Application of MOF (Metal Organic Framework) material encapsulated solanine in preparation of medicine for preventing and treating osteoarthritis

Abstract

The invention provides application of solanine encapsulated by an MOF (Metal Organic Framework) material in preparation of a medicine for preventing and treating osteoarthritis. The preparation method of the solanine encapsulated by the MOF material comprises the following steps: mixing and stirring UIO-66-NH2, DI-water and solanine to obtain a solution A; preparing a polyethyleneimine aqueous solution as a solution B; and adding the solution B into the solution A, and continuously stirring to obtain the MOF material encapsulated solanine USP. The MOF material encapsulated solanine material prepared in the invention can obviously alleviate inflammatory response and prevent cartilage cell ferroptosis progress, and provides new feasibility for treatment of osteoarthritis cartilage cell ferroptosis.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to application of solanine encapsulated by MOF materials in the preparation of medicines for preventing and treating osteoarthritis. Background Art

[0002] Osteoarthritis is a degenerative disease and a major cause of disability and malformation in the elderly. Its pathological features include degenerative changes in articular cartilage, abnormal subchondral bone remodeling, and osteophyte formation. Many factors can aggravate cartilage destruction, including synovitis, subchondral remodeling, and cartilage energy metabolism disorders. Therefore, for patients with advanced or end-stage osteoarthritis, there is no other option except joint replacement. Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used in osteoarthritis due to their inevitable side effects. Therefore, it is imperative to further explore the therapeutic targets of osteoarthritis and develop effective treatment strategies.

[0003] Ferroptosis is a massive accumulation of iron-dependent lethal lipid reactive oxygen species (ROS). Ferroptosis of chondrocytes can promote the progression of osteoarthritis. Chondrocytes are the only cell type in articular cartilage and play a decisive role in maintaining cartilage stability. Therefore, the survival of chondrocytes is essential for maintaining the integrity of cartilage. Metabolic disorders can promote the occurrence and development of osteoarthritis, and metabolic regulation plays a key role in the treatment of osteoarthritis. In the development of osteoarthritis, many metabolic pathways are significantly impaired, including glucose metabolism. Part of glucose metabolism provides energy to cells through glycolysis. Glucose metabolism in chondrocytes tends to glycolysis. Hypoxia-inducible factor-1α (HIF-1α) is a highly conserved transcription factor that induces cells to switch from oxidative metabolism to glycolysis and plays a key role in the synthesis of proinflammatory factors. HIF-1α plays a vital role in maintaining chondrocyte activity and integrity, regulating cartilage formation and energy metabolism.

[0004] A wealth of natural bioactive compounds have been found in herbs and plants, showing beneficial biological effects by exerting anti-inflammatory and antioxidant effects in vitro and in vivo. Further research on these substances may provide promising new drug candidates for the treatment of osteoarthritis. One of these compounds is solanine, a bioactive glycoalkaloid extracted from potatoes. GAs, including solanine, have a wide range of pharmacological activities, such as anti-inflammatory, anti-allergic, anti-diabetic, anti-pathogenic, viral, fungal and protozoal activities. Previous studies have shown that solanine can regulate HIF-1α and affect the expression of endothelial growth factor. However, the effect of solanine on HIF-1α regulation of chondrocyte glycolysis and its downstream signaling pathways has not been confirmed, especially in the context of osteoarthritis. Therefore, effective drug delivery carriers are needed to reduce toxicity and improve efficacy. Based on this, the present invention provides the use of solanine encapsulated by MOF materials in the preparation of drugs for the prevention and treatment of osteoarthritis. Summary of the invention

[0005] The purpose of the present invention is to provide the use of solanine encapsulated by MOF material in the preparation of drugs for preventing and treating osteoarthritis. The prepared solanine material encapsulated by MOF material significantly reduces the inflammatory response, prevents the progression of chondrocyte ferroptosis, and provides new feasibility for the treatment of chondrocyte ferroptosis in osteoarthritis.

[0006] In one aspect, the present invention provides the use of solanine encapsulated by MOF materials in the preparation of a drug for preventing and treating osteoarthritis.

[0007] Furthermore, the osteoarthritis is inflammation of articular chondrocytes.

[0008] Furthermore, the articular chondrocyte inflammation is induced by LPS.

[0009] Furthermore, the solanine encapsulated by the MOF material is used to regulate the expression of HIF-1α and / or GLUT1, which are key factors in glucose metabolism.

[0010] Furthermore, the medicine includes tablets, capsules, pills, granules, oral liquids, and solutions.

[0011] Furthermore, the MOF material is UIO-66-NH2.

[0012] On the other hand, the present invention also provides a method for preparing solanine encapsulated by MOF material, the steps comprising: mixing UIO-66-NH2 with DI water and solanine, stirring at room temperature for 22-26 hours to obtain solution A; preparing a polyethyleneimine aqueous solution, stirring at room temperature for 25-35 minutes to obtain solution B; adding solution B to solution A that has been stirred for 22-26 hours, and continuing to stir at room temperature for 22-26 hours to obtain solanine USP encapsulated by MOF material.

[0013] Furthermore, the usage ratio of UIO-66-NH2, DI-water, solanine and polyethyleneimine is (8-12) mg: (4-6) mL: (3-5) mg: (180-220) mg.

[0014] Furthermore, the preparation method of UIO-66-NH2 includes: dissolving F127 in deionized water, and then adding acetic acid and NaClO4·H2O; stirring sufficiently to form a homogeneous solution; adding ZrO(NO3)2·2H2O and 2-aminoterephthalic acid to the homogeneous solution and stirring at 35-40°C for 10-14 hours; obtaining a precipitate by centrifugation; washing the obtained precipitate, resuspending and storing;

[0015] Furthermore, the usage ratio of F127, deionized water, acetic acid, NaClO4·H2O, ZrO(NO3)2·2H2O and 2-aminoterephthalic acid is (45-55) mg: (2-4) mL: (0.3-0.5) mL: (140-160) mg: (110-120) mg: (45-55) mg.

[0016] Furthermore, the washing method is washing once with water and then washing once with ethanol.

[0017] Furthermore, the polyethyleneimine aqueous solution is 10 mg / ml.

[0018] Beneficial effects:

[0019] (1) Due to the presence of organic ligands, MOFs exhibit enhanced biodegradability. After degradation in biological systems, MOFs can be gradually converted into harmless metabolites, thus reducing people's concerns about their cumulative toxicity; compared with other nanocarriers, MOFs are simple to synthesize, biodegradable, controllable in release, and have high drug loading, making them reliable nanocarriers;

[0020] (2) In the present invention, the MOF material UIO-66-NH2 is used as a delivery vehicle for solanine, and the USP obtained after modification with polyethyleneimine can target the cartilage surface and increase the retention time of solanine. Based on the interaction between solanine and HIF-1α, the released solanine can regulate glycolysis through the HIF-1α signaling pathway, thereby alleviating ferroptosis. In vitro, solanine treatment significantly reduced the expression of HIF-1α, GLUT1, and ferroptosis-related GPX4. In the anterior cruciate ligament transection osteoarthritis (ACLT) model, USP greatly reduced articular cartilage damage caused by ACLT and effectively inhibited the progression of osteoarthritis. Therefore, USP successfully improved the efficacy of solanine.

[0021] (3) In the acidic osteoarthritis joint cavity environment, positively charged carriers can target negative ions on the surface of chondrocytes through charge response. Therefore, in order to make UIO-66-NH2 positively charged, the present invention adds polyethyleneimine to modify the surface of UIO-66-NH2 to be positively charged, making it targeted, and successfully verifies the targeting effect of the modified UIO-66-NH2 in human cartilage. Compared with not using UIO-66-NH2, the sustained release of solanine from USP can improve bioavailability, reduce systemic drug toxicity, and reduce the frequency of medication;

[0022] (4) Solanine is used as a drug for the treatment of osteoarthritis, UIO-66-NH2 is used as a carrier, and polyethyleneimine has targeting properties, and their combination provides the best therapeutic effect. The excellent anti-ferroptosis ability of solanine significantly alleviates the symptoms of osteoarthritis, and the addition of UIO-66-HN2@PEI achieves better therapeutic effects. More importantly, the results of the present invention reveal the mechanism by which solanine inhibits ferroptosis by inhibiting HIF-1α-mediated glycolysis, thereby alleviating the progression of osteoarthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Figure 3 Inhibitory effect of solanine on ferroptosis of chondrocytes; chondrocytes were divided into control group, LPS group, 2μM group and 4μM group; (A, B) Cell model construction scheme; (C) Molecular structure of solanine; (D) The optimal concentration of solanine was explored by CCK8 kit; (E, F) The expression levels of xCT, GPX4, 4HNE and ACSL4 proteins were detected by Western blot (the number of samples was 3); (G) The accumulation of reactive oxygen species in cells of each group was detected by ROS detection kit, and the ROS level of chondrocytes was observed by confocal microscopy; green indicates the accumulation of ROS on the cell surface (n=3); scale bar, 100μm; (H) Chondrocytes were stained with GPX4-specific antibody, the actin filaments of chondrocytes were stained with FITC labeled with phalloidin, and the nuclei were stained with DAPI; red is GPX4 protein staining, green is actin filaments, blue is cell nuclei, and the staining was observed under confocal microscopy; scale bar, 5 0μm; (I) The cells in each group were stained with the JC-1 kit, and the mitochondrial damage of chondrocytes was observed under a confocal microscope. Aggregates represent normal membrane potential, and monomers represent damaged membrane potential; scale bar, 100μm; (J) Changes in mitochondria of chondrocytes were observed under a transmission electron microscope; scale bar, 200nm; (K, L) Toluidine blue and safranin O staining, 6 cells in each group; scale bar, 100μm; (M) Flow cytometry was used to detect mitochondrial membrane potential damage; (N and O) Lipid peroxidation and glutathione status of each group were detected (N=6); *p<0.05, **p<0.01, p<0.001, ns indicates not significant.

[0024] Figure 2 The inhibitory effect of solanine on chondrocyte glycolysis; chondrocytes were divided into normal group, injury group, low-dose treatment group and high-dose treatment group; (A, B) Cell model construction scheme; (C, D) Western blot method was used to detect the expression levels of HK1, LDHA and GLUT1 proteins (n=3); (E) Chondrocytes were stained with GLUT1-specific antibodies; FITC stained chondrocyte actin filaments with phalloidin, and DAPI stained cell nuclei; red is GLUT1 protein staining, green is actin filaments, and blue is cell nuclei; scale bar, 50μm. (F) ATP detection in each group; (G) Glucose content detection in rats in each group (n=6); *p<0.05, **p<0.01, p<0.001, ns indicates not significant;

[0025] Figure 3 To investigate the regulatory effect of solanine on chondrocyte ferroptosis through glycolysis, chondrocytes were divided into injury group, treatment group and glycolysis agonist group; (A) Unit model construction scheme; (B, C) Western blot was used to detect the expression levels of xCT, GPX4, 4HNE and ACSL4 proteins (sample number: 3); (D) ROS detection kit was used to detect the aggregation of reactive oxygen species in cells of each group, and confocal microscopy was used to observe the ROS level of chondrocytes; green represents the aggregation of ROS on the cell surface; scale bar, 100μm; (E) JC-1 kit was used to stain cells of each group, and confocal microscopy was used to observe the mitochondrial damage of chondrocytes. Aggregates represent normal membrane potential, and monomers represent damaged membrane potential; scale bar, 100μm (F) Flow cytometry was used to detect the mitochondrial membrane potential damage of each group (n=6); *p<0.05, **p<0.01, p<0.001, ns indicates not significant;

[0026] Figure 4Molecular docking, protein attenuation experiments, Western blot and immunofluorescence experiments were used to verify the relationship between α-solanine and HIF-1α in order to investigate the regulatory effect of solanine on glycolysis through HIF-1α. (A) The interaction pattern between solanine and HIF-1α was analyzed using AutoDockTools1.5.7 and PyMOL2.3.0. (B) Molecular docking pocket pattern diagram predicting the potential binding site of solanine in the hydrophobic domain containing hydrogen bond acceptors and donors. (C) Prediction of specific amino acid names and sites of the binding site between solanine and HIF-1α. (D) Water and hydrogen bonds connected to solanine. (EG) Attenuation of HIF-1α with or without the addition of chlorhexidine (n=3). (H and I) After LPS stimulation, chondrocytes were treated with solanine and proteins were extracted. Western blot was used to analyze the interaction pattern between solanine and HIF-1α. Blot method was used to detect HIF-1α protein expression (n=3); (J) Chondrocytes were stained with HIF-1α specific antibody, FITC cartilage actin filaments were stained with phalloidin marker, and cell nuclei were stained with DAPI. Red represents HIF-1α protein, green represents actin filaments, and blue represents cell nuclei. The staining was observed under a confocal microscope; scale bar, 50μm. (K) A cell model of HIF-1α activation was constructed; (L and M) Chondrocytes were divided into injury group, treatment group and HIF-1α agonist group, and the protein expression of HK1, LDHA and GLUT1 was detected (n=3); *p<0.05, **p<0.01, p<0.001, ns indicates not significant;

[0027] Figure 5 Synthesis and characterization of UIO-66-NH2; (A and B) Schematic diagram showing material synthesis and targeting map; I TEM image of UIO-66-NH2; scale bar, 50 nm; (D) Particle size results of UIO-66-NH2; (E) Zeta potential results of USP; (F and G) UIO-66-NH2 Mapping results showing elemental composition; (H) USP release; (1) Transmission electron microscopy imaging to observe the degradation of UIO-66-NH2 in human synovial fluid; scale bar, 200 nm; (J) Schematic diagram of the co-culture scheme of UIO-66-NH2@CY5.5@PEI and human articular cartilage; frozen section fluorescence diagram (K and L) USP retention time, targeting; scale bar, 50 μm; (M) USP penetration ability; scale bar, 20 μm;

[0028] Figure 6The improvement effect of USP on articular cartilage destruction in rats with ACLT model. Rats were divided into sham operation group, ACLT group, solanine group and USP group. (A) Rat ACLT model, treatment plan and subsequent experimental design; (B) HE staining and safranin O staining of rat joints according to grouping, the results are shown in the figure; scale bar, 100μm; (C) Pathological score and synovitis score of rat arthritis model (n=6); (D) In ​​vivo imaging of small animals to prove the sustained release ability of UIO-66-NH2; (E and F) Immunohistochemical staining of GPX4, ACSL4 and GLUT1 of samples from different experimental groups using ImageJ software (n=6); scale bar, 100μm. (G) Micro CT front and top views of the above groups; scale bar, 3mm. (H) Quantitative analysis of BV, BV / TV, Tb.N, Tb.Sp, Tb.Th (n=6). * p<0.05, ** p<0.01, p<0.001, ns means not significant;

[0029] Figure 7 Schematic diagram of cartilage-targeted USP chondrocyte therapy. DETAILED DESCRIPTION

[0030] The technical solution of the present invention is described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] The specific description of experimental materials and methods is as follows:

[0032] 1. Animal research

[0033] Adult male SD rats (200-250 g, n = 24) were purchased from the Animal Center of Anhui Provincial Hospital. All experimental rats were housed in standard experimental cages with a 12-h light / dark cycle and free access to drinking water and standard food. The rats were randomly divided into 4 groups (n = 6): sham group, ACLT group, solanine group and USP group. Six rats underwent sham surgery and 18 rats underwent ACLT. The 18 ACLT rats were randomly divided into ACLT group, solanine group and USP group. The surgical animals were given corresponding treatment starting 1 week after surgery. The rat knee joint specimens were collected 12 weeks later (6 rats in each group).

[0034] 2. Preparations and Antibodies

[0035] HIF-1α, GLUT1, LDHA, HK1, GPX4, ACSL4, 4HNE, and GAPDH antibodies were purchased from Proteintech Group and Abcam. BCA kit, MDA kit, GSH kit, JC-1 kit, ATP kit, glucose kit, and RIPA were purchased from Beyotime. Calcein-AM / PI double staining kit and cell counting kit were purchased from Yeasen. Mitapivat was purchased from Selleck. Fenbendazole-d3 and solanine were purchased from MCE. Acetic acid, NaClO4·H2O, ZrO(NO3)2·2H2O, 2-aminoterephthalic acid, and polyethyleneimine were purchased from Aladdin. Nonionic copolymer surfactant (F127) and LPS were purchased from Sigma.

[0036] 3. Cell culture

[0037] Human chondrocytes (C28) were purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. The cells were cultured in DMEM supplemented with 10% fetal bovine serum at 37°C in an atmosphere of 5% CO2. After the cells reached 80% to 90%, they were digested with trypsin and subcultured twice a week at a split ratio of 1:3.

[0038] 4. Cell viability assessment

[0039] Cell counting kit (CCK8) was used to detect cell viability; chondrocytes were seeded in 96-well plates at approximately 5,000 cells per well; 12 hours later, fresh culture medium containing 0, 1, 2, 3, 4, 5, and 10 μM solanine was added to each well and cultured at 37°C for 24 hours in a humidified incubator containing 5% CO2. The safe concentration of solanine was detected by CCK8 method, and CCK8 reagent was added to each well and incubated at 37°C for 2 hours. The absorbance of each well was checked at 450 nm using an ELISA reader, and the cell survival rate of the control group was 100%.

[0040] 5. Western Blot Analysis

[0041] Proteins were extracted from cells and tissues by radioimmunoprecipitation. Cells were cultured in 6-well plates. When the cells grew to 80%, solanine (2μM and 4μM) was added for pretreatment for 2h, and blank control group, LPS group, fenbendazole-d3 group and Mitapivat group were added; RIPA lysis buffer containing 10% phosphatase inhibitor and 1% PMSF was added, and protein was extracted after 24h. The protein was boiled and denatured, centrifuged, and the supernatant was harvested. The protein concentration was determined by BCA method and balanced. The extract was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), protein loading buffer, and NC membrane transfer; the NC membrane was blocked for half an hour with protein-free rapid blocking solution, and then different antibodies with different dilutions were stored in a 4℃ refrigerator overnight with the NC membrane. After washing with TBST three times, it was incubated with the corresponding secondary antibody for 1h. Finally, the band signal was detected on the imager using ECL luminescent solution. GAPDH was used as a control, and the protein expression was calculated by ImageJ. Western blotting was used to detect the protein expression of HIF-1α (1:2000, 66630-1-lg, Wuhan Tri-Eagle Biotechnology Co., Ltd.), GLUT1 (1:2000, 21829-1-AP, Wuhan Tri-Eagle Biotechnology Co., Ltd.), LDHA (1:5000, 21799-1-AP, Wuhan Tri-Eagle Biotechnology Co., Ltd.), HK1 (1:2000, 19662-1-AP, Wuhan Tri-Eagle Biotechnology Co., Ltd.), GPX4 (1:1000, AB125066, Abcam), ACSL4 (1:2000, 22401-1-AP, Wuhan Tri-Eagle Biotechnology Co., Ltd.), 4HNE (1:2000, AB46545, Abcam) and GAPDH (1:2000, 60004-1-lg, Wuhan Tri-Eagle Biotechnology Co., Ltd.). All experiments were repeated 3 times.

[0042] 6. Immunofluorescence

[0043] The treated cells were washed with pre-cooled PBS, fixed with pre-cooled 4% paraformaldehyde on ice for 15 minutes, washed with PBS three times, and perforated with immunostaining permeabilization solution Triton X-100 at room temperature for 10 minutes. Then the cells were blocked with goat serum blocking solution for 1 hour, washed with PBS three times, and incubated with the corresponding specific primary antibody overnight; after washing three times, the corresponding secondary antibody was incubated at room temperature in the dark for 1 hour; 4,6-diamidino-2-phenylindole (DAPI) staining solution was added to mark the cell nucleus for 5 minutes, and anti-fluorescence quenching fixative was added as the final drop; the images were observed and acquired under a confocal microscope.

[0044] 7. RNA Sequencing

[0045] To improve data accuracy, chondrocytes were divided into LPS group and LPS + solanine group, with 3 replicates in each group. Total RNA was extracted using TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer's operating procedures. RNA purity and quantification were assessed using NanoDrop2000 spectrophotometer (Thermo Scientific, USA). RNA integrity was assessed using Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The library was constructed using VAHTSUniversal V6 RNA-seq Library Prep Kit. Transcriptome sequencing and analysis were completed by OE Biotechnology Co., Ltd. (Shanghai, China). After quantification, the count file was entered into the R (version 4.0.2) software package DEseq2, and differentially expressed genes were analyzed based on the negative binomial distribution model. The P value was corrected for accuracy using the Benjamini and Hochberg method. Differentially expressed genes were screened for analysis.

[0046] 8. Detection of active oxygen

[0047] Use the reactive oxygen species (ROS) detection kit according to the manufacturer's instructions. 5 Chondrocytes were inoculated into 6-well plates at a density of cells / well. They were divided into a control group, an LPS group and two drug groups with different concentrations. After 24 hours. The drug groups were pretreated with 2μM and 4μM solanine for 2h, respectively, and the supernatant was removed. Except for the control group, 10μM LPS was added to all other groups. The DCFH-DA fluorescent probe was used to detect the levels of intracellular ROS and lipid ROS, and Rosup was used as a positive control. That is, the chondrocytes were washed 3 times with PBS, and then treated with 10μM DCFH-DA at 37℃ in the dark for 20min; after the culture, the cells were washed with PBS and observed under a confocal microscope.

[0048] 9. Detection of glutathione (GSH) and malondialdehyde (MDA)

[0049] The working solution was prepared according to the instructions. After washing the cells once with PBS, the cells were collected by centrifugation and the supernatant was aspirated. The protein removal reagent M solution was added at 3 times the volume of the cell pellet and vortexed thoroughly. The sample was then subjected to two rapid freeze-thaw cycles using liquid nitrogen and a 37°C water bath. The cells were placed at 4°C or in an ice bath for 5 min. The sample was centrifuged at 10,000 g for 10 min at 4°C, and the supernatant was used for the determination of total glutathione.

[0050] Malondialdehyde (MDA) content was determined using a lipid peroxidation MDA assay kit according to the manufacturer's instructions on ice. The samples were added to the cell lysate and collected, centrifuged at 10000-12000 g for 10 min at 4°C, and the supernatant was collected. 200 μL of MDA solution was added to each sample or standard (0.1 mL), heated at 100°C for 15 min, centrifuged at 1000 g for 10 min, and the supernatant was collected. 200 μL of the supernatant was taken and quantified colorimetrically on a microplate colorimeter at OD = 532 nm. The MDA level is expressed in μM.

[0051] 10. Measurement of mitochondrial membrane potential

[0052] Remove the culture medium from the 6-well plate, wash once with PBS, add 1 ml of cell culture medium, add 1 ml of JC-1 staining working solution and mix well, and incubate the cells in a 37°C cell culture incubator for 20 min. During the incubation period, prepare JC-1 buffer according to the manufacturer's instructions and place it in an ice bath. At the end of the 37°C incubation, remove the supernatant by aspiration, wash twice with JC-1 staining buffer, add 2 ml of cell culture medium, and observe under a confocal microscope.

[0053] 11. Flow cytometry analysis

[0054] The JC-1 kit was used to detect mitochondrial membrane potential. The resuspended cells in the culture medium were reacted with the JC-1 working solution at 37°C for 20 min. After incubation, the cells were washed with buffer and the detection results were analyzed by flow cytometry using FlowJo V10.

[0055] 12. ATP detection

[0056] The ATP detection kit was used according to the manufacturer's instructions, and the results were read using a multifunctional microplate reader. The 6-well plate was placed on ice, and 200 μL of cell lysis solution was added to each well of the plate to lyse the cells. The supernatant after lysis was centrifuged at 12,000 × g at 4°C for 5 min. The culture medium was removed by aspiration, and 200 μL of lysate was added to each well of the 6-well plate in proportion. The supernatant after lysis was removed by centrifugation at 12,000 g at 4°C for 5 min for subsequent detection. 100 μL of ATP detection working solution was added to the detection well and allowed to stand at room temperature for 3-5 min; 20 μL of sample was added to the detection well and mixed quickly with a micropipette. After an interval of 2 seconds, the RLU value was measured using a chemiluminometer, and the results were plotted using Graphpad Prism 9.

[0057] 13. Molecular docking

[0058] After downloading the 3D data of solanine and HIF-1α from the PDB database, the AutoDockTools1.5.7 software was used to dehydrate and hydrogenate HIF-1α, and then the data of solanine was imported. The merged data of solanine and HIF-1α were obtained by calculation and saved. The format was converted using Open Babel, and then the format was opened and analyzed using PyMOL2.3.0 software to find out the names of specific amino acids and the size of the docking distance.

[0059] 14. Protein decay test

[0060] Western blot was used to detect the expression of HIF-1α. The cells were inoculated in a 6-well plate and divided into two groups when the cells grew to 80%. Both groups were stimulated with LPS for 24 h, one group was added with cycloheximide (CHX), and the other group was not added with any drugs. After 12 h, the collected samples were processed according to the Western blot analysis method. Finally, the band signal was detected on a visualization instrument using an ECL luminescent solution.

[0061] 15. CT

[0062] After fixation of rat knee joints with 4% paraformaldehyde, the knee joint specimens were scanned by Micro-CT. The scanning resolution was 10μM, the voltage was 55V, and the current was 114μA. The three-dimensional structural parameters of the tibial plateau subchondral bone were evaluated.

[0063] 16. Transmission Electron Microscope

[0064] The treated cells were collected and fixed with 2.5% glutaraldehyde in 0.1M phosphate buffer at 4°C overnight. After washing the samples with phosphate buffer, they were fixed with phosphate buffer containing 1% OsO4, soaked at 4°C for 2h, and rinsed thoroughly with DD water. The whole block was stained with 2% uranyl acetate aqueous solution for 2h, dehydrated with 50%, 70%, 90%, 100% ethanol and 100% acetone in sequence, and embedded in epoxy resin to make a sample block. The sections were sliced ​​with an ultramicrotome, stained with lead citrate and uranyl acetate, and finally observed by electron microscopy.

[0065] 17. Immunohistochemistry

[0066] Bone and muscle tissues were separated, fixed with 10% formalin, then decalcified with 10% EDTA for 4 weeks and dehydrated with partial alcohol. Embedded in paraffin and cut into 5μm sections. Three sections were analyzed for each rat, all from the medial femoral condyle. Antigen retrieval was performed using the e-enzyme method, and endogenous peroxidase activity and nonspecific binding sites were blocked with H2O2 (3%) and BSA (5%), respectively. The primary antibody was incubated overnight at 4°C. Then the appropriate enzyme-labeled secondary antibody was added to the section for incubation, incubated at room temperature for 20 minutes, and DAB color development solution was added. Finally, the sections were re-stained, dehydrated, transparent and sealed.

[0067] The embodiments of the present invention will be described in detail below in conjunction with examples. The following examples take the best examples as illustrations, but those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. The specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0068] Example

[0069] 50 mg of F127 was dissolved in 3 mL of deionized water, and then 0.4 mL of acetic acid and 150 mg of NaClO4·H2O (1.06 mmol) were added and stirred to form a homogeneous solution; ZrO(NO3)2·2H2O (115.6 mg, 0.5 mmol) and 2-aminoterephthalic acid (50 mg, 0.28 mmol) were added to the homogeneous solution and stirred at 40°C for 12 hours. A precipitate was obtained by centrifugation at 10000 rpm. The obtained precipitate was washed once with water and once with ethanol, and then resuspended and stored to obtain UIO-66-NH2.

[0070] 10 mg UIO-66-NH2 was mixed with 5 mL DI-water and 4 mg solanine and stirred at room temperature for 24 h to obtain solution A; 200 mg polyethyleneimine and 20 mL DI-water were prepared into a 10 mg / ml polyethyleneimine aqueous solution and stirred at room temperature for 30 min to obtain solution B. 7 mL of solution B was added to solution A that had been stirred for 24 h and stirred at room temperature for another 24 h; the obtained product was washed twice by centrifugation with DI-water and stored in PBS until use to obtain USP.

[0071] The USP was observed and photographed by electron microscope to detect the elemental composition of UIO-66-NH2. The particle size and zeta potential of the material were also tested.

[0072] The drug-loaded materials were mixed in an environment of pH = 6.6, and samples were collected every 24 hours until 168 hours. The samples obtained at different times were centrifuged, the supernatant was removed, and the samples were detected by a spectrophotometer (Shimadu-LC20A), and the UV wavelength was detected at 214 nm.

[0073] UIO-66-NH2 and USP were used to detect the effects of different concentrations of MOF on chondrocyte viability and the toxic effects of MOF on chondrocytes. Each group was treated with different concentrations of UIO-66-NH2 and USP for 12 hours; live and dead staining solutions were prepared according to the instructions and added to 24-well plates; after incubation at 37°C in the dark for 15 minutes, the cells were divided into two groups. Images were observed using an inverted fluorescence microscope.

[0074] Test example:

[0075] 1. Inhibitory effect of solanine on chondrocyte ferroptosis

[0076] In order to verify the effect of solanine on ferroptosis, the present invention plans to construct a chondrocyte ferroptosis model to test the effect of solanine on chondrocytes ( Figure 1 ,A,B). The chemical structure of solanine is as follows Figure 1 C. In order to evaluate the toxic effect of solanine on chondrocytes, the present invention treated chondrocytes with different concentrations of solanine (1, 2, 3, 4, 5 and 10 μM) for 12 hours ( Figure 1 D). Cell live / dead staining confirmed that solanine significantly reduced cell viability and produced significant cytotoxicity, while solanine at a concentration of 1-4 μM had no cytotoxic effect on chondrocytes. Therefore, safe concentrations of 2 μM and 4 μM solanine were selected for subsequent experiments. Western blot detected the expression of GPX4, xCT, ACSL4 and 4HNE in the LPS-induced osteoarthritis ferroptosis model. After the addition of LPS, GPX4 and xCT were significantly decreased, and ACSL4 and 4HNE were significantly increased. Solanine pretreatment reduced the downregulation of GPX4 and xCT and the upregulation of ACSL4 and 4HNE caused by LPS stimulation in a dose-dependent manner ( Figure 1 , E and F). The present invention further detected the chondrocyte railway death group using ROS. After LPS stimulation, the accumulation of ROS in chondrocytes increased significantly. After treatment with different concentrations of solanine, the ROS accumulation conditions of chondrocytes were improved to varying degrees ( Figure 1 G). Immunofluorescence showed that the expression of GPX4 was significantly decreased after LPS stimulation, while the expression of GPX4 was significantly increased after the addition of α-gentianine ( Figure 1 H). The present invention also detected the degree of mitochondrial damage. Confocal microscopy showed that after adding solanine, the mitochondrial damage was alleviated and the number of normal mitochondria increased ( Figure 1I). Under transmission electron microscopy, the mitochondria in the LPS group were obviously condensed, the color became darker, and the mitochondrial cristae were wrinkled, which were typical characteristics of ferroptosis. After adding solanine, the mitochondria did not condense, the color became lighter, and the mitochondrial cristae returned to normal shape ( Figure 1 J). Toluidine blue and GAGs staining also showed that solanine can effectively reduce the damage of chondrocytes ( Figure 1 , K, L. In addition, the present invention also detected the release of MDA and glutathione (GSH). The release of GSH was significantly reduced in the LPS group, and the release of GSH was improved after the addition of solanine ( Figure 1 M), the results of MDA are opposite to those of GSH ( Figure 1 N). Based on the control group, Q2 was defined as the main group of undamaged cells, and based on the LPS group, Q3 was defined as the damaged area. After treatment with different concentrations of solanine, the main cell populations underwent significant changes. Low concentrations of solanine improved mitochondrial damage, while high concentrations of solanine had a more significant repair effect on mitochondrial damage ( Figure 1 O).

[0077] 2. Inhibitory effect of solanine on chondrocyte glycolysis

[0078] Models in which solanine alleviates excessive glycolysis in chondrocytes Figure 2 , as shown in A, B. Western blot was used to detect the expression of glycolysis-related pathway proteins HK1, LDHA, and GLUT1 to verify the relationship between solanine and energy metabolism. The results showed that the expression of HK1, LDHA, and GLUT1 increased after the addition of LPS. However, with the increase in the concentration of solanine, the expression of HK1, LDHA, and GLUT1 was significantly downregulated ( Figure 2 , C, D). Immunofluorescence staining further verified the role of solanine in glycolysis. The protein expression of GLUT1 was detected, and the results showed that solanine treatment reduced the overexpression of GLUT1 in the LPS group induced by LPS, and the inhibitory effect was more obvious when pretreated with 4μM solanine ( Figure 2 E). In addition, ATP and glucose released from chondrocytes were measured, and the results were consistent with the fluorescence results ( Figure 2 , F and G); these data indicate that solanine has an antiglycolytic effect on human chondrocytes.

[0079] 3. Relationship between glycolysis and ferroptosis

[0080] The glycolysis activator Mitapivat was added to the treatment group to test whether ferroptosis was activated by activating glycolysis ( Figure 3A), in order to verify the relationship between glycolysis and ferroptosis. Western blot was used to detect the expression of GPX4, xCT, ACSL4 and 4HNE proteins. Compared with the LPS group, the expression of GPX4 and xCT was significantly decreased, and the expression of ACSL4 and 4HNE was significantly increased after Mitapivat treatment ( Figure 3 , B, C). Western blot analysis showed that Mitapivat activated glycolysis, while glycolysis was effectively inhibited by α-gentianine. By detecting ROS, after the addition of Mitapivat, the treated chondrocytes expressed damage signals again ( Figure 3 D). The results of JC-1 detection were consistent with ROS. After adding Mitapivat, the mitochondria of the treated chondrocytes also expressed damage signals ( Figure 3 E), and JC-1 flow cytometry also confirmed this ( Figure 3 F) These data suggest that solanine inhibits ferroptosis by inhibiting glycolysis.

[0081] 4. Regulation of glycolysis by solanine via HIF-1α

[0082] In order to clarify the interaction between solanine and HIF-1α, a multi-step method was used to study the affinity between solanine and HIF-1α. Docking calculations showed that solanine had a strong affinity for HIF-1α. The three-dimensional structural data of solanine and HIF-1α were downloaded from the PDB database, and a matching model of solanine and HIF-1α was established ( Figure 4 A). Based on the structural changes of HIF-1α, solanine has a potential binding site in the hydrophobic domain containing hydrogen bond acceptors and donors ( Figure 4 B). Specifically, the binding sites of α-solanine docking are residues LYS297, GLN421, and VAL422 on HIF-1α, which are eventually extended ( Figure 4 C). 3D schematic showing hydrogen bonding between the docked protein and solanine ( Figure 4 D). Taken together, the results suggest that the high affinity between solanine and HIF-1α depends on their complex non-covalent interactions.

[0083] Based on the docking results, a protein decay experiment was conducted, and solanine was added at the same time as CHX. The results showed that after adding solanine, the degree of HIF-1α inhibition was significantly increased ( Figure 4 ,E~G), indicating that solanine was negatively correlated with HIF-1α. Western blot was used to detect the expression of HIF-1α. Compared with the control group, solanine significantly downregulated the expression of HIF-1α in the LPS group ( Figure 4,H and I). Immunofluorescence staining of HIF-1α showed that compared with the control group, α-gentianine treatment reduced the fluorescence intensity of LPS-induced HIF-1α ( Figure 4 J).

[0084] In addition, a HIF-1α activated glycolytic cell model was constructed using fenbendazole-d3 as an activator ( Figure 4 K). The expression of HK1, LDHA, and GLUT1 was detected by Western blot. Compared with the solanine group, the expression of HK1, LDHA, and GLUT1 increased significantly after fenbendazole-d3 treatment ( Figure 4 , L, M). Western blot analysis showed that solanine could effectively inhibit HIF-1α, and fenbendazole-d3 successfully activated the expression of HIF-1α-related glycolytic proteins HK1, LDHA, and GLUT1. These data indicate that solanine can inhibit ferroptosis through the HIF-1α pathway.

[0085] 5. Characterization of UIO-66-NH2

[0086] In order to obtain a good drug-carrying structure, the present invention uses UIO-66-NH2 with good porosity, strong drug-carrying capacity and non-degradable as a drug-carrying carrier. In order to achieve targeted drug delivery, the present invention uses positively charged and non-toxic polyethyleneimine to encapsulate the drug-carrying UIO-66-NH2 ( Figure 5 A). USP targets the negative charges on the surface of articular cartilage ( Figure 5 B). The TEM image of USP, UIO-66-NH2 shows a spherical structure with many pores, which provides a good basis for drug loading ( Figure 5 C). The particle size of UIO-66-NH2 is about 50nm ( Figure 5 D). The results of ZETA potential detection showed that USP was positively charged after mixing with polyethyleneimine ( Figure 5 E). The mapping element analysis results show that the element composition of UIO-66-NH2 is C, N, O and Zr ( Figure 5 , F and G). The present invention tests the drug release characteristics of USP in an acidic environment (pH = 6.6). The sustained release performance ensures continuous administration ( Figure 5 H). The degradation and targeting capabilities of UIO-66-NH2 were verified in human samples. UIO-66-NH2 was mixed with synovial fluid from osteoarthritis patients and was still detectable for at least 14 days ( Figure 5I), indicating that UIO-66-NH2 may be stable in human knee joints. UIO-66-NH2@CY5.5@PEI and articular cartilage of patients were co-cultured. Next, the present invention made rapid frozen sections and detected the corresponding fluorescence signal bands. The present invention found that there was still a weak CY5.5 signal in the cartilage after 14 days ( Figure 5 K). At the same time, CY5.5 aggregated on the surface of chondrocytes, indicating that UIO-66-NH2@CY5.5@PEI has cartilage targeting properties through charge response ( Figure 5 L). In addition, the present invention found a CY5.5 signal band at 500 μM below the surface of cartilage ( Figure 5 M). The above results indicate that UIO-66-NH2@CY5.5@PEI has the ability to penetrate cartilage.

[0087] 6. USP reduces articular cartilage destruction in ACLT model rats

[0088] In order to verify the effect of USP on the osteoarthritis model in vivo, the present invention uses the rat ACLT model to evaluate the therapeutic effect of solanine. The rats were divided into sham operation group, ACLT group, solanine group and material group ( Figure 6 A). Hematoxylin-eosin (HE) staining and safranin staining showed that the joint surface of the rats in the sham operation group was smooth and the cartilage layer was thicker, while the joint surface of the rats in the ACLT group was no longer smooth, the cartilage layer was significantly thinner, the proteoglycan was significantly lost, and the cartilage damage was aggravated. Adding solanine can improve this situation. However, compared with the direct addition of solanine, the treatment with USP added had a better effect, with a smoother surface and a thicker cartilage layer ( Figure 6 B). The osteoarthritis RSI score and synovitis score were consistent with the pathological results, indicating that solanine was effective in treating the ACLT rat model. There was a significant difference in the efficacy of solanine and USP treatment. The USP group showed the best therapeutic effect, and the USP group could significantly reduce the osteoarthritis score and synovitis score compared with the α-gentianine group and the osteoarthritis group ( Figure 6 C). In vivo imaging of small animals showed that compared with simple injection of CY5.5, the addition of UIO-66-NH2 had a better sustained release effect. The retention time was longer and the fluorescence existed near the joints for a longer time. ( Figure 6 d). The results of immunohistochemical staining also showed that the surface of cartilage was smooth after USP treatment, the expression of ACSL4 and GLUT1 decreased compared with the ACLT group, and the increase of GPX4 was consistent with the in vitro results ( Figure 6 , E, F).

[0089] Micro CT results showed that after three-dimensional reconstruction, compared with the USP group and α-gentianine group, the number of osteophytes in the ACLT group increased significantly and hardened, the joint platform structure became dense, and the loose porous structure disappeared. After solanine and USP treatment, the degree of subchondral bone hardening was significantly improved, and the loose structure was restored ( Figure 6 G). BV, BV / TV, Tb.N, Tb.Sp, Tb.Th showed that solanine and USP inhibited bone destruction in vivo ( Figure 6 H).

[0090] like Figure 7 As shown, the schematic diagram of cartilage-targeted USP chondrocyte therapy, chondrocytes can be treated by targeting chondrocytes with MOF loaded with solanine, and solanine can regulate glycolysis and ferroptosis through HIF-1α, thereby treating osteoarthritis cartilage.

[0091] The present invention first clarifies the role of solanine in osteoarthritis. In the present invention, the present invention explores the mechanism by which solanine alleviates the progression of osteoarthritis and proves that solanine improves the progression of chondrocyte ferroptosis by alleviating glycolysis. In addition, the present invention constructs a positively charged MOF material USP loaded with solanine to target chondrocytes and achieves progress in alleviating chondrocyte ferroptosis in rat ACLT model ( Figure 7 ).

[0092] Finally, it should be noted that the above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Those skilled in the art should understand that the present invention can still be modified or replaced by equivalents. All technical solutions and improvements that do not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. Application of solanine encapsulated by a MOF material in the preparation of a drug for preventing and treating osteoarthritis.

2. The use according to claim 1, characterized in that: The osteoarthritis is an inflammation of the cartilage cells in the joints.

3. The use according to claim 1, characterized in that: The articular chondrocyte inflammation is induced by LPS.

4. The use according to claim 1, characterized in that: The solanine encapsulated by the MOF material is used to regulate the expression of HIF-1α and / or GLUT1, key factors of sugar metabolism.

5. The use according to claim 1, characterized in that: The medicine includes tablets, capsules, pills, granules, oral liquids and solutions.

6. The use according to claim 1, characterized in that: The MOF material is UIO-66-NH2.

7. A method for preparing solanine encapsulated by MOF material, characterized in that: The preparation steps of the MOF material encapsulated solanine include: mixing UIO-66-NH2 with DI-water and solanine and stirring to obtain solution A; preparing a polyethyleneimine aqueous solution as solution B; adding solution B to solution A, and continuing to stir to obtain MOF material encapsulated solanine USP.

8. The method for preparing solanine encapsulated by MOF material according to claim 7, characterized in that: The usage ratio of UIO-66-NH2, DI-water, solanine and polyethyleneimine is (8-12) mg: (4-6) mL: (3-5) mg: (180-220) mg.

9. The method for preparing solanine encapsulated by MOF material according to claim 7, characterized in that: The preparation method of UIO-66-NH2 includes: dissolving F127 in deionized water, adding acetic acid and NaClO4·H2O; stirring thoroughly to form a homogeneous solution; adding ZrO(NO3)2·2H2O and 2-aminoterephthalic acid to the homogeneous solution, stirring, centrifuging to obtain a precipitate, and washing to obtain UIO-66-NH2.

10. The method for preparing solanine encapsulated by MOF material according to claim 9, characterized in that: The usage ratio of the F127, deionized water, acetic acid, NaClO4·H2O, ZrO(NO3)2·2H2O and 2-aminoterephthalic acid is (45-55) mg: (2-4) mL: (0.3-0.5) mL: (140-160) mg: (110-120) mg: (45-55) mg.

Images