration via intra-articular injection enables efficient drug delivery and targeted action at the lesion site. Furthermore, leveraging AMD's ferroptosis-inhibiting and antioxidant properties, this approach improves the safety and efficacy of treatment and is expected to reduce the toxicity risks of traditional oral AMD treatments. This approach offers a new, cost-effective, and clinically promising conservative treatment approach for cartilage fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The present invention will be further described below with reference to the accompanying drawings and examples;

[0025] Figure 1 A diagram showing the mechanism of amygdalin in the preparation of anti-cartilage fibrosis drugs and pharmaceutical compositions thereof in inhibiting cartilage fibrosis and promoting hyaline cartilage formation by regulating the IL-17RA / GPX4 axis, as well as a schematic diagram of the cartilage fibrosis process;

[0026] Figure 2 (A) Representative images of human cartilage samples collected from the tibial plateau of patients undergoing total knee arthritis (TKA). Dashed boxes indicate representative areas of intact articular cartilage (n=6) and damaged cartilage (n=6). Scale bar: 2 cm. (B) OARSI grading. (C) Representative images of ABH staining of intact and damaged articular cartilage. Scale bar: 500 μm. Enlarged area in the dashed box: Scale bar: 100 μm. (D) Representative immunohistochemical staining images of COL1, COL2, COL10, and MMP13 in osteoarthritic cartilage (uninjured and damaged). Scale bar: 250 μm. Enlarged area in the dashed box: Scale bar: 50 μm. (E) IHC analysis of COL1, COL2, COL10, and MMP13. The data are expressed as mean ± SD; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0027] Figure 3IL-17A and oxidative stress participate in human fibrocartilage formation. (A) Representative IHC staining images of IL-17A, IL-17RA, and TRAF6 in intact and damaged articular cartilage. Uninjured cartilage (n=6), damaged cartilage (n=6). Scale bar: 250 μm. Enlarged area in the dotted box: Scale bar: 50 μm. (B) Representative IHC staining images of iNOS and GPX4, as well as ROS staining, in intact and damaged cartilage. IHC staining scale bar: 250 μm. Enlarged area in the dotted box: 50 μm. ROS staining scale bar: 200 μm. Enlarged area in the dotted box: 100 μm. (C) Statistical analysis of representative images. Data are expressed as mean ± SD; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0028] Figure 4 AMD inhibits IL-17A-induced fibrosis and improves chondrogenesis. (A) Effects of different AMD concentrations on ATDC5 cell viability assessed by CCK8 assay. (B to C) DCFH-DA staining and statistical analysis of ROS levels in ATDC5 cells following IL-17A and AMD treatment (n = 3). Scale bar: 100 μm. (D) Analysis of COL1, COL2, iNOS, and GPX4 mRNA expression in chondrocytes.

[0029] Figure 5 (A) Protein imprints of COL1, COL2, and GPX4. (B) Ten-week-old C57BL / 6J mice underwent microfracture (MF) surgery on the distal femur. Saline and various concentrations of AMD solution were injected into the facet joint via a microsyringe. Eight weeks after surgery, the mice were sacrificed and the right knee joints were harvested. (C) Representative images of tissues in the repair and regeneration areas after MF surgery, including HE and ABH / OG staining, as well as IHC staining for COL1, COL2, and MMP13, and ROS staining. Scale bar: 250 μm. Scale bar for ROS staining: 50 μm. (D) IHC staining for COL1, COL2, and MMP13 in the repair area and statistical analysis of ROS; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. ns: Not significantly different.

[0030] Figure 6The IL-17A / RA-mediated effects of AMD on cartilage fibrosis; (A to B) Representative images of DCFH-DA staining and statistical graphs of ATDC5 cells treated with AMD and an IL-17A inhibitor or an IL-17RA inhibitor (n = 3). Scale bar: 100 μm. (C to E) ATDC5 cells treated with AMD and an IL-17A inhibitor were analyzed by RT-PCR for the mRNA expression levels of IL-17RA, TRAF6, COL1, COL2, iNOS, and GPX4 (n = 3). (F) Western blotting was used to examine the expression of COL1, COL2, and GPX4 in cells treated with AMD and an IL-17A inhibitor. (G to I) ATDC5 cells treated with AMD and an IL-17RA inhibitor were analyzed by RT-PCR for the mRNA expression levels of IL-17RA, TRAF6, COL1, COL2, iNOS, and GPX4 (n = 3). (J) Western blotting was used to detect the expression of COL1, COL2, and GPX4 in ATDC5 cells after treatment with AMD and IL-17RA inhibitors. Data are expressed as standard deviation ± mean; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0031] Figure 7 AMD inhibits cartilage fibrosis and improves chondrogenesis by suppressing IL-17A-induced ferroptosis. (A to B) Representative images (n = 3) of DCFH-DA staining and statistical graphs of ATDC5 cells treated with AMD combined with Fer-1 or erastin. Scale bar: 100 μm. (C to E) ATDC5 cells treated with AMD and Fer-1 were assayed for mRNA expression levels of GPX4, iNOS, COL1, COL2, 17RA, and TRAF6 by RT-PCR (n = 3). (F) After Fer-1 treatment, protein levels of COL1, COL2, and GPX4 were assessed by Western blotting. (G to I) mRNA expression levels of GPX4, iNOS, COL1, COL2, 17RA, and TRAF6 were assessed by RT-PCR in ATDC5 cells treated with AMD and erastin (n = 3). (J) After treatment with erastin, the protein levels of COL2 and GPX4 in ATDC5 cells were detected by Western blotting. Data are expressed as standard deviation ± mean; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0032] Figure 8Intra-articular injection of amygdalin and magnesium ions promotes cartilage repair and regeneration by inhibiting fibrosis. (A) Schematic diagram of the mechanism. (B) After modeling, mice were randomly divided into four groups according to the different treatments. (C) Representative images of cartilage regeneration and repair areas in each group stained with ABH / OG, Masson's trichrome, Safranin O / Fast Green, and H&E. Scale bar: 250 μm. (D) Sirius red staining under polarized light. Scale bar: 500 μm. Representative images of IHC staining for COL1, COL2, COL10, and MMP13 in cartilage repair areas. Scale bar: 100 μm. (E) Statistical analysis of IHC staining for COL1, COL2 / COL10, and MMP13. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. ns: not significantly different.

[0033] Figure 9 Amygdalin and magnesium ions inhibit fibrosis and promote cartilage repair and regeneration by regulating the IL-17RA / GPX4 axis. (A to B) Representative images of IHC staining for IL-17RA and TRAF6 in the repaired defect area after modeling. (C to E) Representative images of IHC staining for iNOS and GPX4, and representative images of ROS staining in the repaired area. Scale bars, IHC staining: 250 μm. Magnified area: 50 μm. ROS staining: 100 μm. Magnified area: 20 μm. (F) Statistical analysis of IL-17RA, TRAF6, iNOS, GPX4, and ROS staining in the repaired area; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. ns: not significantly different. DETAILED DESCRIPTION

[0034] The present invention will be further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto.

[0035] Example 1 AMD inhibits IL-17A and ferroptosis-induced cartilage fibrosis and improves chondrogenesis

[0036] 1. Experimental animals and materials

[0037] The mice used in this experiment were C57BL / 6J (WT) male mice. The mice were ordered at 6 weeks of age. All animals were adaptively housed until they reached 10 weeks of age before the experiments and surgeries began. The amygdalin used in this example was purchased from Chengdu Munster, catalog number A0093.

[0038] 2. Experimental methods

[0039] 2.1 Processing of human articular cartilage tissue

[0040] Articular cartilage samples were collected from OA patients undergoing total knee replacement surgery and cut into 2mm x 2mm pieces. The subchondral bone was avoided during collection, and the full-thickness cartilage tissue was primarily collected. The damaged cartilage primarily originated from the medial tibial plateau, which was rough and jagged, while the relatively intact cartilage originated from the lateral region. Articular cartilage tissue from different patients was collected from similar locations on the tibial plateau as much as possible to ensure consistency. The collected human articular cartilage tissue samples were fixed with 4% paraformaldehyde. After complete decalcification, the samples were further dehydrated and embedded, and paraffin sections with a thickness of 3μm were prepared.

[0041] 2.2 Pathological staining and immunohistochemistry

[0042] To accurately assess the morphological characteristics of human cartilage samples, pathological staining was performed according to the ABH / OG staining protocol. After dewaxing and rehydration, the sections were differentiated in 1% hydrochloric acid-alcohol solution (25 seconds), then incubated in ABH staining solution for 120 minutes. Excess dye was then washed with double-distilled water, differentiated again with hydrochloric acid-alcohol solution, and then blued with 0.5% ammonia solution. Finally, the sections were immersed in Orange G staining solution, and excess dye was washed off with high-concentration ethanol.

[0043] To investigate the process of fibrocartilage formation, this example also performed immunohistochemistry. After dewaxing and rehydration, sections were placed in sodium citrate buffer and incubated in a 60°C oven for 4 hours for antigen retrieval. An endogenous peroxidase blocker was then used to eliminate nonspecific background staining, and 5% goat serum was used to reduce nonspecific antibody binding. The corresponding primary antibody was then added dropwise and incubated overnight at 4°C. The following day, a secondary antibody matched to the primary antibody species was added and incubated at room temperature for 30 minutes to enhance the signal. DAB was used for color development, hematoxylin counterstained, and hydrochloric acid-alcohol was used for differentiation. Ammonia was then used for blueing, and the sections were mounted.

[0044] All slides were scanned digitally at high resolution using a Hamamatsu Photonics digital slide scanner. Gross and histopathological evaluation of cartilage damage and degeneration was performed according to the Osteoarthritis Research Society International Scoring System (OARSI). Details of the scoring system are shown in Table 1. Subsequently, immunohistochemical analysis was performed using ImageJ / Fiji software (version 2.14.0).

[0045] Table 1 OARSI score

[0046]

[0047] 2.3 Dihydroethidium staining

[0048] Prepare dihydroethidium reactive oxygen species fluorescent probe solution and dilute it to an appropriate multiple, add it dropwise to the slice to cover the sample surface. Subsequently, add 200μl of cleaning solution working solution and let it stand for 3-5 minutes. Next, add 100-200μl of staining probe working solution to the slice, and then place it in a 37°C constant temperature incubator and incubate in the dark for 20-60 minutes. After incubation, remove the slice from the staining solution and wash it 2-3 times with PBS buffer. Afterwards, use a sealing agent to properly seal the slice. Afterwards, use a laser confocal microscope to capture images to obtain high-quality, clear fluorescence images for subsequent data analysis and research.

[0049] 2.4 Experimental animals

[0050] Before modeling, C57BL / 6J mice were anesthetized and a 1-1.5 cm skin incision was made on the medial side of the right knee to expose the joint capsule and patellar ligament. The right lower limb of the mouse was then flexed, and the patella was dislocated to expose the femoral condyle. A micro-handheld cranial drill was used to create a microfracture (MF) injury perpendicular to the cartilage surface approximately 0.2 cm above the junction of the condylar line and the midline of the distal femoral condyle. After the operation, the patella was repositioned, the joint capsule was closed with absorbable sutures, and the incision was sutured with ordinary silk sutures.

[0051] When administering the drug, first prepare the gas anesthesia machine in the operating room and adjust the corresponding gas flow and concentration, observe the state of the mouse, and after the mouse completely loses consciousness and shows muscle relaxation, the operator uses a microsyringe to inject the drug into the mouse's articular cavity. In order to determine the optimal drug dose of amygdalin, this example randomly divided the mice into 4 groups: a control group (injected with normal saline), a low-dose AMD group (1μmol / mL), a medium-dose AMD group (5μmol / mL), and a high-dose AMD group (10μmol / mL). Each group of mice was injected with the corresponding dose of the drug according to the grouping, with an injection volume of 10μL, twice a week. After the experiment, tissue sampling and related analysis were performed.

[0052] 2.5 Cell culture

[0053] ATDC5 cells were purchased from Cell Biosciences (iCell-m084, Shanghai, China). The culture medium was DMEM / F12 (CGM104.05, Cellmax) with 10% FBS (Sa211.02, Cellmax), and the cells were cultured in a 37°C, 5% CO 2 incubator. Cells within passage 10 were used for the experiments.

[0054] 2.6 Reverse transcription polymerase chain reaction

[0055] This example also analyzed the gene expression of chondrocytes by reverse transcription polymerase chain reaction (RT-PCR). First, total RNA was extracted from chondrocyte cultures using TRIzol reagent (Sigma, T9424). Subsequently, the extracted RNA was reverse transcribed into cDNA using a TaqMan reverse transcription kit (AG11728, Accurate Biology, Hunan, China) to prepare for subsequent gene amplification. Next, SYBR Green Pro-Taq HS (AG11701, Accurate Biology, Hunan, China) and specific primers were used in a 2720 thermal cycler PCR instrument to save the experimental results and collect and analyze the relevant data (primer sequences are shown in Table 2).

[0056] Table 2 RT-PCR primer sequences

[0057]

[0058]

[0059] 2.7 Western blotting

[0060] In this study, this example analyzed the protein expression of chondrocytes by Western Blot. The cells were lysed with pre-cooled RIPA buffer containing protease inhibitors to extract proteins from the cells. Subsequently, this example loaded the extracted protein sample onto an SDS-polyacrylamide gel and separated it by electrophoresis. After electrophoresis, this example transferred the proteins on the gel to a polyvinylidene fluoride (PVDF) membrane, followed by BSA blocking treatment. This example incubated the membrane with a specific primary antibody at 4°C overnight to ensure that the antibody was fully bound to the target protein. Afterwards, this example used a horseradish peroxidase-conjugated secondary antibody for further incubation. This example used BIO-RAD's chemiluminescence kit and ChemiDoc XRS+ imaging system to detect protein signals on the PVDF membrane.

[0061] 2.8 Reactive oxygen species detection

[0062] To measure the accumulation of ROS in chondrocytes, this experiment used the DCFH-DA fluorescent probe (Beyotime, China, S0033S). The specific protocol was as follows: First, ATDC5 cells were plated and then treated with the corresponding drugs. Next, the DCFH-DA fluorescent probe was added to the cells and incubated in the dark. After incubation, the fluorescence intensity of DCFH-DA was measured using a fluorescent probe microscope as an indirect indicator of ROS levels. Finally, the captured images were quantitatively analyzed using Image J software to calculate the relative levels of ROS among the treatment groups.

[0063] 3. Experimental results

[0064] 3.1 Gradual formation of fibrocartilage during human articular cartilage degeneration

[0065] In this example, the tibial plateau cartilage samples collected from patients with OA after total knee replacement were divided into damaged and undamaged areas ( Figure 2 A). OARSI score shows that the damaged cartilage score is higher ( Figure 2 B). ABH / OG staining results showed that the matrix of the damaged cartilage area was damaged and thinned, with cracks and vesicular structures, and a significant loss of sulfated glycosaminoglycans ( Figure 2 C). IHC staining results showed that the expression of COL1 (fibrocartilage marker) increased in the damaged cartilage area, while the expression of COL2 (hyaline cartilage marker) decreased, indicating fibrocartilage formation. At the same time, the expression of MMP13 (catabolism factor) and COL10 (hypertrophy marker) increased significantly ( Figure 2 D to E), reflecting the pathological changes of fibrosis and hypertrophy in the damaged cartilage and the imbalance of ECM homeostasis.

[0066] Figure 1 This study demonstrates the mechanism by which amygdalin, in the preparation of anti-chondrogenic drugs and pharmaceutical compositions, inhibits cartilage fibrosis and promotes hyaline cartilage formation by regulating the IL-17RA / GPX4 axis, as well as the process of fibrocartilage formation. Cartilage fibrosis is primarily caused by injury or degeneration. Due to the limited self-regeneration capacity of cartilage, damaged hyaline cartilage cannot effectively regenerate. Therefore, intervention in the cartilage fibrosis process is particularly important. During this process, adjacent normal chondrocytes transform into fibrous chondrocytes and secrete abnormal extracellular matrix, ultimately leading to the formation of fibrous cartilage. However, fibrous cartilage has poor mechanical properties and cannot effectively distribute joint loads. Compared with normal cartilage, the synthesis of COL2 in damaged cartilage is reduced, while the expression of COL1, MMP13, and COL10 is increased, further exacerbating the degeneration of cartilage function.

[0067] 3.2 IL-17A and oxidative stress are involved in the formation of fibrocartilage in human joints

[0068] By performing IHC analysis on sections of damaged and undamaged cartilage, it was found that IL-17A was highly expressed locally on the surface of damaged cartilage, but relatively less expressed in undamaged areas ( Figure 3 A). Compared to uninjured cartilage, interleukin-17 receptor A (IL-17RA) expression was significantly increased in injured cartilage. Furthermore, significant upregulation of TNF receptor-associated factor 6 (TRAF6), a downstream marker of IL-17, was observed in injured cartilage, indicating activation of the IL-17 signaling pathway.

[0069] To explore whether oxidative stress is involved in the formation of fibrocartilage in human samples, this example used IHC staining to detect the expression level of GPX4. The results of IHC staining showed that the expression level of GPX4 in damaged cartilage was significantly lower than that in non-lesioned tissue. In addition, the IHC results also showed that the expression level of inducible nitric oxide synthase (iNOS) was significantly increased in damaged cartilage ( Figure 3 B to C). This example also uses a tissue section reactive oxygen species detection kit to perform ROS staining on human cartilage sections. The results show that the ROS level in damaged cartilage is higher, indicating abnormal accumulation of ROS in the damaged cartilage area.

[0070] AMD inhibits IL17A-induced fibrosis and improves chondrogenesis

[0071] In order to determine the optimal concentration of AMD, this example used different concentrations of AMD to treat ATDC5 cells for 24 hours, and then evaluated the cell viability using the CCK8 assay. Based on the results, a low concentration group (1 μmol / mL), a medium concentration group (5 μmol / mL), and a high concentration group (10 μmol / mL) were set ( Figure 4 A). Subsequently, this example treated ATDC5 cells with different concentrations of AMD and then intervened with IL-17A (20 ng / ml). DCFH-DA detection was used to further demonstrate whether AMD regulates the production of ROS. The results showed that after IL-17A intervention, the green fluorescence intensity increased, indicating an increase in the level of ROS, while AMD treatment significantly reduced the ROS level increased after IL-17A stimulation ( Figure 4To further investigate the relationship between AMD and cartilage fibrosis, this study evaluated the expression trends of fibrocartilage markers (COL1), cartilage anabolic markers (COL2), oxidative stress-related indicators (iNOS), and antioxidant enzymes (GPX4) in ATDC5 cells after intervention with different concentrations of AMD and IL-17A (20 ng / ml). RT-PCR results showed that after IL-17A stimulation, the mRNA expression levels of COL1 and INOS in chondrocytes increased, while the mRNA levels of COL2 and GPX4 decreased, while AMD reversed these changes in a concentration-dependent manner ( Figure 4 D). WB results showed the same trend as the transcription level ( Figure 5 A).

[0072] Then, this example conducted an in vivo experiment. As shown in the schematic diagram, the mice were randomly divided into groups after modeling. Different concentrations of AMD and normal saline were injected twice a week using a microsyringe. The samples were collected after 8 consecutive weeks of administration ( Figure 5 B) HE and ABH / OG staining results showed that compared with the control group, the cartilage surface of mice treated with AMD was more complete and the sGAG content was significantly increased. Through IHC staining analysis, this example found that the expression level of COL2 in the cartilage repair area of the high-dose AMD treatment group was significantly increased, while the expression levels of COL1 and MMP13 were significantly decreased ( Figure 5 C to D). Based on the experimental results, the high-dose group demonstrated the best effect in suppressing the fibrocartilaginous phenotype and promoting the hyaline phenotype. Therefore, the high-dose AMD group was selected as the optimal intervention concentration. These results demonstrate that AMD treatment can effectively promote the repair and regeneration of articular cartilage by promoting cartilage anabolism and inhibiting its catabolism.

[0073] AMD alleviates IL-17A-induced cartilage fibrosis by inhibiting IL-17A / RA

[0074] To explore the potential mechanism of action of AMD, this study set up two experimental groups for comparative studies. The first experimental group included an AMD group, an IL-17A antagonist (secukinumab, Se) group, and an AMD + Se combination group; the second experimental group included an AMD group, an IL-17RA antagonist (brodalumab, Br) group, and an AMD + Br combination group. The anti-fibrotic effects of IL-17A / RA antagonists and AMD were compared and studied. During the experiment, we first treated ATDC5 cells with the corresponding drugs for 24 hours. Subsequently, the cells were treated with 20 ng / mL of IL-17A for 8 hours. To evaluate the effect of drug intervention on intracellular ROS accumulation, we used the DCFH-DA assay. The experimental results showed that the green fluorescence intensity of the IL-17A intervention group increased significantly, indicating that IL-17A stimulation significantly enhanced the accumulation of intracellular ROS. Treatment with IL-17A inhibitors or IL-17RA inhibitors can effectively reverse the increase in ROS levels induced by IL-17. It is worth noting that compared with the AMD alone group, the combined treatment of Se and AMD can significantly reduce the production of intracellular ROS. However, compared with the AMD alone group, the combined treatment of Br and AMD also showed a trend of reducing ROS levels, but the difference did not reach statistical significance ( Figure 6 A, B).

[0075] Further RT-PCR results showed that IL-17A treatment activated the IL-17 signaling pathway and upregulated the expression level of its downstream gene TRAF6. Inhibition with IL-17A or IL-17RA inhibitors could reverse the upregulation trend of IL-17RA and TRAF6. At the same time, compared with the AMD treatment group alone, the combined treatment group of Se and AMD significantly inhibited the expression of IL-17RA and TRAF6 ( Figure 6 C). In contrast, the combined Br and AMD treatment group showed no significant difference in the expression levels of IL-17RA and TRAF6 compared with the AMD treatment group alone ( Figure 6 G). According to the results of RT-PCR and WB, similar trends were observed in the expression of COL1 and COL2 in ATDC5 cells in the two combined groups after intervention ( Figure 6 D, F, H, J). It is worth noting that inhibition of IL-17A / RA inhibited iNOS expression in cells and upregulated Gpx4 levels ( Figure 6 E, I).

[0076] 3.5AMD inhibits cartilage fibrosis by regulating IL-17A-induced ferroptosis

[0077] In order to clarify whether the anti-fibrotic effect of AMD is achieved by regulating ferroptosis, this example used ferroptosis inhibitors (Ferrostatin-1, Fer-1) and ferroptosis inducers (Erastin) to intervene in chondrocytes, and set up AMD treatment groups and AMD+Fer-1 combination groups and AMD+Erastin combination groups. In the experiment, this example first pretreated ATDC5 cells with Fer-1 or Erastin for 24 hours, and then intervened with IL-17A at a concentration of 20 ng / ml for 8 hours. The experimental results showed that compared with the control group using only IL-17A, the fluorescence intensity after Erastin intervention increased significantly, indicating that Erastin-induced lipid peroxidation led to an increase in intracellular ROS levels. Both Fer-1 treatment and AMD groups can effectively reverse the increase in ROS levels induced by IL-17 ( Figure 7 A to B). PCR analysis results further showed that after Erastin intervention, the expression of ferroptosis marker GPX4 was inhibited, while the mRNA expression level of iNOS in ATDC5 cells was increased ( Figure 7 G). Whether AMD is treated alone or in combination with ferroptosis inhibitor (Fer-1), it can effectively reverse the expression changes of GPX4 and iNOS caused by IL-17A ( Figure 7 C). This example further analyzed the expression of fibrocartilage markers (COL1) and hyaline cartilage markers (COL2) at both the transcriptional and translational levels using Western blotting and RT-qPCR techniques. The results showed that compared with the IL-17A-treated group, the expression of COL2 and GPX4 was significantly upregulated after Fer-1 intervention, while the expression of COL1 was downregulated ( Figure 7 D, F). In contrast, when treated with erastin, the expression of COL2 and GPX4 was downregulated ( Figure 7 H, J). However, it is worth noting that neither erastin nor Fer-1 treatment had an effect on the expression of IL-17RA and TRAF6, key genes in IL-17 signal transduction ( Figure 7 E, I).

[0078] 4. Conclusion

[0079] First, this study found that in the process of osteoarthritis, fibrocartilage gradually forms along with the degeneration and wear of cartilage, and the IL-17A signaling pathway and ferroptosis are jointly involved.

[0080] Second, this study confirmed for the first time that the traditional Chinese medicine monomer amygdalin can be used as an anti-cartilage fibrosis drug to promote cartilage repair and regeneration. Moreover, after in vivo and in vitro experimental verification, the results showed that amygdalin can reduce the expression of fibrocartilage marker COL1, while increasing the expression of hyaline cartilage synthesis metabolism marker COL2, significantly promoting the morphological recovery of microfracture-induced cartilage defects in mice.

[0081] Third, this study further revealed the mechanism of action of AMD, which regulates the IL-17A signaling pathway and chondrocyte ferroptosis. By promoting the expression of GPX4, AMD can effectively prevent chondrocyte ferroptosis caused by abnormal accumulation of ROS, thereby inhibiting the occurrence of cartilage fibrosis.

[0082] Example 2: Amygdalin combined with magnesium ions inhibits fibrosis and promotes cartilage repair and regeneration by regulating the IL-17RA / GPX4 axis

[0083] 1. Experimental animals and materials

[0084] The experimental animals purchased in this example were the same as those in the above examples. Magnesium chloride was purchased from Sigma (MgCl, M8266).

[0085] 2. Experimental methods

[0086] 2.1 Experimental Grouping

[0087] Mice with CD induced by MF surgery were randomly divided into groups and received intra-articular injections of saline, AMD solution (10 μmol / L), MgCl solution (0.5 mmol / L), or AMD (10 μmol / mL) plus MgCl solution (0.5 mmol / L). Injections were administered twice weekly until the time of experimental collection. Four and eight weeks after treatment, mice were sacrificed using the same method as previously described, and knee joint samples were collected for further study.

[0088] 2.2 Animal Experimental Modeling, Intervention, and Sample Processing

[0089] The specific method is consistent with that in Example 1.

[0090] 2.3 Tissue sections, pathological staining, and morphometric analysis

[0091] The specific method is consistent with that in Example 1.

[0092] 2.4 Sirius Red Staining

[0093] The 6-micron tissue sections were dewaxed and rehydrated, and then stained with iron hematoxylin stain for 10 minutes. Next, the sections were soaked in double-distilled water for 20 seconds to remove excess stain, and then the sections were stained in Sirius red stain for 30 minutes. The sections were then rinsed with double-distilled water for 20 seconds, and after staining, the sections were sealed. After staining, the stained sections were scanned with a polarized panoramic scanning system for a panoramic view to further observe the microstructure and details.

[0094] 2.5 Safranin Fast Green Staining

[0095] Stain the sections in freshly prepared Weigert stain for 3 minutes, then differentiate them in 1% hydrochloric acid-alcohol solution for 15 seconds and rinse three times with double-distilled water for 3 minutes each. Stain the sections in Fast Green stain for 3 minutes. Then, immerse the sections in a weak acid solution for 15 seconds to remove any residual stain. Remove the sections, let them air dry, and then immerse them in Safranin stain for 5 minutes. After staining, seal the sections.

[0096] 2.6 Masson's trichrome staining

[0097] Incubate the sections with the mordant in a 60°C oven for 1 hour. Then, soak in double-distilled water to remove excess mordant. Stain in lapis lazuli blue for 3 minutes, followed by Mayer's hematoxylin for 3 minutes. Rinse twice with double-distilled water and differentiate with a hydrochloric acid-alcohol solution for 8 seconds. Next, stain with Ponceau fuchsin for 10 minutes. After removing the supernatant, stain with aniline blue for 5 minutes. Finally, rinse with a weak acid solution, air dry, and mount the sections.

[0098] 3. Experimental results

[0099] 3.1 Intra-articular injection of amygdalin and magnesium ions promotes cartilage repair and regeneration by inhibiting fibrosis

[0100] As shown in the mechanism diagram, IL-17A and ferroptosis play an important role in fibrocartilage formation, and the IL-17A / GPX4 axis can serve as a regulatory target for fibrosis ( Figure 8 A). In this example, a full-thickness cartilage defect model was constructed in 10-week-old mice by MF surgery. The mice were randomly divided into four groups: control group (Ctrl group), AMD treatment group, Mg treatment group, and AMD and Mg combined treatment group. At the 4th and 8th weeks after surgery, samples were collected ( Figure 8B). In this example, the histological structure of regenerated cartilage and subchondral bone tissue was characterized in detail by ABH / OG, Masson staining, Safranin O / Fast green and HE staining. Figure 8 C). ABH / OG staining results showed that 4 weeks after surgery, the subchondral bone in the control group showed obvious collapse, and the newly repaired tissue showed fragile fibrous connections and cystic cavities, with significant gaps with the surrounding tissue, indicating that the defect area had not been effectively filled and repaired. In the Mg-treated group, although the defect was filled to a certain extent, it was mainly composed of immature fibrous tissue with an uneven surface and a clearly visible defect boundary. Safranin O staining results showed that the defect area in the AMD-treated group showed brighter safranin staining, indicating that the newly formed tissue contained more abundant proteoglycans, and the boundary between cartilage and bone was clear, confirming the effectiveness of cartilage regeneration. Masson staining results further revealed that the thickness and maturity of collagen fibers in the AMD and Mg combined treatment group were significantly higher than those in the other groups, bone remodeling was visible in the subchondral bone, and the defect area was mainly composed of hypertrophic chondrocytes, suggesting that new bone tissue was formed through endochondral ossification.

[0101] The reconstruction of subchondral bone plays a vital role in the repair of osteochondral defects. Eight weeks after surgery, this example observed that the subchondral bone remodeling in the MF modeling area of each group increased significantly compared with four weeks after surgery. Eight weeks after surgery, the repair tissue height of the control group was lower than that of the surrounding normal cartilage, but the repair and reconstruction of the subchondral bone was relatively more complete than that of the control group. The filling and repair surface of the defect area in the Mg-treated group was still rough, and flocculent fibrous tissue was visible. In contrast, the thickness and staining area of the new cartilage in the AMD and Mg combined treatment group were significantly better than those in the other groups, and the subchondral bone reconstruction effect was the best, showing fibrin deposition close to that of normal cartilage.

[0102] To further explore the overall mechanical properties of cartilage, this study used polarized light to observe Sirius red staining to detect the collagen fiber structure and arrangement of the new cartilage tissue area, and evaluated the expression levels of COL1, COL2, and MMP13 in the new cartilage defect area by IHC staining ( Figure 8DE). The results showed that Sirius red staining of each group under polarized light showed strong orange-yellow or red fluorescence in the control group, indicating that COL1 was the main collagen component of the new tissue and that the repair process formed fibrocartilage rather than stable mature articular cartilage. After Mg treatment, the collagen fibers in the new area showed a loose and colorful network structure, mainly composed of COL2. In the AMD and Mg combined treatment group, the collagen fibers in the repair area were not only dominated by COL2, but also showed a layered characteristic. Their arrangement and orientation were closer to the collagen fiber arrangement and multi-layered structure of normal cartilage, indicating that it has better mechanical properties. Further IHC staining results also showed that compared with the AMD alone group, the combination of AMD and Mg significantly promoted the expression of COL2 in the cartilage regeneration area. In addition, at 4 and 8 weeks after treatment, the expression of MMP13 in the AMD and Mg combined group was significantly reduced compared with the other groups. Of particular note, the orientation of the collagen fibers in the AMD + Mg group was structurally similar to that of natural cartilage, indicating its potential to withstand long-term pressure and joint wear. In summary, the combination of AMD and Mg showed significant advantages in cartilage repair and regeneration compared with single application.

[0103] 3.2 Amygdalin and magnesium ions inhibit fibrosis and promote cartilage repair and regeneration by regulating the IL-17RA / GPX4 axis

[0104] To further investigate the mechanism of the combined effects of AMD and Mg, the following experiments were conducted. IHC staining results showed that the expression level of IL-17RA in the newly formed cartilage of the MF group was significantly higher than that of the other groups, and its downstream signaling marker TRAF6 also showed an expression trend consistent with IL-17RA. This finding further confirms the involvement of the IL-17RA signaling pathway and its downstream indicators in the process of newly formed cartilage repair ( Figure 9 AB). After 4 and 8 weeks of intra-articular injection therapy, this study observed significantly lower IL-17RA and TRAF6 expression levels in repair tissue in both the AMD and Mg groups compared to the Ctrl group. The combined use of AMD and Mg significantly reduced the therapeutic effect, significantly suppressing IL-17RA and TRAF6 expression.

[0105] Furthermore, this example explored the expression levels of iNOS and GPX4 during cartilage repair by IHC staining, and used a DHE kit to detect the content of ROS in the newly repaired tissue. The results showed that the expression level of iNOS in the control group was significantly higher than that in the other experimental groups. At week 4 after treatment, the Mg group showed a more significant effect than the AMD group in inhibiting iNOS expression. However, as time went on, by week 8, the difference in inhibiting iNOS expression between the Mg treatment group and the AMD group became less obvious. At the same time, the results of ROS staining also showed a similar trend ( Figure 9 CD).

[0106] GPX4 is an important antioxidant enzyme, and its expression level is closely related to the antioxidant capacity of cells. IHC results showed that the expression level of GPX4 in the new tissue was significantly increased after AMD treatment alone compared with the Ctrl group. In the combined treatment group of AMD and Mg, the expression of GPX4 in the repair tissue was significantly enhanced at either 4 or 8 weeks ( Figure 9 EF). This finding further supports the contention of this case study that the combined treatment of AMD and Mg can modulate the oxidative stress microenvironment, thereby effectively inhibiting cartilage fibrosis and promoting hyaline cartilage regeneration. In summary, the results of this case study demonstrate that the combination of AMD and Mg intra-articular injection therapy has a significant effect in inhibiting cartilage fibrosis and promoting hyaline cartilage regeneration.

[0107] 4. Conclusion

[0108] This study demonstrated that intra-articular injection of AMD and Mg 2+ It is a potential method for cartilage repair and regeneration. 2+ Such a combined drug delivery strategy can regulate the IL-17A / GPX4 axis, inhibit the formation of fibrocartilage, and promote the regeneration of hyaline cartilage. In the mouse cartilage defect model, it can better achieve the repair and regeneration of cartilage at the defect site, and the morphology is close to that of natural cartilage, which has the potential for clinical application.

[0109] The above embodiments are used to explain the present invention rather than to limit the present invention. Any modifications and changes made to the present invention within the spirit of the present invention and the protection scope of the claims shall fall within the protection scope of the present invention.

Claims

1. Application of amygdalin in the preparation of drugs for inhibiting articular cartilage fibrosis.

2. The use according to claim 1, characterized in that The amygdalin inhibits cartilage fibrosis by inhibiting the ferroptosis of articular chondrocytes.

3. The use according to claim 2, characterized in that The inhibition of ferroptosis is achieved by upregulating the expression or activity of glutathione peroxidase 4 in chondrocytes.

4. The use according to any one of claims 1 to 3, characterized in that The drug is prepared into a dosage form for intra-articular injection. In the solution for injection, the concentration of amygdalin is 1-10 mmol / L, and the solvent includes PBS buffer solution and physiological saline.

5. An anti-cartilage fibrosis pharmaceutical composition, characterized in that: The composition comprises a therapeutically effective amount of amygdalin and a pharmaceutically acceptable carrier, and is used for inhibiting articular cartilage fibrosis in mammals, wherein the inhibitory effect is achieved by inhibiting the ferroptosis of articular chondrocytes.

6. The anti-cartilage fibrosis pharmaceutical composition according to claim 5, characterized in that: The pharmaceutical composition further comprises magnesium ions, which are derived from magnesium chloride. The ratio of magnesium chloride to amygdalin is 1:96-100 in parts by weight.

7. The anti-cartilage fibrosis pharmaceutical composition according to claim 5 or 6, characterized in that: When administered by intra-articular injection, the concentration of amygdalin is 1-10 mmol / L, and the solvents include PBS buffer and normal saline.

8. The anti-cartilage fibrosis pharmaceutical composition according to claim 7, characterized in that: The concentration of amygdalin is 10 mmol / L, and the concentration of magnesium chloride is 0.5 mmol / L.

9. Use of amygdalin in inhibiting ferroptosis and / or preparing ferroptosis inhibitors.

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