CN111096959A

CN111096959A - Application of spermine in preparation of medicine for treating periprosthetic osteolysis

Info

Publication number: CN111096959A
Application number: CN201911250322.0A
Authority: CN
Inventors: 耿德春; 郁彬清; 柏家祥; 葛高然; 徐耀增; 杨惠林
Original assignee: First Affiliated Hospital of Suzhou University
Current assignee: First Affiliated Hospital of Suzhou University
Priority date: 2019-12-09
Filing date: 2019-12-09
Publication date: 2020-05-05

Abstract

The invention proves that spermine can reduce the expression of RANKL, p65, TNF- α and IL-1 β through animal experiments, relieves the osteolysis and bone loss induced by titanium particles, proves that spermine has obvious inhibition effect on the osteolysis induced by wear particles, can be used as a new means for the drug intervention of the osteolysis around the prosthesis after the replacement of the artificial prosthesis, and thus provides a new way for the treatment of the osteolysis around the prosthesis.

Description

Application of spermine in preparation of medicine for treating periprosthetic osteolysis

Technical Field

The invention belongs to the technical field of new application of medicines, and particularly relates to application of spermine in preparation of a medicine for treating periprosthetic osteolysis.

Background

Currently, Artificial Joint Replacement (AJR) is considered to be the most effective surgical procedure for treating various end-stage osteoarticular diseases. However, as the number of cases of artificial joint replacement increases and the time is prolonged, the number of patients who need revision due to joint replacement failure also increases. After the prosthesis is replaced for 15-20 years, 10-15% of the prostheses will fail. Studies have shown that aseptic loosening caused by periprosthetic osteolysis (PPO) is a major cause of shortening of the service life of joint prostheses. PPO is a complex pathology with multifactorial effects, the exact mechanism of occurrence of which has not been fully understood to date, but the biological response caused by wear particles is believed to be the major cause of PPO. The friction of the prosthetic joint results in chronic erosion of the material, producing tiny metal or polyethylene wear particles. These particles accumulate around the prosthesis, causing a chronic inflammatory reaction, increased osteoclastic bone resorption and decreased osteoblastic bone formation, ultimately leading to the development of PPO and loosening of the prosthesis. Since wear of the prosthesis is often unavoidable, inhibition of inflammatory osteolysis has become a major goal of pharmacotherapy for prosthesis loosening.

Spermine (Spermin) of formula C₁₀H₂₆N₄Molecular weight is 202.34, CAS number 71-44-3, and is a small molecule compound of aliphatic amine class widely distributed in tissue cells of prokaryotes and eukaryotes. It exhibits a variety of biological activities in cell growth, proliferation and differentiation by modulating gene expression and intracellular signaling. Animal experiment research in recent years shows that spermine has the effects of resisting inflammation, resisting oxidation, resisting apoptosis and the like. Studies have shown that spermine has inhibitory effects on cartilage and bone destruction in a rat model of collagen-induced arthritis, while spermine has been shown to prevent bone loss in ovariectomized mice by inhibiting osteoclast maturation and differentiation. Although the clinical benefits of spermine on bone metabolism have been severalThe mode of action of the anti-bone-breaking activity and the relationship with inflammatory osteolysis after joint replacement are not completely elucidated, and the present invention is based on the results.

Disclosure of Invention

The invention aims to research the therapeutic effect of spermine on periprosthetic osteolysis and provides a new way for preventing and treating periprosthetic osteolysis.

In order to achieve the purpose, the invention provides the following technical scheme:

the application of spermine in the preparation of medicaments for treating periprosthetic osteolysis.

Further, the periprosthetic osteolysis is periprosthetic osteolysis occurring after the artificial joint replacement.

A pharmaceutical preparation for the treatment of periprosthetic osteolysis comprises spermine or spermine in a therapeutically effective amount and pharmaceutically acceptable adjuvants. The preparation method of the preparation adopts the conventional preparation method in the field to prepare the preparation on the premise of not reacting with the active ingredient of the invention or influencing the curative effect of the medicament.

Further, the pharmaceutical preparation is suitable for pharmaceutical preparations for gastrointestinal or parenteral administration.

Furthermore, the pharmaceutical preparation is a freeze-dried preparation, an injection, a tablet, a granule or a capsule.

According to the technical scheme, whether spermine can have a therapeutic effect on the skull osteolysis of mice induced by abraded particles is researched by a gastric perfusion administration method, and indexes such as nuclear factor kappa B receptor activator ligand (RANKL), nuclear factor kappa B p65 subunit (p 65) and the like are simultaneously measured to research the relation between the effect and osteoclast activation, so that the mechanism and the theoretical basis of the osteolysis after the spermine treatment of joint replacement are discussed.

The method comprises the steps of randomly dividing 80 mice C57BL/J6 into 4 experimental groups, namely a Control group, a Ti group, a spermine low-dose treatment group (L group) and a spermine high-dose treatment group (H group), wherein each group comprises 20 animals, performing general anesthesia downlink routine operation on the animals of the Ti group and the treatment group, placing 50 mu L of prepared 40% Ti particle PBS (20 mg/mouse) on the surface of the skull of the mice, and administering 50 mu L of PBS to the Control group, wherein the mice are perfused with 200 mu L of sterile PBS containing spermine every day from the day after surgery in the treatment group (wherein the administration doses of the low-dose group and the high-dose group are respectively 0.35 mu g/mL and 3.5 mu g/mL), the stomach 200 mu L of sterile PBS is used for the Control group and the Ti group every day, and the animals are subjected to micro-CT and histological detection after the surgery, and the measurement of the bone density, the bone volume fraction, the bone area, the bone maturation and the bone thickness of the skull of the mice are simultaneously detected by a micro PBS-dissolving method such as TNF-36 KL dissolving method and a method for detecting the skull tissue dissolving amount of the animals.

The result data were analyzed by SPSS11.0 software, and all data were averaged. + -. standard deviation: (

) It is shown that statistical analysis was performed using one-way ANOVA test, and homogeneity of variance was tested on all data before analysis. The differences between the different experimental groups were determined by fisher LSD late assay. p is a radical of<A difference of 0.05 is statistically significant.

The invention discloses an application of spermine in preparing a medicine for treating periprosthetic osteolysis, and animal experiments prove that spermine can reduce the expression of RANKL, p65, TNF- α and IL-1 β, relieve osteolysis and bone loss induced by titanium particles, prove that spermine has a remarkable inhibition effect on osteolysis induced by wear particles, and can be used as a new means for medicine intervention of periprosthetic osteolysis after artificial prosthesis replacement.

Compared with the scheme in the prior art, the method has the following advantages:

spermine, as a widely existing biological polyamine, has been proven to be low in toxicity, safe, low in cost and easy to prepare; the invention adopts a titanium particle induced mouse skull bone lysis model, and verifies the influence of spermine on prosthesis peripheral bone lysis after artificial prosthesis replacement by performing mu CT three-dimensional reconstruction analysis on mouse skull and applying methods such as H & E dyeing, TRAP dyeing, immunohistochemical dyeing and the like, wherein the tiny particles generated by long-term abrasion of the artificial prosthesis can up-regulate the expression of RANKL, activate an RANKL passage and cause osteoclast activation.

Drawings

The invention is further described with reference to the following figures and examples.

FIG. 1 is a three-dimensional model of skull bone of each experimental group of mice scanned by micro-CT. A is Control group, B is Ti group, C is spermine low dose treatment group, and D is spermine high dose treatment group.

FIG. 2 shows the measurement values of bone density (BMD) of the skull bone of mice in each experimental group.

FIG. 3 shows the measurement of skull bone volume fraction (BV/TV) of mice in each experimental group.

FIG. 4 shows the measurement of Porosity (Porosity) of skull bone surface of mice in each experimental group.

FIG. 5 shows HE staining results of mouse skull in each experimental group. A is Control group, B is Ti group, C is spermine low dose treatment group, and D is spermine high dose treatment group.

FIG. 6 shows the measurement values of skull thickness (BT) of mice in each experimental group.

FIG. 7 shows the measurement values of the skull osteolytic area (BES) of the mice in each experimental group.

FIG. 8 shows TRAP staining results of skull bone of mice in each experimental group. A is Control group, B is Ti group, C is spermine low dose treatment group, and D is spermine high dose treatment group.

FIG. 9 shows the results of TRAP staining positive cells on the skull of mice in each experimental group.

FIG. 10 shows the RANKL immunohistochemical staining results of the mouse skull in each experimental group. A is Control group, B is Ti group, C is spermine low dose treatment group, and D is spermine high dose treatment group.

FIG. 11 shows the results of counting the cells expressing RANKL positive by immunohistochemistry of the skull of mice in each experimental group.

FIG. 12 shows the p65 immunohistochemical staining results of mouse skull in each experimental group. A is Control group, B is Ti group, C is spermine low dose treatment group, and D is spermine high dose treatment group.

FIG. 13 shows the counting result of the positive cells expressing p65 in the skull immunohistochemistry of the mice of each experimental group.

FIG. 14 shows the results of TNF- α immunohistochemical staining of the skull of mice in each experimental group, wherein A is Control group, B is Ti group, C is spermine low-dose treatment group, and D is spermine high-dose treatment group.

FIG. 15 shows the counting result of the cells expressing TNF- α positively by the skull immunohistochemistry of the mice of each experimental group.

FIG. 16 shows the results of IL-1 β immunohistochemical staining of the skull of mice in each experimental group, wherein A is Control group, B is Ti group, C is spermine low-dose treatment group, and D is spermine high-dose treatment group.

FIG. 17 shows the counting results of the skull immunohistochemical IL-1 β -expressing positive cells of mice in each experimental group.

Detailed Description

The present invention is further described below with reference to specific examples, which are only exemplary and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

Materials and methods

1. Material

1.1 reagent and Experimental Equipment

1.1.1 major drugs and reagents

Spermine (spectmin), available from Sigma, usa, TRAP staining kit, ethylenediaminetetraacetic acid (EDTA), available from Sigma, usa, paraformaldehyde, PBS, DAB developer, hematoxylin, eosin, absolute ethanol, distilled water, 10% chloral hydrate, RT-PCR kit, available from Invitrogen, usa, titanium particles (Ti) available from Johnson mathematics, usa (catalog #00681; Ward Hill, Massachusetts), RANKL, p65, TNF- α, IL-1 β antibody, available from Abcam, uk.

1.1.2 Main Instrument

μ CT (SkyScan 1176, belgium), paraffin microtome (Leica 2135, germany), bake plate machine (Leica 1120, germany), paraffin embedding machine (BMJ-ii, china, changzhou), Axiovert 40C optical microscope (Zeiss, germany), plate reader (Biotec, usa), set of surgical instruments, etc.

1.2 Experimental animals

80 healthy C57BL/J6 mice, male, with a weight of 19-22 g, 6-8 weeks old, clean grade, were provided by the Suzhou university animal testing center. The feeding conditions were as follows: 5 cages are filled in the food, the room temperature is 18-20 ℃, the humidity is 50-60%, the ventilation is good, and the food can be freely taken. All procedures were performed according to the animal care committee and the national institutes of health care guidelines for the care and use of animals.

2. Experimental methods

2.1 treatment of Ti particles

93% of the particles have a diameter <20 μm. To remove endotoxin, the particles are baked in an oven at 180 ℃ for 6h, dissolved in 75% ethanol by volume fraction, shaken at normal temperature for 1h for 4 times, soaked in 100% ethanol overnight, washed with isotonic saline for 3 times, and stored at 4 ℃ for later use.

2.2 groups of Experimental animals

80C 57BL/J6 mice, randomly assigned to the following 4 groups:

(1) control group: modeling is carried out according to a standard method, the titanium particles are changed into the same amount of normal saline, 0.2mL of sterile PBS solution is perfused into the stomach every day after operation, and the patient is killed after 2 weeks;

(2) group Ti: 20 mice are placed with 20mg titanium particles on the surface of the skull, and are killed after 2 weeks after the operation by intragastric administration of 0.2mL sterile PBS solution every day;

(3) and (3) group L: 20 mice, which are a spermine low-dose treatment group, were subjected to intragastric administration of 0.2mL of spermine-containing sterile PBS solution (0.35 mug/mL) per day after surgery after placement of 20mg of titanium particles on the surface of the mouse skull, and sacrificed after 2 weeks;

(4) group H: 20, high dose treatment with spermine: 20mg of titanium particles were placed on the surface of the skull of the mice, and a sterile PBS solution containing spermine (3.5 mug/mL) was gavaged daily after the operation, and the mice were sacrificed after 2 weeks.

2.2 preparation of mouse skull osteolysis model

The invention adopts a titanium particle (Ti) induced skull bone dissolution model of a mouse to simulate the pathological process of periprosthetic bone dissolution (Liu X, et al, Strong mineral dissolution inhibitors by dissolution inducing in vitro biomaterial 2014;10(11): 4912-8). Laboratory mice were anesthetized with a 500mg/kg intraperitoneal injection of 10% chloral hydrate. After the cranial crown skin had been dehaired and 3 hr iodine disinfections, an approximately 1cm median sagittal incision was made at the cranial crown, 1.0cm x 1.0cm periosteum was exposed, and prepared 50 μ L of 400mg/mL Ti pellet PBS solution (20 mg/pellet) was implanted. The skin was closed with 4-0 sutures. All procedures were performed on the same day, during which time the eyes of the mice were protected using sterile lubricant eye ointment.

2.3 specimen Collection

After 2 weeks of operation, 10% chloral hydrate is injected into the abdominal cavity of each group of animals for anesthesia, the animals are fixed on a mouse operating frame outside the supine position, the chest is opened to expose the heart, the animals are perfused into the left ventricle through the apex of the heart, the right auricle is cut, the normal saline is opened after the aorta is ligated and descended to wash the cooling liquid flowing out of the right auricle, and then the animals are perfused by 200-300 mL of 4% neutral paraformaldehyde until the four limbs of the animals twitch and become hard. After the perfusion is finished, the skull is quickly taken out, and the soft tissue attached to the skull base is removed. After 10 skulls in each group are fixed in 4% paraformaldehyde for 24h, micro-CT detection is carried out on 5 skulls in each group, and decalcification is carried out on the other 5 skulls in 10% EDTA for 3 weeks, and then paraffin embedding and histological detection are carried out.

2.4 micro-CT detection

After the mouse skull is fixed for 24h, the μ CT scan is performed. Scanning parameters are as follows: the resolution is 18 mu m, and the voltage is 80 kV; current is 100 muA; each exposure time is 100 ms; 0.9 °/8 images. Adopting a Wedemeyer C method (Wedemeyer C, et al, Particle-induced bone analysis in three-dimensional Micro-computed tomogry. CalcifTissue int. 2007; 81(5): 394-402.), selecting a cylindrical region of interest (ROI; diameter 3mm, height 1 mm), performing 3D analysis on the image by using Micro-CT image analysis software, and recording the bone density (BMD, mg/mm) of the skull in the ROI region²) Bone volume to tissue volume ratio (BV/TV), bone surface Porosity (Porosity).

2.5 histological staining

After decalcification of the skull with 10% EDTA, the skull was embedded in paraffin by conventional methods. And taking a horizontal position of the skull, and continuously slicing the skull at a sagittal suture position, wherein the thickness of the slice is 5 mu m. H & E, TRAP and immunohistochemical staining were performed separately.

2.5.1H & E staining procedure

(1) Dewaxing the paraffin section by dimethylbenzene (10 min multiplied by 2 times), and then sequentially passing 100%, 95%, 90% and 85% ethanol to water, wherein each pass lasts for 10 min;

(2) washing with distilled water for 3min, staining with hematoxylin solution for 5min, and washing with tap water for 2 min;

(3) differentiating with 1% hydrochloric acid alcoholic solution for 30s, washing with tap water for 1 min;

(4) carrying out anti-blue reaction in 10% ammonia water solution for 30s, and washing with tap water for 1 min;

(5) counterstaining with l% eosin solution for 5min, and washing with tap water for 1 min;

(6) and (5) performing conventional dehydration, transparency and sealing.

The morphological change of the skull is observed under the optical lens. Skull thickness (BT) and Bone-dissolved area (BES) were calculated by using a microscope computer Image analysis system (Image-Proplus 6.0) by referring to von Knoch M method (ob/ob) biomaterials 2004; 25: 4675-81).

2.5.2 anti-tartaric acid phosphatase staining

Tartrate-resistant acid phosphatase (TRAP) is characteristic of osteoclasts and is distributed in the osteoclast cytoplasm. Under acidic conditions with tartrate, TRAP hydrolyzes naphthol ASBI phosphate to yield naphthol ASBI which immediately binds to hexaazofuchsin in the dye liquor to form an insoluble red dye at the enzyme active site. The acid phosphatase activity can be indirectly understood by observing this dye. TRAP staining was used to identify osteoclasts. Staining was performed using TRAP staining kit (Sigma 387A).

2.5.2.1 reagent formulation

Preparing 2 test tubes, adding 0.5mL fast Garnet GBC Base Solution (parafuchsin) in one test tube and 0.5mL LSodinium nitrile Solution (sodium Nitrite) in the other test tube, mixing for 30s, and standing for 2 min; preparing 2 100mL beakers, marking A and B, preparing TRAP dye solution (pH5.2):

2.5.2.2 dyeing step

(1) Paraffin sections were deparaffinized and hydrated and washed 3 times for 3min with PBS

(2) Fixing the prepared specimen slices in an acetone solution for 30 s;

(3) washing with distilled water without drying;

(4) incubating TRAP dye liquor for 1h at 37 ℃, and keeping out of the sun;

(5) washing with distilled water for 3 times, counterstaining with hematoxylin for 2min, and washing with PBS to turn blue.

The TRAP staining positive result is purple red color point and sheet area, and the number of mature osteoclasts is counted under 20 Xlight microscope view by taking the sagittal suture of the skull as the center according to the Nich C method (Nich C, et al, complete of direct estrogen receptor signalling in near particulate-induced bone analysis. biomaterials, 2013; 34(3): 641-50.).

2.5.3 immunohistochemical staining

Changes in expression of RANKL, p65, TNF- α, and IL-1 β were detected by immunohistochemical staining.

Immunohistochemistry step:

(1) paraffin section dewaxing to water: xylene (I) 5min → xylene (II) 5min → absolute ethanol 2min → 95% ethanol 1min → 80% ethanol 1min → 75% ethanol 1min → distilled water washing for 2 min;

(2) incubating for 5-10 minutes at room temperature by using 3% H2O2 to eliminate the activity of endogenous peroxidase; washing with distilled water, and soaking in PBS for about 5 minutes;

(3) antigen retrieval: putting the slices into a container containing citrate buffer solution (working solution), and heating in a microwave oven to keep the temperature of liquid in the container between 92 ℃ and 98 ℃ for 10-15 minutes; taking out the container, cooling for 10-20 minutes at room temperature, and washing with PBS;

(4) blocking of non-specific binding sites: 5-10% normal goat serum (PBS diluted) was blocked and incubated for 10min at room temperature.

(5) The rabbit antibody (primary antibody) with affinity purification of anti-RANKL, anti-I kappa B- α, anti-TNF- α and anti-IL-1 β is added dropwise, the working titer is 1: 400, the rabbit antibody is incubated at 37 ℃ for 1 hour, PBS is washed for 3 times, each time is 5 minutes, the IG antibody (secondary antibody) marked by HRP is added dropwise, the working titer is 1: 500, and the rabbit antibody is incubated for 60 minutes in a room-temperature wet box.

(6) Developing with Diaminobenzidine (DAB) developer, dyeing at room temperature for 5-30min, washing with distilled water

(7) The slices are dehydrated by ethanol and xylene, transparent, sealed by neutral resin and covered by a glass slide.

The evaluation method comprises the following steps: and (3) selecting 5 continuous sections, counting the number of positive cells in the ROI area under a 20 Xlight microscope field, and taking the positive cells as brown yellow particles appearing in cytoplasm.

2.7 statistical analysis

The result data is analyzed by SPSS11.0 statistical software, and the data is averaged plus or minus standard deviation (

) It is shown that one-way ANOVA (one-way ANOVA test) is selected for the multiple groups of comparison, and LSD and Dunnett-t methods are selected for the two-by-two comparison under the condition of uniform overall variance.p<A difference of 0.05 is statistically significant.

Second, result in

1. General conditions of the laboratory animals

All animals revive within 30-60 min after operation, can freely move in the cage, normally eat and have no obvious change in mental state. No incision, no inflammation reaction such as red swelling and exudation, and uniform healing. No animal was dead during the experiment.

micro-CT detection results

The skull of the mouse is scanned and tested by micro-CT and three-dimensional image reconstruction and quantitative analysis are carried out, so that the bone mass and the bone microstructure can be described more accurately, and the degree of bone dissolution is judged. Wherein, A is a Control group, B is a Ti group, C is an L group, and D is an H group. The three-dimensional images showed that there were more craters on the surface of the skull bone and significant osteolysis in the Ti group compared to the Control group, and that the craters on the surface of the skull bone were significantly reduced and osteolysis was reduced after spermine administration (fig. 1).

Bone density (BMD) change: after Ti is added, the skull bone density of the mice is obviously reduced, and compared with the Control group (60.62 +/-9.12 gm/cc vs 135.30 +/-9.02 gm/cc), the difference has statistical significance (p is < 0.01); the bone density of the skull bone of the mice in the treated group was significantly increased compared to the Ti group, where p was <0.05 in the low spermine concentration treated group (78.75 ± 8.01 gm/cc) compared to the Ti group, and p was <0.01 in the high spermine dose treated group (120.12 ± 8.15 gm/cc) compared to the Ti group. See fig. 2.

Bone volume fraction (BV/TV): the number of bone volume was significantly reduced in the Ti group compared to the Control group (23.19 ± 5.39% vs 79.90 ± 7.23%),. p < 0.01. After spermine treatment, the number of bone body scores was significantly increased, with p <0.01 in the high dose spermine treated group (62.89 ± 8.68%) compared to the Ti group and p <0.05 in the low dose spermine treated group (29.96 ± 6.77%) compared to the Ti group. See fig. 3.

Bone surface Porosity (Porosity): after Ti is added, the porosity of the surface of the skull of the mouse is obviously increased, and compared with the Control group (0.73 +/-0.12% vs 0.25 +/-0.05%), the difference has statistical significance (p is less than 0.01); the surface porosity of the skull of the mice in the treated group was significantly reduced compared to the Ti group, where p <0.01 in the high spermine dose treated group (0.35 ± 0.04%) and p <0.05 in the low spermine concentration treated group (0.63 ± 0.09%) compared to the Ti group. See fig. 4.

H & E staining results

Under a light mirror, the surface of the bone tissue of the Control group is flat, the thickness of the periosteum is uniform, the number of cells in the periosteum is small, and the cells are arranged in order; the Ti group bone tissue has worm erosion-like change, the periosteum is obviously thickened, the number of cells in the periosteum is increased, and most of the cells are inflammatory cells; in the spermine treatment group, bone tissue was destroyed, but to a lesser extent; the periosteum is slightly thickened, a small amount of inflammatory cells exist, and the arrangement of fibroblasts is regular. See FIG. 5, where A is Control group, B is Ti group, C is L group, and D is H group.

Bone Thickness (BT): compared with the Control group (0.27 +/-0.02 mm), the thickness of the skull of the Ti group (0.08 +/-0.02 mm) is obviously reduced, and the difference has statistical significance (p is less than 0.01). After spermine treatment, the thickness of the skull was 0.10 ± 0.02mm (group L), 0.22 ± 0.03mm (group H), respectively, with statistical differences compared to the Ti group (. p < 0.05). The difference between the high spermine dose group and the Ti group was statistically significant (. p < 0.01), and the difference between the low spermine dose group and the Ti group was statistically significant (. p < 0.05). See fig. 6.

Osteolytic area (BES): the dissolving area of the Ti group bone is (0.15 +/-0.02 mm)²) And Control group (0.03. + -. 0.01 mm)²) In contrast, the difference was statistically significant (. about.p)<0.01), indicating that the Ti particles are capable of causing significant osteolysis; after the addition of spermine, the areas of osteolysis were (L group, 0.013. + -. 0.02 mm) respectively²(ii) a Group H, 0.05. + -. 0.01mm²). The difference between the high-dose spermine treatment group and the Ti group was statistically significant (. about.p)<0.01), the difference between the low dose spermine treatment group and the Ti group was statistically significant (. about.p)<0.05). See fig. 7.

TRAP staining results

The TRAP staining positive area is purple red, and the Control group can see punctate positive changes and mainly focuses on the edge of the medullary cavity; a large mauve purple area can be seen on the lysis side of the skull in the Ti group, which indicates that a large amount of mature osteoclasts exist on the lysis side of the skull; the spermine treated group had only a few positive areas at the lytic margin of the skull. See fig. 8. The counting result under a light microscope shows that the TRAP positive cells of the Ti group are (56.55 +/-5.83 mm)^-2) And Control group (6.37. + -. 1.38 mm)^-2) In comparison, p<0.01; the TRAP cell counts of the low-concentration and high-concentration spermine treatment groups were (51.71 + -4.38 mm)^-2），（22.54±2.24mm^-2). The difference between the high-dose spermine treatment group and the Ti group was statistically significant (. about.p)<0.01), the difference between the OA low dose treatment group and the Ti group was statistically significant (. about.p)<0.05). See fig. 9.

5. Immunohistochemical detection results

The immunohistochemistry results showed that the expression amount of RANKL was observed under an optical microscope, and see fig. 10. Comparison with Control group (9.19 +/-1.80 mm)^-2) Ti group (35.55 + -6.45 mm)^-2) The expression level of RANKL is obviously increased, and the difference has statistical significance (x p)<0.01). After spermine treatment, the expression level of RANKL is 27.06 +/-4.00 mm^-2(group L), 14.36. + -. 1.86mm^-2(group H), the difference between the high spermine dose-treated group and the Ti group was statistically significant (. about.p)<0.01), the difference between the low dose spermine treatment group and the Ti group was statistically significant (. about.p)<0.01). See fig. 11.

The expression level of p65 was further determined, as shown in FIG. 12, in combination with Control (11.34. + -. 1.77 mm)^-2) Comparative, Ti group (48.55. + -. 6.23 mm)^-2) The expression of p65 was significantly increased, and the difference was statistically significant (. about.p)<0.01). After spermine treatment, the expression level of p65 was 43.71 + -4.38 mm respectively^-2(group L), 21.54. + -. 2.64mm^-2(group H), the difference between the high spermine dose-treated group and the Ti group was statistically significant (. about.p)<0.01), the difference between the low dose spermine treatment group and the Ti group was statistically significant (. about.p)<0.05). See fig. 13.

The staining result shows that spermine reduces the expression of the skull local inflammatory factors TNF- α and IL-1 β, as shown in FIGS. 14 and 16, and the counting result under a microscope shows that the expression level of TNF- α in the Control group is 8.22 +/-1.64 mm^-2Number of IL-1 β positive cells 6.16. + -. 1.23mm^-2With Ti group (42.20. + -. 6.87 mm)^-2，56.21±8.66mm^-2) Comparison, the difference is statistically significant (p<0.05), the expression of TNF- α and IL-1 β in the spermine treatment group is obviously reduced, and the difference has statistical significance compared with the Ti groupp<0.05，**p<0.01). See fig. 15, 17.

The experimental result shows that the skull bone loss degree of a treatment group is reduced, the inflammatory bone destruction degree is reduced, the number of mature osteoclasts is reduced, the expression of key factors RANKL and p65 for osteoclast activation is reduced, particularly, the effect is more obvious by a high-dose group, the number of mature osteoclasts can be obviously reduced, the osteolysis caused by wear particles is inhibited, the expression of RANKL in the skull is reduced, and similarly, the inflammatory factors (TNF- α and IL-1 β) playing an important role in the PPO development process are obviously reduced by spermine treatment.

Claims

1. The application of spermine in the preparation of medicaments for treating periprosthetic osteolysis.

2. The use according to claim 1, wherein the periprosthetic osteolysis is periprosthetic osteolysis occurring after a prosthetic joint replacement.

3. A pharmaceutical preparation for treating periprosthetic osteolysis, which is characterized by comprising spermine with a therapeutically effective amount or spermine and pharmaceutically acceptable auxiliary materials.

4. The pharmaceutical formulation of claim 3, wherein the pharmaceutical formulation is suitable for parenteral administration.

5. The pharmaceutical preparation of claim 3, wherein the pharmaceutical preparation is a lyophilized preparation, an injection, a tablet, a granule or a capsule.