CN113827622B

CN113827622B - Application of Proteus mirabilis adventitia vesicles in preparation of medicines for preventing or treating osteolytic diseases

Info

Publication number: CN113827622B
Application number: CN202111015722.0A
Authority: CN
Inventors: 库蒂·塞尔瓦·南达库玛; 王婷婷
Original assignee: Southern Medical University
Current assignee: Southern Medical University
Priority date: 2021-08-31
Filing date: 2021-08-31
Publication date: 2023-11-07
Anticipated expiration: 2041-08-31
Also published as: CN113827622A

Abstract

The invention discloses application of Proteus mirabilis adventitia vesicles in preparation of a medicament for preventing or treating osteolytic diseases. The outer membrane vesicles of Proteus mirabilis inhibit miR96-5p expression to promote Abca1 expression, so that MAPK/ERK channels are inhibited, and osteoclast differentiation is blocked; proteus mirabilis outer membrane vesicles induce release of MPT-associated cytochrome c, which results in disruption of mitochondrial structure, increased reactive oxygen species production, and increased osteoclast apoptosis. The invention discovers that the outer membrane vesicle of the Proteus mirabilis not only remarkably inhibits the differentiation and the function of the osteoclast induced by RANKL in vitro, but also can improve the bone metabolism unbalance caused by OVX and the bone erosion caused by CII in vivo, thereby providing a new thought for preventing or treating the osteolytic disease.

Description

Application of Proteus mirabilis adventitia vesicles in preparation of medicines for preventing or treating osteolytic diseases

Technical Field

The invention belongs to the technical field of bone disease treatment, and particularly relates to application of Proteus mirabilis adventitia vesicles in preparation of a medicament for preventing or treating bone-dissolving diseases.

Background

Bone resorption and bone regeneration processes, called bone remodeling, occur at all times in the body, and the balance of bone remodeling plays an important role in bone homeostasis. Both osteoclasts and osteoblasts are involved in the bone remodeling process, the function of osteoclasts is to take up old bone tissue, and the function of osteoblasts is to produce new bone, both in an equilibrium state to maintain the balance of bone remodeling. Abnormal increases in osteoclast proliferation and activity lead to bone resorption far exceeding bone formation, break the balance of bone remodeling, cause bone tissue to be destroyed and erode, and cause bone diseases such as osteoporosis, rheumatoid arthritis, ankylosing spondylitis, bone sclerosis, systemic lupus erythematosus, alzheimer's disease, diabetes, breast cancer, prostate cancer, and the like.

The pathway of osteoclast differentiation can be divided into the classical pathway mediated by nuclear factor NF-. Kappa.B ligand receptor activators (e.g.TRAF6/RANKL/NF-. Kappa. B, TRAF 6/RANKL/MAPK) and the non-classical pathway in which some inflammatory cytokines (e.g.IL-1, IL-6, INF-. Alpha.) are involved. In the event of disease, osteoblast lineage cells or bone cells produce an activator of nuclear factor NF- κb ligand Receptor Activator (RANKL), which binds to its receptor RANK, activating TRAF6 activated by the adapter protein TRAF6 and initiating pathways such as NF- κb and MAPK, leading to activation of NFATc1, thereby activating and inducing expression of osteoclast-specific genes, such as osteoclast differentiation fusion (DC-STAMP, atp v0d 2) and function (CTSK, TRAP, MMP 9) related genes, promoting osteoclast differentiation and exerting bone resorption function.

There are many factors that induce the overactivation of osteoclasts, resulting in a decrease in bone density in patients, including endogenous factors (hormone levels and autoimmunity) and exogenous factors (bacterial infection). Previous studies have shown homologous sequences between proteus hemolysin and HLA-DR4 and between proteus urease and hyaline cartilage that can cross-react with certain autoantigens present in synovial tissue to produce molecular effects that may be involved in the pathogenesis of rheumatoid arthritis. In addition, staphylococcus aureus is a common cause of osteomyelitis and can increase the abundance of osteoclasts present in the body at the bone surface.

Currently, bone diseases are ameliorated mainly by modulating hormonal levels and anti-inflammatory, while fewer drugs targeting osteoclasts, mainly bisphosphonates and denomab. (1) Glucocorticoids (dexamethasone) and methotrexate inhibit the release of inflammatory factors such as TNF- α in rheumatoid arthritis, but can cause bone loss over time. Such as dexamethasone, can promote osteoclastogenesis in vitro and cause osteoporosis in animals and patients in vivo; methotrexate also promotes osteoclast formation after use, resulting in bone destruction. (2) Bisphosphonates can inhibit osteoclast activity and bone resorption. Among them, amino bisphosphonates exert their inhibitory effect on osteoclast function by inhibiting farnesyl pyrophosphate synthase, but are not frequently used due to their pro-inflammatory effect. On the other hand, non-amino bisphosphonates, when metabolized to non-hydrolyzable ATP analogs, can inhibit ATP-dependent enzymes leading to increased osteoclast apoptosis, but they are essentially ineffective in regulating bone erosion. (3) The use of denoumab (denosumab) to inhibit RANK ligand (RANKL) results in reduced osteoclast-mediated bone resorption and turnover. The antagonism caused by dendritic cells and activated T lymphocytes in large numbers express RANKL and denosumab may affect the immune system, leading to adverse effects including skin eczema, flatulence, cellulitis and jawbone necrosis. Furthermore, the interaction of RANK and RANKL is essential for the development of immune cells and hypocalcemia following denosumab treatment, and therefore, severe renal insufficiency may be one of the major risk factors for treatment. Receptor activators that block RANK signaling or osteoclasts may protect bone, although they may not interfere with synovitis. Blocking upstream cytokines such as TNF- α or IL-1 reduces synovial inflammation and cartilage and bone destruction. Synovial inflammation cannot be completely inhibited, so protecting the joint by further blocking osteoclast number and activation with the addition of drugs may be the best treatment.

The imbalance of flora in humans is associated with a number of diseases, and the transplantation of probiotics Lactobacillus casei into arthritic mice significantly improves the arthritic symptoms, while E.coli is associated with the development of IBD, porphyromonas gingivalis not only causes periodontitis but also exacerbates the occurrence of arthritis. Although bacteria cannot complete long-distance transmission, the secreted and produced metabolites can be transmitted in a long distance, such as SCFAs, secreted toxins and outer membrane vesicles formed by budding can escape from host immunity, and the bacteria can act after penetrating through the inner layer barrier of the human body to reach a proper position so as to influence the health of the host.

Bacterial-derived Outer Membrane Vesicles (OMVs) are derived from gram-negative bacterial cell membranes, are between 20 and 450nm in diameter and contain inner and outer membrane proteins, periplasmic proteins, lipopolysaccharides (LPS), virulence factors, DNA, RNA and other biomolecules of parent bacteria, and can transport and protect these molecules from the external environment over long distances. Studies have shown that OMVs are capable of interacting not only with immune cells such as macrophages, neutrophils, dendritic cells, but also directly with host cells such as epithelial cells, osteoblasts, synovial cells, etc., such as Kingellakingae OMVs can be internalized by osteoblasts and synovial cells, resulting in increased production of GM-CSF and IL-6. In addition, it can also modulate the host immune system to affect the onset of disease, and studies have shown that OMVs can induce dendritic cell maturation, thereby enhancing the uptake and presentation of antigen molecules to develop adaptive immune responses. Clinically, the OMVs vaccine for type B meningitis for invasive meningococcal disease has passed phase I, II and multiple clinical studies. However, the effect of bacterial OMVs on osteoclasts has not been reported.

Disclosure of Invention

In order to solve the defects in the prior art, the invention aims to provide the application of the outer membrane vesicles of the Proteus mirabilis in preparing medicines for preventing or treating osteolytic diseases. The specific technical scheme is as follows:

pathological bone resorption is mainly due to an increase in the number of osteoclasts, which depends on the rate of osteoclast differentiation and apoptosis. Thus, a key factor in preventing bone loss in many osteolytic diseases is the inhibition of osteoclast number and function.

The first aspect of the invention provides the use of Proteus mirabilis outer membrane vesicles in the manufacture of a medicament for the prevention or treatment of osteolytic disorders.

In the above technical solution of the present invention, the osteolytic disease includes osteoporosis, osteolysis, rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, osteogenesis imperfecta, osteosclerosis, multiple myeloma, bone metastatic cancer, hypercalcemia of malignancy, systemic lupus erythematosus, alzheimer's disease, diabetes mellitus, breast cancer, prostate cancer, bone loss caused by immunosuppressive therapy, bone loss caused by glucocorticoid drug therapy, bone loss caused by methotrexate therapy, bone loss induced by ovariectomy, and bone erosion induced by type II collagen.

In the above technical solution of the present invention, the osteolytic disease includes osteoporosis, osteolysis, rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, osteogenesis imperfecta, osteosclerotic disease, bone loss caused by immunosuppressive therapy, bone loss caused by glucocorticoid therapy, bone loss caused by methotrexate therapy, ovariectomy-induced bone loss, and type II collagen-induced bone erosion;

preferably, the osteolytic disease includes osteoporosis, osteolysis, rheumatoid arthritis, immunosuppression therapy induced bone loss, glucocorticoid therapy induced bone loss, methotrexate therapy induced bone loss, ovariectomy induced bone loss, and type II collagen induced bone erosion.

In the technical scheme of the invention, the mechanism for preventing or treating the osteolytic disease by using the outer membrane vesicles of the Proteus mirabilis is as follows,

the outer membrane vesicles of the Proteus mirabilis inhibit miR96-5p expression to promote Abca1 expression, so that MAPK/ERK channels are inhibited, and osteoclast differentiation is blocked;

the outer membrane vesicles of Proteus mirabilis induce the release of MPT-related cytochrome c, which results in the destruction of mitochondrial structures, increase of active oxygen production, and increase of osteoclast apoptosis.

The Proteus mirabilis outer membrane vesicles inhibit miR96-5p expression to promote Abca1 expression, so that MAPK/ERK pathway is inhibited, and MAPK pathway activation causes activation of NFATc1 to induce differentiation of osteoclasts and expression of specific genes, so that inhibition of MAPK/ERK pathway can cause differentiation inhibition of osteoclasts.

Both osteoclastogenesis and osteolysis require a large energy supply and consumption, and studies have demonstrated that the energy required for osteoclast differentiation is mainly derived from the oxidative metabolism of mitochondria. Thus ATP synthesis is reduced when mitochondrial structures are disrupted, rendering osteoclasts unable to complete the fusion and maturation process due to insufficient energy supply. The destruction of mitochondrial structure following P.M OMVs treatment resulted in a significant decrease in ATP production, MPT-related cytochrome c release, increased Reactive Oxygen Species (ROS) levels, and a significant decrease in mitochondrial membrane potential, thereby inducing apoptosis.

In a second aspect, the invention provides a pharmaceutical composition for preventing or treating osteolytic disorders comprising Proteus mirabilis outer membrane vesicles.

In the technical scheme of the invention, the pharmaceutical composition also comprises pharmaceutically acceptable auxiliary materials.

In a third aspect, the invention provides the use of an outer membrane vesicle of Proteus mirabilis in the preparation of an agent or medicament for inhibiting the formation and/or activation of osteoclasts.

In a fourth aspect, the invention provides a pharmaceutical composition for inhibiting osteoclast formation and/or activation, comprising a Proteus mirabilis outer membrane vesicle;

preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

In a fifth aspect, the invention provides an osteoclast formation and/or activation inhibitor comprising an outer membrane vesicle of Proteus mirabilis.

In a sixth aspect, the invention provides a kit for inhibiting osteoclast formation and/or activation comprising an inhibitor as described above. Further, the kit may further include a buffer, a diluent, a pH adjuster, and the like.

Pharmaceutically acceptable auxiliary materials in the invention comprise pharmaceutically acceptable carriers, excipients, lubricants, colorants and the like. The pharmaceutical composition of the present invention may be administered orally or parenterally, and may be administered parenterally, for example, by intravenous injection, intramuscular injection, or intraoral administration.

The invention has the beneficial effects that:

1. the invention discovers that the outer membrane vesicle of the Proteus mirabilis not only remarkably inhibits the differentiation and the function of the osteoclast induced by RANKL in vitro, but also can improve the bone metabolism unbalance caused by OVX and the bone erosion caused by CII in vivo, thereby providing a new thought for preventing or treating the osteolytic disease.

2. On one hand, the Proteus mirabilis outer membrane vesicle realizes the protection of bones through a miR96-Abca1-MAPK pathway, and the Proteus mirabilis outer membrane vesicle inhibits miR96-5p expression so as to promote Abca1 expression, thereby inhibiting a MAPK/ERK pathway and leading to the blocking of osteoclast differentiation. On the other hand, the apparent inhibition of miR96-5p expression induced by proteus mirabilis outer membrane vesicles causes up-regulation of ATP-binding cassette subfamily a member 1 (Abca 1) involved in mitochondrial function, thereby down-regulating ERK phosphorylation, which is crucial for osteoclast activation. Abca1 promotes the reduction of mitochondrial cholesterol and participates in the release of cytochrome c (Cyto c), resulting in a disruption of mitochondrial structure and a reduction in the number, while significantly reducing the production of ATP during osteoclast formation. OMVs cause mitochondrial destruction by inducing intracellular ROS, decrease mitochondrial membrane potential, and regulate Bax, bcl-2, caspase-3 and cytoc levels leading to increased mitochondrial-dependent apoptosis, leading to reduced ATP production ultimately leading to osteoclast differentiation and functional destruction.

Drawings

Fig. 1 is a TEM image of P.M OMVs.

Figure 2 is a graph showing the size and number statistics of P.M OMVs. Experimental results were obtained from three independent replicates.

FIG. 3 is a graph showing the effect of P.M OMVs on osteoclast differentiation and function. (A) Cell viability after BMMs 48,72,96h were treated with different concentrations of P.M OMVs and P.M LPS, respectively; (B) 0.15,0.3 μg/ml P.M OMVs, P.M LPS treated RANKL inducedRepresentative pictures of osteoclast differentiation from sources and trap+ cell number statistics with more than three nuclei; (C) 0.15,0.3 μg/ml of P.M OMVs and P.M LPS stimulated representative pictures of inflammatory-derived RANKL-induced osteoclast formation and more than three nuclear Trap ⁺ Counting the number of cells; (D) P.M influence of OMVs on RANKL-induced osteoclast-related gene expression; (E) Confocal representative pictures of the effect of P.M OMVs on RANKL-induced osteoclast F-actin ring formation; (F) P.M influence of OMVs on RANKL-induced osteoclast-related gene expression; (G) P.M representation of bone resorption after OMVs stimulation, 3D image presentation of the representation, and every μm ² Is a statistical information of pit areas (n=6). (E) scale bar = 50 μm; (B), (C) and (G) scale bar = 200 μm. Experimental results were obtained from three independent replicates and expressed as mean ± SD. * P, p<0.05；**,p<0.01；***,p<0.001；****,p<0.0001。

Fig. 4 shows P.M OMVs protect against OVX induced bone loss. (A) an experimental procedure; (B) Micrococt images of Sham and OVX (with or without P.M OMV treatment); (C) mouse uterus to weight ratio; (D-H) mouse bone parameters BMD, tb.bv/TV, tb.N, tb.Th, tb.Sp; serum (I) CTX-1 and (J) OCN levels (n=5/group); (K) BMD: bone density, tb.bv/TV: trabecular bone volume per tissue volume, tb.n: bone small Liang Shuliang, tb.th: bone trabecular thickness, tb.sp: bone trabecular gap. Sham surgery group (n=5), OVX group, remaining group n=5, ovx+pbs group and ovx+ P.M OMV group, respectively, experimental results were obtained from three independent replicates and expressed as mean ± sd.#, p <0.05 #, p <0.01 #, p <0.001 #, p <0.0001, p <0.05 #, p <0.01 #, p <0.001 #, p <0.0001 #, p.

Fig. 5 is the effect of P.M OMVs on CIA inflammation and bone erosion. (A) an experimental procedure; (B) representative pictures of hind paws of different groups of mice; (C-D) average arthritis and hind paw arthritis scores for each group after the second immunization; (E-F) HE staining of the hind paw sections of the mice at day 107; (G) anti-CII antibody levels in serum from each group on days 0,21,75 and 107 (normal group n=3, other group n=7); serum (H) CTX-1 and (I) OCN levels; (J) Representative image of μct (normal group n=3, other group n=5); (K-O) different sets of bone parameters. BMD: bone density, tb. Bv/TV: trabecular bone volume per tissue volume, tb.n: bone small Liang Shuliang, tb.th: bone trabecular thickness, tb.sp: bone trabecular clearance. Arthritis scores are expressed as mean ± SEM, and the remaining data are expressed as mean ± SD. # p <0.05; # p <0.01, # p <0.001, # p <0.0001 compared to the normal group; * P <0.05; * P <0.01; * P <0.001; * P <0.0001 compared to CIA group.

FIG. 6 is a graph of the volcanic images of the DEGs after P.M OMVs treatment, a KEGG chordal image, and a MCODE cluster analysis. (a) P.M OMVs post-treatment differential expression gene volcanic patterns; (B) KEGG-enriched chords representing up-and down-regulated genes caused by P.M OMVs, respectively, and (C); (D) And (E) a MCODE cluster analysis map representing P.M OMVs-induced up-regulated differential genes; (F) (G) MCODE cluster analysis map showing P.M OMVs down-regulated differential genes. The darker the color in the MCODE cluster analysis map, the stronger the effect of the gene with other genes, and the greater the effect. Three biological replicates per group.

FIG. 7 is a volcanic plot, a heat map, and a KEGG enrichment pathway plot of predicted target genes for differential expression microRNAs after P.M OMVs treatment. (a) a volcanic pattern of microRNA differential expression caused by P.M OMVs; (B) And (C) heat maps of the first 20 differentially expressed micrornas in P.M OMVs, respectively; (D) And (E) KEGG enrichment pathway maps respectively representing up-regulation and down-regulation of microRNA predicted target genes. Three biological replicates per group.

FIG. 8 is the effect on osteoclast formation and related gene expression following microRNA transfection. (A) And (B) represent NC, miRNA and inhibitor transfected osteoclast Trap, respectively, and right side represents NC, miRNA and inhibitor transfected Trap, respectively ⁺ Osteoclast number; (C) - (E) effect of NC and three microRNA chemicals on osteoclast-related genes after transfection; (F) - (H) effect of NC and three microRNA inhibitors transfection on osteoclast-related genes. Experimental results were obtained from three independent replicates and expressed as mean ± SD. Scale = 200 μm, p<0.05,**,p<0.01,***,p<0.001,****,p<0.0001。

FIG. 9 is the effect of mmu-miR-96-5p on Abca1 expression. (A) TargetScan, RNA22 and miRDB predict the Wen map of the mmu-miR-96-5p target gene; (B) A wien diagram of the intersection of the mmu-miR-96-5p target gene and mRNA in the up-or down-regulation of mRNA in the sequencing; (C) And (D) represent expression of Abca1 by RT-qPCR and WB after treatment with P.M OMVs, respectively; (E) miR-96-5p target site in 3' UTR of Abca1 mRNA and mutation sequence schematic diagram thereof; (F) Relative fluorescence activity after 48 hours of co-transfection of HEK293T cells with reporter constructs containing Abca1WT or Mut 3' UTR and NC or miR-96-5p mic; (G) And (H) RT-qPCR and WB respectively detect expression of Abca1 gene and protein after transfection of miR-96-5p mic and inhibitor. Experimental results were obtained from three independent replicates and expressed as mean ± SD. * P <0.05, p <0.01, p <0.001, p <0.0001.

Fig. 10 is the effect of P.M OMVs on mitochondrial and osteoclast apoptosis. (a) TEM images of mitochondria; (B) ATP levels after BMMs were cultured in M-CSF+RANKL medium with or without 0.15. Mu.g/ml P.M OMV; (C-D) Mean Fluorescence Intensity (MFI) of ROS and DCF in osteoclasts after 5 days of stimulation in medium containing M-csf+rankl (with or without 0.15/0.3 μg/ml P.M OMV); (E) Mitochondrial membrane potential and (F) change in JC-1 aggregate/monomer fluorescence ratio; (G-H) osteoclast apoptosis flow chart, (I) total protein western blot analysis of caspase-3, bcl-2 and Bax; (J-K) expression of cytochrome c in cytoplasm and mitochondria. Experimental results were obtained from three independent replicates and expressed as mean ± SD. # p <0.05; # and p <001; # #, p <0.001 compared to M-CSF, p <0.05, p <0.01, p <0.001, p <0.0001 compared to M-csf+rankl.

In the figure, CIA score data are expressed as mean ± SEM, the remaining statistical data are expressed as mean ± SD, plotted on GraphPad Prism 8.0 and statistically analyzed, and Student's t test compares the two groups, and is considered statistically significant at p < 0.05.

Fig. 1-10, experimental results were obtained from three biological replicates with P <0.05, P <0.01, P < 0.001.

Detailed Description

For a clearer understanding of the present invention, the present invention will now be further described with reference to the following examples and drawings. The examples are for illustration only and are not intended to limit the invention in any way. In the examples, each of the starting reagent materials is commercially available, and the experimental methods without specifying the specific conditions are conventional methods and conventional conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

1. Materials and reagents

Proteus mirabilis (P.mirabilis) was purchased from ATCC (USA). BHI broth, MRS medium was purchased from guangdong-cyciware microbiology limited. LB solid Medium Invitrogen corporation. alpha-MEM was purchased from TRAP staining kit from Sigma (united states). 96-well hydroxyapatite plates were purchased from corning company (Corning Osteoassay). NFATc1 was purchased from CST, c-Fos, CTSK from Abcam (usa), MMP9 from wuhan three eagle biotechnology limited (wuhan), ikb- α from signalwayanti body (usa), GAPDH, β -actin from beijing boossen biotechnology limited (beijing), bax, bcl-2, caspase-3, ERK, p-ERK from Jiangsu-philic biological research center limited (Jiangsu). Alizarin red and ALP staining kits were purchased from bi yun-tian biosciences (Shanghai).

The names and sequences of the primers used in the present invention are shown in Table 1.

TABLE 1 names and sequences of Gene primers

2. Abbreviations

M-CSF, macrophage-Colony Stimulating Factor, macrophage colony stimulating factor

RANKL, receptor activator of nuclear factor kappa B ligand, nuclear factor NF-kappa B ligand receptor activators

BMMs, bone marrow derived monocytes/macrophages, bone marrow derived mononuclear/macrophages

LPS, lipopolysaccharide

ROS, reactive oxygen species, reactive oxygen species

Abca1, ATP binding cassette subfamily A member 1

Cyto c, cytochrome c

Example 1: extraction and identification of Proteus mirabilis outer membrane vesicles (P.M OMVs)

1. Bacterial culture

A monoclonal colony of Proteus mirabilis (P.mirabilis) was picked up and placed in BHI broth and incubated at 37℃for 16-18 hours under aerobic conditions. After the cultivation is finished, the enzyme-labeled instrument detects the absorbance (OD) at the wavelength of 600nm, and when the OD value is 0.6-0.8, bacteria are collected.

2. Extraction, identification and quantification of bacterial outer membrane vesicles

After the collected bacteria are centrifuged for three times at 4 ℃ and 8000rpm, the supernatant is collected, a 0.22 mu m filter is used for filtering, 200 mu l of bacterial liquid is taken to be coated on an LB agar culture plate before and after the filtering and is put into a constant temperature incubator for culturing for 12 hours at 37 ℃, the supernatant after the filtering has no bacterial growth on the culture plate, and when the bacteria before the filtering have bacterial growth on the culture plate, the collected supernatant is put into a refrigerator at 4 ℃ for use. The bacterial serum was ultracentrifuged (150,000 g,1.5h,4 ℃) to obtain a precipitate, which was the Outer Membrane Vesicles (OMVs). The vesicles obtained after the PBS dissolution precipitation were filtered using a 0.22 μm syringe filter on a sterile operating table, and the OMVs obtained after filtration were packed into a plurality of EP tubes and stored at-80℃until use.

Bacterial culture, pretreatment and ultracentrifugation gave a white pellet which was dissolved using PBS, a nanoparticle tracking analyzer (nanoparticle tracking analysis, NTA) captured OMV samples diluted with PBS (1:5000) at 11 positions and the data analyzed using built-in ZetaView 8.02.31 software to determine OMV size and number. OMV morphology was observed by transmission electron microscopy (Transmission electron microscope, TEM). TEM shows a bilayer membrane structure (FIGS. 1A & B), NTA shows that the particle size of the obtained precipitate is between 50-450nm (FIG. 2), these results show that the obtained precipitate is OMV.

Protein quantification was performed on P.M OMVs by BCA method, with P.M OMVs having an initial concentration of 475.6 + -56.7 μg/ml, and the volume of P.M OMVs required to be added at a final concentration of 0.15 μg/ml was calculated prior to use.

Example 2: P.M OMVs inhibit RANKL-induced differentiation, fusion and resorption activity of osteoclasts

1. The quantitative chromogenic endotoxin (LAL) kit determines P.M OMVs with 2.64EU endotoxin per 0.15g protein. The effect on BMMs after 48, 72 and 96 hours of treatment with various concentrations of P.M OMVs and standard P.M LPS was examined by CCK 8. The specific operation is as follows:

BMMs cells were isolated at 5X 10 ⁴ A density of/ml was seeded in 96-well plates with 100. Mu.l of system per well, prepared OMVs and LPS at each concentration gradient were added to the cells and incubated for 48, 72 and 96 hours with 10. Mu.l CCK-8,5% CO per well ₂ OD was measured at 450nm after incubation for 3 hours at 37 ℃. The experiment was repeated three times and the percentage of cell viability was used as a positive control without stimulation.

Cell viability percentage (%) = [ (experimental group OD value-blank group OD value)/(positive control group OD value-blank control group OD value) ]100%

P.M OMVs significantly reduced the cell viability of BMMs at higher concentrations, while at protein concentrations of 0.15 and 0.3 μg/ml and varying concentrations of P.M LPS (0-1000 ng/ml) had a slight effect on BMM viability (FIG. 3A).

2.0.15,0.3 μg/ml P.M OMVs and 5,10ng/ml P.M LPS treated with RANKL induced respectivelyDifferentiation of osteoclast from source and differentiation of osteoclast induced by RANKL of DBA/1 male mouse of inflammatory source (CIA-induced arthritis), trap staining, detection of related gene expression and detection of related protein expression, related gene expression was detected by RT-qPCR experiments, and related protein expression was detected by western blotting experiments. The RNA primer sequences of the related genes are shown in Table 1.

The experiments were divided into three groups: trap staining of the formation of osteoclasts was observed with M-CSF, M-CSF+RANKL, M-CSF+RANKL+OMVs, RANKL-induced osteoclasts stimulated with 0.15. Mu.g/ml P.M OMVs and 5,10ng/ml P.M LPS, respectively. Meanwhile, the effect of 0.15. Mu.g/ml P.M OMVs on the expression of osteoclast-associated genes Acp5, MMP9, CTSK, itg β3, NFATc1 and c-Fos was determined by RT-qPCR. The specific operation is as follows:

1) RANKL-induced osteoclast differentiation: adherent BMMs were digested with 0.25% pancreatin for 5 min, centrifuged at 800rpm for 5 min, and cells were counted after 3ml complete media was resuspended, according to 4X 10 ⁵ 25ng/ml M-CSF was added simultaneously with each ml plating, 37℃and 5% CO ₂ After 24 hours of culture in the incubator, 50ng/ml of RANKL and/or P.M OMVs are added, only 25ng/ml of M-CSF is added into a control group, the control group is put into the incubator for continuous culture, the culture medium, the inducer (M-CSF and RANKL) and P.M OMVs are replaced every two days, continuous induction is carried out for 3-5 days, more fusion and larger volume occur, and the multinuclear cells with visible vacuoles are osteoclasts.

2) Trap staining: the old culture medium in the 96-well plate is sucked away, 80 μl of the prepared fixative is slowly added into each well (the preparation method is operated according to the instruction) and the mixture is fixed at 37 ℃ for 15 minutes; after removing the fixing solution and washing with double distilled water, 80 μl of staining solution is added to each well, and the wells are stained at 37 ℃ in a dark place for 60 minutes; after the dyeing is finished, washing for 1 time by double distilled water, adding 50 mu l of hematoxylin staining solution into each hole, washing the hematoxylin staining solution after 45 seconds to 1 minute of dyeing, washing for three times by double distilled water, photographing the dyed cells under an inverted fluorescent microscope, and counting the number of the cells with the number of cell nuclei being more than 3.

FIG. 3B is 0.15,0.3 μg/ml P.M OMVs stimulated RANKL inductionBMMA differentiation of origin into osteoclasts, representative images of Trap staining and TRAP ⁺ Counting the number of multinuclear cells. FIG. 3C 0.15,0.3 μg/ml P.M OMVs stimulate RANKL-induced differentiation of inflammatory derived BMMs to osteoclasts, trap staining representative images and Trap ⁺ Counting the number of multinuclear cells.

3) RNA extraction and RT-qPCR assay

Cells were lysed using Trizol and RNA was obtained by sequentially using chloroform, isopropanol, 75% ethanol and enzyme-free water, the concentration of sample RNA was measured using a microplate reader, RNA was reverse transcribed into cDNA according to 1. Mu.g of total mass, RNA was reverse transcribed into cDNA according to kit instructions (DBI, germany), statistical data were obtained by RT-qPCR reaction in a Light Cycler480 instrument using Promega fluorescent quantitation kit (USA) and 2 was calculated ^-ΔΔct The value calculates the variation difference. The RNA primer sequences of the osteoclast-associated genes Acp5, MMP9, CTSK and itg beta 3 are shown in table 1.

FIG. 3D is a graph of the effect of 0.15,0.3 μg/ml P.M OMVs on osteoclast-associated gene expression, qPCR results showing that P.M OMVs significantly down-regulate RANKL-induced expression of osteoclast-associated genes Acp5, MMP9, CTSK, itg.beta.3, NFATc1 and c-Fos.

The western blot experiment was performed as follows:

Using 150. Mu.l of RIPA protein lysate containing PMSF, osteoclasts were lysed on ice for 30 min, centrifuged at 4℃for 10 min at 12000g to obtain the supernatant, which was protein quantified by BCA method, the volume required for a total protein loading mass of 20. Mu.g was calculated, and the following proteins: adding a protein loading buffer solution at a ratio of 5×loading buffer=4:1, heating in a metal bath at 100 ℃ for 5 minutes, adding the protein into a 10% sds-PAGE gel, carrying out 120V electrophoresis for 80 minutes, activating a PVDF film in methanol for 2 minutes and placing the PVDF film on the gel, carrying out constant-current 200mA electrophoresis for 90 minutes, placing the PVDF film in 5% skim milk powder, sealing a shaker at room temperature for 2 hours, incubating the shaker at 4 ℃ overnight, adding a proper amount of TBST after taking out the primary antibody, washing the film for 5 minutes×6 times, adding a corresponding secondary antibody, incubating the horizontal shaker for 1 hour, washing the horizontal shaker for 10 minutes×3 times by using TBST after the secondary antibody incubation is completed, preparing an a solution and a B solution of ECL luminescent solution according to a ratio of 1:1, selecting a chemiluminescence mode for exposure, analyzing a grey value of a strip by Image J software, and analyzing data by Graphpad Prism.

FIG. 3F is 0.15 μg/ml P.M OMVs expressing the osteoclast-associated proteins MMP9, CTSK, NFATc1 and c-Fos, but not inhibiting the IκBα protein.

3. Mature osteoclasts have an intact F-actin loop and multiple nuclei following RANKL induction. RANKL-induced osteoclasts were stimulated with 0.15 μg/ml P.M OMVs, respectively, and the effect of P.M OMVs on osteoclast F-actin ring formation was observed and detected by osteoclast F-actin ring imaging. At the same time, osteoclast bone resorption activity was examined. The specific operation steps are as follows:

(1) Osteoclast F-actin loop imaging

1) Induction of osteoclast: according to 2X 10 ⁵ BMMs were induced by culture on confocal dishes and RANKL-induced osteoclasts were stimulated with P.M OMVs for 5 days as in example 2;

2) Fixing, permeabilizing and blocking cells: after washing the successfully induced osteoclasts with PBS and fixing with 4% paraformaldehyde for 15 min, permeabilizing with 0.1% Triton X-100 for 30 min and blocking with 5% BSA for 1h;

3) Staining and imaging: the phalloidin is used for preparing the following components: PBS=1:40 is diluted and becomes a phalloidin working solution, the working solution is dyed for 60 minutes in a dark place, the working solution is washed for three times by PBS, 50 μl of anti-fluorescence quenching agent containing DAPI is added, the working solution is incubated for 15 minutes in a dark place at room temperature, and the dyeing is finished, and an inverted laser confocal microscope is used for photographing.

(2) Osteoclast bone resorption activity assay

BMMs were plated in osteoassay stripwell plates using M-CSF, M-CSF+RANKL induction, and cells were treated with OMVs simultaneously with RANKL induction. After 5 days, the formation of the osteoclast is observed, and the bone absorption function of the osteoclast is fully exerted by prolonged culture for 5-7 days. After the culture is finished, PBS is washed for three times, 0.3% hypochlorous acid is added, the mixture is stood for 10 minutes to remove cells adhered in the holes, the holes are washed by distilled water for three times to sufficiently remove the fallen cells, distilled water is sucked as clean as possible, then the plate is left at room temperature for airing, and finally an inverted fluorescence microscope is used for observing the bone absorption condition of the osteoclast cells. Bone resorption area was analyzed using Image-pro plus software. For 3D visualization, the topography of the absorption region was reconstructed using Image J software.

FIG. 3E is a confocal representation of the effect of P.M OMVs on RANKL induced osteoclast F-actin ring formation. FIG. 3G is a representation of bone resorption after P.M OMVs stimulation, 3D image presentation of the representation and per μm ² Is a statistical information of pit areas (n=6). The results indicated that P.M OMVs reduced F-actin ring formation (FIG. 3E) and significantly reduced osteoclast uptake area (FIG. 3G). The F-actin ring maintains the morphology of osteoclasts so that osteoclasts develop their bone resorption and thus when the structure is damaged or fails to form, it influences the formation and bone resorption of osteoclasts, and the data of example 2 indicate that P.M OMV has the effect of inhibiting the differentiation and function of osteoclasts.

Example 3: P.M OMV can improve bone loss due to osteoporosis caused by OVX

The C57 female mice of 8-10 weeks were intraperitoneally injected with 4% chloral hydrate volume (μl) =mouse weight (g) ×10), the mice were operated after 10 minutes in deep anesthesia, the positions of the ovaries were judged on the backs, the skin, fat and muscle layers of the mice were cut off, the ovaries were found, the ovaries were ligated and removed, the muscle layers, fat layer and skin layers of the mice were sutured, and the other side of the ovaries were removed by using an alcohol cotton ball to stop bleeding, the Sham operation (Sham) group did not remove ovaries, the mice were kept under an infrared treatment lamp after removing ovaries, the mice were routinely fed after 12 hours, and after two weeks ovaries were started to inject PBS and OMVs into the joint cavities once a week, and three groups were divided: sham+pbs group, ovx+pbs group, ovx+ P.M OMVs group, after 8 weeks, mouse serum, uterine and femoral samples were collected, femoral samples were fixed in 4% paraformaldehyde for 48h microCT scans, and HE and Trap staining was performed after 30 days decalcification on a shaker.

The experimental procedure and results are shown in fig. 4. The trabecular bone of OVX group became sparse and improved after P.M OMV treatment (fig. 4B). Successful ovariectomy was confirmed by the uterine/body weight ratio, which was significantly reduced after OVX surgery (FIG. 4C). Bone Mineral Density (BMD) and other trabecular bone parameters Tb.BV/TV, tb.N, tb.Th were significantly reduced in PBS-treated OVX mice as shown in FIGS. 4D-H, but improved upon P.M OMV treatment. CTX-1 is a marker of serum bone resorption, CTX-1 levels were significantly reduced after P.M OMVs treatment (fig. 4I), while Osteocalcin (OCN) levels were unchanged (fig. 4J), and HE staining showed a decrease in trabecular bone number and thickness (fig. 4K). TRAP staining showed a significant decrease in n.oc/b.pm after P.M OMVs treatment (fig. 4L and M). These data indicate that P.M OMVs have protective effects on OVX-induced bone loss.

Example 4: P.M OMVs can reduce bone erosion in type II collagen-induced arthritis (CIA)

The preparation method comprises the steps of injecting 100 mu l of CII emulsifier with the concentration of 0.5mg/ml into DBA/1 mice of 9-11 weeks, calculating the required volumes of CII glacial acetic acid and CFA, adding the CII glacial acetic acid and Freund's complete adjuvant solution into a precooled mortar according to the volume ratio of 1:1, placing the mice at a position about 3cm above the exposed tail root on a fixed frame after grinding and emulsifying successfully on ice, injecting 100 mu l of the emulsifier into the mice intradermally through the tail root, performing secondary immunization after 21 days, wherein the concentration of immunization is 100 mu l of CII emulsifier with the concentration of 0.5mg/ml into each mouse, the Freund's incomplete adjuvant is adopted as an adjuvant, and the immunization method is the same, and PBS and OMVs are injected into joint cavities of the mice on the 3 rd day after the secondary immunization, and the total of 3 groups are divided into the following weekly: normal, cia+pbs, cia+ P.M OMVs, starting scoring at day 5 after secondary immunization, joint swelling and redness were defined as joint inflammation, scoring criteria as follows: each inflamed toe or knuckle is 1 point, giving 1-5 points depending on the severity of the inflammation of the wrist or ankle. Each paw of each mouse has a score of 0 to 15, and the total score of each mouse is 0 to 60 points; serum and a mice post-transmission sample are collected after 14 weeks of secondary immunization, paws are placed into 4% paraformaldehyde for fixation for 48 hours, microCT scanning is carried out, and HE staining is carried out after decalcification on a shaker for 30 days.

The experimental procedure and results are shown in fig. 5. Fig. 5A shows a brief course of this experiment, P.M OMVs affect joint inflammation only at the initial stage (fig. 5B-D), and HE staining of joint sections demonstrates the results of the invention (fig. 5E and F). The anti-CII antibody levels decreased in the post-inflammatory phase following omv treatment (fig. 5G). In contrast to increasing serum OCN levels, P.M OMVs significantly reduced CTX-1 levels (FIGS. 5H and I). As shown in FIG. 5J-O, P.M OMV significantly improved the CIA-induced BMD, tb.N, tb.Th decrease, but had no effect on Tb.BV/TV and Tb.sp.

Example 5: P.M OMVs modulate MAPK pathways

P.M OMVs significantly affected the function of osteoclasts in vitro and in vivo, the present invention compared P.M OMVs with RANKL groups to obtain up-and down-regulated differential genes, respectively (FIG. 6A), and they were subjected to KEGG enrichment analysis, respectively, as shown by the KEGG enrichment chord diagram of FIG. 6B, the up-regulated differential genes were mainly concentrated in cytokine interactions, viral infection and NOD-like receptor signaling pathways, while the down-regulated genes were mainly concentrated in ECM receptor interactions, protein digestion and absorption and MAPK pathways (FIG. 6C), protein-protein interaction network diagrams were drawn by using String, and the up-and down-regulated key genes of P.M OMVs were found by using MCODE, respectively, showing that the up-regulated key genes were CCL5 and IL-1β (FIG. 6D, E), and the down-regulated key genes were Pdgfb, EGFR, pgf, fos, acp5, ATc1, DC-STAMP (FIG. 6F, G), wherein Pgfdb, EGFR Pgf, foPK 1 participate in the pathways. From the sequencing results, RANKL induced osteoclast formation with the MAPK pathway activated, but P.M OMVs treated with MAPK pathway inhibited, suggesting P.M OMVs may affect osteoclast formation by interfering with MAPK pathway.

Through analysis of RANKL and P.M OMVs group miRNA sequencing results, the differential expression fold greater than 2 is shown in fig. 7A in volcanic diagram, the results show that there are 44 micrornas up-regulated, 48 down-regulated in P.M OMVs, fig. 7b, c respectively show the microRNA names with up-down regulation fold of change of 20, the miRanda, targetScan and RNAhybrid are used to predict the target genes up-regulated and down-regulated micrornas and KEGG pathway enrichment analysis is performed on the target genes respectively, as shown in fig. 7d, e, the pathways with the most significant enrichment of microRNA target genes are MAPK pathways, indicating that P.M OMVs can influence the MAPK pathway, which is consistent with the mRNA sequencing results.

In this embodiment, the sequencing analysis of the transcription unit is specifically as follows:

1) mRNA sequencing: after RNA extraction, the purity and integrity of the RNA were separately detected by NanoDrop2000 agarose gel electrophoresis, and after meeting the requirements, mRNA sequencing was performed using IlluminaNovaseq6000 to obtain paired end reads. SeqPrep and sibkle prune the raw paired-end reads for quality control. TopHat2 was used for sequence alignment analysis to obtain mapping reads, which were assembled and spliced using Cufflinks from the existing reference genome. Samples were subjected to differential expression analysis using transcripts per million reads (TPM) and DEseq2, |log2FC| >2 and padjust <0.05 were considered as Differentially Expressed Genes (DEG). Performing KEGG channel analysis on the DEG, and searching for a key gene by utilizing cytoscape;

2) Sequencing miRNA: the preparation kit of the Illumina TruSeq small RNA library constructs a miRNA library, the high-throughput sequencing is carried out on the small RNA fragments of 18-32nt which are enriched by utilizing an Illumina Novaseq6000 platform, a mirtakey 2 software package is used after mapped data is obtained, differential expression miRNA is obtained through TPM and DEseq2, I log2FC I >1 and padjust <0.05 are used as DEG, and KEGG enrichment analysis is carried out on target genes after the target genes of differential miRNA are predicted by using MiRanda, targetScan and RNAhybrid, so that a target gene enrichment path is obtained.

Example 6: effects of microRNA micrometers and inhibitors on osteoclast formation and related genes

micrornas can affect the expression and function of a target gene by binding to the target gene, so studying the effect of micrornas has an important role in understanding genes and pathways downstream thereof. miR-155-5p, miR-96-5p and miR-653-5p are selected for research in the invention. NC, miR-155-5p, miR-96-5p, miR-653-5p micrometers and inhibitor are transfected into RANKL-induced osteoclasts, and the formation of the osteoclasts and the expression of related genes after the transfection of 3 microRNA micrometers are respectively detected, so that the results show that the number of the osteoclast formation is basically unchanged after the miR-96-5p micrometers transfection (figure 8A) but the expression of Acp5 and MMP9 is obviously up-regulated (figure 8C). miR-155-5p mimic decreased osteoclast numbers after transfection (FIG. 8A), and was able to significantly down-regulate the expression of Acp5, CTSK and c-Fos and (FIG. 8D). Inhibition of osteoclast formation following miR-653-5p mimic transfection (fig. 8A) significantly reduced expression of Acp5, MMP9, CTSK, c-Fos but significantly upregulated expression of itg beta 3 (fig. 8E). The results after transfection of miR inhibitors showed that miR-96-5p inhibitor significantly inhibited osteoclast formation (FIG. 8B) and significantly inhibited Acp5 and CTSK expression (FIG. 8F), miR-155-5p inhibitor had no effect on osteoclast formation (FIG. 8B) but significantly upregulated Acp5, MMP9, CTSK, itg beta 3, NFATc1, c-Fos expression (FIG. 8G) in osteoclasts, and miR-653-5p inhibitor significantly inhibited osteoclast formation (FIG. 8B) but enhanced c-Fos, itg beta 3 expression (FIG. 8H). These results indicate that up-regulation of miR-155-5p can inhibit osteoclast formation, which is consistent with previous research results, while decreasing the interaction of miR-96-5p with the target gene can result in inhibition of osteoclast formation, while up-regulation of miR-653-5p expression or inhibition of the effect can inhibit osteoclast formation, so that miR-96-5p is selected for research.

Example 7: mmu-miR-96-5p can target and regulate Abca1 expression

Targetscan, RNA22 and miRDB predict the target genes of mmu-miR-96-5p, 200 target genes are obtained after intersection (FIG. 9A), then the 200 target genes are respectively intersected with the up-regulated or down-regulated DEGs of the RNA sequencing result, 13 common genes are in the up-regulated genes, 6 genes are in the down-regulated genes, and the gene of interest is Abca1 (FIG. 9B) finally determined. RT-qPCR and WB detection P.M OMVs significantly up-regulated Abca1 gene and protein expression following treatment (FIGS. 9C and D), consistent with sequencing results. Double luciferase reports demonstrated whether mmu-miR-96-5p could directly target Abca1, first a wild-type (WT)/mutant (Mut) Abca1-psiCheck2 vector (FIG. 9E) was constructed and transfected into HEK293T cells with mma-miR-96-5 p, respectively, as shown in FIG. 9F that luciferase activity of Abca1 WT was significantly reduced after miR-96-5p mimic treatment, whereas change in luciferase activity of Abca1-Mut was essentially unchanged, indicating that miR-96-5p could target Abca1. To further verify the targeting relationship of miR-96-5p mimic and Abca1, miR-96-5p mimic and inhibitor were transfected into osteoclasts, respectively, and the results indicate that expression of Abca1 gene and protein was significantly inhibited after mimic transfection (FIG. 9G), and expression of Abca1 gene and protein was significantly up-regulated after inhibitor transfection (FIG. 9H).

In this example, the effect of the dual-luciferase reporter assay and miRNA on osteoclast differentiation was as follows:

(1) Double luciferase reporter assay

Construction of Abca1 WT 3'UTR and Abca1 Mut 3' UTR plasmid vectors, digestion and counting of HEK293T cells in good growth conditions followed by 5X 10 ⁴ And (3) carrying out transfection on cells according to the instruction when the cells grow to about 80% in a 24-well plate, adding 100 mu L of a reporter gene cell lysate into the 24-well plate after fully mixing, centrifuging at 12,000g for 5 minutes after fully lysing, sampling 100 mu L of the sample, adding 100 mu L of a luciferase detection reagent, measuring RLU (relative light unit), adding an equivalent amount of a renilla luciferase detection working solution to measure RLU after the measurement is completed, calculating the ratio of two luciferases, and comparing the ratio differences of different groups.

(2) Effect of miRNA on osteoclast differentiation

BMMs cells are paved in a 96-well plate, NC miics and inhibitors are respectively transfected into the cells according to the method after the cells are attached, RANKL is added after 24 hours, liquid is changed after 48 hours, the formation of the osteoclasts is observed after 48 hours, trap staining is carried out, an inverted fluorescence microscope is used for photographing, and the number of cell nuclei is greater than three and is defined as osteoclasts, and the number of the cell nuclei is counted.

BMMs are paved in a 12-hole plate, 50nM NC and miRNA micrometers, 100nM NC inhibitor and miRNA inhibitor are respectively transfected into cells after 24 hours according to the steps of the instruction book, and the expression change of the related genes of the osteoclast after the miRNA micrometers and the inhibitor are transfected is detected.

Example 8: P.M OMVs inhibit osteoclast formation by mitochondrial-dependent apoptosis

Abca1 promotes mitochondrial cholesterol lowering and is involved in cytochrome c (Cyto c) release, which may lead to disruption of mitochondrial structure. The energy required for osteoclast differentiation is mainly derived from mitochondrial oxidative metabolism. To study the effect of P.M OMVs on mitochondria, mitochondrial structures were first observed with transmission electron microscopy, as shown in fig. 10A, P.M OMVs destroyed the mitochondrial structure of RANKL-induced osteoclasts and significantly reduced ATP production on days 1 and 5 (fig. 10B), indicating P.M OMVs not only destroyed mitochondrial structure, but reduced ATP production. Mitochondria are the major source of intracellular Reactive Oxygen Species (ROS), and excessive ROS production may lead to loss of Mitochondrial Membrane Potential (MMP) and impaired ATP synthesis, which in turn lead to apoptosis, thus further examining ROS levels and apoptosis after P.M OMVs treatment. As shown in fig. 10C, DCFH-DA fluorescence percentage and Mean Fluorescence Intensity (MFI) increased significantly after 5 days of treatment with P.M OMVs at two different concentrations (fig. 10D). Accumulation of ROS destroys the mitochondrial membrane potential (Δψm), and thus changes in cellular mitochondrial membrane potential were measured (fig. 10e & f). P.M OMVs significantly increased intracellular ROS but decreased the level of Δψm. As shown in fig. 10g & h, the rate of apoptosis significantly increased in a concentration-dependent manner following treatment with P.M OMVs, and the Bax/Bcl-2 ratio and clear caspase-3 significantly increased following treatment with P.M OMVs (fig. 10I) and promoted release of mitochondrial Cyto c (fig. 10j & k), suggesting that P.M OMVs could significantly promote osteoclast apoptosis via a mitochondrial-dependent pathway.

In this example, mitochondrial structure observation, intracellular active oxygen and apoptosis detection were performed as follows:

(1) Mitochondrial structure

BMMs were prepared at 6X 10 ⁵ Uniformly spreading the cells/ml into a 6-hole plate, uniformly spreading 50ng/ml RANKL after 24 hours to induce osteoclasts, simultaneously adding or not adding P.M OMVs, collecting the cells after three days of induction, centrifuging at 1500rpm for 5 minutes to enable the cells to become 0.5-1mm high cell sediment, slowly adding 500 μl of 2.5% glutaraldehyde along the pipe wall, standing at room temperature for 1 hour, placing into a refrigerator at 4 ℃ for standing for 3 hours, sucking out glutaraldehyde, filling PBS into an EP pipe, placing into the refrigerator at 4 ℃ for standing overnight, sending the samples into an electronic microscope chamber, observing the ultra-microstructure of mitochondria under a transmission electronic microscope after preparing the cells into ultra-thin slices, and taking 5-6 visual fields for each sample for photographing.

(2) Intracellular reactive oxygen species and apoptosis assays

1) RANKL induced osteoclast differentiation for 5 days in presence/absence of OMV stimulation, 1 hour before collecting cells, positive control group added with roup, pre-chilled PBS washed cells twice, after adding 400 μl of DCFH-DA diluted with serum-free medium per well and incubating for 20 min in dark place, washed twice with serum-free medium, collected cells were centrifuged at 1500rpm for 5 min to retain pellet, 200 μl PBS resuspended and transferred to flow tube for on-machine detection, flowJo V10 software processed analysis of results.

2) RANKL induced osteoclast differentiation for 5 days in presence/absence of OMV stimulation, cells were collected after washing with pre-chilled PBS, pellet was collected after centrifugation at 1500rpm for 5 min, 100 μl of 1 x Binding buffer resuspended cells were added to pellet, 5 μl of FITC Annexin V and 5 μl of PI were sequentially added, incubation was performed for 15 min at room temperature in the dark, 200 μl of 1 x Binding buffer resuspended was added and transferred to flow tube for on-machine detection, and after detection was completed, the results were analyzed by FlowJo V10 software.

Example 9:

the present embodiment provides a pharmaceutical composition for preventing or treating osteolytic diseases, the main active ingredient of which comprises Proteus mirabilis outer membrane vesicles.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and may be administered parenterally, for example, by intravenous injection, intramuscular injection, or intraoral administration. The preparation form is selected from oral preparation, injection preparation, mucosa administration preparation, inhalant, and external preparation, and pharmaceutically acceptable adjuvants can be further selected according to the preparation form.

In a specific embodiment, the dosage form of the pharmaceutical composition is an oral liquid, and common auxiliary materials of the oral liquid comprise solvents, aromatic agents, corrigents, clarifying agents, preservatives and the like, and can be added simultaneously or alternatively, wherein the solvents are necessary auxiliary materials, and the solvents can be water.

In one embodiment, the dosage form of the pharmaceutical composition is a granule, and the common auxiliary materials of the granule are one or more of a filler, a binder, a wetting agent, a disintegrating agent, a lubricant and a film coating material.

Example 10:

this example provides an osteoclast formation and/or activation inhibitor whose main component comprises Proteus mirabilis outer membrane vesicles.

In a specific embodiment, the Proteus mirabilis outer membrane vesicles, buffers, diluents, pH modifiers, etc. may be assembled into a kit for inhibiting the formation and/or activation of osteoclasts.

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.

Claims

1. Use of proteus mirabilis adventitia vesicles in the manufacture of a medicament for the treatment of osteolytic disorders, wherein the osteolytic disorders are ovariectomy-induced osteoporosis and type II collagen-induced arthritis bone erosion;

The medicine is a medicine for inhibiting the differentiation and the function of osteoclast by using outer membrane vesicles of Proteus mirabilis.