Application of deer-horn glue in zebra fish bone injury treatment

Abstract

The invention discloses application of deer-horn glue in zebra fish bone injury treatment, and belongs to the technical field of fish bone injury. According to the invention, deer-horn glue is prepared, and a zebra fish bone injury model is established by using LPS induction so as to carry out comprehensive evaluation. The IC50 of the deer-horn glue in the zebra fish is 206.3 mu g / mL; the deer-horn glue treatment can relieve the inhibition effect of LPS on cartilage development and bone formation in a dose-dependent manner; the green fluorescence intensity of macrophages can be obviously reduced, and LPS-induced macrophage activation is inhibited; excessive secretion of inflammatory factors IL-6, IL-1beta and NO is inhibited; the mRNA (messenger Ribonucleic Acid) level of the bone related genes COL2 alpha 1 and SOX9a can be obviously recovered. The research proves that the deer-horn glue can relieve zebra fish bone injury caused by LPS (lipopolysaccharide) and inhibit macrophage-mediated inflammatory cascade reaction at the same time, and a basis is provided for treating fish bone diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of fish bone injuries, and particularly to the application of Cornus Cervi Colla in the treatment of zebrafish bone injuries. Background Art

[0002] The skeletal system undertakes the dual missions of structural support and metabolic regulation in fish development and physiological homeostasis, and bone injuries have a profound impact on fish populations. In the aquaculture environment, infections by pathogenic microorganisms (such as Aeromonas hydrophila) can induce chronic cartilage inflammation, leading to the dissolution of gill arch cartilage and a 30%-50% decrease in the feeding efficiency of fish. In wild populations, abnormal bone mineralization caused by heavy metal exposure or poor self-development can lead to morphological deformities of otoliths or vertebrae, disrupting spatial orientation and locomotor abilities and significantly reducing their success rate in evading natural enemies. Bone injuries have gradually become an important issue restricting the healthy aquaculture and ecological protection of fish, and there is an urgent need to develop natural products with both anti-inflammatory and bone protection functions.

[0003] Antler is a traditional Chinese medicine. Its main components are amino acids, polypeptides, phospholipids, and growth factors, and it also contains various trace elements such as manganese, zinc, and calcium. Cornus Cervi Colla (CCC) is obtained from sika deer or red deer, and is a solid glue made by decocting and concentrating the horn bases that fall off in the following spring after ossification or sawing of the antlers. Cornus Cervi Colla contains animal proteins and is also rich in various amino acids. It has been found that Cornus Cervi Colla contains 19 kinds of amino acids, among which 8 are essential amino acids for the human body; Cornus Cervi Colla also contains polysaccharide components, and the polysaccharides in Cornus Cervi Colla can combine with proteins to further form proteoglycans, which have good regulatory functions. Books and studies have recorded that Cornus Cervi Colla has the effects of "benefiting essence and blood, strengthening bones and muscles". Modern research shows that Cornus Cervi Colla can induce a large amount of new bone growth in cranial defects. However, the conclusions in existing studies are mostly based on mammalian models, and there are significant differences between mammals and fish in terms of species, living environment, physiological structure, administration methods, and drug absorption. Moreover, the current therapeutic drugs for fish bone injuries are relatively limited. Therefore, the role of Cornus Cervi Colla in the fish skeletal system is still unclear, and existing studies mostly focus on the repair of mammalian bone defects, and the immunity and inflammatory responses related to bones in fish have not been analyzed. Summary of the Invention

[0004] In view of the above-mentioned prior art, the object of the present invention is to provide the application of deer horn glue in the treatment of zebrafish bone injury. Bones, as important mechanical support structures and immune regulatory organs, affect the growth, development and survival of fish. Deer horn is a natural medicine, and deer horn glue is its extract. This study explored the protective effect of deer horn glue on lipopolysaccharide (LPS)-induced bone injury based on the zebrafish model. By using LPS induction to establish a zebrafish bone injury model, techniques such as Alcian blue staining, Alizarin red staining, fluorescence tracing, ELISA and qPCR were used to comprehensively evaluate the pharmacological effects of deer horn glue.

[0005] To achieve the above object, the present invention adopts the following technical solutions: In the first aspect of the present invention, there is provided the application of deer horn glue in the preparation of a drug for treating zebrafish bone injury. The drug includes deer horn glue, and the deer horn glue is prepared by the following method: Wash and segment the deer horn, soak it and then heat it; Filter, collect the filtrate and concentrate it to obtain deer horn glue.

[0006] Further, after filtration, add water to the filter residue, continue heating, filter, and collect the filtrate; Repeat 1-5 times.

[0007] Further, when soaking, the ratio of deer horn to water is 1:(1-10).

[0008] Further, heat to boiling, and the heating time is 1-3 h.

[0009] Further, the dosage forms of the drug include powder, granule, capsule, solution, emulsion.

[0010] Further, when in use, dissolve the drug in the water body where the zebrafish is located.

[0011] Further, the concentration of deer horn glue in the water body is 6.25-150 μg / mL.

[0012] Further, deer horn glue treats zebrafish bone injury through at least one of the following pathways: (1) Promote cartilage development and bone formation; (2) Inhibit macrophage activation; (3) Inhibit the excessive secretion of inflammatory factors IL-6, IL-1β and NO; (4) Restore the mRNA levels of bone-related genes COL2α1 and SOX9a.

[0013] The beneficial effects of the present invention: The present invention found that the IC50 of deer horn glue in zebrafish was 206.3 μg / mL, and no death or obvious malformation occurred after treatment in the range of 0 - 150 μg / mL; Alcian blue and Alizarin red staining showed that deer horn glue treatment could relieve the inhibitory effect of LPS on cartilage development and bone formation in a dose-dependent manner. The fluorescence distribution of the Tg(mpeg1:EGFP) macrophage gene showed that the deer horn glue administration group could significantly reduce the green fluorescence intensity of macrophages and inhibit the activation of macrophages induced by LPS; the detection of inflammatory factors inhibited the excessive secretion of inflammatory factors IL-6, IL-1β, and NO. The results of RT-qPCR detection showed that the mRNA levels of bone-related genes COL2α1 and SOX9a were also significantly restored after the intervention of deer horn glue. This study established a zebrafish bone injury model using LPS, systematically evaluated the effects of deer horn glue on zebrafish bone formation, immune inflammation regulation, and gene expression network, revealed its potential role in fish bone injury, and provided a new strategy for the treatment of bone diseases in aquaculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 shows the effects of different concentrations of deer horn glue on the survival rate of zebrafish.

[0015] Figure 2 shows the effects of deer horn glue on LPS-induced chondrodysplasia; among them, the lower rightmost figure is the histogram statistics of the cumulative optical density, and the rest are representative images of zebrafish Alcian blue staining; # indicates comparison with the blank group, ## P < 0.01; * indicates comparison with the bone injury model group, * P < 0.05, ** P < 0.01.

[0016] Figure 3 shows the effects of deer horn glue on the attenuation of LPS-induced bone formation ability; among them, the lower rightmost is the histogram statistics of the cumulative optical density, and the rest are representative images of Alizarin red staining; # indicates comparison with the blank group, ## P < 0.01; * indicates comparison with the bone injury model group, * P < 0.05, ** P < 0.01.

[0017] Figure 4 shows the effects of deer horn glue on LPS-induced macrophage activation, Figure 4 in which A is a representative image of zebrafish macrophage fluorescence, Figure 4In B, it is the histogram statistics of the fluorescence intensity of macrophages. Among them, # indicates comparison with the blank group, ## P <0.01; * indicates comparison with the bone injury model group, * P <0.05, ** P <0.01.

[0018] Figure 5 It is the effect of antler glue on the expression of inflammatory factors induced by LPS, Figure 5 In A, it is the content of IL-6, Figure 5 In B, it is the content of IL-1β, Figure 5 In C, it is the content of NO. Among them, # indicates comparison with the blank group, # P <0.05, ## P <0.01; * indicates comparison with the bone injury model group, * P <0.05, ** P <0.01.

[0019] Figure 6 It is the effect of antler glue on the expression of bone-related genes COL2α1 and SOX9a, Figure 6 In A, it is the mRNA level of COL2α1, Figure 6 In B, it is the mRNA level of SOX9a. Among them, # indicates comparison with the blank group, ## P <0.01; * indicates comparison with the bone injury model group, ** P <0.01. Specific embodiments

[0020] It should be noted that the following detailed description is exemplary and is intended to provide further illustration of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs.

[0021] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below with reference to specific embodiments.

[0022] The test materials not specifically described used in the embodiments of the present invention are all conventional test materials in the art and can be obtained through commercial channels. The NaCl, KCl, CaCl used in the present invention 2 , and MgSO 4Purchased from Aladdin, methylene blue was purchased from Sinopharm Chemical Reagent Co., Ltd., dimethyl sulfoxide (DMSO) was purchased from Shanghai Sangon Biotech Co., Ltd., alizarin red and Alcian blue were purchased from Shanghai Yuanye Bio-Technology Co., Ltd., LPS was purchased from Sigma-Aldrich, ELISA kits were purchased from Nanjing Jiancheng Bioengineering Institute, ready-to-use BCA protein concentration test kits were purchased from Sevenbio, RNA extraction kits, reverse transcription kits and qPCR kits were from Nanjing Vazyme Biotech. The deer antlers were purchased from Jinan Dapeng Agricultural Technology Co., Ltd. The E3 water (also known as E3 medium) used in the present invention was self-prepared, and the ratio was 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl 2 and 0.33 mM MgSO 4 . The zebrafish used in the present invention were provided by the zebrafish drug screening platform of the Institute of Biology, Shandong Academy of Sciences and were raised in the zebrafish drug screening platform of the Institute of Biology, Shandong Academy of Sciences. During the experiment, the "Animal Management Regulations" and the 3R principle were strictly observed (animal experiment ethics number: SWS20240407).

[0023] Example 1: Preparation of deer antler glue and its freeze-dried powder The deer antlers used in the present invention were the horn bases shed in the spring of the following year after sawing the antlers of Cervus nippon Temminck. The preparation was carried out according to the method of the 2020 edition of the "Chinese Pharmacopoeia", specifically as follows: The deer antlers were washed and cut into sections, placed in a round-bottomed flask, added with distilled water 5 times the mass of the deer antlers, soaked for 2 hours and then heated. After boiling, continue heating for 3 hours, then filter and save the filtrate. The solid was put back into the round-bottomed flask, supplemented with distilled water of the same mass as the filtrate and continued to decoct. After repeating the above steps twice, the filtrates were combined; the combined filtrate was concentrated to a paste and condensed to obtain deer antler glue. The deer antler glue was freeze-dried in a freeze dryer to obtain freeze-dried powder of deer antler glue. In subsequent experiments, the freeze-dried powder of deer antler glue was dissolved to obtain deer antler glue with different concentrations.

[0024] Example 2: Safety study of deer antler glue To determine the dosage of deer antler glue and explore its safe concentration, normal zebrafish that had developed normally to 5 dpf after fertilization were randomly divided into a blank control group and 9 deer antler glue groups, and placed in six-well plates. Each group had 3 wells as replicates, with 10 zebrafish in each well. Among them, the blank control group was only added with E3 water for zebrafish culture; in the deer antler glue groups, the deer antler glue was dissolved in E3 water and then added. The only difference among the groups in the deer antler glue groups was the final concentration of the deer antler glue, which were 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 75 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, and 250 μg / mL respectively; the volume of the liquid in each well was 3 mL, and they were cultured in a constant temperature incubator at 28 °C, and the corresponding culture medium was changed every 24 h. After 9 days, the body views of the zebrafish were obtained, and the survival rate was counted.

[0025] The results are as Figure 1 shown. Compared with the blank group, there were no deaths and obvious malformations in the zebrafish in the deer antler glue groups with deer antler glue concentrations of 6.25, 12.5, 25, 50, 75, 100, and 150 μg / mL; when the deer antler glue concentration was 200 μg / mL, the zebrafish began to die, and the surviving zebrafish showed malformations; when the deer antler glue concentration reached 250 μg / mL, all the zebrafish died. After calculation, the IC50 result of the deer antler glue was 206.3 μg / mL, and thus the safe dosage of the deer antler glue (CCC) was confirmed.

[0026] Example 3: Using Alcian blue staining to study the effect of deer antler glue on LPS-induced chondrodysplasia The morphology of cartilage is the result of the combined action of cell proliferation, differentiation, matrix synthesis and external regulation during development, and can directly reflect its maturity, functional state and pathological abnormalities. Therefore, Alcian blue staining was used to explore the effect of deer antler glue on the cartilage morphology of LPS-treated zebrafish.

[0027] 3.1 Model establishment Normal zebrafish that developed normally to 5 dpf after fertilization were randomly divided into a blank control group (Control), a bone injury model group (LPS), and three groups administered with different concentrations of deer antler glue (CCC). They were placed in six-well plates, with three wells set as duplicates for each group and 10 zebrafish in each well. Only E3 water used for zebrafish culture was added to the blank control group; for the bone injury model group, LPS was dissolved in E3 water and added to a final concentration of 100 μg / mL; the only difference among the groups in the deer antler glue administration groups was the final concentration of deer antler glue. Each group contained LPS at a final concentration of 100 μg / mL, and different concentrations of deer antler glue (50 μg / mL, 100 μg / mL, 150 μg / mL) were added, with the solvent being E3 water. The volume of the liquid in each well was 3 mL, and they were cultured in a constant temperature incubator at 28 °C, with the corresponding culture medium being changed every 24 h.

[0028] 3.2 Alcian blue staining When the culture reached the 9th day, zebrafish body views were obtained. After each group of zebrafish was fixed with 4% paraformaldehyde solution for 24 h, they were rinsed three times with PBS buffer; transferred to 0.1% Alcian blue staining solution for overnight staining, and then dehydrated with gradient ethanol; after being treated with a 0.05% trypsin-sodium tetraborate solution mixture for 90 min, they were rinsed three times again with PBS buffer; the samples were treated with a 1% KOH - 3% H 2 O 2 mixture for 90 min and then stored in 50% glycerol. The cartilage staining was observed under a microscope (Zeiss AXIOzoom V16) and images were obtained, and the fluorescence intensity (Integrated Optical Density, IOD) of the head was statistically analyzed.

[0029] The results are shown in Figure 2 , compared with the blank control group, the staining of Meckel's cartilage, palatoquadrate cartilage and other parts in the bone injury model group decreased significantly ( P < 0.01), and there were phenomena such as cartilage atrophy and dysplasia; the deer antler glue administration groups could increase the cartilage staining intensity and fullness in a dose-dependent manner, indicating that deer antler glue could reverse LPS-induced cartilage dysplasia.

[0030] Example 4: Using Alizarin red staining to study the effect of deer antler glue on the attenuation of LPS-induced bone formation ability Bone formation plays a crucial role in maintaining bone health and function. Alizarin red staining was used to explore the effect of deer antler glue on the bone mineralization ability of LPS-treated zebrafish.

[0031] 4.1 Model establishment Normal zebrafish that developed normally to 5 dpf after fertilization were randomly divided into a blank control group (Control), a bone injury model group (LPS), and three groups of deer horn glue administration groups (CCC) with different concentrations. They were placed in six-well plates, with three wells set as parallels in each group, and 10 zebrafish in each well. Only E3 water for zebrafish culture was added to the blank control group; for the bone injury model group, LPS was dissolved in E3 water and added, with a final concentration of 100 μg / mL; the difference among the groups in the deer horn glue administration groups was only the final concentration of deer horn glue. Each group contained LPS with a final concentration of 100 μg / mL, and different concentrations of deer horn glue (50 μg / mL, 100 μg / mL, 150 μg / mL) were added, and the solvent was E3 water. The volume of the liquid in each well was 3 mL, and they were cultured in a constant temperature incubator at 28 °C, and the corresponding culture medium was changed every 24 h.

[0032] 4.2 Alizarin red staining When culturing to the 9th day, obtain the zebrafish body view. Collect ten 9-dpf zebrafish from each group and soak them in a mixture of 2% paraformaldehyde and PBS for 1 hour. Soak the zebrafish in 50% ethanol for 1 hour and rinse three times with ddH 2 2O, 20 minutes each time. After digesting with trypsin until the bones in the fish body are clearly visible, soak the zebrafish in 0.1 g / L alizarin red solution overnight. After staining, store the zebrafish in glycerol. Select 10 zebrafish larvae that have developed to 9 days, place them in 2% paraformaldehyde tissue fluid for fixation for 60 minutes.

[0033] Transfer the fixed samples to 50% ethanol solution and soak for 60 minutes in sequence, and rinse three times with ddH 2 2O (20 minutes each time). Treat the samples by the trypsin digestion method. After the bone outline is clearly revealed, transfer them to 0.1 g / L alizarin red staining solution for overnight staining. After staining is completed, transfer the samples to glycerol medium for storage. Observe the bone mineralization situation with a microscope (Zeiss AXIO zoom V16) and obtain images, and statistically analyze the IOD.

[0034] The results are shown in Figure 3 , compared with the blank control group, the staining intensity of mineralized bone in the bone injury model group was significantly weakened ( P <0.01), indicating that LPS inhibits bone formation; the deer horn glue treatment intervention can increase the staining intensity and fullness of mineralized bone ( P <0.01), indicating that deer horn glue can alleviate the decrease in bone mineralization ability induced by LPS and promote bone formation.

[0035] Example 5: Detection of macrophage distribution and expression After joint injury, the inflammatory response promotes the differentiation of monocytes into mature macrophages and polarizes them into M1 macrophages, which secrete inflammatory cytokines and matrix metalloproteinases to promote the degradation of type II collagen and proteoglycans, thereby accelerating the degeneration of articular cartilage. In addition, LPS-induced M1 macrophage polarization further exacerbates the inflammatory response by releasing reactive oxygen species (ROS) and inducing overexpression of NO. Therefore, in the present invention, the distribution and expression of macrophages are detected, and by recording the Tg(mpeg1:EGFP) fluorescent expression of transgenic zebrafish after administration, the effect of antler glue on macrophage activation is clarified.

[0036] Using Tg(mpeg1:EGFP) transgenic simple macrophage fluorescent zebrafish, zebrafish that had developed normally to 5 dpf after fertilization were randomly divided into a blank control group (Control), a bone injury model group (LPS), and three antler glue administration groups with different concentrations (CCC). They were placed in a six-well plate, with three wells set as parallels for each group, and 10 zebrafish in each well. Only E3 water for zebrafish culture was added to the blank control group; for the bone injury model group, LPS was dissolved in E3 water and added, with a final concentration of 100 μg / mL; the difference between the groups in the antler glue administration groups was only the final concentration of antler glue. Each group contained LPS with a final concentration of 100 μg / mL, and different concentrations of antler glue (50 μg / mL, 100 μg / mL, 150 μg / mL) were added, with the solvent being E3 water. The volume of the liquid in each well was 3 mL, and they were cultured in a constant temperature incubator at 28 °C, and the corresponding culture medium was changed every 24 hours. On the 9th day, a microscope (Zeiss AXIO zoom V16) was used to observe the distribution of macrophages, and images were obtained and the IOD was counted.

[0037] The results are as Figure 4 shown. Compared with the blank control group, the fluorescence intensity of macrophages in the bone injury model group increased significantly ( P < 0.01), indicating that LPS can cause an inflammatory response in zebrafish, leading to an increase in macrophages. Treatment with antler glue can dose-dependently reduce the green fluorescence intensity of zebrafish, and the effect is significantly enhanced when the concentration of antler glue is 100 and 150 μg / mL. This indicates that antler glue can inhibit the immune activation of zebrafish caused by LPS and reduce the number of macrophages.

[0038] Example 6: Detection of the content of inflammatory factors in zebrafish Inflammatory factors are an important cause of cartilage damage and can trigger apoptosis, pyroptosis and other cascade reactions.

[0039] 6.1 Model establishment Normal zebrafish that developed normally to 5 dpf after fertilization were randomly divided into a blank control group, a bone injury model group, and three groups treated with different concentrations of deer antler glue. They were placed in six-well plates, with three wells set as duplicates for each group, and 10 fish in each well. The blank control group was only added with E3 water for zebrafish culture; for the bone injury model group, LPS was dissolved in E3 water and added, with a final concentration of 100 μg / mL; the only difference among the groups in the deer antler glue treatment groups was the different final concentrations of deer antler glue. Each group contained LPS with a final concentration of 100 μg / mL, and different concentrations of deer antler glue (50 μg / mL, 100 μg / mL, 150 μg / mL) were added, with the solvent being E3 water. The volume of the liquid in each well was 3 mL, and they were cultured in a constant temperature incubator at 28 °C, and the corresponding culture medium was changed every 24 h.

[0040] 6.2 Detection of inflammatory factor content When cultured until the 9th day, for each group, 10 9-dpf larvae were randomly selected and placed in 1.5 mL centrifuge tubes. PBS was added at a ratio of 1:9 and homogenized on ice. After centrifugation at 3000 rpm for 20 minutes at 4 °C, the supernatant was taken for detection. The levels of IL-1β and IL-6 were measured according to the kit instructions.

[0041] For zebrafish samples, physiological saline was added at a ratio of 1:9 and homogenized on ice. After centrifugation at 3500 rpm for 10 minutes, the supernatant was taken, and the protein concentration of each group of samples was measured using a ready-to-use BCA protein concentration assay kit; the NO assay was divided into a blank well, a standard well, and a measurement well; in the blank well, 0.16 mL of double-distilled water and 0.08 mL of chromogenic reagent were added, in the standard well, 0.16 mL of 20 μM sodium nitrite standard solution and 0.08 mL of chromogenic reagent were added, and in the measurement well, 0.16 mL of supernatant and 0.08 mL of chromogenic reagent were added; after mixing and standing for 15 minutes, the absorbance value at 550 nm was detected using an enzyme-linked immunosorbent assay (ELISA) reader (BMG Labtech).

[0042] The results were as Figure 5 shown. Compared with the blank control group, the levels of cellular inflammatory factors (IL-6, IL-1β, NO) in the bone injury model group were increased to varying degrees. Treatment with deer antler glue could inhibit the expression of inflammatory factors and reduce the levels of inflammatory factors. Among them, the inhibitory effect of deer antler glue on NO was the most significant (P < 0.01), indicating that deer antler glue could inhibit the inflammatory response induced by LPS and reduce the expression of inflammatory factors.

[0043] Example 7: Using RT-qPCR to detect the effect of deer antler glue on the expression of bone-related genes COL2α1 is the main gene encoding the synthesis of type II collagen, which is involved in the regulation of intramembranous ossification and endochondral ossification. Its mutation can cause abnormal structure of type II collagen, leading to various skeletal dysplasia diseases. SOX9a is a core transcription factor for osteocyte differentiation and extracellular matrix (ECM) synthesis, and is a necessary condition for the expression of COL2a1. During normal skeletal development, SOX9a directly activates its transcription by binding to the enhancer of the COL2a1 gene, promoting the synthesis of type II collagen; while during osteoporosis, the decrease in SOX9a expression leads to a reduction in COL2α1 synthesis, accelerating cartilage degeneration, resulting in bone loss and bone damage. COL2α1 and SOX9a play a core role in skeletal development, homeostasis and repair, and their coordinated regulation is the key to maintaining skeletal structure and function. Therefore, the present invention studies the expression of the above bone-related genes.

[0044] 7.1 Model establishment Normal zebrafish that had developed normally to 5 dpf after fertilization were randomly divided into a blank control group, a bone injury model group, and 3 groups treated with different concentrations of deer horn glue, and placed in a six-well plate. Each group had 3 wells as duplicates, with 10 zebrafish in each well. The blank control group was only added with E3 water for zebrafish culture; for the bone injury model group, LPS was dissolved in E3 water and added, with a final concentration of 100 μg / mL; the difference between the groups in the deer horn glue treatment groups was only the final concentration of deer horn glue. Each group contained LPS with a final concentration of 100 μg / mL, and different concentrations of deer horn glue (50 μg / mL, 100 μg / mL, 150 μg / mL) were added, and the solvent was E3 water. The volume of the liquid in each well was 3 mL, and they were cultured in a constant temperature incubator at 28 °C, and the corresponding culture medium was changed every 24 h.

[0045] 7.2 Detection of the expression of related genes Zebrafish that had been modeled with LPS for 9 days and treated with deer horn glue were homogenized and RNA was extracted, and then reverse transcribed into cDNA. RPL13a with relatively stable expression was selected as the internal reference gene. Using cDNA as a template, the expression levels of COL2α1 and SOX9a were detected by RT-qPCR technology, the relative expression levels of each gene were calculated, and the anti-osteoarthritis efficacy of deer horn glue was evaluated by statistical analysis.

[0046] The expression of related genes COL2α1 and SOX9a was measured by RT-qPCR, and the results were as Figure 6 shown. Compared with the blank control group, the mRNA gene expressions of COL2α1 and SOX9a in the bone injury model group were significantly down-regulated ( P < 0.01); after treatment with deer horn glue, the mRNA expression levels of COL2α1 and SOX9a both increased significantly (vs. Model,P (<0.01), and the results showed that deer antler glue could inhibit the decrease of COL2α1 and SOX9a-related genes induced by LPS, alleviate the skeletal damage of zebrafish caused by LPS, and promote bone development.

[0047] This study explored the therapeutic potential of deer antler glue on LPS-induced skeletal damage in zebrafish, revealing its dual ability to enhance bone regeneration and inhibit macrophage-mediated inflammatory cascades. Our study confirmed that deer antler glue has a protective effect on bone injury in the fish animal model and can alleviate LPS-induced cartilage dysplasia and bone mineralization inhibition. These findings not only expand the pharmacological effects of deer antler glue but also provide new insights into the management of fish skeletal diseases and a basis for the treatment of fish bone diseases.

[0048] The above are only the preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. The use of antler glue in the preparation of a drug for treating zebrafish bone injury, characterized in that: The medicine comprises deer antler glue, and the deer antler glue is prepared by the following method: washing and segmenting the deer antlers, soaking and then heating; filtering, collecting and concentrating the filtrate to obtain the deer antler glue.

2. The use according to claim 1, characterized in that: After filtration, add water to the residue, continue heating, filter, and collect the filtrate; repeat 1-5 times.

3. The use according to claim 1, characterized in that: When soaking, the ratio of deer antlers to water is 1:(1-10).

4. The use according to claim 2, characterized in that: Heat to boiling for 1-3 hours.

5. The use according to claim 1, characterized in that: The dosage forms of the drug include powders, granules, capsules, solutions, and emulsions.

6. The use according to claim 1, characterized in that: When used, the drug is dissolved in the water where the zebrafish are located.

7. The use according to claim 6, characterized in that: The concentration of antler gum in water is 6.25-150μg / mL.

8. The use according to claim 1, characterized in that: Deer antler glue treats zebrafish bone injuries through at least one of the following pathways: (1) Promote cartilage development and bone formation; (2) inhibiting macrophage activation; (3) Inhibit excessive secretion of inflammatory factors IL-6, IL-1β, and NO; (4) Restore the mRNA levels of bone-related genes COL2α1 and SOX9a.

Images