Application of didymoside in medicine for promoting postoperative tendon-bone interface fibrous cartilage regeneration

Abstract

The invention discloses an application of didymoside in a medicine for promoting postoperative tendon-bone interface fibrous cartilage regeneration. According to the application, an important way for treating the tendon-bone interface injury and preventing the tendon-bone healing disorder is obtained through the research of macrophages on fibrous cartilage tendon-bone interface repair and the influence of didymoside on interface bone formation and structure, fibrous cartilage formation and macrophage polarization regulation, so that the tendon-bone interface injury is further treated.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to an application of melissa officinalis in a drug for promoting postoperative tendon-bone interface fibrocartilage regeneration. Background Art

[0002] The tendon-bone interface (TBI) is an important structure for muscle to transmit force to bones. It is a special structure that transitions from soft tissue to hard tissue. Due to stress concentration, it is easy to rupture, making tendon-bone interface injury one of the most common diseases in sports injuries. The tendon-bone interface is composed of four layers of tissue: gradient bone, calcified fibrocartilage, non-calcified fibrocartilage and tendon. It has the function of transmitting and buffering muscle contraction force and avoiding stress concentration in mechanical conduction. Due to the formation of a large amount of scar tissue at the tendon-bone interface (BTI) during tendon-bone healing (Tendon union), the biomechanical strength is far lower than the normal level. Clinically, the re-rupture rate of tendon-bone interface injury after surgery is high, which increases the burden of treatment. Therefore, rapid and high-quality regeneration after tendon-bone interface injury has always been a difficult problem to be solved in the fields of orthopedics, sports medicine and tissue regeneration. A series of studies at home and abroad have confirmed that the regeneration of the fibrocartilage layer after tendon-bone interface injury is closely related to the quality of its injury repair. Promoting the formation of fibrocartilage during tendon-bone healing is the key to improving the healing quality. Therefore, it is crucial to clarify the regeneration mechanism of the fibrocartilage layer after tendon-bone interface injury.

[0003] At present, the research on tendon-bone interface repair mainly includes the following strategies:

[0004] (1) Stem Cell Therapy

[0005] Stem cells (such as mesenchymal stem cells, MSCs) are widely used in tendon-bone repair due to their multidirectional differentiation potential and paracrine effects. Studies have shown that MSCs can promote fibrocartilage regeneration, angiogenesis, and inflammation regulation, but their clinical application faces the following limitations:

[0006] Low survival rate: The survival rate of transplanted stem cells is low at the site of injury, which is affected by microenvironment such as local hypoxia, inflammation and mechanical stress.

[0007] Insufficient differentiation efficiency: The efficiency of stem cells differentiating into target cells (such as fibrochondrocytes) in vivo is limited and is easily disturbed by the microenvironment.

[0008] (2) Biomaterial scaffolds

[0009] Biomaterial scaffolds (such as collagen and decalcified bone matrix) provide a microenvironment for cell growth by simulating the gradient structure of the tendon-bone interface. However, existing scaffold materials have the following problems:

[0010] Poor degradability: The degradation rate of some scaffold materials does not match tissue regeneration, which may lead to inflammatory response or insufficient mechanical support.

[0011] Immunogenicity: Scaffold materials of xenogeneic or allogeneic origin may induce immune rejection reactions and affect the repair effect.

[0012] (3) Exosome technology

[0013] Exosomes, as an important medium for intercellular communication, have anti-inflammatory, angiogenic and cell differentiation regulating effects. However, the application of exosomes still faces the following challenges:

[0014] Insufficient targeting: The distribution of natural exosomes in the body lacks specificity, making it difficult to accurately act on the site of injury.

[0015] Poor stability: Exosomes have a short half-life in the body and are unable to continuously play a repairing role.

[0016] Therefore, rapid and high-quality regeneration of tendon-bone interface after injury has always been a difficult problem to be solved in the fields of orthopedics, sports medicine and tissue regeneration. Studies have confirmed that promoting the formation of fibrocartilage is the key to improving the quality of tendon-bone healing. For this reason, we proposed the use of melissa glycoside in a drug to promote the regeneration of fibrocartilage at the tendon-bone interface after surgery. Summary of the invention

[0017] The present application provides an application of melissa officinalis in a drug for promoting postoperative regeneration of fibrocartilage at the tendon-bone interface, which solves the problem that the biomechanical strength of existing tendon-bone interface injury treatments is far below normal levels, and the re-rupture rate of tendon-bone interface injuries after surgery is clinically high, increasing the burden of treatment.

[0018] The present application provides an application of melissa officinalis in a drug for promoting postoperative regeneration of fibrocartilage at the tendon-bone interface.

[0019] Preferably, the melissa officinalis promotes the transformation of macrophages from M1 type to M2 type and is applied to the treatment of tendon-bone interface injury.

[0020] Preferably, the melissa officinalis promotes the transformation of macrophages from M1 type to M2 type through fatty acid oxidation.

[0021] Preferably, the melissa officinalis promotes the formation of fibrocartilage for the treatment of tendon-bone interface injuries.

[0022] Preferably, the melissa officinalis affects macrophage M2 polarization by activating the PPAR-γ signaling pathway.

[0023] Preferably, the melissa officinalis promotes the transformation of macrophages from a pro-inflammatory phenotype (M1, marker CD86 + / IL-1β + ) towards the repair phenotype (M2, marker CD206 + / IL-10 + ) polarization.

[0024] Preferably, the dosage of melittoside is 1 - 4 mg / kg / d, and the usage period is four weeks.

[0025] As can be seen from the above technical solutions, the present application provides an application of melittoside in a drug for promoting the regeneration of fibrocartilage at the tendon-bone interface after surgery. The repair process of the fibrocartilaginous tendon-bone interface can be roughly divided into 4 stages: the initial inflammatory reaction stage, the stage of new bone formation and the regeneration of fibrocartilage-like structures, the stage of woven bone remodeling and the maturation of the regenerated fibrocartilage layer, and the final remodeling stage. Among them, macrophages play an important role in the whole process of tendon-bone interface repair. In the early stage of healing, a large number of M1 macrophages infiltrate locally, secrete pro-inflammatory factors such as TNF-α, IL-1β, and IL-6 to enhance the local inflammatory reaction, stimulate cell division, and chemotactically attract fibroblasts to aggregate at the damaged site. During the fibrocartilage regeneration stage, macrophages regulate the recruitment, proliferation, and differentiation of fibroblasts, osteoblasts, mesenchymal stem cells, etc. by releasing cytokines such as TGF-β and BMP, providing "materials" for the repair of the tendon-bone interface. In addition, the new collagen matrix produced by fibroblasts is conducive to the attachment of the damaged site and surrounding soft tissues to VEGF, and VEGF provides a nutritional basis for the regeneration of the fibrocartilage layer, promoting angiogenesis at the damaged tendon-bone interface. With the progress of repair, the woven bone at the tendon-bone healing interface gradually matures, and macrophages induce the proliferation and differentiation of various cells by secreting cytokines (IL-1, IL-10, NO, etc.), promoting the orderly arrangement of collagen fibers, and further maturing the composition and structure of the repair tissue.

[0026] M1 macrophages play a negative role in cartilage regeneration and repair. It can inhibit the expression of genes related to cartilage matrix production, and the pro-inflammatory factors and matrix metalloproteinases secreted will induce the degeneration of chondrocytes, accelerate cartilage degeneration, and lead to the formation of a large amount of scar tissue, hindering the development of the fibrocartilage layer. However, the transformation of macrophages into the M2 type helps the repair of damaged cartilage. The cytokines such as IL-10 and TGF-β3 secreted by M2 macrophages have strong effects on promoting cartilage repair or chondrogenic differentiation. Increasing the polarization of M2 macrophages in tendon-bone healing can promote fibrocartilage formation and improve biomechanical properties.

[0027] Compared with the prior art, the beneficial effects of the present invention are:

[0028] 1. Immunomodulatory microenvironment: Apigenin 7 - O - glucoside inhibits the expression of pro - inflammatory factors (such as IL - 1β, IL - 6) in M1 macrophages, promotes the secretion of anti - inflammatory factors (such as IL - 10, TGF - β) in M2 macrophages, thereby reducing the inflammatory response, decreasing scar formation, and promoting the regeneration of fibrocartilage;

[0029] 2. Endogenous repair: Apigenin 7 - O - glucoside activates the PPAR - γ signaling pathway, enhances the FAO metabolism of macrophages, provides energy support for cells, and further promotes the formation of fibrocartilage and new bone;

[0030] 3. Low immunogenicity: As a natural compound, apigenin 7 - O - glucoside has extremely low immunogenicity, avoiding the risk of immune rejection.

[0031] 4. Easy availability: Apigenin 7 - O - glucoside is widely present in Rutaceae plants (such as oranges, lemons), with rich sources and low extraction costs.

[0032] This application has obtained an important approach for treating tendon - bone interface injuries and preventing tendon - bone healing disorders through the study of macrophage - mediated repair of the fibrocartilaginous tendon - bone interface and the effects of apigenin 7 - O - glucoside on interface bone formation and structure, fibrocartilage formation, and macrophage polarization regulation, which further advances the treatment of tendon - bone interface injuries. Brief Description of the Drawings

[0033] To more clearly illustrate the technical solutions of this application, the following will briefly introduce the drawings required in the implementation cases. Obviously, for those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0034] Figure 1 Shows the effect of apigenin 7 - O - glucoside on macrophage polarization in a tendon - bone healing mouse model in this application.

[0035] Figure 2 Shows the effect of apigenin 7 - O - glucoside on macrophage polarization and the PPAR - mediated fatty acid oxidation pathway in this application.

[0036] Figure 3 Verifies that the PPAR - mediated fatty acid oxidation pathway is involved in DID - induced macrophage polarization in bone marrow - derived macrophages in this application. Detailed Description of the Embodiments

[0037] To enable those skilled in the art to better understand the technical solutions in this application, the following will clearly and completely describe the technical solutions in the embodiments of this application in conjunction with the drawings.

[0038] See Figures 1 - 3, The application of didymin in a drug for promoting the regeneration of fibrocartilage at the tendon-bone interface after surgery. Further, didymin promotes the formation of fibrocartilage for the treatment of tendon-bone interface injuries. In this application, through the study of the use of didymin in treating tendon-bone interface injuries, mice were gavaged with didymin at a dose of 1-4 mg / kg / d for a period of four weeks. Using this model, we studied the effect of didymin on the regulation of macrophage polarization. Cell experiments confirmed that didymin affects macrophage M2 polarization by activating the PPAR-γ signaling pathway. The experimental results revealed an important approach for the treatment and prevention of tendon-bone interface injuries.

[0039] In this application, didymin has the effect of activating fatty acid oxidation and promoting the transformation of macrophages from the M1 type to the M2 type. Didymin is a naturally abundant flavonoid glycoside that is widely distributed in the fruits of the Rutaceae family, such as oranges, lemons, and bergamots.

[0040] In this application, the promotion of the transformation of macrophages from the M1 type to the M2 type by didymin is applied to the treatment of tendon-bone interface injuries.

[0041] Example

[0042] To study the promoting effect of didymin on Achilles tendon-bone healing, an in vivo experiment was conducted to establish a tendon-bone healing model of the Achilles tendon-calcaneus in mice, and the mice were treated with low, medium, and high doses of didymin respectively. The tendon-bone healing condition, overall osteogenic activity, biomechanical strength at the tendon / bone junction, fibrocartilage formation at the tendon-bone interface, changes in macrophages and macrophage-related factors of the model mice were observed to explore the feasibility of didymin as a potential therapeutic agent for tendon-bone injuries.

[0043] In this example, a total of 36 SPF-grade male C57BL / 6J mice (weighing 18 - 22 g) were from Chengdu Dashuo Laboratory Animal Co., Ltd. After 1 week of acclimation and feeding, the mice were randomly divided into 6 groups according to the principle of random grouping: control group (sham); model group (Model); low-dose group (L-DID, L-apigenin-7-O-glucoside, 1 mg / kg / d); medium-dose group (M-DID, M-apigenin-7-O-glucoside, 2 mg / kg / d); high-dose group (H-DID, H-apigenin-7-O-glucoside, 4 mg / kg / d); positive drug group (DSF, 50 mg / kg / d) (DSF). A mouse Achilles tendon-bone healing model was established. Specifically, the mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital, and the hind limbs and hind feet were disinfected bilaterally with 75% alcohol. The skin incision was longitudinally concentrated on the Achilles tendon, and the tissue was separated to expose the Achilles tendon. The Achilles tendon was cut with a blade close to the upper edge of the Achilles tendon. After the Achilles tendon was repaired, the surgical incision was closed and disinfected. The wound was disinfected with iodophenol every day for 3 days after the operation, and penicillin was injected locally. The control group only exposed the Achilles tendon without incision. One day after the operation, the mice were gavaged with the corresponding doses of apigenin-7-O-glucoside and DSF every day for 4 weeks, while the control operation group and the model group were gavaged with an equal amount of normal saline. After the last administration, the mice were euthanized, and tendon-bone connection tissue samples were collected.

[0044] The mouse tendon-bone connection tissue was rinsed with phosphate buffer (PBS), the bone marrow was collected, and a bone marrow cell suspension was prepared. The bone marrow cell suspension was filtered through a 200-mesh sieve to obtain a single-cell suspension. The single-cell suspension was centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The cells were resuspended with erythrocyte lysate at 4°C and centrifuged at 1000 rpm for 5 min to finally obtain bone marrow cells. A complete medium containing macrophage colony-stimulating factor (M-CSF) was prepared [DMEM (containing 4.5 g of sugar, L-glutamine, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) + 15% fetal bovine serum + 20 ng / mL macrophage colony-stimulating factor + 50 μM 2-mercaptoethanol]. The above-extracted bone marrow cells were resuspended in 10 mL of the complete medium containing macrophage colony-stimulating factor, and the cell concentration was adjusted to 2×10 6 cells / mL; 5 mL of the cell suspension was added to a 60-mm cell culture dish and cultured in an incubator at 37°C with 5% CO2 to obtain mature mouse bone marrow-derived macrophages (BMDMs).

[0045] Set up cell groups:

[0046] (I) BMDMs (M0) group, BMDMs + LPS / IFN-γ (M1) group, BMDMs + LPS / IFN-γ + apigenin-7-O-glucoside (30 μM) group;

[0047] (2) BMDMs (M0) group, BMDMs + IL-4 / IL-13 (M2) group, BMDMs + IL-4 / IL-13 + melittoside (30 μM) group;

[0048] (3) BMDMs (M0) group, BMDMs + LPS / IFN-γ (M1) group, BMDMs + LPS / IFN-γ + melittoside (30 μM) group, BMDMs + LPS / IFN-γ + melittoside (30 μM) + PPAR-γ antagonist (GW9662, 5 μM) group, BMDMs + LPS / IFN-γ + melittoside (30 μM) + fatty acid oxidation inhibitor (Etomoxir, 50 μM) group.

[0049] BMDMs in each group were treated with LPS (100 ng / mL) + IFN-γ (20 ng / mL) or IL-4 (20 ng / mL) + IL-13 (20 ng / mL) for 24 hours. Then, the corresponding melittoside, GW9662, and Etomoxir were added and the treatment was continued for 24 hours.

[0050] Enzyme-linked immunosorbent assay (ELISA)

[0051] The concentrations of osteocalcin (OCN), calcium (Ca), alkaline phosphatase (ALP), and acetyl coenzyme A were detected using ELISA kits and polymerase chain reaction kits.

[0052] Flow cytometry

[0053] Mouse tendon-bone junction tissues were rinsed with phosphate-buffered saline (PBS), homogenized, and centrifuged to obtain cell pellets. The cells were resuspended with phosphate-buffered saline (PBS), F4 / 80 and CD86 were added to each test tube, and the cells were cultured in the dark at 4°C, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. 200 μL of fixation buffer was added to each tube, and the cells were cultured at room temperature for 20 min, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The cells were resuspended for analysis using a cell flow analyzer.

[0054] Immunofluorescence (IF) evaluation

[0055] The dewaxed sections were first blocked in 3% hydrogen peroxide to block endogenous peroxidase, followed by serum blocking. The sections were incubated overnight at 4°C with primary antibodies [Sox9 (1:100 dilution), CollagenII (1:100 dilution)], then incubated with secondary antibody (FITC-labeled goat anti-rabbit antibody, 1:100 dilution) for 30 minutes at room temperature, and then DAPI staining was performed. Images were captured using a VS200 Olympus scanner (Japan) and analyzed using the Image-J data image analysis system. In addition, the expressions of F4 / 80, CD86, and CD206 in cell smears were detected in the same manner.

[0056] Real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR)

[0057] Tissues were homogenized and pulverized in a high-speed and low-temperature mode, total RNA was separated, and cell samples were extracted for RNA. cDNA was synthesized using a reverse transcription kit. The mRNA expression levels of each gene were quantitatively detected by real-time fluorescence quantitative PCR method. Primers were designed and synthesized by Shanghai Sangon Biotech Co., Ltd. Using β-actin as an internal reference, the expression levels of IL-1β, IL-6, Agr1, IL-10, TGF-β1, TGF-β3, Cpt1a, and ACS were detected. The primer sequences are shown in Table 1

[0058] Table 1: Primer sequences used for RT-qPCR

[0059]

[0060] Western blot analysis

[0061] Total protein was obtained from cells using RIPA lysis buffer and quantified using a BCA kit (Beyotime, China). Then, it was separated by 10% SDS-PAGE gel electrophoresis and transferred to a PVDF membrane (Sigma-Aldrich, USA). Subsequently, it was blocked in 5% skim milk at room temperature for 1 hour and incubated with the following primary antibodies: peroxisome proliferator-activated receptor γ (PPAR-γ, ratio 1:1000) and retinoid X receptor α (RXRA, ratio 1:2000). After washing the membrane, it was incubated with a biotinylated goat anti-rabbit IgG (H+L) horseradish peroxidase (HRP) secondary antibody (ratio 1:5000, China, Abbexa) overnight at 4°C and then incubated for 1 hour. Finally, protein bands were detected using an ECL detection kit and analyzed using Image J software. The relative protein expression levels were calculated with reference to the expression of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

[0062] Statistical analysis

[0063] The data obtained were analyzed using Statistical Product and Service Solutions (SPSS) software (version 20.0) and expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used for between-group comparisons, and the least significant difference (LSD) test was used for post hoc mean analysis. P < 0.05 was considered statistically significant;

[0064] In summary

[0065] Effect of apigenin-7-O-glucoside on macrophage polarization during tendon-bone healing in mice

[0066] By analyzing the levels of macrophage-related factors, we investigated the effect of apigenin-7-O-glucoside on macrophage polarization during tendon-bone healing in mice. It was found that the levels of F4 / 80 + CD86 + , interleukin-1β (IL-1β), and interleukin-6 (IL-6) were increased in the model group mice, while administration of low, medium, and high doses of apigenin-7-O-glucoside could reduce the levels of these factors in mice. In addition, administration of medium and high doses of apigenin-7-O-glucoside could also increase the levels of F4 / 80 + CD206 + , arginase-1 (Agr1), and interleukin-10 (IL-10) in mice (P < 0.05, Figure 1 ), indicating that the application of apigenin-7-O-glucoside promoted the polarization of macrophages at the tendon-bone interface to the M2 type and reduced their polarization to the M1 type.

[0067] Effect of apigenin-7-O-glucoside on macrophage polarization and peroxisome proliferator-activated receptor (PPAR)-signaling-mediated fatty acid oxidation (FAO) pathway in bone marrow-derived macrophages (BMDMs)

[0068] To evaluate the effect of apigenin-7-O-glucoside on macrophage polarization, we measured the levels of F4 / 80 + CD86 + and F4 / 80 + CD206 + . In bone marrow-derived macrophages (BMDMs) co-stimulated with lipopolysaccharide (LPS) / interferon-γ (IFN-γ), the level of F4 / 80 + CD206 + was increased (P < 0.01, Figure 2 A, B), while in BMDMs induced by interleukin-4 (IL-4) / interleukin-13 (IL-13), the level of F4 / 80 + CD206 + was increased (P < 0.01, Figure 2 C, D). Apigenin-7-O-glucoside treatment significantly reduced the level of F4 / 80 +CD86 + levels (P < 0.01). In addition, we also analyzed the levels of the fatty acid oxidation pathway mediated by peroxisome proliferator-activated receptor (PPAR) signaling. Compared with unstimulated BMDMs (M0 group), the levels of coenzyme A (CoA), carnitine palmitoyltransferase 1a (Cpt 1a), acyl-CoA synthetase (ACS), PPAR-γ, and retinoid X receptor α (RXRA) were significantly decreased in the M1-type BMDMs group, while DID treatment promoted the levels of the above factors (P < 0.05, Figure 2 E-J), indicating that apigenin-7-O-neohesperidoside induced macrophage polarization to the M2 type and promoted the fatty acid oxidation pathway mediated by PPAR signaling.

[0069] Verification of the role of the fatty acid oxidation (FAO) pathway mediated by peroxisome proliferator-activated receptor (PPAR) signaling in apigenin-7-O-neohesperidoside-induced macrophage polarization of bone marrow-derived macrophages (BMDMs)

[0070] To verify whether the fatty acid oxidation (FAO) pathway mediated by peroxisome proliferator-activated receptor (PPAR) signaling is involved in apigenin-7-O-neohesperidoside-induced macrophage polarization of bone marrow-derived macrophages (BMDMs), PPAR-γ antagonists and FAO inhibitors were used in the experiment. As Figure 3 shown in A, the inhibitory effect of the PPAR-γ antagonist was first verified. GW9662 significantly inhibited the promoting effect of apigenin-7-O-neohesperidoside on the expression of PPAR-γ and RXRA in BMDMs (M1) (P < 0.01). Compared with the BMDMs (M1) + apigenin-7-O-neohesperidoside group, the CoA levels ( Figure 3 B), F4 / 80 + CD206 + levels ( Figure 3 C), Agr1 and IL-10 levels ( Figure 3 D, P < 0.05) were significantly decreased in the BMDMs (M1) + apigenin-7-O-neohesperidoside + GW9662 group and the BMDMs (M1) + apigenin-7-O-neohesperidoside + Etomoxir group, while the F4 / 80 + CD86 + levels ( Figure 3 C), IL-1β and IL-6 levels ( Figure 3 D, P < 0.05) were significantly increased, indicating that apigenin-7-O-neohesperidoside may be involved in the macrophage polarization process of BMDMs by promoting the FAO pathway mediated by PPAR signaling.

[0071] In this application, mice were selected as the experimental animals for this study. The above experiments showed that the above model was similar to the change pattern after rotator cuff reconstruction, and the process of tendon-bone healing could be reflected through morphological observations at different postoperative time points. In this application, verbascoside significantly increased the levels of cyanate, ALP, and Ca in the serum. In addition, HE and safranin-O / fast green staining of tendon-bone connective tissue showed that verbascoside promoted the formation of fibrocartilage. This demonstrated that verbascoside had a promoting effect on tendon-bone healing.

[0072] Verbascoside can effectively induce M2 macrophages (CD206 + ), and increase the expression of anti-inflammatory factors (Arg1, IL-10) in tendon-bone connective tissue, while the expression of M1 macrophages (CD86+) and pro-inflammatory factors (IL-1β, IL-6) decreased significantly, suggesting that verbascoside induced the polarization of macrophages at the tendon-bone healing site into M2 type.

[0073] The results of in vivo studies showed that verbascoside could simultaneously down-regulate Ml macrophages and up-regulate M2 macrophages, indicating that verbascoside promoted the transformation process of macrophages. Once activated, macrophages can maintain plasticity and can transform from one phenotype to another according to environmental signals. Thus, it was demonstrated that verbascoside promoted the transformation of M1 to M2 macrophages. In this application, verbascoside significantly down-regulated M1 (CD86 + ) and up-regulated M2 macrophages (CD206+), as well as the expression of related factor mRNAs under M1 polarization conditions, proving that verbascoside promoted the transformation of M1 to M2. In addition, PPAR-γ directly promoted the differentiation of bone marrow mesenchymal stem cells into osteoblasts and indirectly led to a decrease in osteogenesis. The activation of PPAR-γ and its regulatory genes contributed to an increase in fatty acid oxidation (FAO), and M2 macrophages relied on fatty acid oxidation to obtain energy. In the application, verbascoside increased the levels of PPAR-γ, RXRA, and CoA. After adding PPAR-γ antagonists and FAO inhibitors, verbascoside up-regulated M2 macrophages. These results showed that verbascoside could drive macrophage polarization to M2 by promoting the PPAR-mediated FAO pathway, thereby promoting tendon-bone healing, revealing that verbascoside promoted the polarization of macrophages from a pro-inflammatory phenotype (M1, marker CD86 + / IL-1β) to a repair phenotype (M2, marker CD206 + / IL-10).

[0074] From the above technical solutions, it can be seen that this application reveals that verbascoside can promote the formation of fibrocartilage in tendon-bone connective tissue, drive macrophage polarization to M2 type, and affect macrophage polarization by promoting the fatty acid oxidation (FAO) pathway mediated by PPAR signals.

[0075] Other embodiments of the present application will be readily apparent to those skilled in the art upon consideration of the specification and practice of the application disclosed herein. The present application is intended to cover any variations, uses, or adaptations of the present application, which follow the general principles of the present application and include known common general knowledge or conventional technical means in the technical field not disclosed in the present application. The specification and examples are only illustrative, and the true scope of the present application is pointed out by the claims.

[0076] It should be understood that the present application is not limited to the exact structures described above and shown in the drawings, and various modifications and changes can be made without departing from its scope. The embodiments of the present application described above do not constitute a limitation on the protection scope of the present application.

Claims

1. Application of melittoside in drugs for promoting the regeneration of fibrocartilage at the tendon-bone interface after surgery.

2. Use of verbascoside according to claim 1 in a medicament for promoting the regeneration of fibrocartilage at the tendon-bone interface after surgery, characterized in that, The verbascoside promotes the polarization of macrophages from a pro-inflammatory phenotype (M1, marker CD86 + / IL-1β + ) to a reparative phenotype (M2, marker CD206 + / IL-10 + ).

3. Use of verbascoside according to claim 2 in a medicament for promoting the regeneration of fibrocartilage at the tendon-bone interface after surgery, characterized in that The application of melittoside in promoting the transformation of macrophages from M1 type to M2 type is used for the treatment of tendon-bone interface injury.

4. Use of verbascoside according to claim 3 in a medicament for promoting the regeneration of fibrocartilage at the tendon-bone interface after surgery, characterized in that, Melittoside promotes the transformation of macrophages from M1 type to M2 type through fatty acid oxidation.

5. Use of verbascoside according to claim 4 in a medicament for promoting the regeneration of fibrocartilage at the tendon-bone interface after surgery, characterized in that, Melittoside affects macrophage M2 polarization by activating the PPAR-γ signaling pathway.

6. Use of verbascoside according to claim 3 in a medicament for promoting the regeneration of fibrocartilage at the tendon-bone interface after surgery, characterized in that, The dosage of melittoside is 1 - 4 mg / kg / d, and the usage period is four weeks.

7. Use of verbascoside according to claim 3 in a medicament for promoting the regeneration of fibrocartilage at the tendon-bone interface after surgery, characterized in that Melittoside promotes the formation of fibrocartilage and is used for the treatment of tendon-bone interface injury.

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