Cerium oxide nanoparticles and preparation method and application thereof

Abstract

The invention relates to cerium oxide nanoparticles as well as a preparation method and application thereof, and belongs to the technical field of biology, the contents of Ce < 3 + > and Ce < 4 + > in the cerium oxide nanoparticles (CNPs) are 51.2% and 48.8% respectively, the particle size is 3-5nm, and the surfaces of the cerium oxide nanoparticles are modified with succinic acid (SA), the preparation method comprises the following steps: using cerium nitrate hexahydrate, trioctylphosphine oxide, absolute ethyl alcohol, octadecene, oleylamine and other raw materials to prepare the cerium oxide nanoparticles. CNPs are obtained through a series of reactions and calcination treatment, then succinic acid is modified through ligand preparation and reaction, cerium oxide nanoparticles have the characteristic of catalyzing enzyme activity, and CNPs can effectively relieve bone loss induced by glucocorticoid and maintain bone homeostasis by enhancing the activity of bone formation related factors, inhibiting osteoclast activity and resisting oxidation. And a new solution is provided for the treatment of the hormone-induced osteoporosis.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to cerium oxide nanoparticles and a preparation method and application thereof. Background Art

[0002] Osteoporosis is a systemic metabolic disease characterized by decreased bone mass, destruction of bone tissue microstructure, and increased bone brittleness, making patients susceptible to fractures. With the arrival of an aging society, osteoporosis has become an important health problem for middle-aged and elderly people in my country. The causes of osteoporosis are multifaceted, and organ dysfunction caused by aging is the main factor.

[0003] Glucocorticoid-induced osteoporosis (GIO) is currently the most common cause of secondary osteoporosis. Epidemiological studies have shown that the increased risk of fractures (especially vertebral fractures) is closely related to the cumulative dose and duration of glucocorticoid (GC) use, and the risk of fractures can be partially saved after stopping medication. Although the harm of long-term use of glucocorticoids to bones is well known, the understanding and treatment of this disease are still insufficient. Therefore, revealing the pathological mechanism of GIO and finding new intervention measures have important clinical significance and social value. Previous studies have pointed out that long-term high-dose GC can inhibit the differentiation of mesenchymal stem cells into osteoblasts, promote the apoptosis of osteoblasts and osteocytes, and prolong the life of osteoclasts. Reduced bone formation and increased bone resorption lead to the destruction of bone turnover balance, which in turn promotes the development of osteoporosis.

[0004] The maintenance of bone homeostasis is regulated by two important links of bone remodeling: the dynamic balance between bone formation and bone resorption. When the effect of either side is enhanced or weakened, bone homeostasis will be unbalanced. However, excessive or long-term exposure to GC disrupts this dynamic balance, which is manifested as increased bone resorption and reduced bone formation. Glucocorticoids have direct and indirect effects on bone remodeling. On the one hand, GC increases the differentiation of osteoblast precursor cells into adipocytes rather than osteoblasts by upregulating the expression of peroxisome proliferator-activated receptor gamma (PPAR-γ), thereby reducing the differentiation and formation of osteoblasts. At the same time, osteoblastogenesis mediated by the Wnt / β-catenin signaling pathway is also inhibited by GC. GC inhibits Wnt binding by increasing the expression of Dickkopf-1 protein, which leads to a decrease in osteoblastogenesis. In addition, GCs also have an apoptotic effect on osteocytes, reducing bone mass and promoting the development of osteoporosis. On the other hand, GC enhances bone resorption by stimulating the generation and activity of osteoclasts. It has been reported that high doses of GC increase osteoclastogenesis by upregulating the expression of membrane-bound M-CSF, a key regulator of osteoclast differentiation. In addition to M-CSF, studies have shown that GC upregulates the level of RANKL in osteocytes and osteoblasts, but downregulates the expression of OPG, thereby increasing the RANKL / OPG ratio to enhance osteoclast formation and activity. In addition, GC is also able to affect the apoptotic process of osteoclasts, reducing their apoptosis, thereby extending their lifespan and enhancing bone resorption.

[0005] Metal nanomaterials have shown great potential in simulating various enzyme activities. For example, gold nanoparticles exhibit RNase and oxidase activities, and V2O5 nanoparticles and Fe2O3 show peroxidase activities. Previous studies have reported that cerium oxide (CNPs) have excellent SOD and CAT mimic enzyme activities, which has led to their application in many biological fields. Since nanoparticles used in biological applications must usually be hydrophilic, most of the CNPs used previously were synthesized by water-based wet chemical methods. However, due to the lack of sufficient active agent protection on the surface of cerium oxide nanoparticles, their stability, enzyme activity, and biocompatibility are all affected to a certain extent. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a cerium oxide nanoparticle with good stability, enzyme activity and biocompatibility and a preparation method thereof. The second purpose of the present invention is to use cerium oxide as a new type of antioxidant in the preparation of hormone-induced osteoporosis drugs.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a cerium oxide nanoparticle (CNPs), wherein Ce 3+ The content of Ce is 51.2%, 4+ The content of cerium oxide nanoparticles is 48.8%, the surface of the cerium oxide nanoparticles is modified with succinic acid (SA), and the size of the cerium oxide nanoparticles is 3-5nm.

[0009] Further, the preparation method of the cerium oxide nanoparticles is as follows:

[0010] S1: Ce nitrate hexahydrate and trioctylphosphine oxide (TOPO) (mass ratio 7:10) are placed in a flask, anhydrous ethanol is added, and the mixture is fully dissolved at a constant temperature of 90°C, and then octadecene (ODE) is added, and the mixed system is vacuum treated;

[0011] S2: The mixed solution was transferred to a glass bottle, 100 μL of oleylamine was added with slow stirring, the reaction started at 80°C, and the temperature was gradually increased to 190°C for 15 minutes;

[0012] S3: washing the nanoparticles with ethanol or acetone, drying them, and calcining the nanoparticles at 300° C. to obtain the cerium oxide nanoparticles.

[0013] S4: The surface of the cerium oxide nanoparticles is modified with succinic acid (SA).

[0014] The method for modifying the surface of the cerium oxide nanoparticles with succinic acid (SA) is as follows:

[0015] A1: Ligand preparation: 109 mg SA, 105 mg NHS and 175 mg EDC were completely dissolved in 10 mL dichloromethane solvent and stirred continuously for 8 h at room temperature. The solvent in the solution was effectively removed by rotary evaporation.

[0016] A2: Weigh a mixture of 100 mg of sodium alendronate and 250 mg of Na2CO3, dissolve in water, and add to the ligand prepared in step A1. React at room temperature for 12 hours.

[0017] A3: 1 mL of CNPs was diluted in 5 mL of tetrahydrofuran and mixed evenly with 5 mL of ligand solution. The mixed solution was stirred at 80°C for 8 h. The solution was cooled to room temperature, and the water layer containing cerium oxide nanomaterials was separated. Acetone was added to the separated water layer to precipitate the nanomaterials. The surface-modified CNPs were dialyzed in ultrapure water for 24 hours to obtain CNPs-SA.

[0018] The present invention provides an application of cerium oxide nanoparticles in the preparation of a drug for hormone-induced osteoporosis;

[0019] The concentration of the cerium oxide nanoparticles in the preparation of the medicine is 100 uM.

[0020] The beneficial effects of the present invention are:

[0021] The invention adopts a thermal decomposition method to synthesize cerium oxide nanoparticles. Compared with a water-based wet chemical method, the inorganic nanoparticles prepared by the thermal decomposition method have higher crystallinity, better morphology and narrower particle size distribution.

[0022] As a new type of antioxidant, cerium oxide nanoparticles can effectively reduce GC-induced bone loss and maintain bone homeostasis by enhancing the activity of bone formation-related factors, inhibiting osteoclast activity and anti-oxidation, providing a new strategy for the treatment of osteoporosis.

[0023] Other advantages, objectives and features of the present invention will be described in the following description to some extent, and to some extent, will be obvious to those skilled in the art based on the following examination and study, or can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be described in detail below in conjunction with the accompanying drawings, wherein:

[0025] Figure 1 For the synthesis and characterization of CNPs, Figure 1 a is the TEM image of CNPs. Figure 1 b is the particle size distribution of CNPs, Figure 1 c is the thermogravimetric analysis result of CNPs, Figure 1 d is the XPS result of CNPs;

[0026] Figure 2 The results of the CNPs scavenging activity test on ROS;

[0027] Figure 3 To construct and evaluate the GIO model in mice. Figure 3 a is micro-CT analysis and three-dimensional reconstruction of the distal femur of mice (upper scale: 1 mm; lower scale: 500 μm). Figure 3 bf Quantitative analysis of bone mineral density (BMD), bone volume ratio (BV / TV), trabecular number (Tb.N), trabecular thickness (Tb.Th) and trabecular space (Tb.Sp);

[0028] Figure 4 The staining results of mouse tissue sections;

[0029] Figure 5 TUNEL was used to determine the viability of osteocytes in the distal femur of each group of mice;

[0030] Figure 6 Immunohistochemistry was used to detect the changes in the levels of ferroptosis-related proteins GPX4 and ACSL4;

[0031] Figure 7 ELISA was used to measure the serum PINP, OC / BGP, and CTX levels of mice in each group;

[0032] Figure 8 CCK-8 was used to detect cell activity;

[0033] Fig. 9 Flow cytometry was used to detect changes in cellular ROS levels;

[0034] Fig.10 To evaluate the osteogenic differentiation and mineralization ability of CNPs on MC3T3-E1 cells;

[0035] Fig.11 Western blot was used to detect the expression of osteogenic function-related proteins ALPL, RUNX2, OPN, and COL I;

[0036] Fig.12 The changes of GSH / GSSG, SOD and MDA in MC3T3-E1 cells under the action of DEX by CNPs;

[0037] Fig.13 DHE staining was used to detect the changes in ROS levels in MC3T3-E1 cells affected by DEX by CNPs;

[0038] Fig.14 The effects of different cell death inhibitors on the activity of MC3T3-E1 cells under the action of DEX;

[0039] Fig.15 Annexin V / PI double staining method was used to determine the changes of CNPs in the apoptosis of MC3T3-E1 cells under the action of DEX;

[0040] Fig.16 Annexin V / PI double staining method was used to determine the changes of CNPs in the apoptosis of MC3T3-E1 cells under the action of DEX;

[0041] Fig.17 Western blot was used to detect the changes of caspase3, a marker of apoptosis;

[0042] Fig.18 Western blot was used to detect the expression of ferroptosis-related proteins GPX4 and ACSL4;

[0043] Fig.19 Effects of CNPs on lipid peroxidation level and Fe 2+ changes;

[0044] Fig. 20 To observe the changes of cell mitochondria by TEM;

[0045] Fig.21 The effect of CNPs on peroxisomes of MC3T3-E1 cells under the action of DEX;

[0046] Fig. 22 CCK-8 was used to detect the cell activity of BMMs;

[0047] Fig.23 TRAP staining was used to evaluate the osteoclast differentiation of BMMs cells under the action of DEX;

[0048] Fig.24 TRAP staining was used to evaluate the osteoclast differentiation of BMMs cells under the action of CNPs;

[0049] Fig.25 The effect of CNPs / DEX on osteoclast differentiation of BMMs cells and the formation of skeletal protein F-actin;

[0050] Fig.26 The effect of CNPs / DEX on the changes of ROS during osteoclast differentiation of BMMs cells;

[0051] Fig. 27 The effect of CNPs / DEX on the expression of cell osteoclast differentiation marker proteins. DETAILED DESCRIPTION

[0052] The following describes the embodiments of the present invention by specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments only illustrate the basic concept of the present invention in a schematic manner, and the following embodiments and features in the embodiments can be combined with each other without conflict.

[0053] Among them, the drawings are only used for illustrative explanations, and they only represent schematic diagrams rather than actual pictures, and should not be understood as limitations on the present invention. In order to better illustrate the embodiments of the present invention, some parts of the drawings may be omitted, enlarged or reduced, and do not represent the size of actual products. For those skilled in the art, it is understandable that some well-known structures and their descriptions in the drawings may be omitted.

[0054] The same or similar numbers in the drawings of the embodiments of the present invention correspond to the same or similar parts; in the description of the present invention, it should be understood that if the terms "upper", "lower", "left", "right", "front", "rear", etc. indicate the orientation or position relationship, they are based on the orientation or position relationship shown in the drawings, which is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operate in a specific orientation. Therefore, the terms describing the position relationship in the drawings are only used for illustrative purposes and cannot be understood as limiting the present invention. For ordinary technicians in this field, the specific meanings of the above terms can be understood according to specific circumstances.

[0055] Example 1

[0056] Synthesis of CNPs nanoparticles (4 nm)

[0057] 700 mg of cerium nitrate hexahydrate and 1 g of trioctylphosphine oxide (TOPO) were weighed and placed in a flask with a capacity of 100 mL, and then 3 mL of anhydrous ethanol was used to fully dissolve the above mixture under a constant temperature of 90 ° C. After the dissolution was complete, 5 mL of octadecene (ODE) was added, and the mixed system was vacuum treated for 15 minutes. Subsequently, the treated mixed solution was transferred to a glass bottle with a capacity of 20 ml, and 100 μL of oleylamine was slowly added under stirring. The reaction started from 80 ° C, gradually heated to 190 ° C, and maintained at this temperature for 15 minutes to complete the reaction. After the reaction was completed, the generated nanoparticles were washed with ethanol or acetone, and the solvent was removed by a drying step. In order to remove the ligands and impurities adsorbed on the surface of the nanoparticles, they were placed at 300 ° C for calcination, and finally pure cerium oxide nanoparticles (CeO2-NPs, CNPs) were obtained. In order to comprehensively evaluate the performance parameters of the prepared CeO2 nanoparticles, transmission electron microscopy, X-ray photoelectron spectrometer and other instruments were used to study their morphology, size and Ce 3+ / Ce 4+ The key indicators such as ratio were characterized and analyzed in detail.

[0058] Surface modification

[0059] A certain amount of ligand, specifically comprising 109mg succinic acid (SA), 105mg N-hydroxysuccinimide (NHS) and 175mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), is completely dissolved in 10mL dichloromethane solvent. Subsequently, continuous stirring is carried out for 8h at room temperature to ensure that the ligand is fully dissolved and mixed. Afterwards, the solvent in the solution is effectively removed by rotary evaporation. Subsequently, a mixture containing 100mg alendronate (AL) and 250mg Na2CO3 is dissolved in an appropriate amount of water and added to the previously activated ligand. The reaction process is carried out at room temperature for a whole night to ensure that the reaction is fully carried out.

[0060] Subsequently, 1 mL of the synthesized CNPs was diluted in 5 mL of tetrahydrofuran and mixed evenly with 5 mL of the ligand solution. Then, the mixed solution was stirred for 8 hours at a temperature of 80 ° C. After the reaction was completed, the solution was cooled to room temperature, and the water layer containing cerium oxide nanomaterials was successfully separated by separation technology. For further purification, acetone was added to the separated water layer to precipitate the nanomaterials. After that, the surface-modified CNPs were dialyzed in ultrapure water using a dialysis bag with a molecular weight of 30 kD for 24 hours. Finally, the modified CNPs-SA was successfully obtained, providing high-quality nanomaterials for subsequent research and applications.

[0061] ROS scavenging ability detection

[0062] 800 μL EDTA-Na2 (0.1M), 300 μL nitro blue tetrazolium chloride (NBT) (2mM), 200 μL riboflavin (0.6mM) and 11.4 mL sodium phosphate buffer (10mM, pH 7.8) were mixed to prepare a working solution. The surface-modified CNPs were diluted to the specified concentration in sodium phosphate buffer (10mM, pH 7.8) to prepare the test solution. The test solution (50 μL) and the working solution (100 μL) were mixed in a 96-well plate and shaken for 60 seconds, and the samples were irradiated under white light for 90 seconds. Excess ROS reacted with NBT to produce blue, which was quantified by detecting the absorbance at 560nm with an enzyme reader. The ROS inhibition rate was determined by calculating the decrease in absorbance at 560nm. High Ce 3+ Concentration is one of the main reasons why cerium oxide materials have super high ROS scavenging activity. Subsequent ROS scavenging activity experiments confirmed this view ( Figure 2 ), the experimental results showed that CNPs could achieve 80% ROS inhibition rate at a concentration of 5 μg / mL.

[0063] Characterization analysis

[0064] Transmission electron microscope (TEM): The morphology and size of CNPs were determined using a HT770 transmission electron microscope. The samples were prepared by dropping CNPs dispersed in toluene onto ultrathin copper grids. The size distribution of CNPs was measured on Nano Measurer software.

[0065] X-ray photoelectron spectroscopy (XPS): The synthesized CNPs were tested on a SCIENTIFIC ESCALAB 250 instrument. The binding energy (875 eV to 930 eV) was recorded and calibrated by the Ce3d peak of 883.20 eV. XPS Peak Fit 4.0 software was used for spectrum analysis. 3+ The binding energy peaks set by the concentration determination software are 885.53 and 903.95 eV, respectively. 4+ The binding energy peaks set by concentration are 881.79 and 899.98 eV respectively

[32] .

[0066] Thermogravimetric analysis (TGA): The surface-modified CNPs were tested on a thermogravimetric analyzer with a heating rate of 20°C / min to obtain the specific gravity of the surface-modified ligands.

[0067] like Figure 1 As shown in a, TEM results show that the synthesized CNPs have a regular spherical morphology and uniform size. The size of individual nanoparticles was counted using the particle size analysis software Nano Measure, with a total statistical sample of 300, and the average size of the CNPs was about 4.2nm ( Figure 1 b). In order to make the cerium oxide nanoparticles have better dispersibility and stability, we modified them with succinic acid (SA). The thermogravimetric analysis results showed that the weight loss was 23% at 100℃-450℃, indicating that SA was successfully modified on CNPs. In addition, in order to more accurately define the phase composition of the material and the generation of oxygen vacancies, this study used XPS technology to systematically characterize the chemical composition and element valence state of the material surface. The results are as follows Figure 1 As shown in d, Ce in CNPs 3+ The content of Ce is 51.2%, 4+ The content is 48.8%.

[0068] Example 2

[0069] Construction of mouse GIO model

[0070] Male C57BL / 6J mice, 6 weeks old, were selected for the experiment. After 1 week of adaptive culture, the mice were randomly divided into 3 groups (5 in each group): control group (Control), model group (dexamethasone DEX), and CNPs treatment group (DEX+CNPs). The model group was constructed by subcutaneous injection of dexamethasone water-soluble preparation, 3 times a week, at a dose of 10 mg / Kg. In the treatment group, CNPs were administered by tail vein injection, 3 times a week, at a dose of 1 mg / Kg. All groups of mice had normal diet and water, and were raised for a total of 28 days. After 28 days, the mice were anesthetized with 3% isoflurane, and blood was collected from the eye sockets. After the blood was collected, the mice were euthanized, and the bilateral femoral tissues were aseptically removed, one part was frozen at -80°C for standby use, and the other part was fixed in 4% paraformaldehyde. The blood samples were centrifuged at 4000r / min for 10 minutes, and the supernatant for bone formation and bone resorption ELISA detection was separated from the blood samples.

[0071] micro-CT analysis

[0072] The distal femur of mice was scanned by high-resolution micro-CT with a resolution of 5 μm using an ex vivo conical micro-focus CT (Skyscan1272). The scanning parameters were set to an X-ray power of 40 kV and a tube current of 0.25 mA. The average of three photos was taken at each angle (0.9°) to generate the final image, which was analyzed by NRecon software (Micro-Scanning Photonics Inc., Allentown, PA, USA). Bone microstructure parameters, such as trabecular parameters, include trabecular BMD, trabecular volume / total volume (BV / TV), trabecular thickness (Tb.N), trabecular thickness (Tb.N), trabecular spacing (Tb.Sp), etc. Trabeculae are determined by a fixed threshold. The micro-CT results show ( Figure 3 ), compared with the normal group (Control group), the model group (i.e., DEX group) showed significantly sparse trabecular tissue at the distal end of the femur. The bone mineral density (BMD), bone volume ratio (BV / TV), trabecular number (Tb.N), trabecular thickness (Tb.Th) of the mice in the DEX group were significantly lower than those in the control group, and the trabecular gap (Tb.Sp) was significantly increased, with statistically significant differences (p < 0.05). The results showed that the glucocorticoid-induced osteoporosis mouse model was successfully constructed, and the bone loss of GIO mice was significantly reduced after intervention with CNPs.

[0073] Tissue section staining

[0074] (1) H&E staining:

[0075] The femoral tissue was fixed in 4% paraformaldehyde for more than 24 hours; then it was immersed in bone tissue decalcification solution for about 2-3 weeks, and then routine paraffin embedding, sectioning, and HE staining were performed. The tissue sections were dewaxed to water, and then placed in xylene (15min, 2 times), 95% ethanol solution (5min, 2 times), 85% ethanol solution (5min, 1 time), 75% ethanol solution (5min, 1 time), and 50% ethanol solution (5min) in sequence. The double-distilled water shaker was washed 3 times, each time for 5min. Stained with hematoxylin for 3-5min, rinsed with double-distilled water for 2min, and 7% hydrochloric acid solution was added for differentiation, and the control time was 20s under the microscope. Rinse with running water for about 2min, and then add eosin staining solution for 1min. After rinsing with running water, follow the reverse steps of dewaxing to water, and finally put into xylene for 30min, and dry the slices after 2 times. Seal the slides, scan, and analyze the results.

[0076] (2) TRAP staining:

[0077] Dewax the paraffin sections and wash with pure water for several minutes. Circle the sections with a histochemical pen and place them in a wet box (with a certain amount of pure water to prevent the sections from drying up), and incubate them with pure water at 37°C for 2 hours. After the sections are incubated, pour off the pure water, add filtered TRAP working solution to cover the tissue, and place it at 37°C in the dark for 20-30 minutes. Restain the cell nuclei: pour off the incubation solution and wash with water, and stain the nuclei with hematoxylin staining solution. Dehydrate, make it transparent, and seal the sections with neutral gum. TRAP staining showed that in terms of bone resorption, the number of active osteoclasts in the DEX group mice was significantly higher than that in the control mice and the mice co-administered with DEX and CNPs, indicating that CNPs can reduce bone resorption by inhibiting osteoclasts. In addition, TUNEL assay was performed to evaluate the viability of osteocytes, and CNPs can protect osteocytes from DEX-induced cell death ( Figure 5 ).

[0078] (3) Masson staining:

[0079] Dewaxing of paraffin sections to water: sequentially place the sections in xylene (15min, 2 times), 95% ethanol solution (5min, 2 times), 85% ethanol solution (5min, 1 time), 75% ethanol solution (5min, 1 time), and 50% ethanol solution (5min). Wash on a shaker with double distilled water for 3 times, 5min each time. Hematoxylin staining of cell nuclei: Stain with Weigert's iron hematoxylin in the Masson staining kit for 5min, wash with tap water, differentiate with 1% hydrochloric acid alcohol for a few seconds, rinse with tap water, and rinse with running water for a few minutes to return to blue. Ponceau staining: Stain with ponceau acid fuchsin solution in the Masson staining kit for 5-10min, and rinse quickly with distilled water. Phosphomolybdic acid treatment: Treat with phosphomolybdic acid aqueous solution in the Masson staining kit for about 3-5min. Aniline blue staining: Do not wash with water, directly counterstain with aniline blue solution in the Masson staining kit for 5min. Differentiation: Treat with 1% ice CH3COOH for 1min. Dehydration and sealing: After washing with running water, follow the reverse steps of dewaxing to water, and finally put into xylene for 30 minutes. After 2 times, dry the slices, take out the slices from the xylene and dry them slightly, and seal them with neutral gum. Microscope examination, image acquisition and analysis. The micro-CT results were further verified by H&E and Masson staining. The trabeculae of mice in the DEX group were sparse and smaller than those in the control group, and this pathological change was significantly inhibited in mice co-administered with DEX and CNPs ( Figure 4 ).

[0080] (4) Immunohistochemistry:

[0081] 1) Dewaxing and hydration: Place the paraffin sections in a 60° oven for 30-60 minutes. Then place them in xylene I, II, and III for 10 minutes each, ethanol gradient (high to low: 100%, 95%, 80%, 70%) for 2 minutes each, and wash with water for 5 minutes. Wash with PBS 3 times, 3 minutes each time.

[0082] 2) Cell permeabilization and blocking: Infiltrate the slices with preheated blocking permeabilization solution (40 mL PBS + 120 uL TritonX-100 + 400 μL 30% H2O2) for 30 min (RT, avoid light) to reduce the activity of endogenous peroxidase. Wash with PBS 3 times, 3 min each time.

[0083] 3) Antigen repair: Soak the sections in 0.01M sodium citrate buffer at pH 6.0, and heat in a microwave oven for 4 minutes until boiling. Remove and allow to cool naturally to room temperature. Repeat twice, replenishing the liquid each time to prevent the sections from drying out. Wash with PBS 3 times, 3 minutes each time.

[0084] 4) Serum blocking: Use serum from the same source as the secondary antibody to block some non-specific binding sites, incubate at 37°C for 30 minutes, and remove excess serum with filter paper.

[0085] 5) Incubate with primary antibody: Add 20 μL of diluted primary antibody to the tissue in the circle (area 1×1) and incubate at 4°C overnight or at 37°C for 1-2 hours. Wash with PBS 3 times, 3 minutes each time.

[0086] 6) Incubation with secondary antibody: Add 20 μL of diluted secondary antibody to the tissue in the circle (area 1×1), incubate at 37°C for 1-2 hours, and wash 3 times with PBS, 3 minutes each time.

[0087] 7) Slice color development: Use DAB-H2O2 for 10 minutes, observe whether the staining is obvious under a microscope, and use distilled water to stop the color development within 10 minutes.

[0088] 8) Counterstaining and sealing: Stain with Mayer's hematoxylin for 30 seconds, wash with water, differentiate with hydrochloric acid alcohol for 2 seconds, and immerse in running water for 15 minutes. When dehydrating, use ethanol gradient (from low to high: 50%, 70%, 95%, 100%) for 2 minutes each. Use xylene for 5 minutes to make the slices transparent, and finally add neutral gum to seal the slices.

[0089] The results show that Figure 6 ), the GPX4 content in the bone tissue of the DEX group mice was significantly lower than that in the normal rat group; while CNPs could reverse this effect, indicating that CNPs can alleviate the ferroptosis of GIO mice.

[0090] ELISA test

[0091] Before killing the animals, 1 mL of blood was collected from mice in different groups by retroorbital bleeding. The whole blood from each mouse was transferred to a 2 mL tube and centrifuged at 4000 r / min for 10 min to obtain as much clarified serum as possible. The supernatant was removed, transferred to another 2 mL tube, and stored at -80 ° C for future use. According to the corresponding protocol, PINP, OC / BGP, and CTX serum levels were detected using ELISA detection kits (Elabscience, China).

[0092] The results show that Figure 7 ), CNPs can increase the content of bone formation marker proteins OC / BGP and PINP, and reduce the content of bone resorption marker protein CTX-I.

[0093] Example 3

[0094] Cell culture

[0095] Mouse osteoblast cell line MC3T3-E1 cells were obtained from the National Authenticated Cell Culture Collection (Shanghai, China). MC3T3-E1 cells were cultured in α-minimum essential medium (α-MEM, Gibco) supplemented with 10% (vol / vol) fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C in a humidified atmosphere of 5% CO2. During mineralization, MC3T3-E1 cells were cultured in an incubator containing 4 mmol·L-1β-glycerophosphate and 50 μg·mL -1 The cells were cultured in osteogenic differentiation medium supplemented with ascorbic acid.

[0096] Cell viability and DCFH-DA detection

[0097] MC3T3-E1 cells were plated in 96-well plates at 10,000 cells per well or 400,000 cells per well in 6 cm culture dishes. Dex at different concentrations (0.01-200 μM) was added to the culture medium for 24 hours, and the changes in cell activity were determined using CCK-8 reagent. The OD at a wavelength of 450 nm was measured using an ELISA reader (Bio-Phase Tek, USA). In addition, the cells were incubated in the dark according to the instructions for use of the DCFH-DA (Biyuntian, China) reagent and then detected on a flow cytometer using an emission wavelength of 525 nm and an excitation wavelength of 488 nm. The average fluorescence intensity of each group of cells represents the level of ROS in the cells.

[0098] Alkaline phosphatase (ALP) and alizarin red (ARS) staining to evaluate cell osteogenic differentiation and mineralization ability

[0099] MC3T3-E1 cells were resuspended, plated and plated at 1×10 5 / well were cultured in 24-well plates. After 48h of different treatments, the cells were cultured in osteogenic induction medium for 2 weeks (ALP staining) or 3 weeks (ARS staining). For ALP staining, the cells were washed with phosphate-buffered saline (PBS) and fixed with 4% (v / v) paraformaldehyde for 30min. After washing three times with PBS, the cells were stained with ALP staining solution (Biyuntian, China) prepared according to the manufacturer's instructions. Subsequently, they were observed under a microscope and photographed. The ARS staining method was carried out according to the instructions for use, and the same treatment, staining and photography were performed in the corresponding time period.

[0100] Western blot

[0101] After the cells in each group to be treated reach the corresponding time, the residual culture medium is discarded and washed twice with PBS, and then 200 μL RIPA lysis buffer is added to each dish for lysis on ice for 30 minutes. Then the cell protein is scraped off with a cell scraper and transferred to an EP tube, centrifuged at 12000rpm, 4℃ for 15 minutes, and the supernatant is retained. Use PBS to prepare gradient standard protein, so that the concentrations are 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL respectively. At the same time, prepare BCA protein detection working solution. Add gradient standard protein and 20 μL of the protein to be tested after 5 times dilution to the 96-well plate, add 200 μL BCA protein detection working solution, and after 37℃ water bath for 30 minutes, the absorbance of each well is detected by an enzyme reader at 562nm and the protein concentration of the sample to be tested is calculated. Add appropriate amount of loading buffer (4×) and PBS to make the final protein concentration of each sample 1 μg / μL. Heat at 100°C for 10 min using a heating module, cool and place at -20°C for later use.

[0102] Glue preparation: Take a clean glass plate for gel preparation and clamp it on the gel preparation device. Select the concentration of separation gel according to the molecular weight of the protein to be tested. According to the instructions of the SDS-PAGE gel preparation kit, add each component in proportion, add it to the gel preparation device quickly after mixing, and add anhydrous ethanol to seal the upper layer of separation gel. When the separation gel solidifies and a dividing line appears between it and ethanol, carefully pour off the upper layer of ethanol, add the pre-prepared concentrated gel and insert the 15-hole comb. After the concentrated gel solidifies, you can proceed to the next step of the experiment.

[0103] Electrophoresis: Remove the prepared gel and put it into the electrophoresis tank. Carefully fill the inner tank with the newly prepared electrophoresis solution and remove the comb. Add 20μL protein to each well (total protein is 20ug). Add 5uL of color pre-stained protein marker to the hole at the edge of the gel. After the outer tank is filled with electrophoresis solution, start electrophoresis at a voltage of 50V. When the protein moves downward into the separation gel, increase the voltage to 100V. Stop electrophoresis when the protein moves to 1cm above the bottom edge of the glass plate.

[0104] Transfer: Remove the device where the electrophoresis is finished and take out the gel, carefully place it on the transfer clip that has been pre-padded with a pad and three layers of filter paper, with the black side facing down. Cut a PVDF membrane of appropriate width and length and soak it in methanol for a while. Carefully lay the PVDF membrane flat on the gel, paying attention to the size of the membrane should include the target protein, and remove the bubbles, cover with three layers of filter paper and pad. Close the transfer clip and put it into the transfer device, check the positive and negative poles, and pour in the transfer solution. Transfer the membrane in a 4℃ constant temperature refrigerator, constant voltage 100V, 80min.

[0105] Blocking: After the transfer, remove the PVDF membrane with tweezers, put it into TBST to wash off the transfer solution, and then put it into 5% blocking solution, shake on a 60rpm platform, and block for 60 minutes at room temperature.

[0106] Primary antibody: Use primary antibody diluent (Biyuntian) to dilute the antibody according to the antibody manual. Wash the PVDF membrane with TBST to remove the residual blocking solution and then put it into an appropriate amount of antibody to ensure that the membrane can be completely immersed in the antibody. Refrigerate at 4℃ overnight.

[0107] Secondary antibody: Use secondary antibody diluent (Biyuntian) to dilute the antibody according to the antibody manual. Remove the PVDF membrane from the primary antibody with tweezers, put it into an appropriate amount of TBST, and wash it on a shaker at room temperature at 60rpm for 10 minutes, and continue three times. Select the appropriate secondary antibody according to the source species of the primary antibody of the target protein. Put the washed membrane into the secondary antibody and incubate it on a shaker at room temperature for 60 minutes.

[0108] Luminescence: Take out the PVDF membrane, put it into an appropriate amount of TBST, and wash it for 10 minutes on a shaker at room temperature at 60rpm, and continue three times. Mix AB solution 1:1 according to the instructions of the ECL luminescence kit to obtain luminescent solution, and protect it from light. Carefully put the membrane into the luminescence machine, drop the luminescent solution, adjust the machine parameters, select the appropriate exposure time, adjust the brightness and contrast, obtain a clear Western band image, and save it.

[0109] Analysis: Three appropriate Western band images were selected from each group and processed and analyzed using Image J software to obtain the grayscale value, perform statistical analysis and draw graphs.

[0110] Intracellular GSH / GSSG, SOD, MDA level detection

[0111] After the cells in each group to be treated reached the corresponding time, the residual culture medium was discarded and washed twice with PBS, and then 200 μL RIPA lysis solution was added to each dish for lysis on ice for 30 minutes. Then the cell protein was scraped off with a cell scraper and transferred to an EP tube, centrifuged at 12000 rpm and 4°C for 15 minutes, and the supernatant was retained. After the cell protein content was detected using the BCA protein quantification kit, the operation was performed according to the instructions of the GSH / GSSG, SOD, and MDA detection kits.

[0112] DHE staining to detect intracellular ROS levels

[0113] MC3T3-E1 cells were resuspended, plated and plated at 1×10 5 / well were cultured in a 24-well plate. After 24 hours of different treatments, the DHE staining solution was used according to the instructions. After incubation at 37°C in the dark for 30 minutes, the red emission images of the cells were observed and photographed under a fluorescence microscope with blue or green light excitation. ROS-positive cells were stained red in the entire nuclear region; when excited with ultraviolet light, the unoxidized DHE in the cytoplasm could emit blue fluorescence.

[0114] Annexin V / PI apoptosis staining

[0115] Aspirate the cell culture medium into a suitable centrifuge tube, wash the adherent cells once with PBS, and add an appropriate amount of trypsin cell digestion solution (without EDTA) to digest the cells. Incubate at room temperature until the adherent cells can be blown down by gentle blowing, add complete culture solution to terminate digestion, centrifuge, and remove the supernatant. Avoid excessive digestion with trypsin. Add the collected cell culture medium, mix slightly, transfer to a centrifuge tube, centrifuge at 1000g for 5 minutes, discard the supernatant, collect the cells, gently resuspend the cells with PBS and count. Take 100,000 resuspended cells, centrifuge at 1000g for 5 minutes, discard the supernatant, add 195μL buffer and gently resuspend the cells. Add 5μL AnnexinV-FITC and mix gently. Incubate at room temperature (20-25℃) in the dark for 10 minutes. Aluminum foil can be used to protect from light. Centrifuge at 1000g for 5 minutes, discard the supernatant, add 190μL buffer and gently resuspend the cells. Add 10μL propidium iodide staining solution, mix gently, and place in an ice bath in the dark. Then flow cytometry was performed, Annexin V-FITC was green fluorescence and PI was red fluorescence.

[0116] Analysis of lipid peroxidation levels

[0117] Using BODIPY TM 581 / 591 C11 (D3861; Thermo Fisher Scientific), a lipid peroxidation sensor, was used to examine the level of lipid peroxidation in vitro according to the manufacturer's instructions. Briefly, cells were incubated with BODIPY581 / 591C11 (2 μM) in serum-free medium in the dark for 30 min. The fluorescence of the sensor was examined using a flow cytometer to collect green (484 / 510 nm) and red signals (581 / 610 nm), thereby providing a ratio index of lipid peroxidation.

[0118] Fe 2+ Concentration detection

[0119] The intracellular ferrous iron content was assessed using a ferrous ion colorimetric test kit (E-BC-K773-M, Elabscience). After the specified treatment, the treated cells were harvested and lysed in an iron assay buffer. The whole cell lysate was centrifuged at 12000 × g for 5 min, and only the supernatant was used for the iron assay. The experimental procedure strictly followed the manufacturer's instructions.

[0120] Transmission electron microscopy (TEM) analysis

[0121] In order to study the morphological changes of cells, MC3T3-E1 cells exposed to different treatments were collected, pre-fixed with 2.5% ice-cold glutaraldehyde overnight, and then post-fixed in 1% osmium tetroxide at 4 ° C for 1 hour. After dehydration in a graded ethanol series, the samples were embedded in an epoxy-acetone mixture for 2 hours and then immersed in a pure resin solution at 37 ° C overnight. Uranyl acetate and lead citrate were detected by microscopy as negative controls. Electron micrographs were observed by TEM (JEM 1400, JEO).

[0122] Immunofluorescence staining

[0123] MC3T3-E1 cells were cultured in α-MEM supplemented with 10% FBS for 24 hours in 12-well plates, and then treated with drugs for a specified period of time. MC3T3-E1 cells were incubated with anti-PMP70 antibodies at 4 ° C overnight, and then incubated with the corresponding fluorescent secondary antibodies conjugated with fluorescein isothiocyanate (FITC) in the dark for 1 hour. DAPI was used to stain the nucleus. Images were observed and photographed using a fluorescence microscope.

[0124] Results Analysis

[0125] One-way analysis of variance (Fisher's LSD test) was used to evaluate statistical significance among multiple groups, and p ≤ 0.05 was considered significant. All results were analyzed using GraphPad Prism 7 software, and mean ± SD was used to present all data.

[0126] The effects of DEX and CNPs on the activity of MC3T3-E1 cells were evaluated by CCK-8 assay. It was found that DEX at a concentration of 100 μM could significantly reduce cell activity (p < 0.01), while CNPs at a concentration of 50 μM could significantly alleviate the DEX-induced decrease in cell activity ( Figure 8 In addition, when CNPs acted alone on MC3T3-E1 cells, even at a concentration of up to 500 μM, they did not show obvious cytotoxic effects. By detecting the changes in cellular ROS ( Fig. 9 ), CNPs can significantly reduce the level of ROS induced by DEX (100μM). DEX will induce cells to produce ROS, thereby reducing cell activity; CNPs can play a role in scavenging ROS and alleviate the negative effects of DEX. 100μM DEX was used as the induction concentration, and 100μM CNPs was used as the rescue concentration of the experiment.

[0127] The effects of CNPs on osteogenic differentiation and mineralization of MC3T3-E1 cells under the influence of DEX were shown in the results of alkaline phosphatase staining and Alizarin Red S staining. Fig.10As shown in the figure, DEX reduced the osteogenic differentiation ability of MC3T3-E1 cells, and its mineralized nodules were significantly reduced; after CNPs intervention, the osteogenic differentiation ability of MC3T3-E1 cells was restored, and the number of mineralized nodules was significantly increased. Western blot was used to detect ( Fig.11 ) osteogenic function-related proteins ALPL, RUNX2, OPN, and COLⅠ, further confirming that CNPs can restore the damage of DEX to the ability of successful cell differentiation.

[0128] CNPs can reduce the level of cellular ROS induced by DEX. A series of related oxidative stress levels, such as GSH / GSSG, SOD, MDA and other indicators ( Fig.12 ): The antioxidant capacity of MC3T3-E1 cells under the action of DEX, GSH, SOD decreased significantly, and the lipid peroxidation product MDA increased, indicating that DEX induced oxidative stress in cells; CNPs can reverse the above indicators, restore redox homeostasis, and reduce the level of cellular oxidative stress. DHE staining further confirmed that CNPs have the ability to reduce cellular ROS levels ( Fig.13 ).

[0129] What kind of cell death or self-rescue process occurs in MC3T3-E1 cells under the action of DEX? Fig.14 ), apoptosis inhibitor (Z-VAD-FMK), ferroptosis inhibitor (Ferrostatin-1, Fer-1), necroptosis inhibitor (Necrostatin-1, Nec-1) and autophagy inhibitor (3-Methyladenine, 3-MA) / agonist (Rapamycin, Rap) were applied. Z-VAD or Fer-1 can rescue the cell activity under the action of DEX, while Nec-1 and 3-MA / Rap cannot restore the cell activity. MC3T3-E1 cells mainly die in the form of apoptosis and ferroptosis under the action of high doses of DEX.

[0130] DEX can promote apoptosis of MC3T3-E1 cells. Annexin V / PI double staining was used to determine cell apoptosis, and it was found that CNPs could significantly reduce the apoptosis rate induced by DEX ( Fig.15 ). Detection of mitochondrial membrane potential and cell apoptosis staining further revealed that DEX induced a decrease in cell mitochondrial membrane potential and an increase in the number of apoptosis-positive cells ( Fig.16 ). The changes of caspase3, a marker of apoptosis, were detected by Western blot, and it was found that the expression of caspase3 (34kD) was significantly downregulated under the action of DEX ( Fig.17 ).

[0131] The effect of CNPs on ferroptosis of MC3T3-E1 cells under the action of DEX was investigated by Western blot to detect the expression of ferroptosis-related proteins GPX4 and ACSL4. RSL3 was used as a positive control. RSL3 is an inhibitor of glutathione peroxidase 4 (GPX4) that specifically induces ferroptosis in cells. Fer-1 is an effective and selective ferroptosis inhibitor that prevents damage to membrane lipids through a reduction mechanism, thereby inhibiting cell death. Fig.18 As shown in the figure, DEX induced ferroptosis in MC3T3-E1 cells, while CNPs inhibited ferroptosis by increasing GPX4 protein expression and downregulating ACSL4 expression. The C11 BODIPY 581 / 591 probe was used to detect the level of cellular lipid peroxidation ( Fig.19 a), found that DEX increased the level of cellular lipid peroxidation, and CNPs could reverse this effect. Another hallmark feature of ferroptosis is Fe 2+ The increase of ferrous ion concentration in cells after DEX treatment was detected. 2+ CNPs can reduce intracellular Fe 2+ content( Fig.19 b).

[0132] TEM images further confirmed the protective effect of CNPs on mitochondria ( Fig. 20 ), in which the mitochondrial ultrastructure of cells treated with DEX and RSL3 showed typical features of ferroptosis, including mitochondrial shrinkage, membrane rupture, and reduced cristae morphology (red star: normal mitochondria; green triangle: abnormal mitochondria), while cells treated with CNPs or Fer-1 showed mitochondrial ultrastructure similar to that of control cells.

[0133] Immunofluorescence and WB detection showed that DEX or RSL3 could reduce the number of intracellular peroxisomes ( Fig.21 ), the catalase in the peroxisome also decreases, resulting in an imbalance in the ROS homeostasis of the cell. CNPs or Fer-1 can alleviate the reduction of peroxisomes and restore ROS homeostasis. At the same time as ferroptosis occurs, the expression level of lipoxygenase ALOX15 increases. CNPs or Fer-1 can reduce the expression of ALOX15 and inhibit the occurrence of ferroptosis.

[0134] Example 4

[0135] Cell culture

[0136] Mouse bone marrow mononuclear cells (BMMs) were prepared from whole bone marrow of femurs of 6-week-old mice. Briefly, bilateral mouse femurs were obtained aseptically, and the bone marrow cavity was flushed three times with sterile phosphate buffered saline (PBS) using a sterile syringe, and the liquid was collected in a 15 mL centrifuge tube, centrifuged at a radius of 3 cm and 1200 r / min for 3 min; the supernatant was discarded, 5 mL of red blood cell lysis buffer was added, and lysed on ice for 2 min; centrifuged, the supernatant was discarded, and the cells were resuspended with α-MEM medium; centrifuged, the supernatant was discarded, and the cells were resuspended with 5 mL of α-MEM complete medium containing 10% fetal bovine serum, and inoculated in a 25 cm2 culture flask, placed in a 5% CO2, 37°C cell culture incubator overnight (about 24 hours); the supernatant was collected and centrifuged, the supernatant was discarded, and the cells were resuspended with 5 ml of complete medium containing 50 ng / mL M-CSF, and inoculated in a 25 cm2 culture flask. After 2 days, the adherent cells were BMMs; the supernatant was discarded, and the cells were washed twice with sterile PBS. After trypsin digestion and repeated blowing, the liquid was collected, centrifuged, and the supernatant was discarded. After resuspension, according to the needs of subsequent experiments, select the appropriate cell density after cell counting and inoculate them into different cell culture dishes; add complete culture medium containing 50ng / mL M-CSF and 100ng / ml RANKL, and mature osteoclasts can be induced after 4-6 days.

[0137] TRAP staining

[0138] BMMs cells were seeded at 2000 / cell in a 96-well plate, and complete medium containing 50 ng / mL of M-CSF and RANKL was added for osteoclast induction. After the fifth day of culture, the operation was performed according to the instructions of the kit. In brief, preheat enough deionized water at 37°C; fix with 4% paraformaldehyde for 20 minutes and wash with deionized water; prepare two 1.5mL ep tubes, add 50uL Fastgarnet GBC Base solution and 50uL Sodium Nitrite Solution to each, mix gently for 30 seconds, and let stand at room temperature for 2 minutes; the mixture of the two is the solution mixed in step 3; 100uL Naphthol AS-BI Phosphate Solution 50uL Acetate Solution 200uL of the solution mixed in step 3; 4500uL deionized water preheated at 37°C Tartrate Solution 100uL; heat the staining solution prepared in step 4 to 37°C, and then incubate at 37°C in the dark for 1 hour; after incubation for 1 hour, wash with deionized water; observe under a microscope.

[0139] F-actin fluorescence staining

[0140] TraKine F-actin (Abbkine, USA) was used to stain the osteoclast cytoskeleton protein F-actin. Briefly, after culturing in osteoclast induction medium containing CNPs / DEX for 5 days, the cells were fixed in 4% formaldehyde for 15 minutes and then permeabilized with 0.5% Triton X-100 solution. The experiment was performed in the dark, stained with TraKine F-actin for 1 hour, and then the cell nucleus was stained with DAPI for 5 minutes, and then washed with cold PBS. The actin ring and DAPI staining of mature osteoclasts were observed by fluorescence microscopy.

[0141] Results Analysis

[0142] One-way analysis of variance (Fisher's LSD test) was used to evaluate statistical significance among multiple groups, and p ≤ 0.05 was considered significant. All results were analyzed using GraphPad Prism 7 software, and mean ± SD was used to present all data.

[0143] The effect of DEX / CNPs on BMMs cell activity. The CCK-8 test showed that lower doses of DEX (0.01-0.1 μM) promoted BMMs cell activity. As the concentration of DEX increased, cell activity decreased. The effect of CNPs on BMMs cell activity showed a promoting effect within the concentration range (50-500 μM). Fig. 22 ).

[0144] TRAP staining was used to evaluate the effect of different concentrations of DEX on osteoclast differentiation of BMMs cells. It was found that 0.01-0.1 μM DEX significantly promoted the formation of osteoclasts. As the concentration increased, osteoclast differentiation was inhibited, which was consistent with the previous cell activity test results ( Fig.23 ). CNPs showed an effect of gradually inhibiting osteoclast differentiation as the dose increased ( Fig.24 ). In order to better conform to the bone homeostasis environment of GIO in vivo, 0.1 μM DEX was selected as the stimulation concentration and 100 μM CNPs was selected as the treatment concentration in subsequent experiments.

[0145] It was further confirmed by TRAP staining and F-actin fluorescence staining ( Fig.25 ), CNPs can inhibit the formation of F-actin actin ring, affecting the differentiation and maturation of osteoclasts. Especially under the enhancement of DEX, CNPs still have a strong inhibitory effect on osteoclast activity.

[0146] ROS can significantly regulate osteoclast differentiation and regulate bone homeostasis ( Fig.26 ). In order to explore the effect of CNPs / DEX on ROS during osteoclast differentiation of BMMs cells, the fluorescence changes of DCFH-DA were analyzed by flow cytometry, and it was found that RANKL stimulation would increase the level of cellular ROS, and the addition of DEX would further increase the level of ROS; while CNPs alone could reduce the level of cellular ROS, which could not be reversed even in the presence of DEX. CNPs have the effect of inhibiting the generation of ROS, thereby affecting the differentiation of osteoclasts.

[0147] RANKL activates several transcription factors involved in osteoclast differentiation ( Fig. 27 ). Among them, c-Fos activates NFATc1, a major regulator of osteoclast differentiation. Compared with RANKL-induced differentiated osteoclasts, CNPs-treated cells showed reduced expression of these transcriptional proteins, NFATc1 and c-Fos. In addition, ROS plays a role as a powerful multifunctional signaling molecule in the intercellular pathway that regulates osteoclast differentiation. CNPs-treated cells significantly reduced the expression of NOX1, thereby inhibiting ROS production and inhibiting osteoclast differentiation.

[0148] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution, which should be included in the scope of the claims of the present invention.

Claims

1. Cerium oxide nanoparticles, characterized in that: The Ce in the cerium oxide nanoparticles (CNPs) 3+ The content of Ce is 51.2%, 4+ The content of is 48.8%, and the surface of the cerium oxide nanoparticles is modified with succinic acid (SA).

2. The cerium oxide nanoparticles according to claim 1, characterized in that The size of the cerium oxide nanoparticles is 3-5 nm.

3. The method for preparing cerium oxide nanoparticles according to claim 1, characterized in that: The following steps are involved: S1: Synthesis of cerium oxide: Ce nitrate hexahydrate and trioctylphosphine oxide (TOPO) (mass ratio 7:10) are placed in a flask, anhydrous ethanol is added, and the mixture is fully dissolved at a constant temperature of 90°C, and then octadecene (ODE) is added, and the mixed system is vacuum treated; S2: The mixed solution was transferred to a glass bottle, oleylamine was added with slow stirring, and the reaction started at 80°C and gradually heated to 190°C for 15 minutes; S3: washing the nanoparticles with ethanol or acetone, drying them, and calcining the nanoparticles at 300° C. to obtain the cerium oxide nanoparticles; S4: The surface of the cerium oxide nanoparticles is modified with succinic acid (SA).

4. The method for preparing cerium oxide nanoparticles according to claim 3, characterized in that: The method for modifying the surface of the cerium oxide nanoparticles with succinic acid (SA) is as follows: A1: Preparation of ligand solution: SA, NHS and EDC (mass ratio 109:105:175) were completely dissolved in dichloromethane solvent and stirred continuously for 8 h at room temperature. The solvent in the solution was effectively removed by rotary evaporation. A2: Weigh a mixture of sodium alendronate (AL) and Na2CO3 (mass ratio 10:25), dissolve it in water, and add it to the ligand solution prepared in step A1. React at room temperature for 12 hours. A3: CNPs were diluted in tetrahydrofuran and mixed evenly with the ligand solution (volume ratio of 1:5:5). The mixed solution was stirred at 80°C for 8 hours. The solution was cooled to room temperature, and the water layer containing cerium oxide nanomaterials was separated. Acetone was added to the separated water layer to precipitate the nanomaterials. The surface-modified CNPs were dialyzed in ultrapure water for 24 hours to obtain CNPs-SA.

5. Use of the cerium oxide nanoparticles according to claim 1 in preparing a drug for hormone-induced osteoporosis.

6. The use of cerium oxide nanoparticles according to claim 5 in preparing a drug for steroid-induced osteoporosis, characterized in that: The concentration of the cerium oxide nanoparticles in the preparation of the medicine is 100 uM.

Images