阅读说明：本技术 奇异变形杆菌外膜囊泡在制备预防或治疗骨溶解性疾病药物中的应用 (Application of proteus mirabilis outer membrane vesicle in preparation of medicine for preventing or treating osteolytic diseases ) 是由 库蒂·塞尔瓦·南达库玛 王婷婷 于 2021-08-31 设计创作，主要内容包括：本发明公开了奇异变形杆菌外膜囊泡在制备预防或治疗骨溶解性疾病药物中的应用。奇异变形杆菌外膜囊泡抑制miR96-5p表达而促进Abca1表达,从而抑制MAPK/ERK通路,导致破骨细胞分化受阻；奇异变形杆菌外膜囊泡诱导MPT相关细胞色素c的释放导致线粒体结构被破坏,增加活性氧产生,导致破骨细胞凋亡增加。本发明发现奇异变形杆菌外膜囊泡不仅在体外显著抑制RANKL诱导的破骨细胞的分化和功能,也能够在体内改善OVX引起的骨代谢失衡和CII引起的骨侵蚀,为预防或治疗骨溶解性疾病提供了新思路。(The invention discloses an application of proteus mirabilis outer membrane vesicles in preparation of a medicine for preventing or treating osteolytic diseases. The proteus mirabilis outer membrane vesicle inhibits miR96-5p expression and promotes Abca1 expression, so that MAPK/ERK pathway is inhibited, and osteoclast differentiation is hindered; proteus mirabilis outer membrane vesicles induce release of MPT-associated cytochrome c, resulting in disruption of mitochondrial structure, increased reactive oxygen species production, and increased osteoclast apoptosis. The invention finds that the proteus mirabilis outer membrane vesicle not only can remarkably inhibit the differentiation and the function of the RANKL-induced osteoclast in vitro, but also can improve the bone metabolism imbalance caused by OVX and the bone erosion caused by CII in vivo, and provides a new idea for preventing or treating osteolytic diseases.)

1. Application of proteus mirabilis outer membrane vesicles in preparation of medicines for preventing or treating osteolytic diseases.

2. The use according to claim 1, wherein the osteolytic disease comprises osteoporosis, osteolysis, rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, osteogenesis imperfecta, osteopetrosis, multiple myeloma, bone metastatic cancer, hypercalcemia of malignancy, systemic lupus erythematosus, alzheimer's disease, diabetes, breast cancer, prostate cancer, bone loss due to immunosuppressive therapy, bone loss due to glucocorticoid drug therapy, bone loss due to methotrexate therapy, ovariectomy-induced bone loss, and type II collagen-induced bone erosion.

3. The use according to claim 1, wherein the osteolytic disease comprises osteoporosis, osteolysis, rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, osteogenesis imperfecta, osteopetrosis, bone loss due to immunosuppressive therapy, bone loss due to glucocorticoid drug therapy, bone loss due to methotrexate therapy, ovariectomy-induced bone loss, and type II collagen-induced bone erosion;

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

4. The use according to claim 1, wherein the proteus mirabilis outer membrane vesicle is used for preventing or treating a bone-soluble disease by a mechanism,

the proteus mirabilis outer membrane vesicle inhibits the expression of miR96-5p and promotes the expression of Abca1, so that the MAPK/ERK channel is inhibited, and osteoclast differentiation is hindered;

the proteus mirabilis outer membrane vesicle destroys a mitochondrial structure, induces the release of MPT related cytochrome c, increases the generation of active oxygen, and leads to the increase of osteoclast apoptosis.

5. A pharmaceutical composition for preventing or treating osteolytic diseases, comprising proteus mirabilis outer membrane vesicles.

6. The pharmaceutical composition of claim 5, further comprising a pharmaceutically acceptable excipient.

7. Use of proteus mirabilis outer membrane vesicles in the manufacture of an agent or medicament for inhibiting the formation and/or activation of osteoclasts.

8. A pharmaceutical composition for inhibiting osteoclastogenesis and/or activation, comprising an proteus mirabilis outer membrane vesicle;

preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

9. An inhibitor of osteoclastogenesis and/or activation, comprising an proteus mirabilis outer membrane vesicle.

10. A kit for inhibiting osteoclastogenesis and/or activation, comprising the inhibitor of claim 9.

Technical Field

The invention belongs to the technical field of bone disease treatment, and particularly relates to application of proteus mirabilis outer membrane vesicles in preparation of a medicine for preventing or treating osteolytic diseases.

Background

The bones in the living body are constantly undergoing a bone resorption and bone neogenesis process called bone remodeling, the balance of which has an important role in bone homeostasis. Both osteoclasts, which function to resorb old bone tissue, and osteoblasts, which function to generate new bone, are involved in the bone remodeling process, and are in equilibrium to maintain the balance of bone remodeling. Abnormal increase in osteoclast proliferation and activity causes bone resorption at a rate far exceeding that of bone formation, breaking the balance of bone remodeling, and causing bone tissue destruction and erosion, thereby causing bone diseases such as osteoporosis, rheumatoid arthritis, ankylosing spondylitis, osteopetrosis, systemic lupus erythematosus, alzheimer's disease, diabetes, breast cancer, prostate cancer, and the like.

The osteoclast differentiation pathway can be divided into a classical pathway mediated by nuclear factor NF-kB ligand receptor activator (such as TRAF 6/RANKL/NF-k B, TRAF6/RANKL/MAPK) and a non-classical pathway involved by some inflammatory cytokines (such as IL-1, IL-6 and INF-alpha). When the disease occurs, osteoblast lineage cells or osteocytes generate activating nuclear factor NF-kB ligand Receptor Activators (RANKL), the RANKL is combined with receptor RANK thereof, TRAF6 activated by adaptor TRAF6 is activated, pathways such as NF-kB and MAPK are started, NFATc1 is activated, and therefore osteoclast specific genes, such as osteoclast differentiation fusion (DC-STAMP, Atp6v0d2) and expression of related genes of functions (CTSK, TRAP and MMP9), are activated, osteoclast differentiation is promoted, and bone resorption functions are exerted.

There are many factors that induce over-activation of osteoclasts, leading to a decrease in bone density in patients, including endogenous factors (hormone levels and autoimmunity) and exogenous factors (bacterial infection). Previous studies have shown that there are homologous sequences between proteoliposysin and HLA-DR4 and between proteoliposomic urease and hyaline cartilage, which are capable of cross-reacting with certain autoantigens present in synovial tissue to produce related molecular effects that may be associated with the pathogenesis of rheumatoid arthritis. In addition, staphylococcus aureus is a common cause of osteomyelitis and can increase the abundance of osteoclasts present on the surface of bones in vivo.

Currently, bone disease is mainly ameliorated by modulating hormone levels and anti-inflammation, while fewer drugs targeting osteoclasts, mainly bisphosphonates and denosumab. (1) Glucocorticoid drugs (dexamethasone) and methotrexate can inhibit the release of inflammatory factors such as TNF-alpha in rheumatoid arthritis, but all cause bone loss after long-term use. Dexamethasone, for example, can promote osteoclastogenesis in vitro and cause osteoporosis in animals and patients in vivo; methotrexate also promotes osteoclast formation after use, causing 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 action. On the other hand, non-amino bisphosphonates inhibit ATP-dependent enzymes after metabolism to non-hydrolysable ATP analogues leading to increased osteoclast apoptosis, but they are essentially ineffective in regulating bone erosion. (3) Denosumab (denosumab) used to inhibit RANK ligand (RANKL) resulted in a decrease in osteoclast-mediated bone resorption and turnover. While RANKL is abundantly expressed by dendritic cells and activated T lymphocytes, antagonism by denosumab may affect the immune system, leading to adverse effects including skin eczema, flatulence, cellulitis and jaw necrosis. Furthermore, the interaction of RANK and RANKL is essential for the development of immune cells and hypocalcemia following denosumab treatment, and thus, severe renal insufficiency may be one of the major risk factors for treatment. Receptor activators that block RANK signaling or osteoclasts, while possibly not interfering with synovitis, can protect bone. Blocking upstream cytokines such as TNF-alpha or IL-1 reduces synovial inflammation and cartilage and bone destruction. Synovial inflammation cannot be completely inhibited, and therefore protection of joints by further blocking osteoclast number and activation by the addition of drugs may be the best treatment.

The dysbacteriosis in human body is related to many diseases, the transplantation of probiotic Lactobacillus casei into arthritic mice can obviously improve arthritic symptoms, while the E.coli is related to the development of IBD, and the porphyromonas gingivalis can not only cause periodontitis but also aggravate the generation of arthritis. Although the bacteria can not complete long-distance transmission, the secreted and produced metabolites can be transmitted long-distance, such as SCFAs, secreted toxins and outer membrane vesicles formed by budding can escape from host immunity, and the SCFAs, the secreted toxins and the outer membrane vesicles penetrate through the inner layer barrier of a human body to reach proper positions to play roles, so that the health of the host is influenced.

Bacterially 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 the parent bacterium 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, etc., but also directly with host cells such as epithelial cells, osteoblasts, synoviocytes, etc., e.g., kingllaking OMVs can be internalized by osteoblasts and synoviocytes, leading to increased GM-CSF and IL-6 production. In addition, it can also modulate the host immune system and thus affect the pathogenesis of the disease, studies have shown that OMVs can induce dendritic cell maturation, thereby enhancing the uptake and presentation of antigenic molecules to develop an adaptive immune response. Clinically, type B meningitis OMVs vaccines for invasive meningococcal disease have 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 proteus mirabilis outer membrane vesicles in preparing a medicament 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, depending on the rate of osteoclast differentiation and apoptosis. Thus, one key factor in preventing bone loss in many osteolytic diseases is the inhibition of osteoclast number and function.

The invention provides an application of proteus mirabilis outer membrane vesicles in preparation of a medicine for preventing or treating osteolytic diseases.

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

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

In the technical scheme of the invention, the mechanism for preventing or treating osteolytic diseases by proteus mirabilis outer membrane vesicles is that,

the proteus mirabilis outer membrane vesicle inhibits the expression of miR96-5p and promotes the expression of Abca1, so that the MAPK/ERK channel is inhibited, and osteoclast differentiation is hindered;

the proteus mirabilis outer membrane vesicle induces the release of MPT related cytochrome c, so that the mitochondrial structure is damaged, the generation of active oxygen is increased, and the apoptosis of osteoclast is increased.

The proteus mirabilis outer membrane vesicle inhibits miR96-5p expression and promotes Abca1 expression, so that the MAPK/ERK pathway is inhibited, and the activation of the MAPK pathway causes the activation of NFATc1 so as to induce the differentiation of osteoclasts and the expression of specific genes, so that the inhibition of the MAPK/ERK pathway can cause the differentiation of osteoclasts to be hindered.

Both osteoclastogenesis and osteolysis require a large supply and expenditure of energy, and studies have shown that the energy required for osteoclast differentiation comes primarily from mitochondrial oxidative metabolism. ATP synthesis is therefore reduced when the mitochondrial structure is disrupted, rendering osteoclasts unable to complete the fusion and maturation process due to insufficient energy supply. P.M disruption of mitochondrial structure after OMVs treatment resulted in a significant decrease in ATP production, release of MPT-associated cytochrome c, and an increase in Reactive Oxygen Species (ROS) levels, which resulted in a significant decrease in mitochondrial membrane potential and induction of apoptosis.

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

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

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

In a fourth aspect, the present invention provides a pharmaceutical composition for inhibiting osteoclastogenesis and/or osteoclastogenesis, comprising an proteus mirabilis outer membrane vesicle;

preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

In a fifth aspect, the present invention provides an inhibitor of osteoclastogenesis and/or activation, comprising an proteus mirabilis outer membrane vesicle.

In a sixth aspect, the present invention provides a kit for inhibiting osteoclastogenesis and/or activation, comprising the inhibitor. Further, the kit may further include a buffer, a diluent, a pH adjuster, and the like.

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

The invention has the beneficial effects that:

1. the invention discovers for the first time that the proteus mirabilis outer membrane vesicle not only can remarkably inhibit the differentiation and the function of the RANKL-induced osteoclast in vitro, but also can improve the bone metabolism imbalance caused by OVX and the bone erosion caused by CII in vivo, and provides a new idea for preventing or treating the osteolytic diseases.

2. The proteus mirabilis outer membrane vesicle realizes the protection of bones through a miR96-Abca1-MAPK pathway on one hand, and the proteus mirabilis outer membrane vesicle inhibits the expression of miR96-5p to promote the expression of Abca1, so that the MAPK/ERK pathway is inhibited, and the differentiation of osteoclasts is hindered. On the other hand, the proteus mirabilis outer membrane vesicle induces the remarkable inhibition of miR96-5p expression, so that the up-regulation of an ATP binding cassette subfamily A member 1(Abca1) participating in mitochondrial function is caused, and the ERK phosphorylation is further reduced, which is important for osteoclast activation. Abca1 promotes the lowering of mitochondrial cholesterol and is involved in the release of cytochrome c (cyto c), resulting in the destruction and reduction of mitochondrial structure, while significantly reducing ATP production during osteoclastogenesis. 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, resulting in decreased ATP production ultimately leading to disruption of osteoclast differentiation and function.

Drawings

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

FIG. 2 shows the size and number statistics of P.M OMVs. Experimental results were obtained in three independent replicates.

FIG. 3 is a graph of the effect of P.M OMVs on osteoclast differentiation and function. (A) Cell viability after 48,72,96h treatment of BMMs with P.M OMVs and P.M LPS at different concentrations, respectively; (B)0.15, 0.3. mu.g/ml P.M OMVs, RANKL-treated P.M LPS inducedRepresentative pictures of the differentiation of the osteoclasts and the number statistics of Trap + cells with more than three cell nucleuses; (C) representative pictures of P.M OMVs and P.M LPS at 0.15, 0.3. mu.g/ml stimulating RANKL-induced osteoclast formation of inflammatory origin and Trap with more than three nuclei⁺Counting the number of cells; (D) P.M effects of OMVs on RANKL-induced osteoclast-associated gene expression; (E) and P.Confocal representations of the effect of M OMVs on RANKL-induced osteoclast F-actin looping; (F) P.M effects of OMVs on RANKL-induced osteoclast-associated gene expression; (G) P.M representative graph of bone resorption after OMVs stimulation, 3D image presentation representing pictures and per μm²The statistical information (n ═ 6) of the pit area. (E) Scale bar 50 μm; (B) the scale bar of (C) and (G) is 200 μm. Experimental results were obtained in three independent replicates and are expressed as mean ± SD. A, p<0.05；**,p<0.01；***,p<0.001；****,p<0.0001。

FIG. 4 shows that P.M OMVs protected against OVX-induced bone loss. (A) An experimental process; (B) microcht images of Sham and OVX (with or without P.M OMV treatment); (C) uterus to body weight ratio of mice; (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) HE (n ═ 5) and (L TRAP (n ═ 5) stained femoral sections and (M) the ratio n.oc/b.pm, BMD: bone density, tb.bv/TV: trabecular bone volume per tissue volume, tb.n: number of trabecular bone, tb.th: thickness of trabecular bone, tb.sp: gap between trabecular bone, sham group (n ═ 5), OVX group, the remaining groups n ═ 5, OVX + PBS group and OVX + P.M OMV group, respectively, the experimental results were obtained in three independent replicates and are expressed as mean ± SD #, p < 0.05;, p < 0.01;, p #, p < 0.001;, p #, p < 0.0001;. p < 0.05;, p < 0.01;. p, p < 0.001;. p, p < 0.0001. c.

FIG. 5 is a graph of the effect of P.M OMVs on CIA inflammation and bone erosion. (A) An experimental process; (B) representative pictures of hind paws of different groups of mice; (C-D) mean arthritis and hind foot arthritis scores for each group after the second immunization; (E-F) HE staining of the mouse hindpaw sections on day 107; (G) anti-CII antibody levels in each group of sera at days 0,21,75 and 107 (normal group n-3, other groups n-7); serum (H) CTX-1 and (I) OCN levels; (J) μ CT representative images (normal group n is 3, other groups n is 5); (K-O) different sets of bone parameters. BMD: bone density, Tb.BV/TV: trabecular bone volume per tissue volume, tb.n: trabecular bone number, tb.th: trabecular bone thickness, tb.sp: trabecular bone space. 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 compare with the normal group; p < 0.05; p < 0.01; p < 0.001; p <0.0001 compared to group CIA.

FIG. 6 is a volcano graph, KEGG chord-rich graph and MCODE cluster analysis graph of DEGs after P.M OMVs treatment. (A) P.M differential expression gene volcano plot after OMVs treatment; (B) and (C) KEGG enrichment chords representing P.M OMVs-induced up-and down-regulated genes, respectively; (D) and (E) MCODE cluster analysis plots showing up-regulated differential genes caused by P.M OMVs; (F) (G) MCODE cluster analysis graph showing that P.M OMVs down-regulated differential genes. The darker the color in the MCODE cluster analysis chart represents that the gene has stronger effect with other genes, and the effect is larger. Three biological replicates per group.

Fig. 7 is a volcanic map of differentially expressed micrornas after P.M OMVs treatment, a heat map, and a KEGG enrichment pathway map of predicted target genes. (A) P.M volcano plots of microRNA differential expression by OMVs; (B) and (C) heat maps representing the first 20 differentially expressed micrornas from P.M OMVs, respectively; (D) and (E) KEGG enrichment pathway maps representing up-regulation and down-regulation of microRNA predicted target genes, respectively. Three biological replicates per group.

FIG. 8 shows the effect of microRNA transfection on osteoclastogenesis and expression of related genes. (A) And (B) represent images of the osteoclast Trap stained after transfection of NC, miRNA mix and inhibitor, respectively, and the right side represents the Trap after transfection of NC, miRNA mix and inhibitor, respectively⁺The number of osteoclasts; (C) - (E) effects of NC and of the three microRNA mimics on osteoclast-associated genes after transfection; (F) (H) Effect of NC and three microRNA inhibitors on osteoclast-associated genes after transfection. Experimental results were obtained from three independent replicates and are expressed as mean ± SD. Scale bar 200 μm<0.05,**,p<0.01,***,p<0.001,****,p<0.0001。

FIG. 9 is a graph of the effect of mmu-miR-96-5p on Abca1 expression. (A) TargetScan, RNA22 and miRDB predict the wien diagram of the mmu-miR-96-5p target gene; (B) a Wien diagram of mRNA intersection up-regulated or down-regulated in sequencing of mmu-miR-96-5p target gene and mRNA; (C) and (D) RT-qPCR and WB detect the expression of Abca1 after treatment with P.M OMVs, respectively; (E) schematic diagrams of miR-96-5p target sites in 3' UTR of Abca1 mRNA and mutant sequences thereof; (F) relative fluorescence activity after cotransfection of HEK293T cells with a reporter gene construct containing Abca1WT or Mut 3' UTR and NC or miR-96-5p mimic for 48 hours; (G) and (H) RT-qPCR and WB are used for respectively detecting the expression of Abca1 gene and protein after miR-96-5p mimic and inhibitor transfection. Experimental results were obtained from three independent replicates and are expressed as mean ± SD. P <0.05, p <0.01, p <0.001, p < 0.0001.

Figure 10 is a graph of the effect of P.M OMVs on mitochondrial and osteoclast apoptosis. (A) TEM images of mitochondria; (B) ATP levels after culturing BMMs in M-CSF + RANKL medium with or without 0.15 μ g/ml P.M OMV; (C-D) ROS and DCF Mean Fluorescence Intensity (MFI) in osteoclasts after 5 days of stimulation in medium containing M-CSF + RANKL (with or without 0.15/0.3. mu.g/ml P.M OMV); (E) changes in mitochondrial membrane potential and (F) 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) cytochrome c expression in the cytoplasm and mitochondria. Experimental results were obtained from three independent replicates and are expressed as mean ± SD. #, p < 0.05; #, p < 001; # #, p <0.001 compared to M-CSF, # p <0.05, # p <0.01, # p <0.001, # p, p <0.0001 compared to M-CSF + RANKL.

In the figures, CIA score data are presented as mean. + -. SEM and the remaining statistical data are presented as mean. + -. SD, plotted on GraphPad Prism 8.0 and statistically analyzed, and Student's t test compared between groups, and p <0.05 was considered statistically significant.

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

Detailed Description

In order that the invention may be more clearly understood, it will now be further described with reference to the following examples and the accompanying drawings. The examples are for illustration only and do not limit the invention in any way. In the examples, each raw reagent material is commercially available, and the experimental method not specifying the specific conditions is a conventional method and a conventional condition well known in the art, or a condition recommended by an instrument manufacturer.

Materials and reagents

Proteus mirabilis (p. mirabilis) was purchased from ATCC (usa). BHI broth, MRS medium was purchased from Kyork, Guangdong, Microbiol technologies, Inc. LB solid Medium Invitrogen. alpha-MEM was purchased from TRAP staining kit from Sigma (USA). The 96-well hydroxyapatite plate was purchased from Corning corporation (Corning ostoeassay). NFATc1 was purchased from CST, c-Fos, CTSK from Abcam (USA), MMP9 from Sanying biotech Limited, Wuhan, I κ B- α from Signalway Antibody (USA), GAPDH, β -actin from Beijing Booshen Biotech Limited (Beijing), Bax, Bcl-2, pasase-3, ERK, p-ERK from Jiangsu Kogyo research center, Inc. (Jiangsu). Alizarin red and ALP staining kits were purchased from bi yun tian bio ltd (shanghai).

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

TABLE 1 Gene primer names and sequences

Second, abbreviation

M-CSF, Macrophage-Colony Stimulating Factor, Macrophage Colony Stimulating Factor

RANKL, Receptor activator of nuclear factor kappa B ligand and nuclear factor NF-kappa B ligand Receptor activator

BMMs, bone marrow derived monocytes/macrophages

LPS, Lipopolysaccharide, Lipopolysaccharide

ROS, 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 and placed in BHI broth and cultured aerobically at 37 ℃ for 16 to 18 hours. After the culture is finished, detecting the absorbance (OD) of the culture solution at the wavelength of 600nm by using an enzyme-labeling instrument, and collecting bacteria when the OD value is 0.6-0.8.

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

Centrifuging the collected bacteria at 4 ℃ and 8000rpm for three times, collecting the supernatant, filtering with a 0.22 mu m filter, taking 200 mu l of bacterial liquid before and after filtering, coating the bacterial liquid on an LB agar culture plate, putting the LB agar culture plate into a constant-temperature incubator, culturing for 12 hours at 37 ℃, when the filtered supernatant has no bacteria growth on the culture plate, and when the bacteria before filtering have bacteria growth on the culture plate, putting the collected supernatant into a 4 ℃ refrigerator for use. The bacterial supernatant was ultracentrifuged (150,000g,1.5h,4 ℃) to obtain a pellet, which was an Outer Membrane Vesicle (OMV). After the PBS dissolution precipitation, the resulting vesicles were filtered on a sterile bench using a 0.22 μm needle filter, and the filtered OMVs were loaded into multiple EP tubes and stored at-80 ℃ until use.

After bacterial culture, pretreatment and ultracentrifugation, white pellets were obtained, solubilized using PBS, and OMV samples diluted with PBS (1:5000) were captured at 11 positions by Nanoparticle Tracking Analyzer (NTA) and the data were analyzed using built-in ZetaView 8.02.31 software to determine the size and number of OMVs. The OMV morphology was observed with a Transmission Electron Microscope (TEM). TEM showed a bilayer membrane structure (FIGS. 1A & B), NTA showed a particle size of the obtained precipitate between 50-450nm (FIG. 2), and these results indicated that the obtained precipitate was OMV.

The BCA method was used to quantitate the protein from P.M OMVs, P.M OMVs at an initial concentration of 475.6. + -. 56.7. mu.g/ml, using the volume of P.M OMVs required to be added at the pre-calculated final concentration of 0.15. mu.g/ml.

Example 2: P.M OMVs inhibit RANKL-induced osteoclast differentiation, fusion and absorption activity

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

BMMs were treated at 5X 10 cells, respectively⁴The cells were incubated in 96-well plates at a density of one ml in 100. mu.l per well, for 48,72 and 96 hours with OMVs and LPS in each concentration gradient formulated, and 10. mu.l of CCK-8, 5% CO in each well₂OD values were measured at 450nm after 3 hours of incubation at 37 ℃. The experiment was repeated three times, with percent cell viability as a positive control with no stimulation.

Percent cell viability (%) < x 100%

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

2.0.15, 0.3. mu.g/ml P.M OMVs and 5,10ng/ml P.M LPS treated with RANKL-inducedOsteoclast differentiation derived from and osteoclast differentiation induced by RANKL derived from inflammation (DBA/1 male mouse of CIA-induced arthritis), Trap staining, detection of related gene expression and detection of related protein expression, wherein the related gene expression is detected by RT-qPCR experiment, and the related protein expression is detected by western blotting experiment. The RNA primer sequences of the relevant genes are shown in Table 1.

The experiment was divided into three groups: M-CSF, M-CSF + RANKL, M-CSF + RANKL + OMVs, RANKL-induced osteoclasts stimulated with 0.15 μ g/ml P.M OMVs and 5,10ng/ml P.M LPS, respectively, osteoclast formation was observed with Trap staining. Meanwhile, the effect of 0.15. mu.g/ml P.M OMVs on the expression of osteoclast-associated genes Acp5, MMP9, CTSK, itg β 3, 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% trypsin for 5 minutes, centrifuged at 800rpm for 5 minutes, 3ml of complete medium was resuspended and the cells counted at 4X 10⁵One/ml plate was plated while adding 25ng/ml M-CSF, 37 ℃ 5% CO₂Adding RANKL and/or P.M OMVs into an incubator after culturing for 24 hours, adding M-CSF of only 25ng/ml into a control group, placing the control group into the incubator for continuous culture, replacing the culture medium, the inducer (M-CSF and RANKL) and the P.M OMVs every two days, continuously inducing for 3-5 days, wherein more fusion and large volume appear, and the multinuclear cells with visible vacuoles are osteoclasts.

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

FIG. 3B is a graph of the induction of RANKL stimulation by 0.15,0.3 μ g/ml P.M OMVsDifferentiation of BMMA from sources into osteoclasts, representative images of Trap staining and Trap⁺Counting the number of multinucleated cells. FIG. 3C is a representative image of the differentiation of RANKL-induced inflammation-derived BMMs into osteoclasts stimulated by 0.15, 0.3. mu.g/ml P.M OMVs, Trap staining and TRAP staining⁺Counting the number of multinucleated cells.

3) RNA extraction and RT-qPCR assay

After lysing cells using Trizol and obtaining RNA using chloroform, isopropanol, 75% ethanol, and enzyme-free water in this order, the concentration of sample RNA was measured using a microplate reader, RNA was reverse-transcribed into cDNA at a total mass of 1. mu.g, RNA was reverse-transcribed into cDNA (DBI, Germany) according to the kit instructions, RT-qPCR was performed in a Light Cycler480 instrument using Promega fluorescent quantitation kit (USA) to obtain statistical data and calculate 2^-ΔΔctThe values calculate the variance difference. The sequence of the RNA primers for the osteoclast-associated genes Acp5, MMP9, CTSK and itg β 3 is shown in Table 1.

Fig. 3D is 0.15,0.3 μ g/ml P.M OMVs effect on osteoclast-associated gene expression, qPCR results showed that P.M OMVs significantly down-regulated expression of RANKL-induced osteoclast-associated genes Acp5, MMP9, CTSK, itg β 3, NFATc1, and c-Fos.

The Western blot experiment comprises the following operation steps:

osteoclast was lysed on ice for 30 minutes using 150. mu.l of RIPA protein lysate containing PMSF, and centrifuged at 12000g for 10 minutes at 4 ℃ to obtain a supernatant, which was subjected to protein quantification by BCA method, and the volume required for 20. mu.g of total protein loading was calculated as protein: adding a protein loading buffer at a ratio of 5 × loading buffer to 4:1, heating in a metal bath at 100 ℃ for 5 minutes, adding the protein to a 10% SDS-PAGE gel, performing 120V electrophoresis for 80 minutes, activating PVDF membrane in methanol for 2 min, placing on the gel, performing constant current 200mA electrophoresis for 90 min, placing the PVDF membrane in 5% skimmed milk powder, sealing on a shaking table at room temperature for 2 hr, incubating overnight at 4 deg.C on a shaking table, taking out the primary antibody, adding appropriate amount of TBST, washing membrane for 5 min × 6 times, adding corresponding secondary antibody, incubating for 1 hr on a horizontal shaking table, after the secondary antibody incubation is completed, the secondary antibody is washed for 10 minutes × 3 times by using TBST on a horizontal shaking table, solution A and solution B of ECL luminescence solution are prepared according to the proportion of 1:1, a chemiluminescence mode is selected for exposure, the gray value of a strip is analyzed by Image J software, and data are analyzed by Graphpad Prism.

FIG. 3F is the expression of 0.15. mu.g/ml P.M OMVs on the osteoclast-associated proteins MMP9, CTSK, NFATc1 and c-Fos, but without inhibiting I κ B α protein.

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

(1) osteoclast F-actin ring imaging

1) Induction of osteoclasts: by 2X 10⁵BMMs were induced in culture on a confocal dish at one/ml and RANKL-induced osteoclasts were stimulated using P.M OMVs for 5 days as described in example 2;

2) fixation, permeabilization and sealing of cells: osteoclasts successfully induced were washed with PBS, fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.1% TritonX-100 for 30 minutes, and blocked with 5% BSA for 1 h;

3) dyeing and imaging: adding phalloidin: after diluted 1:40, the solution was changed to phalloidin working solution, after staining the phalloidin working solution in dark for 60 minutes, washed three times with PBS, 50 μ l of anti-fluorescence quencher containing DAPI was added, and after incubation for 15 minutes in dark at room temperature, the staining was finished, and the photographs were taken with an inverted laser confocal microscope.

(2) Osteoclast bone resorption Activity assay

BMMs plated on osteopass stripwell plates were induced with M-CSF, M-CSF + RANKL, and cells were treated with OMV concurrently with RANKL induction. Osteoclast formation was observed after 5 days, and the osteoclast bone resorption function was fully exerted by prolonged culture for 5 to 7 days. After the culture is finished, PBS is washed for three times, 0.3% hypochlorous acid is added, standing is carried out for 10 minutes to remove cells adhered in the holes, the holes are washed with distilled water for three times to fully remove the fallen cells, the distilled water is sucked to the greatest extent, then the plate is placed at room temperature to be dried, and finally the bone absorption condition of osteoclasts is observed by an inverted fluorescence microscope. Bone resorption area was analyzed using Image-pro plus software. For 3D visualization, the topography of the absorbing 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 looping. FIG. 3G is a representative graph of bone resorption after P.M OMVs stimulation, representing 3D image rendering of pictures and per μm²The statistical information (n ═ 6) of the pit area. The results show that P.M OMVs reduced the formation of F-actin rings (FIG. 3E) and significantly reduced the area of osteoclast resorption (FIG. 3G). The F-actin ring maintains the osteoclast morphology to facilitate the osteoclast's bone resorption function, and thus affects the osteoclast's formation and bone resorption function when the structure is damaged or fails to form, and the data of example 2 indicates that P.M OMV has the effect of inhibiting osteoclast differentiation and function.

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

C57 female mice at 8-10 weeks were intraperitoneally injected with 4% chloral hydrate (chloral hydrate volume (μ l) ═ mouse weight (g) × 10), after 10 minutes, the mice underwent deep anesthesia to start surgery, the position of the ovaries of the mice was determined on the back, the skin, fat and muscle layers of the mice were cut, the ovaries of the mice were found, the ovaries were ligated and removed, the muscle, fat and skin layers of the mice were sutured, hemostasis was performed with alcohol cotton balls, the ovaries of the other side were removed in the same manner, the ovaries were not removed in Sham (Sham) group, the mice were kept warm under an infrared treatment lamp after removal of the ovaries, the mice were raised conventionally after 12 hours, and after two weeks of ovaries removal, injections of PBS and OMVs into the joint cavities were started once a week, and the three groups were divided: after 8 weeks, mouse sera, uterine and femoral samples were collected, fixed in 4% paraformaldehyde for 48h for microCT scans, and HE and Trap stained after 30 days of decalcification on a shaker.

The experimental procedure and results are shown in fig. 4. Trabecular bone in the OVX group became sparse and improved after P.M OMV treatment (fig. 4B). Successful ovariectomy was confirmed by the uterus/body weight ratio, which decreased significantly 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 after P.M OMV treatment. CTX-1 was a marker of serum bone resorption, with significant reductions in CTX-1 levels after P.M OMVs treatment (fig. 4I), while no differences in Osteocalcin (OCN) levels (fig. 4J), HE staining showed a reduction in trabecular bone number and thickness (fig. 4K). TRAP staining showed a significant reduction in n.oc/b.pm after P.M OMVs treatment (fig. 4L and M). These data indicate that P.M OMVs have a protective effect on OVX-induced bone loss.

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

Injecting 100 mu l of 1mg/ml CII emulsifier into DBA/1 mice at 9-11 weeks, calculating the volumes of the required glacial acetic acid CII and CFA, adding the glacial acetic acid CII and Freund's complete adjuvant solution into a precooled mortar according to the volume ratio of 1:1, after grinding and emulsifying successfully on ice, placing the mice at the position of exposing the tail root on a fixed frame by about 3cm, injecting 100 mu l of emulsifier through the tail root in the skin, carrying out secondary immunization after 21 days, wherein the immunization concentration is that each mouse is injected with 100 mu l of 0.5mg/ml CII emulsifier, the adjuvant is Freund's incomplete adjuvant, the immunization method is the same, injecting PBS and OMVs into the joint cavities of the mice on the 3 rd day after the secondary immunization, once per week, and totally dividing into 3 groups: normal, CIA + PBS, CIA + P.M OMVs, starting on day 5 after the second immunization, joint swelling and redness were defined as joint inflammation, with the following scoring criteria: each inflamed toe or knuckle is given a score of 1, which is given a score of 1-5 depending on the severity of the inflammation of the wrist or ankle. Each mouse had a score of 0 to 15 per paw and a total score of 0 to 60 per mouse; serum and a mouse postnatal sample begin to be collected 14 weeks after the secondary immunization, the paw is placed into 4% paraformaldehyde for fixation for 48 hours, then the micCT scanning is carried out, and HE staining is carried out after 30 days of decalcification on a shaking table.

The experimental procedure and results are shown in FIG. 5. Figure 5A shows a brief course of the experiment, P.M OMV affected joint inflammation only in the initial phase (figures 5B-D), HE staining of joint sections confirmed the results of the invention (figures 5E and F). anti-CII antibody levels decreased in the later stages of inflammation following omv treatment (fig. 5G). In contrast to increasing serum OCN levels, P.M OMV significantly reduced CTX-1 levels (FIGS. 5H and I). The results of μ CT analysis are shown in FIG. 5J-O, where P.M OMV significantly improved the reduction of BMD, Tb.N, Tb.Th caused by CIA, but had no effect on Tb.BV/TV and Tb.sp.

Example 5: P.M OMVs modulate MAPK pathways

P.M OMV significantly affects the function of osteoclast in vitro and in vivo, P.M OMVs and RANKL group are compared to obtain up-regulated and down-regulated differential genes (FIG. 6A), they are respectively subjected to KEGG enrichment analysis, the up-regulated differential genes are mainly concentrated on cytokine interaction, viral infection and NOD-like receptor signaling pathways as can be seen from the KEGG enrichment chord chart of FIG. 6B, while the down-regulated genes are mainly concentrated on ECM receptor interaction, protein digestion and absorption and MAPK pathway (FIG. 6C), protein-protein interaction network diagram is drawn by String, after the obtained results are introduced into cytoscap, MCODE is used to find out the up-regulated and down-regulated key genes in P.M OMVs, and the results show that the up-regulated key genes are CCL5 and IL-1 beta (FIGS. 6D, E), and the down-regulated key genes are Pdgg, EGFR, Pgf, Fos, Acp fb 5, NFSTAATc 1, DC-MP (FIG. 6F, G) wherein Pdgfb, EGFR, Pgf, NFATc1, Fos participate in the MAPK pathway. From sequencing results, it was known that MAPK pathway is activated when RANKL induces osteoclast formation, but MAPK pathway is inhibited after P.M OMVs treatment, suggesting that P.M OMVs may influence osteoclast formation by interfering with MAPK pathway.

Through the sequencing result analysis of miRNA in RANKL and P.M OMVs groups, the differential expression multiple is more than 2 and is shown in a volcano diagram in a figure 7A, the result shows that 44 microRNAs in P.M OMVs are up-regulated and 48 are down-regulated, figures 7B and C respectively show the microRNA names with the up-regulation and down-regulation multiples being the first 20, MiRanda, TargetScan and RNAhybrid are used for predicting the up-regulation and down-regulation of the microRNAs, and the target genes are respectively subjected to KEGG channel enrichment analysis, as shown in figures 7D and E, the most significant channels of the microRNAs target gene enrichment are MAPK channels, which shows that P.M OMVs can shadow the MAPK channels, and the MAPK channels are consistent with the mRNA sequencing result.

In this example, the specific operations of sequencing and analyzing transcriptome were as follows:

1) mRNA sequencing: after extracting RNA, the purity and the integrity of the RNA are respectively detected by NanoDrop2000 agarose gel electrophoresis, and mRNA sequencing is carried out by IlluminaNovaseq6000 to obtain paired end reading after the purity and the integrity of the RNA meet the requirements. SeqPrep and simple prune the original paired end reads for quality control. TopHat2 is used for sequence alignment analysis to obtain mapping reads, and Cufflinks is used for assembling and splicing the mapping reads according to the existing reference gene group. Differential expression analysis of the samples was performed using transcripts per million reads (TPM) and DEseq2, | log2FC | >2 and padjust <0.05 were considered Differentially Expressed Genes (DEG). Carrying out KEGG pathway analysis on DEG, and searching a key gene by using cytoscape;

2) sequencing miRNA: an Illumina TruSeq small RNA library preparation kit constructs a miRNA library, performs high-throughput sequencing on the enriched 18-32nt small RNA fragments by utilizing an Illumina Novaseq6000 platform, obtains mapped data, uses a miRDeep2 software package, obtains differentially expressed miRNA through TPM and DESeq2, | log2FC | >1 and padjust <0.05 as DEG, predicts target genes of the differential miRNA by using miRanda, targetScan and RNAhybrid, and performs KEGG enrichment analysis on the target genes to obtain a target gene enrichment pathway.

Example 6: effect of microRNA mimics and inhibitors on osteoclastogenesis and related genes

The microRNA can influence the expression and the function of a target gene by combining with the target gene, so that the research on the action of the microRNA plays an important role in understanding the downstream genes and the passages of the microRNA. In the invention, miR-155-5p, miR-96-5p and miR-653-5p are selected for research. NC, miR-155-5p, miR-96-5p, miR-653-5p mimicres and inhibitor are transfected into RANKL induced osteoclasts, and the formation of osteoclasts and the expression of related genes after the transfection of 3 kinds of microRNA mimicres are respectively detected, and the result shows that the formation amount of osteoclasts after the transfection of miR-96-5p mimicres is basically unchanged (figure 8A) but the expression of Acp5 and MMP9 is remarkably adjusted (figure 8C). miR-155-5p mimic reduces the number of osteoclasts after transfection (FIG. 8A), and can significantly down-regulate the expression of Acp5, CTSK and c-Fos (FIG. 8D). Inhibition of osteoclast formation after miR-653-5p mimic transfection (FIG. 8A) significantly reduced the expression of Acp5, MMP9, CTSK, c-Fos but significantly up-regulated the expression of itg β 3 (FIG. 8E). Results after transfection with miR-96-5p inhibitor showed that miR-96-5p inhibitor significantly inhibited osteoclast formation (fig. 8B) and significantly inhibited expression of Acp5 and CTSK (fig. 8F), miR-155-5p inhibitor had no effect on osteoclast formation (fig. 8B) but significantly up-regulated the expression of Acp5, MMP9, CTSK, itg β 3, NFATc1, c-Fos in osteoclasts (fig. 8G), whereas miR-653-5p inhibitor significantly inhibited osteoclast formation (fig. 8B) but enhanced the expression of c-Fos, itg β 3 (fig. 8H). These results show that miR-155-5p can inhibit the formation of osteoclasts through up-regulation, which is consistent with the previous research results, the reduction of the interaction of miR-96-5p and a target gene can lead the formation of osteoclasts to be inhibited, and the up-regulation or the inhibition of the action of the expression of miR-653-5p can inhibit the formation of osteoclasts, so the 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 and the DEGs which are up-regulated or down-regulated in the RNA sequencing result are respectively intersected, 13 common genes are included in the up-regulated genes, 6 down-regulated genes are included in the down-regulated genes, and finally the gene of interest is determined to be Abca1 (fig. 9B). RT-qPCR and WB detection P.M OMVs treatment significantly upregulated Abca1 gene and protein expression (fig. 9C and D), consistent with sequencing results. The dual luciferase report verifies whether mmu-miR-96-5p can directly target Abca1, a Wild Type (WT)/mutant (Mut) Abca1-psiCheck2 vector (FIG. 9E) is firstly constructed and transfected into HEK293T cells together with mmu-miR-96-5p respectively, as shown in FIG. 9F, the luciferase activity of Abca1WT is obviously reduced after miR-96-5p micom treatment, but the luciferase activity of Abca1-Mut is not changed basically, which indicates that miR-96-5p can target Abca 1. In order to further verify the targeting relationship between miR-96-5p mimic and Abca1, miR-96-5p mimic and inhibitor are transfected into osteoclasts respectively, and the results show that the Abca1 gene and protein expression are remarkably inhibited after the mimic transfection (FIG. 9G), and the Abca1 gene and protein expression are remarkably increased after the inhibitor is transfected (FIG. 9H).

In this example, the dual-luciferase report experiment and the influence of miRNA on osteoclast differentiation were performed as follows:

(1) dual luciferase reporter assay

Abca1WT 3'UTR and Abca1 Mut 3' UTR plasmid vectors were constructed, and HEK293T cells in good growth state were digested and counted to 5X 10⁴The method comprises the steps of planting cells in a 24-well plate, transfecting the cells according to an instruction when the cells grow to about 80%, fully and uniformly mixing reporter gene cell lysate, adding 100 mu L of the reporter gene cell lysate into the 24-well plate, fully lysing, centrifuging for 5 minutes at 12,000g, sampling 100 mu L of the sample, adding 100 mu L of luciferase detection reagent, determining RLU (relative light unit), after the determination is finished, adding equivalent renilla luciferase detection working solution to determine the RLU, calculating the ratio of two luciferases, and comparing the ratio difference of different groups.

(2) Effect of miRNA on osteoclast differentiation

The BMMs are paved in a 96-well plate, NC mix and inhibitor, miRNA mix and inhibitor are respectively transfected into cells according to the method after the cells adhere to the wall, RANKL is added after 24 hours, the liquid is changed after 48 hours, the osteoclast formation condition is observed after 48 hours, Trap staining is carried out, the cells are photographed by an inverted fluorescence microscope, the number of the cell nucleus is more than three, the osteoclast is defined, and the number is counted.

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

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

Abca1 promotes a decrease in mitochondrial cholesterol and is involved in the release of cytochrome c (cyto c), which may lead to disruption of the mitochondrial structure. The energy required for osteoclast differentiation comes mainly from mitochondrial oxidative metabolism. To investigate the effect of P.M OMVs on mitochondria, mitochondrial structure was first observed with transmission electron microscopy, as shown in fig. 10A, P.M OMV disrupted the mitochondrial structure of RANKL-induced osteoclasts and significantly reduced ATP production on days 1 and 5 (fig. 10B), indicating that P.M OMV not only disrupted mitochondrial structure but also reduced ATP production. Mitochondria are the major source of Reactive Oxygen Species (ROS) in cells, and excessive production of ROS may lead to loss of Mitochondrial Membrane Potential (MMP) and impaired ATP synthesis, which in turn leads to apoptosis, and therefore, ROS levels and apoptosis were further examined after P.M OMVs treatment. As shown in fig. 10C, the percentage of DCFH-DA fluorescence and the 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 (Δ ψ m), and thus changes in the mitochondrial membrane potential of cells were measured (fig. 10E & F). P.M OMVs significantly increased intracellular ROS but decreased levels of Δ ψ m. As shown in fig. 10G & H, the apoptosis rate significantly increased in a concentration-dependent manner after P.M OMVs treatment, and the Bax/Bcl-2 ratio and clear caspase-3 significantly increased (fig. 10I) and promoted the release of mitochondrial cytoc (fig. 10J & K) after P.M OMVs treatment, indicating that P.M OMVs could significantly promote osteoclast apoptosis via a mitochondria-dependent pathway.

In this example, the observation of mitochondrial structure and the detection of intracellular reactive oxygen species and apoptosis were performed as follows:

(1) mitochondrial structure

The BMMs are arranged according to 6 x 10⁵Uniformly spreading the cells/ml in a 6-well plate, inducing osteoclast by 50ng/ml RANKL after 24 hours, adding or not adding P.M OMVs, collecting the cells after inducing for three days, centrifuging at 1500rpm for 5 minutes to enable the cells to become cell sediment with the height of 0.5-1mm, slowly adding 500 mu l of 2.5% glutaraldehyde along the tube wall, standing for 1 hour at room temperature, placing in a refrigerator with the temperature of 4 ℃ for standing for 3 hours, sucking out the glutaraldehyde, filling PBS in an EP tube, placing in the refrigerator with the temperature of 4 ℃ for standing overnight, sending a sample into an electric microscope chamber, preparing the cells into ultrathin slices, observing the ultrastructure of mitochondria under a transmission electron microscope, and taking a picture of 5-6 visual fields for each sample.

(2) Intracellular reactive oxygen species and apoptosis assays

1) The differentiation of osteoclast induced by RANKL under the stimulation of OMV/no OMV for 5 days, 1 hour before collecting cells, adding Rosu into a positive control group, washing the cells twice by precooled PBS, adding 400 mul of DCFH-DA diluted by serum-free culture medium into each hole, incubating for 20 minutes in a dark place, washing twice by serum-free culture medium, collecting the cells, centrifuging at 1500rpm for 5 minutes, retaining the precipitate, resuspending 200 mul of PBS, transferring to a flow tube, detecting by an upper machine, and processing and analyzing the result by FlowJo V10 software.

2) RANKL induces osteoclast differentiation for 5 days with/without OMV stimulation, cells are collected after precooled PBS is washed, the precipitates are collected after centrifugation for 5 minutes at 1500rpm, 100 mu l of 1 XBinding buffer is added into the precipitates for resuspension, 5 mu l of FITC Annexin V and 5 mu l of PI are sequentially added, the precipitates are incubated for 15 minutes at room temperature in a dark place, 200 mu l of 1 XBinding buffer is added for resuspension and is transferred to a flow tube for onboard detection, and after the detection is finished, the results are processed and analyzed by FlowJo V10 software.

Example 9:

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

The pharmaceutical composition of the present invention can be administered orally or parenterally, and the parenteral administration may be, for example, intravenous injection, intramuscular injection, or oral 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 oral liquid, and the oral liquid usually contains a solvent, an aromatic, a flavoring agent, a clarifying agent, a preservative and the like as auxiliary materials, and can be added simultaneously or optionally, wherein the solvent is an essential auxiliary material, and the solvent can adopt water.

In one embodiment, the pharmaceutical composition is in the form of granules, and the granules are usually supplemented with one or more of fillers, binders, wetting agents, disintegrants, lubricants and film coating materials.

Example 10:

the present embodiment provides an osteoclastogenesis and/or activation inhibitor, the main component of which comprises proteus mirabilis outer membrane vesicles.

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

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.