Application of Cornu Cervi Colla in the Treatment of Zebra Fish Bone Injury

Abstract

The present invention discloses the application of deer horn glue in the treatment of zebrafish bone injury, belonging to the technical field of fish bone injury. The present invention prepared deer horn glue and used LPS induction to establish a zebrafish bone injury model for comprehensive evaluation. The IC50 of deer horn glue in zebrafish was 206.3 μg / mL; the treatment with deer horn glue could alleviate the inhibitory effects of LPS on cartilage development and bone formation in a dose-dependent manner; it could significantly reduce the green fluorescence intensity of macrophages and inhibit the activation of macrophages induced by LPS; it inhibited the excessive secretion of inflammatory factors IL-6, IL-1β and NO; it significantly restored the mRNA levels of bone-related genes COL2α1 and Sox9a. This study confirmed that deer horn glue could alleviate zebrafish bone injury caused by LPS and at the same time inhibit the macrophage-mediated inflammatory cascade reaction, providing a basis for the treatment of fish bone diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of fish bone injuries, and specifically relates 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 the spatial orientation ability and locomotor ability, and significantly reducing the success rate of escaping from natural enemies. Bone injuries have gradually become an important problem restricting the healthy aquaculture of fish and ecological protection, 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, and its main components are amino acids, polypeptides, phospholipids and growth factors, and also contain various trace elements such as manganese, zinc and calcium. Cornus Cervi Colla (CCC) comes from sika deer or red deer, and is a solid glue made by decocting and concentrating the horn bases shed in the spring of the following year after ossification or sawing off the antlers. Cornus Cervi Colla contains animal protein and is also rich in various amino acids. It is found that Cornus Cervi Colla contains 19 amino acids, of 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 research records show that Cornus Cervi Colla has the effects of "benefiting essence and blood, strengthening tendons and bones". Modern research shows that Cornus Cervi Colla can induce a large amount of new bone growth in cranial defects. However, the conclusions in existing research 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 research mostly focuses 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. As an important mechanical support structure and immune regulatory organ, bones 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 pharmacodynamic effects of deer horn glue.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] 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, wherein the drug comprises deer horn glue, and the deer horn glue is prepared by the following method: washing and segmenting deer horns, soaking them and then heating; filtering, collecting the filtrate and concentrating it to obtain deer horn glue.

[0007] Further, after filtering, water is added to the filter residue, and heating is continued, followed by filtering and collecting the filtrate; this is repeated 1 - 5 times.

[0008] Further, when soaking, the ratio of deer horn to water is 1:(1 - 10).

[0009] Further, heating is carried out until boiling, and the heating time is 1 - 3 h.

[0010] Further, the dosage forms of the drug include powder, granule, capsule, solution, emulsion.

[0011] Further, when in use, the drug is dissolved in the water body where the zebrafish is located.

[0012] Further, the concentration of deer horn glue in the water body is 6.25 - 150 μg / mL.

[0013] Further, deer horn glue treats zebrafish bone injury through at least one of the following pathways:

[0014] (1) Promoting cartilage development and bone formation;

[0015] (2) Inhibiting macrophage activation;

[0016] (3) Inhibiting the excessive secretion of inflammatory factors IL - 6, IL - 1β and NO;

[0017] (4) Restoring the mRNA levels of bone - related genes COL2α1 and SOX9a.

[0018] The beneficial effects of the present invention:

[0019] The present invention found that the IC50 of deer antler 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 antler glue treatment could alleviate the inhibitory effect of LPS on cartilage development and bone formation in a dose-dependent manner. The fluorescence distribution of Tg(mpeg1:EGFP) macrophage gene showed that the deer antler 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 antler glue. This study established a zebrafish bone injury model using LPS, systematically evaluated the effects of deer antler 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

[0020] Figure 1 shows the effects of different concentrations of deer antler glue on the survival rate of zebrafish.

[0021] Figure 2 shows the effects of deer antler glue on LPS-induced chondrodysplasia; among them, the right-bottommost figure is the histogram statistics of 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.

[0022] Figure 3 shows the effects of deer antler glue on the attenuation of bone formation ability induced by LPS; among them, the right-bottommost is the histogram statistics of 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.

[0023] Figure 4 shows the effects of deer antler 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.

[0024] Figure 5 It is the effect of deer horn 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.

[0025] Figure 6 It is the effect of deer horn 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

[0026] It should be noted that the following detailed description is illustrative and is intended to provide further explanation 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.

[0027] 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.

[0028] The test materials not specifically described 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, CaCl2, and MgSO4 used in the present invention were purchased from Aladdin, methylene blue was purchased from Sinopharm Chemical Reagent Co., Ltd., dimethyl sulfoxide (DMSO) was purchased from Sangon Biotech Co., Ltd., alizarin red and Alcian blue were purchased from Shanghai Yuanye Bio-Technology Co., Ltd., LPS was purchased from Sigma-Aldrich, the ELISA kit was purchased from Nanjing Jiancheng Bioengineering Institute, the ready-to-use BCA protein concentration test kit was purchased from Sevenbio, the RNA extraction kit, reverse transcription kit, and qPCR kit 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, with a ratio of 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, and 0.33 mM MgSO4. 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).

[0029] Example 1: Preparation of deer antler glue and its freeze-dried powder

[0030] The deer antlers used in the present invention are the horn bases shed in the following spring after sawing the antlers of Cervus nippon Temminck. Preparation was carried out according to the method in the 2020 edition of the "Chinese Pharmacopoeia", specifically as follows: Wash the deer antlers, saw them into sections, place them in a round-bottom flask, add distilled water 5 times the mass of the deer antlers, soak for 2 hours and then heat. After boiling, continue to heat for 3 hours, then filter and save the filtrate. Put the solid back into the round-bottom flask, supplement distilled water with the same mass as the filtrate and continue boiling. Repeat the above steps twice and then combine the filtrates; Concentrate the combined filtrates to a paste, condense, and obtain deer antler glue. Freeze-dry the deer antler glue in a freeze-dryer to obtain freeze-dried deer antler glue powder. In subsequent experiments, different concentrations of deer antler glue were obtained by dissolving the freeze-dried deer antler glue powder.

[0031] Example 2: Safety study of deer antler glue

[0032] To determine the dosage of deer antler glue and explore its safe concentration, normal zebrafish at 5 dpf after normal fertilization and development were randomly divided into a blank control group and 9 deer antler glue groups, placed in six-well plates, with 3 wells set as parallels in each group and 10 zebrafish in each well. Among them, only E3 water for zebrafish culture was added to the blank control group; for the deer antler glue groups, deer antler glue was dissolved in E3 water and then added. The only difference among the deer antler glue groups was the final concentration of 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 zebrafish were obtained and the survival rate was counted.

[0033] The results are as Figure 1 shown. Compared with the blank group, there were no deaths or 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, deaths began to occur in zebrafish, and the surviving zebrafish showed malformations; when the deer antler glue concentration reached 250 μg / mL, all zebrafish died. After calculation, the IC50 result of deer antler glue was 206.3 μg / mL, thus confirming the safe dosage of deer antler glue (CCC).

[0034] Example 3: Using Alcian blue staining to study the effect of deer antler glue on LPS-induced chondrodysplasia

[0035] The morphology of cartilage is the result of the combined action of cell proliferation, differentiation, matrix synthesis and external regulation during development, and can intuitively reflect its maturity, functional status 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.

[0036] 3.1 Model establishment

[0037] 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. 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 administration group was the final concentration 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.

[0038] 3.2 Alcian blue staining

[0039] When cultured until the 9th day, the zebrafish body views were obtained. After the zebrafish in each group were 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 dehydrated with gradient ethanol; after being treated with 0.05% trypsin-sodium tetraborate solution mixture for 90 min, they were rinsed with PBS buffer three times again; the samples were treated with 1% KOH - 3% H2O2 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.

[0040] 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 group could increase the cartilage staining intensity and fullness in a dose-dependent manner, indicating that deer antler glue could reverse LPS-induced cartilage dysplasia.

[0041] Example 4: Using Alizarin red staining to study the effect of deer antler glue on the attenuation of bone formation ability caused by LPS

[0042] 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.

[0043] 4.1 Model establishment

[0044] Normal zebrafish that developed normally after fertilization until 5 dpf 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 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, 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, 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.

[0045] 4.2 Alizarin red staining

[0046] When culturing until the 9th day, zebrafish body views were obtained. Ten 9-dpf zebrafish were collected from each group and immersed in a mixture of 2% paraformaldehyde and PBS for 1 hour. The zebrafish were immersed in 50% ethanol for 1 hour and rinsed three times with ddH2O, 20 minutes each time. After digestion with trypsin until clear bones were visible inside the fish, the zebrafish were immersed in 0.1 g / L alizarin red solution overnight. After staining, the zebrafish were stored in glycerol. Ten zebrafish larvae at 9 days of development in each group were selected and fixed in 2% paraformaldehyde tissue fluid for 60 minutes.

[0047] The fixed samples were successively transferred to 50% ethanol solution and immersed for 60 minutes, and rinsed three times with ddH2O (20 minutes each time). The samples were treated by the trypsin digestion method. After the bone contour was clearly revealed, they were transferred into 0.1 g / L alizarin red staining solution for overnight staining. After staining was completed, the samples were transferred to glycerol medium for storage. The bone mineralization was observed under a microscope (Zeiss AXIO zoom V16) and images were obtained, and the IOD was statistically analyzed.

[0048] 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.

[0049] Example 5: Detection of macrophage distribution and expression

[0050] 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, thus 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, the present invention detects the distribution and expression of macrophages, and by recording the Tg(mpeg1:EGFP) fluorescent expression of transgenic zebrafish after administration, clarifies the effect of deer horn glue on macrophage activation.

[0051] Using Tg(mpeg1:EGFP) transgenic simple macrophage fluorescent zebrafish, zebrafish that have developed normally to 5 dpf after fertilization are randomly divided into a blank control group (Control), a bone injury model group (LPS), and three deer horn glue administration groups (CCC) with different concentrations. They are placed in a six-well plate, with 3 wells set as parallels for each group and 10 in each well. The blank control group is only added with E3 water for zebrafish culture; the bone injury model group; LPS is dissolved in E3 water and then added, with a final concentration of 100 μg / mL; the only difference among the groups in the deer horn glue administration group is the different final concentrations of deer horn glue. Each group contains 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) are added, and the solvent is E3 water. The volume of the liquid in each well is 3 mL, and they are cultured in a constant temperature incubator at 28 °C, and the corresponding culture medium is changed every 24 hours. On the 9th day, a microscope (Zeiss AXIO zoom V16) is used to observe the distribution of macrophages, and images are obtained and the IOD is counted.

[0052] The results are as Figure 4 shown. Compared with the blank control group, the fluorescence intensity of macrophages in the bone injury model group was significantly increased ( P <0.01), indicating that LPS can cause an inflammatory response in zebrafish, leading to an increase in macrophages. Treatment with deer horn glue can dose-dependently reduce the green fluorescence intensity of zebrafish. Among them, when the concentration of deer horn glue is 100 and 150 μg / mL, the effect is significantly enhanced. It shows that deer horn glue can inhibit the immune activation of zebrafish caused by LPS and reduce the number of macrophages.

[0053] Example 6: Detection of inflammatory factor content in zebrafish

[0054] Inflammatory factors are an important cause of cartilage damage and can trigger apoptosis, pyroptosis and other cascade reactions.

[0055] 6.1 Model establishment

[0056] 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 horn glue. They were placed in six-well plates, 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 only difference among the groups in the deer horn glue treatment groups was the different final concentrations 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, 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.

[0057] 6.2 Detection of inflammatory factor content

[0058] 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 in 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.

[0059] Zebrafish samples were added with normal saline in a ratio of 1:9 and homogenized on ice. After centrifugation at 3500 rpm for 10 minutes, the supernatant was taken. 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; 0.16 mL of double-distilled water and 0.08 mL of chromogenic reagent were added to the blank well, 0.16 mL of 20 μM sodium nitrite standard solution and 0.08 mL of chromogenic reagent were added to the standard well, and 0.16 mL of supernatant and 0.08 mL of chromogenic reagent were added to the measurement well; 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).

[0060] The results are 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. The treatment with deer horn glue could inhibit the expression of inflammatory factors and reduce the levels of inflammatory factors. Among them, the inhibitory effect of deer horn glue on NO was the most significant (P<0.01), indicating that deer horn glue could inhibit the inflammatory response caused by LPS and reduce the expression of inflammatory factors.

[0061] Example 7: Using RT-qPCR to detect the effect of deer horn glue on the expression of bone-related genes

[0062] 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 will 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 will lead 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.

[0063] 7.1 Model establishment

[0064] 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 replicates, 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 an incubator at a constant temperature of 28 °C, and the corresponding culture medium was changed every 24 h.

[0065] 7.2 Detection of the expression of related genes

[0066] After homogenizing the zebrafish that had been modeled with LPS for 9 days and treated with deer horn glue, RNA was extracted and 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.

[0067] The expression of related genes COL2α1 and SOX9a was measured by RT-qPCR. The results are 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), indicating that deer horn glue can 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.

[0068] This study explored the therapeutic potential of deer horn 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 horn glue has a protective effect on bone injury in a fish animal model and can alleviate LPS-induced chondrodysplasia and inhibition of bone mineralization. These findings not only expand the pharmacological effects of deer horn glue but also provide new insights into the management of fish skeletal diseases and a basis for the treatment of fish bone diseases.

[0069] The above are only the preferred embodiments of the present application and are not used to limit the present application. For those skilled in the art, the present application can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the protection scope of the present application.

Claims

1. Use of deer horn glue in the preparation of a drug for treating zebrafish bone injury, characterized in that, The deer antler glue is prepared by the following method: (1) washing and segmenting the deer antlers, adding distilled water 5 times the mass of the deer antlers, soaking for 2 hours and then preparing to heat; (2) heating to boiling and continuing to heat for 3 hours, then filtering and saving the filtrate, returning the solid, and adding distilled water of the same mass as the filtrate to continue decocting; Repeat this step twice and combine the filtrates; (3) concentrate the combined filtrates to a paste, and condense to obtain deer antler glue; 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 the excessive secretion of inflammatory factors IL-6, IL-1β and NO; (4) Restore the mRNA levels of bone-related genes COL2α1 and SOX9a.

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

3. The application according to claim 1, characterized in that, When used, dissolve the drug in the water where the zebrafish is located.

4. The application according to claim 3, wherein The concentration of antler glue in water is 6.25-150μg / mL.

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