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    • 专利摘要:
    The present invention relates to the use of recombinant IFN-M protein in the preparation of medicines for the prevention and/or treatment of inflammatory bone loss. In order to solve the problem of imbalanced bone metabolism caused by excessive activation of osteoclasts triggered by the release of a large number of inflammatory factors in a state of the severe inflammatory bone loss due to bone infection, the applicant found by experiments that recombinant lFN—M protein can regulate the osteoclast differentiation and fusion, and reduce the release of inflammatory factors in the process of osteoclast differentiation, thereby solving the problem of excessive activation of osteoclasts in the inflammatory situation and protecting from inflammatory bone loss.
    公开号:NL2027028A
    申请号:NL2027028
    申请日:2020-12-02
    公开日:2021-08-17
    发明作者:Chen Yueqi；Li Jianmei；Dong Shiwu；Hu Wenhui
    申请人:Univ Army Medical；
    IPC主号:
    专利说明:

    Use of IFN -A1 protein target in the preparation of medicines for the prevention and/or treatment of inflammatory bone loss Technical field The present invention relates to the biological prevention and/or treatment field of orthopedic technology, to the use of recombinant IFN-A1 protein in the preparation of medicines for the treatment of inflammatory bone loss. Background Bone infection is one of the most common and serious complications in fracture and orthopedic surgery, which have troubled clinicians due to the limitation of knowledge and treatment methods. Staphylococcus aureus is the main pathogenic bacteria, accounting for 65% - 70% of the pathogenic bacteria detected in traumatic osteomyelitis. Bone infection often causes excessively inflammatory bone destruction and non-union, resulting in nonunion or delayed healing of fractures. Osteoblasts (OB) and osteoclasts (OC) are the main cells involved in bone remodeling, and they can keep the dynamic balance of bone tissue by joint regulation. In the state of infection, acute and chronic inflammatory stimulation can change this dynamic balance and affect bone healing. Therefore, it is a feasible strategy to develop a medicine that can regulate the balance of bone matrix through its strong anti-inflammatory effect.
    Normal bone tissue remodels its own microlesion through bone repair to maintain the stable balance of structure, load and calcium ions. Osteoblasts and osteoclasts are two major types of cells in bone remodeling. In the physiological state, osteoblasts and osteoclasts work together to maintain the dynamic balance of bone formation and resorption. In the state of bone infection, the sustained effect of staphylococcus aureus and virulence factors can promote the release of a large number of inflammatory factors, which may change the normal biological behaviors of osteoblasts and osteoclasts and upset the dynamic balance.
    Osteoclasts derive from monocyte/macrophage differentiation, and are the only physiological multinucleated giant cells in the human skeletal system with the capacity of absorbing and reshaping bone morphology. The excessive bone loss caused by bone infection is not only related to the inhibition of osteoblast function, but also to the excessive activation of osteoclasts. The two cell factors of RANKL and M-CSF play an important role in the process of osteoclast differentiation.
    Activated NFATc1 promotes the expression of osteoclast marker genes, such as tartrate-resistant acid phosphatase (TRAP) and cathepsin K (CTSK) which regulates osteoclast production and phagocytosis of bone matrix. In the case of Staphylococcus aureus infection, a large number of inflammatory cells are recruited to the infected site, releasing a large number of inflammatory media to possibly promote osteoclast differentiation. In general, proinflammatory factors including TNF-a, IL-18, IL-6, IL-11, IL-17, etc. can promote bone resorption by increasing osteoclast differentiation. Recently, it has been reported that Staphylococcus aureus can directly promote osteoclast differentiation. In addition, virulence factors of Staphylococcus aureus such as peptidoglycan and lipopolysaccharide (LPS) can directly act on osteoclasts to promote their formation. In the bone infection due to LPS released by Staphylococcus aureus, a series of changes in the normal biological behaviors of osteoblasts and osteoclasts will occur, and the change of the normal bone remodeling process leads to bone non-union or excessive bone loss. Staphylococcus aureus and its toxin directly or indirectly upset the dynamic balance of bone matrix maintained by the joint effect of osteoblasts and osteoclasts, thereby inhibiting the activity of osteoblasts and inducing their apoptosis to curb the process of osteogenic differentiation; meanwhile, enhance bone resorption capacity of osteoclasts by promoting their differentiation.
    Hematopoietic cells of different origins can be induced to express interferon A1 (IFN-A1) after being infected by bacteria. IFN-A1 initiates signal transduction and plays a biological role by binding to a special heterodimeric receptor complex, and it shares the same JAK-STAT signal transduction pathway with Type | IFN and promotes the expression of a group of collaborating genes. Therefore, IFN-A1 shows some of the same properties as Type | IFN, such as anti-inflammation, anti- virus, anti-proliferation, in vivo anti-tumor, immune regulation and other biological activities. In the literature, the different monocytes were compared. !t is found that only IFN-A1 was produced when peripheral blood monocytes was stimulated by LPS, and the expression level of IFN-A1 was lower than that of immature or mature dendritic cells stimulated by LPS. However, there has been no report about the use of IFN-A1 for the treatment of inflammatory bone loss.
    Contents of the present invention For that reason, the object of the present invention is to provide the use of recombinant IFN-A1 protein in the preparation of medicines for the prevention and/or treatment of inflammatory bone loss.
    To achieve the above objective, the present invention provides the following technical proposals: The use of recombinant IFN-A1 protein in the preparation of medicines for the prevention and/or treatment of inflammatory bone loss.
    Preferably, the mediating factors of inflammatory bone loss are selected from one or more of the following: osteoclast fusion or differentiation and maturation, enhanced bone resorption capacity of mature osteoclasts, the expression of gene related to the inhibition of the different stages of osteoclast fusion, differentiation or maturation, the release of inflammatory factors, the formation of NF-kB signaling pathway, the formation of NLRP3 inflammasome mediated by HMGB1, and the activation of JAK-STAT signaling pathway in the process of osteoclast differentiation and maturation.
    A medicine for the prevention and/or treatment of inflammatory bone loss, and its active ingredient is recombinant IFN-A1 protein. The beneficial effects of the present invention are as follows: in order to solve the problem of imbalanced bone metabolism caused by excessive activation of osteoclasts triggered by the release of a large number of inflammatory factors in a state of the severe inflammatory bone loss due to bone infection, the applicant found by experiments that recombinant IFN-A1 protein can regulate the differentiation and fusion process of osteoclast and reduce the release of inflammatory factors in the process of osteoclast differentiation, thereby solving the problem of excessive activation of osteoclast in the inflammatory situation and protecting from inflammatory bone loss.
    The results showed that the recombinant IFN-A1 protein has the following characteristics within the effective dose: have no toxic effect on the monocyte macrophage line (RAW264.7) in mice; inhibit the maturation and differentiation of mouse primary bone marrow mononuclear macrophages (BMMs) and monocyte macrophage lines (RAW264.7) into osteoclasts induced by RANKL and LPS; inhibit the bone resorption capacity of matured osteoclasts in mouse primary BMMs and monocyte macrophage lines (RAW264.7) induced by RANKL and LPS; inhibit osteoclast fusion in a concentration dependent manner; significantly down-regulate the marker genes in different stages of osteoclast differentiation and maturation at mRNA level, such as CD9, PU1, etc. in the early stage, CTR, CTSK, etc.in the mature stage; inhibit NF-k B p65 nuclear translocation and negative time- dependent regulate NF-kB signaling pathway; significantly inhibit the formation of HMGB1 mediated NLRP3 inflammasome and activate JAK-STAT signaling pathway in the process of osteoclast differentiation; inhibit the release of inflammatory factors in the process of osteoclast differentiation and maturation induced by LPS; significantly protect from inflammatory bone loss caused by LPS local injection and inhibit the release of inflammatory factors in animal model in vivo. Description of drawings The following figures are provided for clear illustration of the object, technical proposals and beneficial effects of the present invention: Figure 1: the expression of IFN-A1 in infectious bone tissue is higher than that in normal bone tissue. A: the KEGG pathway analysis shows that the immune- related signaling pathway has changed significantly in the comparison between infectious and normal bone tissues; B: the result of RNA-Seq shows that the expression of IFN-A1 specific receptor IFNLR1 in infectious bone tissue is higher than that in normal bone tissue; C: the immunohistochemical staining results show that the number of IFN-A1 positive cells in infectious bone tissue is greater than that in normal bone tissue; D: the content of IFN-A1 in peripheral blood of patients with chronic hematogenous osteomyelitis is higher than that in the peripheral blood of ordinary fracture patients without infection.
    Figure 2: local injection of recombinant IFN-A1 protein can protect from inflammatory bone loss. A: the implementation step diagram of the specific animal experiments; B: the results of micro-CT imaging; C: the result of micro-CT bone parameter analysis; D: the staining result of HE and Masson slices; E: Quantitative statistical results of the staining bone tissue area in HE slice; F: the content of inflammatory factors (TNF-a, IL-18, IL-6) in peripheral blood of mice in different groups.
    Figure 3: the effect of different concentrations of recombinant IFN-A1 protein on the proliferation of RAW264.7 cells. A: the flow cytometry detection of different concentrations of recombinant IFN-A1 protein induced by RANKL and M-CSF for 72 hours; B: statistics of the flow cytometry detection result; C, D: results of cytotoxicity after 24 and 72 hours’ action, respectively. The horizontal axis is the final sampling concentration of the recombinant IFN-A1 protein, and the vertical axis is the absorbance value at 450 nm. Figure 4: the negative regulation effect of recombinant IFN-A1 protein on 5 osteoclast differentiation and bone resorption capacity. A, C, E : the TRAP staining micrographs of IFN-A1 protein inhibiting osteoclast differentiation induced by RANKL and LPS; B, D, F: the number of TRAP-positive osteoclasts in each well under the influence of recombinant IFN-A1 protein; G, H: the microscopic images and the resorption fraction statistics of the lacunae of the bone matrix collagen plate under the influence of recombinant IFN-A1 protein; I: the effect of the recombinant IFN-A1 protein on the bone resorption capacity of osteoclasts, toluidine blue staining results and statistics of resorption fraction on the bone surface.
    Figure 5: the effect of recombinant IFN-A1 protein on osteoclast formation and multinucleated osteoclast formation. A, B: the FAK immunofiuorescence staining images of recombinant IFN-A1 protein on RANKL and LPS induced osteoclast formation and the count statistics of the number of mature osteoclasts and osteoclast nuclei.
    Figure 8: the recombinant IFN-A1 protein regulates NFATc1 in the process of osteoclast differentiation. A: the immunofluorescence staining image of the nuclear translocation of NFATc1 by recombinant IFN-A1 protein; B: the quantitative statistics of fluorescence intensity in the nucleus; C: the recombinant IFN-A1 protein inhibition of osteoclast differentiation specific genes CD9, c-Fos, CTSK, PUT, NFATc1 at the mRNA levels in early stage of osteoclast differentiation; D: the recombinant IFN-A1 inhibition of osteoclast differentiation gene c-Fos, NFATct at protein level in the early stage of osteoclast differentiation; E: the recombinant IFN- A1 protein inhibition of osteoclast differentiation genes c-Fos, NFATct, CD9, CTSK, and MMP9 at the protein level in the mature osteoclast differentiation stage; F: the recombinant IFN-A1 protein inhibition of osteoclast differentiation genes c-Fos, NFATcet, mitf, CTSK, and CTR at the mRNA level in the mature osteoclast differentiation stage; G: the recombinant IFN-A1 protein inhibition of LPS induced osteoclast differentiation specific genes CTR, CTSK, OC-STAMP, mitf at the mRNA level; H: the effect of the recombinant IFN-A1 protein on LPS-induced osteoclast differentiation inflammatory factors (TNF-a, IL-1B, IL-8) at mRNA level.
    Figure 7: the recombinant IFN-A1 protein inhibit the NF-kB signaling pathway, the formation of NLRP3 inflammasomes and promote the JAK-STAT signaling pathway in the process of osteoclast differentiation. A: the immunofluorescence staining image of the recombinant IFN-A1 protein for nuclear translocation of NF-kB p65; B: the proportion of nuclear translocation positive cells; C: the quantitative statistics of the fluorescence intensity in the nucleus; D: the expression of IKB, p-IkB, p65 and p-p65 induced by RANKL for 15min, 30min, 45min and 60min with the intervention of the recombinant IFN-A1 protein; E, F: the expression of HMGB1, RAGE and NLRP3 for the recombinant IFN-A1 at the protein level in the formation of osteoclasts induced by RANKL and LPS; G, H : the expression of HMGB1 and NLRP3 for the recombinant IFN-A1 at the mRNA level in the formation of osteoclasts induced by RANKL and LPS; I: the expression of Jak, p-Jakt, Tyk2, p-Tyk2, Stat1, p-Stat1, Stat2 and p-Stat2 induced by RANKL for 15min, 30min, 45min, 60min with the intervention of the recombinant IFN-A1 protein.
    Figure 8: the schematic diagram of the signaling pathway related to the recombinant IFN-A1 protein inhibiting the fusion and differentiation of osteoclasts to protect from inflammatory bone loss.
    Detailed description of the preferred embodiment The preferred embodiment of the present invention will be described below in detail in combination with the drawings.
    RAW264.7 is mouse monocyte macrophage line and BMMs are mouse primary monocyte macrophages, which can differentiate into osteoclasts under the stimulation of RANKL and M-CSF, and are common cell models for the study of the biological behaviors of osteoclasts such as differentiation, fusion, maturation and bone resorption. Meanwhile, osteoclasts are closely related and coupled with osteoblasts, osteocyte, stromal cells etc, and are important regulatory elements of the dynamic balance of bone matrix and electrolyte homeostasis in vivo.
    Inflammatory bone loss is generally caused by excessive bone resorption due to the excessive release of inflammatory factors, which is the result of excessive activation of osteoclasts. Therefore, it is suitable to select RAW264.7 and BMMs as cell models to investigate the effect of recombinant IFN-A1 protein on osteoclast formation induced by RANKL and LPS. The recombinant IFN-A1 protein inhibition of the release of inflammatory factors and inflammation related signaling pathways can be observed to evaluate whether it inhibits the expression of osteoclast specific related genes and affects the bone resorption capacity of osteoclasts, so as to elaborate the use and mechanism of recombinant IFN-A1 protein in the preparation of medicines for the prevention and/or treatment of inflammatory bone loss. 1) Gene detection at transcriptome level in infectious and normal bone tissues Infected bone tissues (3 cases) of Staphylococcus aureus osteomyelitis and uninfected bone tissues (3 cases) of long bone fractures were collected; TRIzol was added in the presence of liquid nitrogen, and total RNA was extracted.
    TruSeq PE Cluster kit (v3-cBot-HS, lllumia) was used to generate DNA clusters on the cBot Cluster Generation system. Please refer to the instructions for specific methods. The Illumina Hiseq 4000 platform was used to sequence the library; HTSeq v0.6.1 was used to calculate the number of reads for each gene, and calculate the number of reads per kilobase length of a gene per ten thousand reads (RPKM), and the RPKM value represented the gene expression level; the differential genes between the two groups were analyzed by DE Seq R package (1.10.1), and the generated P value controlled the false positive rate by the Benjamini and Hochberg method. Genes with a change ratio greater than 1.5 and a difference probability value more than 0.8 were considered as differentially expressed genes (DEGs); the signaling pathway regulatory network was constructed based on the KEGG database.
    The results are shown in A and B of Figure 1. The KEGG pathway analysis shows that the immune-related signaling pathway changed significantly in the comparison between the infectious bone tissue and the normal bone tissue; the expression of IFN-A1 specific receptor IFNLR1 in the infectious bone tissue is higher than that in the normal bone tissue.
    2) Detection of the expression of IFN-A1 in peripheral blood and histological level (1) Immunohistochemical staining (IFN-A1): the tissue was taken out and sliced with a microtome, with the thickness of about 30 microns. Subsequently, the slices were rinsed 3 times with 0.1M phosphate buffer (PBS), for 10 minutes each time, cleaned with 50% (volume concentration) ethanol solution on the shaker for 30 minutes, washed with a mixture solution of 50% ethanol and 30% H2O2 (mass concentration) for 30 minutes, and sealed with 5% BSA blocking solution (mass concentration) for 30 minutes. After diluting the primary antibody in the antibody diluent (PBH), the slices were incubated with the primary antibody overnight at room temperature with shaking, and then incubated at 4 °C for two nights with shaking. The slices were washed 3 times with 0.1 M phosphate buffer, 10 minutes each time. The secondary antibody was diluted proportionally according to the product specification, and incubated for 2 h in the dark. The slices were washed 3 times with 0.1 M phosphate buffer, for 15 minutes each time, and incubated with DAB mixture solution for 10 minutes. 2 to 3 minutes after addition of glucose oxidase, observe the staining under the microscope; wash the slices with Tris buffer 3 times, for about 10 minutes each time. 4% gelatin (mass concentration) was applied to the slices and dried overnight. The slices were kept in 95% ethanol solution and absolute ethanol successively for 15 minutes each time, then washed with Histolene for 20 minutes, and finally covered with the slides. (2) ELISA detection of human peripheral blood serum (IFN-A1): from September 2018 to December 2018, the peripheral blood in patients with staphylococcus aureus osteomyelitis and long bone fractures without complicated infections who were treated in the Department of Orthopedics, First Affiliated Hospital of the Third Military Medical University were collected and centrifuged at 12000g, 4 °C to collect the upper serum. IFN-A1 ELISA kit of Boiss Biotechnology Limited was used for detection, and the standard curve was drawn to get the IFN-A1 content of the patients in different groups.
    Results: As shown in C and D of Figure 1, the number of IFN-A1 positive cells at the histological level was more in infectious bone tissue than in normal bone tissue; in terms of quantitative analysis, IFN-A1 content in peripheral blood of patients with chronic blood-borne osteomyelitis was higher than that in the peripheral blood of ordinary fracture patients without infection. 3) Recombinant IFN-A1 protein significantly improves inflammatory bone loss caused by local injection of LPS
    3.1 Construction of an animal model of inflammatory bone loss caused by LPS Take 18 8-week-old C57BL/6J female mice from the Animal Experiment Center of the Third Military Medical University, with body weight of 18-22 g. The recombinant IFN-A1 was purchased from Sigma in the United States. The mice were anesthetized with pentobarbital sodium (50mg/kg) is, injected with the related medicines locally in the skull and administered for 14 consecutive days. The mice were randomly divided into the following three groups, each with 6 mice; PBS group: administration of 100 uL for each mouse; LPS group: administration of 100 pL (10mg / kg) for each mouse; LPS+ IFN-A1: administration of 100 pl (0.2mg/kg IFN-A1 + 10mg / kg LPS) for each mouse;
    3.2. Detection of bone quality parameters After 14 days of administration, the skull of each mouse was separated, the extra intracranial tissue and surrounding soft tissue were removed and fixed. Blood samples were collected from eyeballs. The bone microstructure was detected by micro CT in small animals. The tube voltage was 50kV, and the tube current was
    0.1mA, with the resolution of 8mm. The scanning area of skull was defined as a circle with a radius of 3cm, and the middle point of cranial sutures was the center of the circle. 3D parameters of the selected region of interest (ROI) were used for data analysis, including the number, thickness, separation degree of bone trabecula and relative bone volume fraction. Blood samples were used to analyze the levels of inflammatory factors including TNF-a, IL-18 and IL-6. Results: As shown in Figure 2, compared with the recessive vehicle group (PBS group), the bone mineral density and bone volume fraction of mice in LPS group decreased significantly, meanwhile, the number of bone trabecula and relative bone volume fraction also decreased. However, the number of bone trabecula and the relative bone volume fraction of mice treated with IFN-A1 were significantly higher than those of LPS group. Moreover, the release of inflammatory factors in IFN-A1 intervention conditions was significantly inhibited. The results were statistically significant (P < 0.05). Therefore, it was concluded that IFN-A1 can protect from inflammatory bone loss induced by LPS and inhibit the release of inflammatory factors. 4) Different concentrations of recombinant IFN-A1 protein are not toxic to osteoclast precursors Flow cytometry: RAW264.7 cells were exposed to different concentrations of IFN-A1 (0 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL) for 48 hours, and the apoptosis level was detected after phagocytosis; 100X buffer was diluted to 1X in advance and pre-cooled at 4 °Caccording to the instructions of BD flow cytometry kit; the supernatant of culture medium in the well plate was collected and put into a
    15ml centrifuge tube, and 1 mi TrypLE was added into each well, digesting at 37 °C for 3 min; blow the bottom of the well plate to disperse the cells, move the digested cell suspension into the corresponding 15 mi centrifuge tube, and centrifuge at 200 g / min for 5 min; discard the supernatant and add the pre-prepared 1xbuffer for resuspending; count by Trypan blue and each sample was unified into 108 / mi with 1xbuffer; 100 pL of each sample was incubated with 5 pL propidium iodide (Pl) and 5 pL annexin V for 15 minutes at room temperature in the dark; after the incubation, 300 pL 1 x buffer was added to each sample for terminating the reaction; 96-mesh copper screen was used to filter, and the fluorescence intensity Pl and APC of each sample were detected by flow cytometry within 1 hour.
    The debris in the lower left corner was removed on FSC/SSC interface by Flow Jo software.
    The fluorescence intensity of PI and APC channels was analyzed, and the proportion of cells in each quadrant was calculated.
    CCK-8 detection: RAW264.7 cells were inoculated in a 96-well plate with 1 x 103 / well, and the medium was discarded after the cells were full.
    The medium components were DMEM high glucose culture solution + 10% volume percentage of fetal bovine serum + 1% mass concentration of double antibody (mycillin). IFN- A1 with the final concentration of 0 (blank vehicle group), 10 ng/mL, 25ng/mL, 50 ng/mL,100 ng/mL and 200 ng/mL was added to each well plate containing fresh medium, and incubated for 24h and 72h, respectively.
    The cell viability was detected by CCK-8 method, and the effect of IFN-A1 on proliferation of RAW264.7 cells was observed.
    Results: As shown in A and B of Figure 3, the {otal number of apoptotic cells detected by flow cytometry did not change significantly under different concentrations of IFN-A1. In CCK-8 cytotoxicity detection, IFN-A1 with different concentrations (less than 200 ng/mL) had no significant effect on the cell proliferation in the process of osteoclasts differentiation in 24h or 72h groups. 5) Recombinant IFN-A1 protein reduces the generation of TRAP-positive cells induced by RANKL and LPS Grouping: Group 1: RANKL (0 ng/mL) induction, vehicle group without IFN-A1; Group 2: RANKL (50 ng/mL) induction, vehicle group without IFN-A1; Group 3: RANKL (50 ng/mL) induction, 100 ng/mL IFN-A1 experimental group;
    Group 4: LPS (100 ng/mL) induction, experimental group without IFN-A1; Group 5: LPS (100 ng/mL) induction, 100 ng/mL IFN-A1 experimental group; Inoculate RAW264.7 and BMM cells in a 96-well plate with 3x10%/well. After the cells grow up, discard the culture medium. The components of the culture medium were DMEM high-glucose culture solution and a-MEM + 10% volume percentage of fetus bovine serum + 1% mass concentration of double antibody (myecillin). IEN-A1 with a final concentration of 100 ng/mL was added to each well plate containing fresh medium. Meanwhile, RAW264.7 or BMM cells were induce to differentiate into osteoclasts by RANKL (50 ng/mL), M-CSF (50 ng/mL) or LPS (100 ng/mL).
    TRAP staining was performed after 72h or 120h of induction culture. After discarding the medium, wash with PBS 3 times, each time for 3 minutes. Fix with 4% mass concentration of paraformaldehyde for 20 minutes, wash with PBS 3 times, each time for 3 minutes. Dye with TRAP staining solution (0.1 mg/ml of naphthol AS-MX phosphate, 0.3 mg/ml of Fast Red Violet LB counterstain) for 1h in the dark, and discard the staining solution. Rinse with PBS 3 times, and add 100 pL PBS to each well for observation. With the light microscope, it can be found that TRAP-positive cells were stained purple (A, C, E of Figure 4). Count the number of TRAP-positive cells based on the quantity of nuclei, and separately count the total number of TRAP-positive cells (B, D, F of Figure 4).
    Results: With the intervention of IFN-A1, the number of TRAP-positive mature osteoclasts showed the same trend of inhibitory effect, demonstrating that IFN-A1 inhibits the formation of osteoclasts in a concentration-dependent manner (**P<0.01).
    6) Recombinant IFN-A1 protein inhibits the bone resorption capacity of mature osteoclasts induced by RANKL and LPS Grouping: Group 1: RANKL (0 ng/mL) induction, vehicle group without IFN-A1; Group 2: RANKL (50 ng/mL) induction, vehicle group without IFN-A1; Group 3: RANKL (50 ng/mL) induction, 100 ng/mL IFN-A1 experimental group; Group 4: LPS (100 ng/mL) induction, experimental group without IFN-M1; Group 5: LPS (100 ng/mL) induction, 100 ng/mL IFN-A1 experimental group; RAW264.7 and BMM cells were inoculated on a bone matrix collagen plate and a 48-well plate covered with 200 um calf bone slices with 4x103%/well. The culture medium was discarded after the cells grew up. The components of the medium were DMEM high-glucose culture solution + 10% volume percentage of fetal bovine serum + 1% mass concentration of double antibody (mycillin). 1FN-A1 with a final concentration of 100 ng/mL was added to each well plate containing fresh medium. Meanwhile, RAW264.7 and BMM cells were induced to differentiate into osteoclasts by RANKL {50 ng/mL), M-CSF {50 ng/mL) or LPS (excluding the blank vehicle group). After 96 hours, take out the 48-well plate containing calf bone slices and discard the medium. Bleach with the mass concentration of 10% bleaching agent (HCIO) for 5 minutes at room temperature. Wash with double distilled water 3 times, each time for 5min, Dry at room temperature for 3-5 hours. Dye with toluidine blue staining solution for 5 min. Rinse with double distilled water 3-5 times, each time for Smin. Add 200 ul of double distilled water or 1xPBS to each well and observe with the light microscope. The bone surface can be dyed blue by toluidine blue after being absorbed by osteoclasts, therefore, the bone resorption capacity of osteoclasts is evaluated according to the size of the blue bone resorption area and the percentage of the total surface area of bone slice. The bone matrix collagen plate was bleached with a mass concentration of 10% bleach (HCIO) for 5 minutes at room temperature, washed with double distilled water 3 times, 5min each time, and observed with the light microscope at room temperature under dry conditions.
    Results: The blank vehicle group without RANKL or LPS induction had no bone resorption. After addition of RANKL (50 ng/mL), M-CSF (50 ng/mL) or LPS induction, the proportion of bone resorption area in the vehicle group without IFN- A1 was the largest compared with the experimental group (**P <0.01). With the intervention of IFN-A1, the bone resorption capacity of osteoclasts decreased significantly in each experimental group (**P<0.01). 7) Recombinant IFN-A1 protein inhibits the fusion of RANKL and LPS induced multinucleated mature osteoclasts Grouping: Group 1: RANKL {0 ng/mL) induction, vehicle group without IFN-A1; Group 2: RANKL (50 ng/mL) induction, vehicle group without IFN-A1; Group 3: RANKL (50 ng/ml) induction, 100 ng/mL IEN-A1 experimental group; Group 4: LPS (100 ng/mL) induction, experimental group without IFN-A1;
    Group 5: LPS (100 ng/mL) induction, 100 ng/mL IFN-A1 experimental group; RAW264.7 and BMM cells were inoculated in a 96-well plate with 2x10%well. The culture medium was discarded after the cells grew up. The components of the culture medium were DMEM high-glucose culture solution + 10% volume percentage of fetus bovine serum + 1% mass concentration of double antibody (mycillin). IFN-A1 with a final concentration of 0, 100 ng/mL was added to each well plate containing fresh medium. Meanwhile, RAW264.7 or BMM cells were induce to differentiate into osteoclasts by RANKL (50 ng/mL), M-CSF (50 ng/mL) or LPS. FAK (Actin Cytoskeleton and Focal Adhesion Staining) immunotfluorescence staining was performed after 72h or 120h of induction culture. The culture medium was discarded, and the cells were taken out, washed twice with 1x PBS, fixed in 4% paraformaldehyde for 20 min at room temperature, and washed twice with 1x PBS. Penetrate the cells with a mass concentration of 0.1% Triton X-100 for 5 minutes, and wash twice with 1xPBS. Fix in blocking buffer (mass concentration 1% BSA in 1xPBS) for 30 min. The primary antibody (Anti- Vincpling was diluted to the working concentration (volume ratio 1:300) in blocking buffer, incubated the cells for 1h at room temperature, Wash three times with 1x washing buffer (mass concentration 0.05% Tween-20 in 1x PBS), each time for 5- 10min. The secondary antibody (Alexa Fluor 488 Goat Anti-Mouse IgG (H+L) antibody, Invitrogen) was diluted to working concentration (volume ratio 1:500), and TRITC-labeled phalloidin (volume ratio 1:500) was added. Incubate together for th at room temperature. Wash three times with 1x wash buffer, each time for 5-10min. Counterstain of the nuclei with DAPI (volume ratio 1:1000} was conducted for 5 min at room temperature. Wash three times with 1x washing buffer, each time for 5-10 min. Observe the cells with a fluorescence microscope. Count the number of osteoclasts and the average nucleus number of osteoclasts in the field of view.
    Results: With the intervention of 100 ng/mL IFN-A1, the formation of multinucleated osteoclasts (nucleus number was greater than 3) was significantly inhibited, “*P<0.01 {Figure 5). Moreover , the number of mature osteoclast nuclei decreased, "P<0.01 (Figure 5).
    8) Recombinant IFN-A1 protein inhibits RANKL-induced nuclear translocation of NFATc1 Grouping: Group 1: RANKL (0 ng/mL) induction, vehicle group without IFN-A1;
    Group 2: RANKL (50 ng/mL) induction, vehicle group without IFN-A1; Group 3: RANKL (50 ng/mL) induction, 100 ng/mL IFN-A1 experimental group.
    Inoculate RAW264.7 cells in a 48-well plate (5x10%well.) covered with glass slides. After the cells grow up, discard the culture medium. The components of the culture medium were DMEM high-glucose culture solution + 10% volume percentage of fetus bovine serum + 1% mass concentration of double antibody (mycillin). IFN-A1 with a final concentration of 0, 100 ng/mL was added to each well plate containing fresh medium. Meanwhile, RAW264.7 cells were induced by RANKL (50 ng/mL), M-CSF (50 ng/mL). The immunofluorescent staining was performed after 24h induction culture. The culture medium was discarded, and the cells were taken out, washed twice with 1x PBS, fixed in 4% mass concentration of paraformaldehyde for 20 min at room temperature, and washed twice with 1x PBS. Penetrate the cells with 0.1% mass concentration of Triton X-100 for 5 minutes, and wash twice with 1xPBS. Fix in blocking buffer (1% mass concentration of BSA in 1xPBS) for 30 min. The primary antibody (Anti-NFATc1) was diluted to the working concentration (volume ratio 1:300) in blocking buffer, incubated the cells for 1h at room temperature. Wash three times with 1x washing buffer (0.05% mass concentration of Tween-20 in 1x PBS), each time for 5-10min. The secondary antibody (Alexa Fluor 488 Goat Anti-Mouse IgG (H+L) antibody, Invitrogen) was diluted to working concentration (volume ratio 1:500), incubated together for 1h at room temperature. Wash three times with 1x wash buffer, each time for 5-10min. Counterstain of the nuclei with DAPI (volume ratio 1:1000) was conducted for 5 min at room temperature. Wash three times with 1x washing buffer, each time for 5-10 min. Observe with laser confocal microscope. Count the number of the nucleus containing NFATc1 and the average fluorescence intensity in the nucleus.
    Results: As shown in A and B of Figure 6, the average fluorescence intensity in the nucleus showed a strong inhibitory effect with the intervention of 100 ng/mL IFN-A1.
    9) Recombinant IFN-A1 protein inhibits the expression of specific genes at different stages of osteoclast differentiation Grouping: Group 1: RANKL (0 ng/mL) induction, vehicle group without IFN-A1; Group 2: RANKL (50 ng/mL) induction, vehicle group without IFN-A1;
    Group 3: RANKL (50 ng/mL) induction, 100 ng/mL IFN-A1 experimental group; Group 4: LPS (100 ng/mL) induction, experimental group without IFN-A1; Group 5: LPS (100 ng/mL) induction, 100 ng/mL IFN-A1 experimental group.
    Inoculate RAW264.7 cells in a 6-well plate with 1x10%well, and discard the medium after the cells grow up.
    The components of the culture medium were DMEM high-glucose culture solution + 10% volume percentage of fetus bovine serum + 1% mass concentration of double antibody (mycillin). IFN-A1 with a final concentration of 0, 100 ng/mL was added to each well plate containing fresh medium.
    Meanwhile, RAW264.7 cells were induced to differentiate into osteoclasts by RANKL (50 ng/mL), M-CSF (50 ng/mL) or 100 ng/mL LPS.
    After induction culture for 24h and 72h, take out the cells, discard the medium.
    Lyse the cells with Trizol, extract total RNA in the cells.
    Detect the target genes Ctsk, c-Fos, NFATc1, CTR, mitf, OC-STAMP, PU1, CD9 by real-time quantitative RT-PCR and Western Blot, and B-actin was used as internal reference control.
    The amplification was performed with CFX96 touch quantitative PCR system.
    For each well, add 7.5 pL SYBR (1), 5.1 uL water (PCR), 1.2 yl. cDNA, 0.6 yl of each upper and lower primers, and the reaction system was 15 yl.
    Each group had 3 replicate wells.
    Reaction conditions: (1) 95°C, 30s; (2) 40 cycles, 95°C, 5s; 60°C, 30s; (3) 80 cycles, 60°C, 30s; 55°C, 10s; the relative content of the target gene was automatically calculated by PCR instrument, and analyzed by the 2-22CT method.
    The primer sequence is shown in Table 1. Genes Forward Reverse PU.1 5'-GATGGAGAAGCTGATGGCTTGG-3' S-TTCTTCACCTCGCCTGTCTTGC-3' CTR 5-CGCATCCGCTTGAATGTG-3' 5.TC TGTCTTTCCCCAGGAAATGA-3' cD9 5-CGGTCAAAGGAGGTAG-3' 5-GGAGCCATAGTCCAATA-3' mitf S-AACTCCTGTCCAGCCAACCTTC-3' 5-TCTGCCTCTCTTTAGCCAATGC-3' OC-STAMP ~~ 5-GGGCTACTGGCATTGCTCTTAGT-3 5-CCAGAACCTTATATGAGGCGTCA-3' CTSK 5-GAAGAAGACTCACCAGAAGCAG-3 5'-TCCAGGTTATGGGCAGAGATT-3° c-Fos 5-CGGGTTTCAACGCCGACTA-3' 5 -TTGGCACTAGAGACGGACAGA-3' NFATc1 5 -CCCGTCACATTCTGGTCCAT-3' 5 -CAAGTAACCGTGTAGCTGCACAA-3' B-actin 5 -TCCCTGTATGCCTCTG-3 5- ATGTCACGCACGATTT-3' Table 1 Primer sequence
    Results: As shown in C and D of Figure 6, 100 ng/mL IEN-A1 inhibited the expression of early osteoclast markers Cisk, c-Fos, PU1, CD9, and NFATct at the mRNA level and inhibited the expression of c-Fos and NFATc1 at the protein level
    (”P<0,01). E and F of Figure 6 showed that 100 ng/mL IFN-A1 inhibited the expression of mature osteoclast markers Cisk, c-Fos, NFATc1, CTR and mitf at the mRNA level (**P < 0.01), and inhibited the expression of Cisk, CD9, MMPS, c-Fos and NFATc1 at the protein level.
    It can be concluded that IFN-A1 inhibits the differentiation, formation, maturation and bone resorption capacity of osteoclasts by curbing the expression of osteoclast differentiation-specific genes. 10) Recombinant IFN-A1 protein inhibits NF-kB signaling pathway in the process of osteoclast differentiation
    (1) Recombinant IFN-A1 protein inhibits RANKL-induced nuclear translocation of NF-kB p65
    Grouping:
    Group 1: RANKL (0 ng/ml) induction, vehicle group without IFN-A1;
    Group 2: RANKL (50 ng/mL) induction, vehicle group without IFN-A1;
    Group 3: RANKL (50 ng/mL) induction, 100 ng/mL IFN-A1 experimental group,
    Inoculate RAW264.7 cells in a 48-well plate (5x 103/well.) covered with glass slides.
    After the cells grow up, discard the culture medium.
    The components of the culture medium were DMEM high-glucose culture solution + 10% volume percentage of fetus bovine serum + 1% mass concentration of double antibody {mycillin). IFN-A1 with a final concentration of 0, 100 ng/mL was added to each well plate containing fresh medium.
    Meanwhile, RAW264.7 cells were induced by RANKL (50 ng/mL), M-CSF (50 ng/mL). The immunofluorescent staining was performed after 24h induction culture.
    The culture medium was discarded, and the cells were taken out, washed twice with 1x PBS, fixed in 4% mass concentration of paraformaldehyde for 20 min at room temperature, and washed twice with 1x PBS.
    Penetrate the cells with 0.1% mass concentration of Triton X-100 for 5 minutes, and wash twice with 1xPBS.
    Fix in blocking buffer (1% BSA in 1xPBS) for 30 min.
    The primary antibody (Anti-NF-kB p65) was diluted to the working concentration
    {volume ratio 1:300} in blocking buffer, incubated the cells for 1h at room temperature. Wash three times with 1x washing buffer (0.05% mass concentration of Tween-20 in 1x PBS), each time for 5-10min. The secondary antibody (Alexa Fluor 647 Goat Anti-Mouse IgG (H+L) antibody, Invitrogen) was diluted to working concentration (volume ratio 1:500), incubated together for 1h at room temperature. Wash three times with 1x wash buffer, each time for 5-10min. Counterstain of the nuclei with DAPI (volume ratio 1:1000) was conducted for 5 min at room temperature. Wash three times with 1x washing buffer, each time for 5-10 min. Observe with laser confocal microscope. Count the number of positive cells containing NF-kB p65 in the nucleus and the average fluorescence intensity in the nucleus.
    Results: As shown in A, B and C of Figure 7, with the intervention of 100 ng/mL IFN-A1, the average fluorescence intensity in the nucleus showed a strong inhibitory effect and the number of positive cells containing NF-kB p65 in the nucleus was gradually decreased.
    (2) Recombinant IFN-A1 protein inhibits NF-kB p65 signaling pathway Grouping: Group 1: RANKL (50 ng/mL) induction for15min; Group 2: RANKL (50 ng/mL) induction for 30min; Group 3: RANKL (50 ng/mL) induction for 45min; Group 4: RANKL (50 ng/mL) induction for 60min; Group 5: RANKL (50 ng/mL) + 100 ng/mL IFN-A1 induction for 15min; Group 6: RANKL (50 ng/mL) + 100 ng/mL IFN-A1 induction for 30min; Group 7: RANKL (50 ng/mL) + 100 ng/mL IFN-A1 induction for 45min; Group 8: RANKL (50 ng/mL) + 100 ng/mL IFN-A1 induction for 60min RAW264.7 cells were inoculated in 6-well plates containing RNAKL and stimulated with 100 ng/mL RANKL. Lyse the cells with radioimmunoprecipitation (RIPA) lysis buffer. The protein was separated by SDS-polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride (PVDF) membrane, sealed in 5% mass concentration of skim milk for 1 hour, and then gently shaken with various specific primary antibodies (IkB, p-IkB, p65, p-p65) at 4 °C overnight, followed by incubating the membrane with a secondary antibody conjugated with horseradish peroxidase (HRP). The antibody reactivity was detected by the enhanced chemiluminescence reagent (United States Pharmacopoeia
    Biotechnology Company, Piscataway, NJ), and displayed on the image quantification LAS 4000. Results: As shown in D of Figure 7, the expression of p-IkB and p-p65 showed a time-dependent inhibitory effect with the intervention of 100 ng/mL IFN-
    AL 11) Recombinant IFN-A1 protein inhibits HMGB1-mediated NLRP3 inflammasome formation and the mechanism of inflammatory factor release Grouping: Group 1: RANKL (0 ng/mL) induction, vehicle group without IFN-A1; Group 2: RANKL (50 ng/mL) induction, vehicle group without IFN-A1; Group 3: RANKL {50 ng/ml.) induction, 100 ng/mL IFN-A1 experimental group; Group 4: LPS (100 ng/mL) induction, experimental group without IFN-A1; Group 5: LPS (100 ng/mL) induction, 100 ng/mL IFN-A1 experimental group; inoculate RAW264.7 cells in a 6-well plate with 1x10%well, and discard the medium after the cells grow up. The components of the culture medium were DMEM high-glucose culture solution + 10% volume percentage of fetus bovine serum + 1% mass concentration of double antibody (mycillin). IFN-A1 with a final concentration of 0, 100 ng/mL was added tc each well plate containing fresh medium. Meanwhile, RAW264.7 cells were induced to differentiate into osteoclasts by RANKL (50 ng/mL), M-CSF (50 ng/mL) or 100 ng/mL LPS. After induction culture for 24h and72h, take out the cells, discard the medium. Lyse the cells with Trizol, extract total RNA in the cells. Detect the target genes NLRP3, HMGB, IL-6, 1-18, TNF-a by real-time quantitative RT-PCR and Western Blot, and B-actin was used as internal reference control. The amplification was performed with CFX96 touch quantitative PCR system. For each well, add 7.5 uL SYBR (ll), 5.1 uL water {PCR}, 1.2 uL cDNA, 0.6 uL of each upper and lower primers, and the reaction system was 15 uL. Each group had 3 replicate wells. Reaction conditions: (1) 95°C, 30s; {2} 40 cycles, 95°C, 5s; 60°C, 30s; (3) 80 cycles, 60°C, 30s; 55°C, 10s; the relative content of the target gene was automatically calculated by PCR instrument, and analyzed by the 22ACT method. The primer sequence was shown in Table 2.
    Table 2 Primer sequence Genes Forward Reverse HMGB1 5 -TATCTAAATACGGATTGCTCAGGAA-3 5 -AGGGACAAACCACAATATAGGARAA-3 NLRP3 5'-GATCTTCGCTGCGATCAACAG-3 5 -CGTGCATTATCTGAACCCCAC-3 TNF-a 5 -AGGCGGTGCTTGTTCCTCA-3 5’ -AGGCGAGAAGATGATCTGACTGCC-3 IL-18 5'-CTOAACTGTGAAATGCCACC-3' 5 -TGTCCTCATCCTGGAAGGT-3 IL-6 5 -TGGGAAATCGTGGAAATGAGA-3 5 -ACTCTGGCTTTGTCTTTCTTGT-3 B-actin 5 -TCCCTGTATGCCTCTG-3 5’- ATGTCACGCACGATTT-3' Results: As shown in E, F, G and H of Figure 7, the expression of NLRP3 and HMGB1 showed a potent inhibitory effect at the mRNA and protein levels with the intervention of 100 ng/mL IFN-A1. Moreover, the expression of inflammatory factor IL -6, IL-18, TNF-a was also down-regulated at the mRNA level. 12) Recombinant IFN-A1 protein activates JAK-STAT signaling pathway in the process of osteoclast differentiation Grouping: Group 1: RANKL (50 ng/mL) induction for15min; Group 2: RANKL (50 ng/mL) induction for 30min; Group 3: RANKL (50 ng/ml) induction for 45min; Group 4: RANKL (50 ng/mL) induction for 60min; Group 5: RANKL (50 ng/mL) + 100 ng/mL IFN-A1 induction for 15min; Group 6: RANKL (50 ng/ml) + 100 ng/mL IFN-A1 induction for 30min; Group 7: RANKL (50 ng/mL) + 100 ng/mL IFN-A1 induction for 45min; Group 8: RANKL (50 ng/mL) + 100 ng/mL IEN-A1 induction for 60min RAW284.7 cells were inoculated in 6-well plates containing RNAKL and stimulated with 100 ng/mL RANKL.
    Lyse the cells with radicimmunoprecipitation (RIPA) lysis buffer.
    The protein was separated by SDS-polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride (PVDF) membrane, sealed in 5% mass concentration of skim milk for 1 hour, and then gently shaken with various specific primary antibodies (Jak1, p-Jaki, Tyk2, p-Tyk2, Stall, p-Stati, Stat2, p-Stat2) at 4°C overnight, followed by incubating the PVDF membrane with a secondary antibody conjugated with horseradish peroxidase (HRP). The antibody reactivity was detected by the enhanced chemiluminescence reagent (United States Pharmacopoeia Biotechnology Company, Piscataway, NJ), and displayed on the image quantification LAS 4000.
    Results: As shown in | of Figure 7, the expression of p-Jak1, p-Tyk2, p- Stat1 and p-Stat2 showed potent promoting effect at the protein level with the intervention of 100 ng/mL IFN-A1, indicating that IFN-A1 can activate the JAK-STAT signaling pathway in the formation of osteoclasts.
    Results: As shown in Figure 8, the differentiation and fusion of osteoclasts are inhibited through the JAK-STAT and NF-kB signaling pathway with the intervention of IFN-A1, thus achieving the effect of protecting from inflammatory bone loss.
    Finally, it should be noted that the above preferred embodiment is only used to illustrate the technical proposals of the present invention, not a limitation.
    Although the present invention has been described in detail through the above preferred embodiment, the technicians in this field should understand that it is possible to make various changes to it in form and detail, which is covered in the protective scope of the claims of the present invention
    PCC200172NL-QINGF -Gene sequence list
    SEQUENCE LISTING <110> Army Medical University <120> Use of IFN -|E1l protein target in the preparation of medicines for the prevention and/or treatment of inflammatory bone loss <140> 2027028 <141> 02 December 2020 <160> 28 <170> PatentIn version 3.5 <21e> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PU.1 <400> 1 gatggagaag ctgatggctt gg 22 <2105 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PU.1 <400> 2 ttcttcacct cgcctgtctt gc 22 <2105 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> CTR <400> 3 cgcatccgct tgaatgtg 18 <2105 4 <211> 22 Pagina 1
    PCC200172NL-QINGF -Gene sequence list <212> DNA <213> Artificial Sequence <220> <223> CTR <400> 4 tctgtctttc cccaggaaat ga 22 <216> 5 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> (D9 <400> 5 cggtcaaagg aggtag 16 <210> 6 <211> © <212> DNA <213> Artificial Sequence <220> <223> GGAGCCATAGTCCAATA <400> 6 000 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> mitf <400> 7 aactcctgtc cagccaacct tc 22 <2105 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> mitf
    Pagina 2
    PCC200172NL-QINGF -Gene sequence list <400> 8 tctgcctctc tttagccaat gc 22 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> OC-STAMP <400> 9 gggctactgg cattgctctt agt 23 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> OC-STAMP <400> 10 ccagaacctt atatgaggcg tca 23 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CTSK <400> 11 gaagaagact caccagaagc ag 22 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CTSK <400> 12 tccaggttat gggcagagat t 21 Pagina 3
    PCC200172NL-QINGF -Gene sequence list <210> 13 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> c-Fos <400> 13 cgggtttcaa cgccgacta 19 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> c-Fos <400> 14 ttggcactag agacggacag a 21 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NFATcl <400> 15 cccgtcacat tctggtccat 20 <210> 16 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> NFATcl <400> 16 caagtaaccg tgtagctgca caa 23 <210> 17 <211> 25 <212> DNA
    Pagina 4
    PCC200172NL-QINGF -Gene sequence list <213> Artificial Sequence <220> <223> HMGB1 <400> 17 tatctaaata cggattgctc aggaa 25 <210> 18 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> HMGB1 <400> 18 agggacaaac cacaatatag gaaaa 25 <210> 19 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NLRP3 <400> 19 gatcttcgct gcgatcaaca g 21 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NLRP3 <400> 20 cgtgcattat ctgaacccca c 21 <210> 21 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> TNF-|Á
    Pagina 5
    PCC200172NL-QINGF -Gene sequence list <400> 21 aggcggtgct tgttcctca 19 <210> 22 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> TNF-|Á <400> 22 aggcgagaag atgatctgac tgcc 24 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 1IL-1,A <400> 23 ctcaactgtg aaatgccacc 20 <210> 24 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 1IL-1,A <400> 24 tgtcctcatc ctggaaggt 19 <210> 25 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> IL-6 <400> 25 tgggaaatcg tggaaatgag a 21 Pagina 6
    PCC200172NL-QINGF -Gene sequence list <210> 26 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> IL-6 <400> 26 actctggctt tgtctttctt gt 22 <210> 27 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> |Â-actin <400> 27 tccctgtatg cctctg 16 <210> 28 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> |Â-actin <400> 28 atgtcacgca cgattt 16 Pagina 7
    权利要求:
    Claims (3)
    [1]
    Use of recombinant IFN-A1 protein in the manufacture of medicaments for the prevention and/or treatment of bone loss due to inflammation.
    [2]
    Use according to claim 1, wherein the inflammatory bone loss factors are selected from one or more of the following, i.e. osteoclast fusion or differentiation and maturation, increased bone resorption capacity of mature osteoclasts, expression of a gene related to the different stages of osteoclastius, differentiation or maturation, release of inflammatory factors, activation of an NF-kB signaling pathway, NLRP3 inflammasome formation acting by HMGB1, and the change in the activation of a JAK-STAT signaling pathway during the process of osteoclast differentiation and maturation.
    [3]
    3. Medications for preventing and/or treating bone loss due to inflammation, wherein recombinant IFN-A1 protein is the active ingredient.
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    同族专利:
    公开号 | 公开日
    CN110882381A|2020-03-17|
    CN110882381B|2020-11-17|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    AU2003243223A1|2002-05-10|2003-11-11|The Trustees Of The University Of Pennsylvania|Stimulation by toll-like receptors inhibits osteoclast differentiation|
    CN1962873B|2005-11-09|2010-12-08|中国医学科学院基础医学研究所|Expression method of IFN-lambada 1 and its special expression vector and engineering bacterium|
    CN102512666A|2011-12-15|2012-06-27|武汉大学|Application of interferon lambda1 in preparation of anti-enterovirus 71 medicines|
    KR101704589B1|2016-07-12|2017-02-08|주식회사 뉴트라팜텍|Composition comprising extracts of Magnolia flower and Magnolia officinlis for preventing or treating periodentitis as an active ingredient|
    GB201621728D0|2016-12-20|2017-02-01|Ucb Biopharma Sprl|Methods|
    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    CN201911310840.7A|CN110882381B|2019-12-18|2019-12-18|Application of recombinant IFN-lambda 1 protein in preparation of medicines for preventing and treating inflammatory bone loss|
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