Application of ombarizumab in preparation of medicine for treating bone destruction

Abstract

The invention discloses an application of omaszumab in preparation of a medicine for treating bone destruction, discovers that a spinal proliferation type schizocyst patient accompanied by serious bone destruction is the first case in the world, and discovers that IgE is combined with Fc epsilon RII on OPCs cell membranes to mediate an AKT / ERK signal channel through sequencing and experiments, so that OPCs are promoted to be differentiated into osteoclasts, and the osteoclast differentiation is promoted. The omaszumab can inhibit the osteoclast promoting effect of SPS-derived plasma, can inhibit bone destruction of SPS patients, and can delay the disease progression of the patients.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of omalizumab in preparing a drug for treating bone destruction. Background Art

[0002] Spinal proliferative sparganosis (SPS) is a parasitic disease caused by the larvae of the tapeworm Spirometra mansoni, the sparganosis. It is a type of sparganosis (the "spinal" indicates that the infection primarily affects spinal tissues). Essentially, sparganosis invades the perivertebral area or the spinal canal, causing local tissue damage and nerve compression. Although clinically rare, the consequences can be serious. Currently, there have been 28 reports of intraspinal sparganosis, and two cases of bone-involving SPS have been reported: one in the thoracic vertebra 10 and the other in the sacrum. Neither patient had the proliferative form; the lesions were localized, with minimal bone destruction. Surgery combined with medication (antiparasitic drugs, such as albendazole or praziquantel) was sufficient to cure the disease.

[0003] However, the proliferative sparganosis has the ability to reproduce asexually and can reproduce by budding in the bone. Neither drugs nor surgery can eradicate the sparganosis. Summary of the Invention

[0004] The purpose of the present invention is to overcome the defect in the prior art of lacking drugs for treating bone destruction caused by spinal proliferative sparganosis.

[0005] In order to achieve the above-mentioned object, the present invention provides a use of omalizumab in the preparation of a drug for treating bone destruction.

[0006] Optionally, the bone destruction is caused by infection with Spinae diabrosa.

[0007] Optionally, the bone destruction is caused by infection with sparganosis.

[0008] Optionally, the bone destruction is characterized as bone destruction caused by specific increase in IgE.

[0009] Optionally, the bone destruction is characterized by aberrant activation of plasma cells.

[0010] Optionally, said bone destruction is induced by IgE.

[0011] Optionally, the IgE can promote the differentiation of OPCs into osteoclasts.

[0012] Optionally, the bone destruction is mediated by the IgE through binding to FcεRII on the cell membrane of OPCs, thereby promoting the differentiation of OPCs into osteoclasts.

[0013] Optionally, the bone destruction at least includes osteoporosis and / or pathological fractures caused by hyper-IgEemia.

[0014] Optionally, the drug further comprises a pharmaceutically acceptable carrier.

[0015] Compared with the prior art, the beneficial effects of the present invention include at least:

[0016] The present invention discovered the world's first patient with spinal proliferative sparganosis infection, who presented with severe bone destruction. Through sequencing and experiments, it was found that IgE mediated the AKT / ERK signaling pathway by binding to FcεRII on the OPCs cell membrane, promoting the differentiation of OPCs into osteoclasts. Omalizumab can inhibit the pro-osteoclast effect of SPS-derived plasma, thereby inhibiting bone destruction in SPS patients and delaying disease progression. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Figures showing severe vertebral bone destruction caused by SPS in patients identified in this study. A shows lumbar X-ray and CT scans; B shows lumbar MRI scans; C shows PET-CT scans; D shows a schematic diagram of some parasite bodies and eggs removed during decompression surgery; E shows HE staining of the lesion's bone tissue; Bar scale: 1000μm and 100μm; F shows postoperative X-rays and CT scans.

[0018] Figure 2 This is a schematic diagram of the affected segments of SPS bone destruction from the sacrum to the cervical spine over a period of ten years.

[0019] Among them, AB are imaging data showing that the internal fixation of an SPS patient loosened due to vertebral erosion by parasites in 2017; CD are preoperative imaging examinations in 2021; E is the sparganosis exposed during surgery; F is metagenomic sequencing showing sparganosis infection; G is the HE staining image of the lesion bone tissue; Bar Scale: 2000 μm and 200 μm; HI are imaging examinations in 2023 (H) and 2024 (I).

[0020] Figure 3Figure 1 shows that SPS-derived monocytes of the present invention are more likely to differentiate into osteoclasts. Figure A shows the t-SNE plot of the single-cell transcriptome of PBMCs from SPS (right) and a healthy control group of the same age and sex (left); Figure B shows the fold change in the proportion of all cell subpopulations in PBMCs (SPS / Ctrl), with plasma cells (group 7) increasing in the SPS group and being the cell subpopulation with the largest change; Figure C shows the KEGG enrichment analysis of differentially expressed genes between classical monocytes in the SPS group and the healthy control group, showing significant expression in the "Osteoclast Differentiation" signaling pathway; and Figure D shows the expression levels of differentially expressed genes in the "Osteoclast Differentiation" pathway.

[0021] Figure 4 This is a schematic diagram showing that the SPS-derived OPCs of the present invention have a strong ability to promote osteoclast differentiation.

[0022] Figure 5 The SPS plasma of the present invention breaks the balance diagram of bone resorption and bone formation by enhancing the activity of OPCs.

[0023] Figure 6 Figure 1 shows the results of high IgE expression in SPS patients. Figure A shows Human Phenotype Ontology enrichment analysis of differentially expressed genes in SPS-derived plasma cells; Figure B shows ELISA analysis of the expression levels of four immunoglobulins in SPS plasma; and Figure C shows IgE expression in bone tissue of SPS and control subjects using IHC.

[0024] Figure 7 FIG. 1 is a diagram showing that the IgE of the present invention promotes the differentiation of OPCs into osteoclasts.

[0025] Figure 8 This is a diagram showing that the IgE Fc of the present invention promotes osteoclast differentiation through FcεRII on the cell membrane of OPCs.

[0026] Figure 9 FIG. 1 is a diagram showing that the IgE Fc of the present invention activates the AKT / ERK signaling pathway to promote osteoclast differentiation.

[0027] Figure 10 Schematic diagram of the present invention's omalizumab inhibiting IgE-induced bone destruction. A is a schematic diagram of the bone resorption mouse model established by injection of rhIgEFc; B is a TRAP staining and micro-CT image of the mouse skull; C is micro-CT analysis of whole skull bone mass; and D is micro-CT analysis of the number of osteolytic lesions.

[0028] Figure 11This figure shows that Omalizumab of the present invention specifically neutralizes the osteoclastic activity of SPS plasma. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is further described below with reference to the accompanying drawings and examples. Experimental materials not mentioned herein are all commercially available, and experimental methods not mentioned herein are all commonly used methods in the art.

[0030] (1) SPS progresses rapidly and causes osteolytic destruction

[0031] 1) Severe bone destruction was found when the patient first visited the hospital in 2014

[0032] The inventor team found that a 29-year-old female patient was first admitted to the hospital in April 2014 due to low back pain and intermittent claudication. Figure 1 A and B) show destruction of the L5 vertebral body and its appendages, accompanied by pathological fractures, vertebral collapse, and spinal cord compression. PET-CT ( Figure 1 Figure C) shows multiple lymph nodes around the L5 vertebral body and retroperitoneum, with increased glucose metabolism, suggesting infection or solitary plasmacytoma. Percutaneous puncture biopsy was negative. During the operation, a large number of pea-sized, cotton-like parasites and parasitic eggs were removed from the L5 lesion ( Figure 1 D). HE staining showed that the trabeculae were filled with a large number of parasites / parasite eggs with different cross-sectional shapes ( Figure 1 E), and ELISA test showed that it was infected with sparganosis. Postoperative X-ray and CT showed that the surgical results of lesion decompression and internal fixation were satisfactory ( Figure 1 F).

[0033] 2) Sparganosis migrates from the lumbar sacral region to the cervical spine in the SPS patient's spine. Three years after the first surgery, the patient was admitted to the hospital again due to back pain. Imaging examination showed that in addition to the sacral lesions, the right connecting rod of L5 was broken and the broken ends were dislocated ( Figure 2 A and B). After revision surgery, the patient's symptoms improved. However, the parasite continued to migrate toward the cervical spine and brain, and by 2021, it had spread to L5-T6 ( Figure 2 C and D), parasite bodies can be seen during surgery ( Figure 2 E), the parasites were collected and sent for metagenomic testing to confirm that they were infected with sparganosis ( Figure 2 HE staining results still showed that the trabeculae were filled with a large number of parasites / parasite eggs with different cross-sectional shapes ( Figure 2 During this period, the Department of Infectious Diseases and the Institute of Parasitic Diseases Control consulted and adjusted the medication several times. Imaging in 2023 and 2024 showed that the parasites were still migrating upward to the cervical vertebrae level ( Figure 2 H and I).

[0034] (2) SPS drives abnormal activation of OPCs by reshaping the peripheral immune microenvironment

[0035] The core of bone metabolic homeostasis lies in the functional balance between osteoclasts and osteoblasts, and the local immune microenvironment plays a key regulatory role in this process through cellular interactions and metabolic signaling networks. To understand the regulatory mechanism of the immune microenvironment on bone metabolism during the pathological process of SPS, this study, based on single-cell transcriptome sequencing (scRNA-seq), revealed for the first time the dynamic imbalance characteristics of immune cell subsets in the peripheral blood PBMCs of SPS patients ( Figure 3 Among them, the plasma cell subset showed significant expansion in the SPS group, with its proportion changing by as much as 4.1 times ( Figure 3 B), suggesting that humoral immune abnormalities may be involved in the pathological process of SPS. Based on the classic theory that OPCs mostly originate from monocytes, this study focused on the classical monocyte subsets. KEGG enrichment analysis found that the differentially expressed genes of SPS-derived classical monocytes were enriched in the Osteoclast Differentiation signaling pathway ( Figure 3 These results suggest that SPS may induce functional polarization of classical monocytes by reshaping the peripheral immune microenvironment, thereby enhancing their tendency to differentiate into osteoclasts.

[0036] (3) Enhanced osteoclast differentiation potential of SPS-derived monocytes and its clinical pathological verification

[0037] To verify the osteoclast differentiation tendency revealed by scRNA-seq, this study further isolated PBMCs and CD14 + Monocytes were differentiated in vitro under a standardized osteoclast induction system. Quantitative analysis showed that the number of osteoclasts in the SPS group was greater than that in the control group ( Figure 4Figures A and B show the Ctrl1 and Ctrl2 groups (control 1 and control 2, both patients with intervertebral disc herniation undergoing surgery). To correlate in vitro experiments with clinical pathological phenotypes, this study performed TRAP staining on bone lesions in patients with SPS and normal controls of the same age and sex. The following steps were performed: Bone tissue sections were removed from a 37°C incubator and baked on a 60°C oven for 45 minutes. Dewaxing and rehydration were performed by sequentially immersing sections in the following solutions: xylene for 115 minutes, xylene for 215 minutes, alcohol: xylene in a ratio of 1:1:1:1:1:1:1:1:1:1:1:100% alcohol for 5 minutes, 90% alcohol for 5 minutes, 75% alcohol for 5 minutes, 50% alcohol for 5 minutes, and double-distilled water for 5 minutes. The entire dewaxing and rehydration process must be continuous, and the sections must not be allowed to dry out. Fixation: 4% FPA fixation for 30 minutes; Permeabilization: 0.1% X-Triton permeabilization for 6 minutes; Renaturation: PBST renaturation for 30 minutes, double-distilled water washing, and absorbent paper on one side of the slice to remove moisture; TRAP staining: Use a histochemical pen to draw a closed circle around the tissue, add the freshly prepared TRAP stain solution into the closed circle to completely cover the bone tissue, place the slice in a wet box, incubate in a 37°C constant temperature box for 1 hour, dry the slice, seal the slide with neutral resin, count the TRAP activity under a microscope, and take pictures to record.

[0038] The results showed that the area of ​​osteoclast infiltration in the lesions of the SPS group was significantly higher than that in the control group ( Figure 4 These results, based on both in vitro functional experiments and in vivo pathological evidence, confirm that remodeling of the peripheral immune microenvironment in SPS patients can endow monocytes with enhanced osteoclast differentiation potential, and that this abnormal differentiation process is closely related to the degree of clinical bone destruction.

[0039] (4) SPS plasma disrupts bone metabolism homeostasis by specifically activating osteoclast precursor function

[0040] Extraction of CD14 from blood + Monocytes and detection of their osteoclast differentiation ability:

[0041] Blood was drawn from patients or controls and stored in EDTA anticoagulant tubes at 4°C. Within 48 hours, the blood was transferred to a 50ml centrifuge tube and diluted 1:1 with PBS. PBMCs were extracted using FICOLL lymphocyte separation medium. CD14 positive monocytes were isolated using a CD14 positive monocyte isolation kit. +Monocytes. Count and plate monocytes in a 96-well plate at 80,000 cells / well. Add recombinant human macrophage colony-stimulating factor (rhM-CSF) to a final concentration of 40 ng / ml in 100 μl of the culture medium. The next day, after the monocytes adhere, the culture medium is changed. 20 ng / ml rhM-CSF and 80 ng / ml rhRANKL are added to stimulate monocyte differentiation into osteoclasts. The culture medium is changed every two days. Osteoclast differentiation is observed daily. After differentiation is complete, fix with 4% paraformaldehyde for 30 minutes, permeabilize with 0.1% X-triton for 6 minutes, and renature with PBST for 30 minutes before TRAP staining. Osteoclasts are counted and photographed under a microscope.

[0042] To understand the molecular basis of SPS immune microenvironment regulating bone metabolism, this study established a cross-system validation model: SPS plasma was compared with healthy donor CD14 + Monocyte co-culture. Obtain CD14 + After mononuclear cells are counted, they are plated into 96-well plates at 80,000 cells / well. The culture medium is supplemented with 20 ng / ml rhM-CSF and 3% plasma, or with various concentrations of rhIgEFc. The medium is changed daily. After several days, 10 μl of CCK8 reagent is added to each well. The cells are incubated in a 37°C incubator for 1 hour, and the OD values ​​are measured using a microplate reader. Ctrl to Ctr3 represent control groups 1 to 3, respectively, of the same age and sex, all of whom underwent surgery for intervertebral disc herniation.

[0043] TRAP staining results showed that the number of osteoclasts in the SPS plasma stimulation group was greater ( Figure 5 A), and can promote CD14 + Monocyte proliferation and migration ability ( Figure 5 The inventors confirmed through alkaline phosphatase (ALP) staining, Alizarin red (ARS) staining and Von Kossa staining that SPS plasma had no significant effect on the osteogenic differentiation of BMSCs and the mineralization ability of osteoblasts ( Figure 5 The above results indicate that there may be specific factors in SPS plasma that promote osteoclast differentiation, which can aggravate bone metabolism imbalance by directly enhancing the proliferation, migration and osteoclast differentiation of OPCs, leading to a breakdown in the bone resorption-bone formation coupling, which is highly consistent with the clinical phenotype of progressive bone destruction in SPS patients.

[0044] (5) IgE is a potential key molecule in SPS osteolytic destruction

[0045] Single-cell sequencing analysis showed that plasma cells were the most significantly altered cell subset in the SPS immune microenvironment. Gene set enrichment analysis further revealed that multiple immunoglobulin synthesis-related pathways were significantly activated in SPS-derived plasma cells ( Figure 6A), suggesting that humoral immune abnormalities may be a potential characteristic of SPS. To clarify the humoral immune characteristics of SPS patients and their association with bone metabolism, this study conducted multi-dimensional research and found the following: 1) Specific increase in circulating IgE: ELISA testing showed that the plasma IgE concentration of SPS patients reached 642.00 IU / mL, which is 6.4 times the normal high value, while no significant difference was found in IgG / IgM / IgA levels ( Figure 6 B); 2) Local bone tissue IgE deposition: Immunohistochemistry showed that the number and intensity of IgE-positive cell infiltration in SPS bone destruction lesions were higher than those in normal bone tissue ( Figure 6 C); 3) Clinical epidemiological support: Retrospective analyses have shown that the incidence of osteoporosis / pathological fractures in patients with hyperIgEemia (such as allergic diseases and hyperIgE syndrome) is significantly higher than in the normal population, suggesting a potential association between abnormal IgE levels and imbalanced bone metabolism. Combined with these findings, this study found abnormal plasma cell activation accompanied by specific elevation of IgE in SPS patients, with significant IgE accumulation both in the circulation and at sites of bone destruction. Therefore, it is hypothesized that IgE may be a key potential regulator of osteoclast differentiation and hyperfunction in SPS.

[0046] (6) IgE promotes the differentiation of OPCs into osteoclasts

[0047] Isolation and osteoclast differentiation of mouse BMM cells:

[0048] BMMs were obtained from 6-8 week old C57BL / 6 wild-type mice. Primary bone marrow cells were removed and cultured for 3 days. BMMs were digested with versene and centrifuged, resuspended, counted, and plated. 12,000 cells were plated per well of a 96-well plate, and 100 μl of rmM-CSF was added to a final concentration of 40 ng / ml. The next day, 20 ng / ml rmM-CSF and 80 ng / ml rmRANKL were added to stimulate BMM differentiation into osteoclasts, with the medium changed every two days. To investigate the effect of rmIgE Fc on osteoclast differentiation, various concentrations of rmIgE Fc were added to the complete differentiation medium. Osteoclast differentiation was observed daily. After differentiation was complete, cells were fixed with 4% paraformaldehyde for 30 minutes, permeabilized with 0.1% X-triton for 6 minutes, and renatured with PBST for 30 minutes before TRAP staining. Osteoclasts were counted and photographed under a microscope.

[0049] Based on the fact that IgE recognizes and binds to Fc receptors (FcεRI and FcεRII) or Galectin-3 through Fc fragments to mediate allergic reactions or interactions with immune cells, this application uses rmIgE Fc and rhIgE Fc to study the role of IgE Fc in the differentiation of OPCs into osteoclasts. BMMs of 6-8 week old mice were extracted, and different concentrations (0, 100, 200, 400 ng / ml) of rmIgE Fc were added to the osteoclast differentiation system, 50 ng / ml rmRANKL, and the medium was changed every two days. TRAP staining was performed on the 7th day. PBMCs from healthy donors were obtained by density gradient centrifugation, and CD14 + Different concentrations (0, 100, 200, 400 ng / ml) of rhIgE Fc were added to the osteoclast differentiation system, along with 50 ng / ml rhRANKL. The medium was changed every two days, and TRAP staining was performed on the 7th day. The results showed that under the stimulation of rmIgE Fc or rhIgE Fc, mouse BMMs ( Figure 7 A) and human peripheral blood-derived CD14 + Monocytes ( Figure 7 The number of osteoclasts differentiated from B) increased in a dose-dependent manner. These results indicate that IgE can enhance the osteoclastogenic differentiation of OPCs, and that the Fc domain is the key functional domain of its osteoclastogenic activity.

[0050] (7) IgE Fc specifically activates the osteoclast differentiation signaling axis through the low-affinity receptor FcεRII

[0051] There are three IgE Fc receptors on the surface of OPCs, including high-affinity FcεRI, low-affinity FcεRII and Galectin-3. This study screened key receptors through a three-level validation strategy: 1) Receptor expression spectrum analysis: Using RAW264.7 cells, rmIgE Fc 200ng / ml+rmRANKL 100ng / ml was added to the IgE group, and rmRANKL 100ng / ml was added to the control group. After 48 hours of stimulation, the proportion of FcεRI, FcεRII, and Galectin-3 positive cells was detected by flow cytometry. Flow cytometry showed that rmIgE stimulation significantly upregulated the expression of FcεRII on the surface of RAW264.7 cells, while Galectin-3 was only slightly upregulated, and FcεRIα expression did not change ( Figure 82) Direct interaction verification: His-rmIgE Fc was added to RAW264.7 cell lysate, and His-rmIgE Fc was IPed using Anti-His magnetic beads to detect whether FcεRII interacted with rmIgE Fc. Co-IP experiments confirmed that rmIgE-Fc specifically bound to FcεRII protein in RAW264.7 cell lysate ( Figure 8 3) Functional receptor identification: Different concentrations (10, 100 μg / ml) of FcεRI neutralizing antibody were added to the BMMs osteoclast differentiation system. The control group was added with the same volume of solvent (PBS) and 50 ng / ml rmRANKL. The medium was changed every two days and TRAP staining was performed on the 7th day. Lumiliximab (10, 100 μg / ml) was added to the CD14 + In the monocyte osteoclast differentiation system, the control group received the same volume of solvent (100 mM Pro-AC + 20 mM Arg, pH 5.0) and 50 ng / ml rhRANKL. The medium was changed every two days, and TRAP staining was performed on day 7. Different concentrations (1 and 10 μM) of FcεRI neutralizing antibody were added to the BMM osteoclast differentiation system. The control group received the same volume of solvent (DMSO) and 50 ng / ml rmRANKL. The medium was changed every two days, and TRAP staining was performed on day 7. Bar Scale: 35 μm. **P < 0.01, ***P < 0.001, ns, not statistically significant. Three replicate wells per group were used, and the mean ± SD is presented. Using a receptor-specific blocking strategy—FcεRIα neutralizing antibody, FcεRII blocker Lumiliximab, and Galectin-3 inhibitor GB1107—it was found that only FcεRII blockade could eliminate the pro-osteoclastogenic effect of IgEFc in a dose-dependent manner ( Figure 8 Therefore, these results suggest that in the bone microenvironment, IgE Fc triggers the downstream osteoclast differentiation signaling axis through the low-affinity receptor FcεRII rather than the traditional high-affinity receptor.

[0052] (8) IgE Fc drives osteoclast differentiation by selectively amplifying the RANKL-mediated MAPK pathway. Extraction of nuclear and plasma proteins from RAW264.7 cells:

[0053] RAW264.7 cells were divided into four groups. Two groups were stimulated with rmRANKL alone at a final concentration of 100 ng / ml for 48 or 72 hours, and the other two groups were stimulated with rmRANKL at a final concentration of 100 ng / ml plus rmIgE Fc at 200 ng / ml for 48 or 72 hours. After stimulation, cells were rinsed 2-3 times with PBS and detached by pipetting. Centrifugation was performed at 1000 rpm for 5 minutes. Proteins were then extracted using a nuclear and cytoplasmic protein extraction kit. After centrifugation, the supernatant was discarded as much as possible. 200 μl of CERA solution supplemented with protease inhibitors was added. Vortex vigorously at maximum speed for 5 seconds to completely suspend and disperse the cell pellet (if the cell pellet is still not fully dispersed, the vortexing time can be extended appropriately). Incubate on ice for at least 30 minutes. Add 10 μl of CER B solution. Vortex vigorously at maximum speed for 5 seconds and incubate on ice for 2 minutes. Vortex vigorously at top speed again for 5 seconds, and centrifuge at 14,000-16,000g for 5 minutes at 4°C. Prepare and label new microcentrifuge tubes during this time, and keep them chilled on ice. Immediately after centrifugation, aspirate the supernatant into a pre-chilled microcentrifuge tube. This is the cytoplasmic protein. It can be used immediately or frozen at -80°C. For the pellet, completely aspirate any remaining supernatant, add 50 μl of NER solution supplemented with protease inhibitors, and vortex vigorously at top speed for 5 seconds to completely resuspend and disperse the cell pellet. Return the pellet to the ice bath. Repeat this "ice bath for 1 minute - vortex vigorously at top speed for 20 seconds" cycle 25 times. Finally, centrifuge at 14,000-16,000g for 10 minutes at 4°C. Prepare and label new microcentrifuge tubes during this time, and keep them chilled on ice. Immediately after centrifugation, aspirate the supernatant into a pre-chilled microcentrifuge tube. This is the nuclear protein. Both the extracted cytoplasmic and nuclear proteins were analyzed by Western blotting.

[0054] Western blot detection of relative expression levels of target proteins:

[0055] Prepare the gel: Select the appropriate gel concentration based on its correlation with protein resolution. Prepare the lower separation gel according to the instructions. Add a coagulant and pour it into the plate. Seal with anhydrous ethanol and flatten the upper surface of the separation gel. Once the separation gel has completely solidified, discard the anhydrous ethanol. Prepare the upper stacking gel. Add a coagulant and pour it into the plate. Quickly insert the sample comb. Allow the upper gel to solidify.

[0056] Sample preparation: Pour off the culture medium from the 6-well plate and wash three times with PBS. Assuming the same number of BMMs are seeded in each well of the plate, add 150 μl of 1X SDS-loading buffer to each well of the 6-well plate to lyse the cells. Transfer the lysate to a 1.5 ml EP tube. The entire sample collection process should be performed on ice to prevent protein degradation, especially when analyzing protein phosphorylation levels.

[0057] Cooking samples: centrifuge the EP tube containing the sample at low speed for 10 seconds, open the dry bath, and cook the sample at 100℃ for 10 minutes.

[0058] Sample loading: After boiling, centrifuge again to allow any water that has evaporated or condensed on the sides of the EP tube to centrifuge to the bottom of the tube, preventing changes in protein concentration. Remove the comb before loading the sample. If there are extra lanes, add a small amount of loading buffer to the edge lanes to ensure uniform band alignment.

[0059] Electrophoresis: After checking and inserting the positive and negative electrodes, run the gel at a constant voltage of 60V first. After the marker runs out of the concentrated gel, increase the voltage to 120V and run the separation gel.

[0060] Transfer: Stop the gel run when the marker reaches the bottom of the separation gel. Soak the sponge, filter paper, and appropriately sized NC membrane in 1X transfer buffer. After cutting the gel with a comb, assemble the transfer cassette in the order "white membrane, black gel": black side of the cassette - sponge 1 - filter paper 1 - gel - NC membrane - filter paper 2 - sponge 2 - white side of the cassette. Once assembled, insert the cassette into the transfer tank in the designated orientation. Place an ice pack and fill it with 1X transfer buffer. After verifying and properly inserting the positive and negative electrodes, transfer the membrane at a constant current of 350mA. Cover the entire transfer apparatus with ice to quickly dissipate heat generated during transfer. The transfer time (min) should roughly correspond to the molecular weight (kDa) of the target sample.

[0061] Membrane cutting and blocking: After transfer, wash the membrane with double-distilled water and stain with 1X Ponceau red for 10 seconds to visualize the bands. Recycle the Ponceau red and wash the membrane twice with double-distilled water. Cut the membrane with a knife at the location of the target protein marker and block with 5% skim milk for 1 hour. If detecting protein phosphorylation sites, block with 5% BSA for 1 hour.

[0062] Primary antibody incubation: After blocking, the membrane was washed once with double-distilled water, then immersed in the primary antibody solution and incubated overnight in a 4°C refrigerator with a shaker at 10 rpm.

[0063] Secondary antibody incubation: After primary antibody incubation, wash with PBST on a shaker at 60 rpm for 6 minutes, and wash six times for a total of 36 minutes. Then, add the corresponding secondary antibody and incubate at room temperature on a shaker at 10 rpm for 1 hour.

[0064] Development: After secondary antibody incubation, wash the sections six times with PBST on a shaker at 60 rpm for 6 min, for a total of 36 min. The sections were then developed using an AIimage 600 instrument.

[0065] Classical RANKL signaling regulates osteoclast differentiation by activating MAPK and NF-κB pathways. In order to analyze the regulatory characteristics of IgE-Fc, this application combined rmIgE-Fc treatment on the basis of RANKL stimulation, plated RAW264.7 cells, and pretreated the IgE group with rmIgE Fc for 1 hour. rmIgE Fc 200ng / ml + rmRANKL 100ng / ml was added to the IgE group, and rmRANKL 100ng / ml was added to the control group, and the treatments were performed for 0, 5, 15, and 30min, respectively. The expression levels of key molecules in the NF-κB and MAPK signaling pathways and their phosphorylation levels were detected. It was found that rmIgE-Fc significantly enhanced the phosphorylation of ERK1 / 2 and AKT induced by RANKL, but had no significant effect on the phosphorylation of p65, a key molecule in the NF-κB pathway, and the degradation of IκBα ( Figure 9 A). As a transcription factor, the translocation of NFATc1 from the cytoplasm to the nucleus and its regulation of target gene expression are necessary conditions for RANKL to induce osteoclast formation and characterize active osteoclasts. RAW264.7 cells were plated, and rmIgE Fc 200ng / ml+rmRANKL 100ng / ml was added to the IgE group, and rmRANKL 100ng / ml was added to the control group, and they were treated for 48h and 72h, respectively. Plasma protein and nuclear protein were extracted, and the expression level of NFATc1 was detected by Western Blot. Subcellular protein analysis showed that rmIgE-Fc combined with RANKL stimulation increased the level of NFATc1 protein in the nucleus of RAW264.7 cells compared with the RANKL alone group, and the total NFATc1 protein amount increased simultaneously, while there was no change in cytoplasmic NFATc1 ( Figure 9 B), suggesting that IgE-Fc enhances NFATc1 transcriptional activity by promoting its nuclear translocation and stable expression. These results indicate that IgE Fc selectively amplifies the RANKL-mediated MAPK pathway, synergistically promoting NFATc1 nuclear translocation, ultimately leading to the differentiation of OPCs into osteoclasts.

[0066] (9) Omalizumab can effectively block the IgE Fc-induced bone destruction phenotype in mice

[0067] Omalizumab is currently the only IgE-targeted therapeutic drug that has been validated by clinical trials and is widely used in clinical practice. Based on the core role of IgE in SPS bone destruction, this application constructed a subscalp rhIgE Fc mouse model to evaluate the intervention potential of targeting IgE ( Figure 10After one month of stimulation with PBS, rhIgE Fc (1 mg / kg / 2 days), and rhIgE Fc (1 mg / kg / 2 days) + Omalizumab (6 mg / kg / 2 days), TRAP staining and micro-CT imaging of the mouse skulls were performed. The results showed that the density of osteoclasts in the suture area of ​​the skull of mice in the rhIgE Fc group was increased compared with the control group, and the number of osteolytic lesions was also significantly increased compared with the control group ( Figure 10 BD); combined with Omalizumab, it can significantly reduce osteoclast density and the number of osteolytic lesions ( Figure 10 These results indicate that omalizumab can effectively neutralize the bone resorption activity of IgE Fc and preliminarily suggest the potential value of omalizumab in the treatment of bone destruction in SPS.

[0068] (10) Omalizumab specifically blocks the pro-osteoclastogenic effect of plasma from SPS patients and demonstrates therapeutic potential

[0069] To verify the intervention effect of Omalizumab on SPS pathological plasma, this study included plasma from SPS patients and healthy controls of the same sex and age, and established an in vitro osteoclast differentiation evaluation system. 3% SPS and control plasma combined with 100 μg / ml Omalizumab were added to CD14 + In the monocyte differentiation system, the medium was changed every two days, and TRAP staining was performed on day 7. Bar Scale: 35 μm. ***P < 0.001, ns, no statistical difference. Three replicates per group, expressed as mean ± SD. Omalizumab was found to inhibit the promotion of osteoclast differentiation of OPCs by SPS plasma, while the same dose of healthy control plasma had no significant effect ( Figure 11 ), confirming its precise neutralization of SPS-specific pathogenic factors. These results demonstrate that omalizumab specifically eliminates the pro-osteoclastic activity of SPS plasma without interfering with normal physiological bone remodeling, demonstrating the precision and safety of targeted therapy.

[0070] In summary, the present invention discovered the world's first case of spinal proliferative sparganosis with severe bone destruction. Through sequencing and experiments, it was found that IgE mediated the Erk / Akt signaling pathway by binding to FcεRII on the cell membrane of OPCs, promoting the differentiation of OPCs into osteoclasts. Omalizumab can inhibit the pro-osteoclast effect of SPS-derived plasma, thereby inhibiting bone destruction in SPS patients and delaying disease progression.

[0071] Although the present invention has been described in detail through the above preferred embodiments, it should be understood that the above description is not intended to limit the present invention. After reading the above description, various modifications and substitutions of the present invention will become apparent to those skilled in the art. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. Application of omalizumab in the preparation of drugs for the treatment of bone destruction.

2. The use according to claim 1, characterized in that The bone destruction is caused by infection with spina bifida larvae.

3. The use according to claim 1, characterized in that The bone destruction is caused by infection with the proliferative form of sparganosis.

4. The use according to claim 1, wherein The bone destruction is characterized by bone destruction caused by specific increase of IgE.

5. The use according to claim 4, characterized in that The bone destruction is characterized by abnormal activation of plasma cells.

6. The use according to claim 1, wherein The bone destruction is induced by IgE.

7. The use according to claim 1, wherein The IgE can promote the differentiation of OPCs into osteoclasts.

8. The use according to claim 7, characterized in that The bone destruction is mediated by the AKT / ERK signaling pathway through the binding of the IgE to FcεRII on the cell membrane of OPCs, thereby promoting the differentiation of OPCs into osteoclasts.

9. The use according to claim 1, wherein The bone destruction at least includes osteoporosis and / or pathological fractures caused by hyper-IgEemia.

10. The use according to claim 1, wherein The drug further comprises a pharmaceutically acceptable carrier.

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