Application of catalpol in preparation of periprosthetic osteolysis inhibitor

Abstract

The invention relates to the field of biological medicine, in particular to application of catalpol in preparation of a periprosthetic osteolysis inhibitor, which is helpful for inhibiting aseptic loosening of a prosthesis, can remarkably reduce the occurrence rate of joint replacement postoperative complications and realizes more effective clinical intervention, thereby accelerating rehabilitation of a patient and improving the daily life quality of the patient.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to the application of catalpol in the preparation of periprosthetic osteolysis inhibitors. Background Art

[0002] Total joint replacement refers to the use of materials such as metal, high molecular polyethylene, and ceramics to make artificial joint prostheses according to the shape, structure, and function of human joints, and implant them into the human body through surgical techniques to replace the function of diseased joints, so as to relieve joint pain and restore joint function. Total joint replacement has good therapeutic effects and can effectively restore joint function and improve the quality of life of patients. However, as an organ substitute, artificial joints inevitably have maintenance problems. At present, periprosthetic osteolysis (PPO) is considered to be one of the main reasons for aseptic loosening of prostheses and joint revision after total joint replacement (TJA).

[0003] Although the pathogenic mechanism of PPO is not yet clear, many scholars believe that after total joint replacement, long-term movement of the joint will cause wear or corrosion of the contact surface, generating nanoparticles such as titanium (Ti), chromium (Cr), cobalt (Co), ceramics, and polyethylene, which accumulate at the implant-bone contact surface and induce PPO. In order to reduce the problems of aseptic loosening of prostheses and joint revision after TJA and extend the maintenance period, it is necessary to develop a drug that can inhibit PPO. Summary of the Invention

[0004] The purpose of the present invention is to provide a substance that can effectively inhibit PPO.

[0005] To achieve the above purpose, the present invention provides the following technical solutions:

[0006] The application of catalpol in the preparation of periprosthetic osteolysis inhibitors.

[0007] Optionally, the catalpol is used to inhibit ferroptosis of osteoblasts induced by titanium particles.

[0008] Optionally, the catalpol is used to inhibit ferroptosis of osteoblasts induced by the ferroptosis inducer erastin.

[0009] Optionally, the catalpol inhibits the decline of osteogenic differentiation ability of bone marrow mesenchymal stem cells.

[0010] Optionally, the catalpol inhibits the decline of osteogenic mineralization ability of bone marrow mesenchymal stem cells induced by titanium particles.

[0011] Optionally, the catalpol exerts a therapeutic effect by activating the Nrf2 / HO-1 signaling pathway.

[0012] Optionally, the periprosthetic osteolysis is the periprosthetic osteolysis that occurs after artificial joint replacement.

[0013] Optionally, the inhibitor is administered by intraperitoneal injection.

[0014] Optionally, the content of catalpol in the inhibitor is any value in the range of 10 mg / kg to 30 mg / kg.

[0015] The beneficial effect of the present invention lies in providing an effective ingredient catalpol for periprosthetic osteolysis and its application in the preparation of inhibitors, which helps to inhibit aseptic loosening of the prosthesis, can significantly reduce the incidence of complications after joint replacement, and achieve more effective clinical intervention, thereby accelerating the recovery of patients and improving the quality of their daily life.

[0016] The above description is only an overview of the technical solution of the present invention. In order to be able to understand the technical means of the present invention more clearly and implement it in accordance with the content of the specification, the following will be described in detail with reference to the preferred embodiments of the present invention and the accompanying drawings. Brief Description of the Drawings

[0017] Figure 1 It is a micro-CT reconstruction image and quantitative analysis chart of a mouse skull sample in Example 1 of the present invention;

[0018] Figure 2 It is a histological staining and quantitative analysis chart of a mouse skull section in Example 1 of the present invention;

[0019] Figure 3 It is an H&E staining chart of a mouse visceral sample in Example 1 of the present invention;

[0020] Figure 4 It is an experimental result chart for verifying the effects of Ti particle and catalpol treatment on the viability and osteogenic ability of rat bone marrow mesenchymal stem cells in Example 1 of the present invention;

[0021] Figure 5 It is a staining chart and quantitative analysis chart for verifying the induction effect of cell Ti particles and the treatment effect of catalpol in Example 1 of the present invention;

[0022] Figure 6 It is an RNA sequence analysis result chart in Example 1 of the present invention;

[0023] Figure 7 It is an experimental result chart for verifying that the cell death induced by Ti particles is ferroptosis in Example 1 of the present invention;

[0024] Figure 8 It is an experimental result chart for verifying the improvement of erastin-induced cell ferroptosis by catalpol in Example 1 of the present invention;

[0025] Figure 9 This is the experimental result graph for verifying the effects of Ti particle induction and catalpol treatment on the expression of cell antioxidant stress-related factors in Example 1 of the present invention;

[0026] Figure 10 This is the experimental result graph for verifying the effects of Ti particle induction and catalpol treatment on the intracellular distribution of Nrf2 in Example 1 of the present invention;

[0027] Figure 11 This is the experimental result graph for verifying the effects of Ti particle induction and catalpol treatment on the corresponding mRNA expression of cell antioxidant stress-related factors in Example 1 of the present invention;

[0028] Figure 12 This is the experimental result graph for verifying the effects of knocking down the intracellular expression of Nrf2 on the expression of antioxidant stress-related factors after catalpol treatment in Example 1 of the present invention;

[0029] Figure 13 This is the experimental result graph for verifying the effects of knocking down the intracellular expression of Nrf2 on the expression of osteogenesis-related factors after catalpol treatment in Example 1 of the present invention;

[0030] Figure 14 This is the experimental result graph for verifying the effects of knocking down the intracellular expression of Nrf2 on the cell differentiation and mineralization ability after catalpol treatment by tissue staining in Example 1 of the present invention;

[0031] Figure 15 This is the experimental result graph for verifying the effects of knocking down the intracellular expression of Nrf2 on the cell differentiation and mineralization ability after catalpol treatment by immunofluorescence staining in Example 1 of the present invention;

[0032] Figure 16 This is the experimental result graph for verifying the effects of knocking down the intracellular expression of Nrf2 on the expression of antioxidant stress-related factors after catalpol treatment in Example 1 of the present invention. Detailed implementation manners

[0033] Next, the technical solutions of the present invention will be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0034] In the description of the present invention, it should be noted that the orientation or positional relationship indicated by the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. is based on the orientation or positional relationship shown in the drawings. It 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 operated in a specific orientation, and thus should not be construed as a limitation to the present invention. In addition, the terms "first", "second", "third" are only used for descriptive purposes and should not be construed as indicating or implying relative importance.

[0035] In the description of the present invention, it should be noted that unless otherwise clearly specified and defined, the terms "installed", "connected", "coupled" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, and it can be the communication inside two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0036] In addition, the technical features involved in different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0037] Studies have shown that catalpol can enhance the osteogenic differentiation ability of bone marrow mesenchymal stem cells in a rat cranial defect model, reduce bone loss in an ovariectomized osteoporosis rat model, promote bone formation, and treat postmenopausal osteoporosis. In addition, catalpol can affect autophagy. Based on these research results, the inventors studied the effect of catalpol on PPO.

[0038] Please refer to the following embodiments for details.

[0039] Example 1:

[0040] A total of 40 six-week-old male C57BL / 6J mice with an average body weight of 20 g ± 2 g were selected for the animal experiment. All animal experiment procedures were authorized by the Animal Ethics Committee of the Affiliated Changshu Hospital (Changshu First People's Hospital) affiliated with Soochow University, and the operations were strictly carried out in accordance with the "Guide for the Care and Use of Laboratory Animals". The mice were randomly divided into 4 groups: Control group, Ti group, Ti+L-CA group, and Ti+H-CA group. Except for the Control group, the mice in the other three groups were subjected to Ti nanoparticle implantation surgery. The operation steps were as follows: After anesthesia with 0.4 mL of 1.25% tribromoethanol by intraperitoneal injection in the mice, the scalp hair was shaved and disinfected with iodophor. A 10-mm midline sagittal incision was made, and the periosteum on the surface of the skull was thoroughly scraped to separate the periosteum from the skull. 40 μl of a suspension of titanium nanoparticles in phosphate buffered saline (PBS) solution with a concentration of 1 g / ml was aspirated and evenly applied on the surface of the skull, and finally the incision was sutured. The mice in the Control group underwent sham surgery, and the surgical steps were the same as those of the Ti nanoparticle implantation surgery except that the titanium nanoparticle suspension was not applied. After the surgery, the mice in the Ti+L-CA group were intraperitoneally injected with catalpol solution at a concentration of 10 mg / kg every day, and the mice in the Ti+H-CA group were intraperitoneally injected with catalpol solution at a concentration of 30 mg / kg every day. The mice in the Control group and the Ti group were intraperitoneally injected with normal saline every day for two weeks. All the mice were euthanized two weeks after the surgery, and then all the fresh skull specimens were collected and fixed with 4% paraformaldehyde. Subsequent micro-computed tomography scanning and histological analysis were performed.

[0041] After fixation with 4% paraformaldehyde for 48 h, we used a high-resolution micro-CT (SkyScan1176, Belgium) to analyze the microscopic structure of the skull at an isometric resolution of 9 μm, an X-ray voltage of 50 kV, and a current of 200 μA. Before scanning, the residual Ti nanoparticles on the surface of the skull were gently wiped off with a gauze to reduce the impact of metal artifacts on subsequent analysis. Please refer to Figure 1 , the NRecon software (Skyscan micro-CT, Aartselaar, Belgium) was used to perform two-dimensional (2D) and three-dimensional (3D) reconstructions and local magnifications of the acquired images, and a circle with a diameter of 4 mm was selected near the sagittal suture as the region of interest (ROI), and the Skyscan software was used to analyze the bone morphology parameters, namely bone volume / total volume (BV / TV, %), bone mineral density (BMD, g / mm 3) The trabecular bone number (Tb.N, 1 / mm), total porosity (Total Porosity, %), and trabecular bone separation (Tb.Sp, mm) were used to draw box plots. The data in the figures are presented as the mean within the group. When compared with the Ti group, * is marked when P < 0.05, ** is marked when P < 0.01, and ns is marked when there is no statistical significance. It can be seen from the figure that catalpol treatment can significantly reduce the bone erosion caused by titanium particles and has a concentration-dependent effect. The high concentration has a significantly better treatment effect than the low concentration. After catalpol treatment, the decrease in bone volume / total volume, bone density, and trabecular bone number caused by titanium particles, as well as the increase in total porosity and trabecular bone separation, were significantly inhibited, demonstrating that catalpol can have a therapeutic effect on the deterioration of bone microstructure caused by titanium particles.

[0042] To further verify the attenuation of osteolysis after catalpol treatment, histological analysis of cranial bone sections was subsequently performed. Decalcification was carried out with 10% ethylenediaminetetraacetic acid (EDTA, Sigma) for 3 weeks, and then 6 cranial bone samples in each group were embedded in liquid paraffin. The paraffin-embedded cranial bone samples were cut into 6-μm-thick slices. Subsequently, hematoxylin-eosin (H&E) staining and Masson's trichrome (Masson) staining were performed according to the instructions of the kit, and images were captured by an EVOS M7000 microscope. H&E staining was used to study the morphological changes of tissues, and the ratio of eroded bone surface to total bone surface (EBS / BS) and bone thickness (Bone Thickness, BT) were measured using Image J. Masson staining was used to evaluate the osteogenicity of new immature collagen. The new immature collagen in the blue area represents new bone formation, and the collagen volume fraction was measured using Image J. Please refer to Figure 2 , it can be seen that the newly formed immature collagen fibers after catalpol treatment were significantly increased compared with the titanium particle group, and the treatment effect was significantly improved with the increase in dose.

[0043] The hearts, livers, spleens, lungs, and kidneys of four groups of mice were taken for H&E staining. Please refer to Figure 3 , it can be seen that there was no damage to the hearts, livers, spleens, lungs, and kidneys of all mice, which demonstrated that catalpol has no obvious toxicity to visceral organs.

[0044] Rat bone marrow mesenchymal stem cells (BMSCs) were taken, and the cytotoxic effects of catalpol and titanium particles on BMSC cells were determined by CCK-8 cell viability assay. Different concentrations of catalpol and titanium particles were added respectively, and cell incubation was carried out. After 1 day, 3 days and 5 days of incubation respectively, the absorbance (OD Value) of the culture medium at a wavelength of 450 nm was detected, and the proliferation rate (inhibitionrate) was calculated to judge whether the cell viability was affected. The BMSC cells were induced to differentiate with osteogenic medium. The cells without special treatment were marked as the Control group, the cells added with 10 μg / cm 2 of titanium particles were marked as the Ti group, the cells added with titanium particles and containing 10 μM of catalpol were marked as the Ti+L-CA group, and the cells added with titanium particles and containing 30 μM of catalpol were marked as the Ti+H-CA group, and they were co-cultured for 5 days. In each cell experiment of this example, the doses of low-dose catalpol and high-dose catalpol were the same as those in this experiment. The contents of osteogenesis-related marker proteins Runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) were detected by Western Blot (WB) experiment, and the expression levels (relative expression) of the corresponding mRNAs of Runx2 and OCN were detected by qRT-PCR experiment. For the test results, please refer to Figure 4 , it can be seen from the results of the CCK-8 experiment that when the concentration of catalpol is lower than 80 μM and the concentration of titanium particles is lower than 10 μg / cm 2 , there is no obvious cytotoxicity within 5 days of co-culture, and the cell viability is not affected; it can be seen from the results of the WB experiment that titanium particles will cause a decrease in the expression of osteogenesis-related marker proteins Runx2 and OCN in cells, that is, the ability of bone marrow mesenchymal stem cells to transform into osteoblasts decreases, and the addition of catalpol can inhibit this change, and the inhibitory ability is positively correlated with the concentration of catalpol; it can be seen from the results of the qRT-PCR experiment that the expression of the two mRNAs of Runx2 and OCN has recovered after catalpol intervention.

[0045] BMSCc cells were co-cultured for 7 days and 21 days in four osteogenic induction media without titanium, with titanium, with titanium and low-dose catalpol, and with titanium and high-dose catalpol, respectively, to form four groups: Control group, Ti group, Ti+L-CA group, and Ti+H-CA group. Alkaline phosphatase (ALP) staining was performed on the samples of each group, and the average staining density (ALPIntDen / Area) was quantitatively analyzed. Then, alizarin red S (ARS) staining was performed, and the recovery rate (ARS RecoveryRatiol) at a wavelength of 420 nm was quantitatively analyzed. By means of cell immunofluorescence staining, the nucleus was labeled by binding of the fluorescent DAPI tag to DNA, and the osteogenic marker proteins Runx2 and OCN in the samples of each group were labeled with different fluorescent markers to study their expression and distribution, and the average optical density (Runx2 IntDen / Area) of Runx2 and the average optical density (OCN IntDen / Area) of OCN were quantitatively analyzed. Please refer to Figure 5 The results of ALP staining showed that compared with the Control group, the treatment with Ti particles significantly inhibited the ALP activity of BMSCc cells. According to the quantitative analysis results, the ALP activity was significantly reduced by approximately 82.67%. After treatment with catalpol, the inhibition of ALP activity by Ti particles was rescued. Quantitative analysis showed that the ALP activity increased by 26.9% and 68.1%, respectively. In addition, the results of ARS staining analysis confirmed that Ti particles significantly inhibited the bone mineralization ability of BMSCc cells. At the same time, the semi-quantitative analysis results of ARS showed that compared with the Control group, the absorbance of the Ti group decreased by approximately 56.7%. After treatment with catalpol, the inhibition of the bone mineralization ability of BMSCc cells by Ti particles was rescued, and it was dose-dependent. The absorbance of the Ti+L-CA group increased by approximately 26.8%, and the absorbance of the Ti+H-CA group increased by approximately 41.9%. In addition, compared with the Control group, the treatment with Ti significantly reduced the average fluorescence intensity of Runx2 and OCN, while catalpol inhibited this change and was dose-dependent. The above results consistently showed that Ti particles significantly inhibited the transcription and protein expression of osteogenic differentiation-related markers, and the treatment with catalpol strongly promoted osteogenic differentiation and mineralization ability after treatment with Ti particles.

[0046] Combined with the above animal experiments and in vitro cell experiments, it can be demonstrated that Ti particles can induce osteoblast death and inhibit osteogenic differentiation and mineralization ability, while catalpol can play a therapeutic role and is dose-dependent. The mechanism of action of catalpol was further explored through experiments.

[0047] It is known from the prior art that catalpol can activate the nuclear factor (erythroid-derived 2)-related factor 2 (Nrf2) / heme oxygenase-1 (HO-1) pathway, increase the expression levels of HO-1 and superoxide dismutase 2 (SOD2), reduce myocardial ischemia-reperfusion injury, and exert an antidepressant effect. There are also studies showing that Nrf2 has a protective effect on erastin-induced ferroptosis in hypoxic-ischemic encephalopathy. Combining the above research results, the inventors speculate that the mechanism by which catalpol inhibits PPO is to inhibit the ferroptosis of osteoblasts by activating the Nrf2 / HO-1 pathway.

[0048] After BMSCc cells were treated with Ti alone or in combination with catalpol for 48 hours, RNA sequence analysis was performed to study the relationship between catalpol treatment and cell ferroptosis. Please refer to Figure 6 , it can be seen that compared with treatment with Ti alone, co-treatment with catalpol significantly up-regulated the expression of SLC7A11 and HOMX1 genes. GO enrichment analysis showed that catalpol can regulate related mechanisms such as oxidative stress, lipid metabolism, and iron transport. Through KEGG enrichment analysis, we found that catalpol can regulate ferroptosis-related pathways.

[0049] Please refer to Figure 7, BMSCc cells in a titanium particle environment were treated with a variety of different inhibitors, and cell viability was measured after 72 hours of culture. Cells without titanium particle treatment formed the Control group, cells treated only with titanium particles without adding inhibitors formed the Ti group, cells treated with the necrosis inhibitor Nec-1 formed the Ti+Nec-1 group, cells treated with the caspase inhibitor Z-VAD-FMK formed the Ti+Z group, cells treated with the autophagy inhibitor 3-MA formed the Ti+3-MA group, cells treated with the ferroptosis inhibitor Fer-1 formed the Ti+Fer-1 group, and cells treated with the iron chelator DFO formed the Ti+DFO group. By comparison, it can be seen that the necrosis inhibitor Nec-1, the caspase inhibitor Z-VAD-FMK, and the autophagy inhibitor 3-MA have a relatively low inhibitory ability on titanium particle-induced cell death, while the ferroptosis inhibitor Fer-1 and the iron chelator DFO have a relatively high inhibitory ability on titanium particle-induced cell death. Then, BMSCc cells without titanium particles were treated with different inhibitors respectively to form the Z group, 3-MA group, Nec-1 group, Fer-1 group, and DFO group, and cell viability was measured respectively. It was found that each inhibitor itself did not affect cell viability. BMSCc cells were treated with erastin to form the erastin group. After 72 hours of culture, the mitochondrial ultrastructure of the Control group, erastin group, and Ti group was observed by transmission electron microscopy. It can be seen that compared with the Control group, the volume of mitochondria in the cells of the erastin group and Ti group shrank and became smaller, the membrane density increased, and the cristae decreased or disappeared. Using GAPDH as an internal reference, the contents of SLC7A11 and GPx4, the ferroptosis markers, in the cells of the Control group, erastin group, Ti group, and Ti+Fer-1 group were detected by WB experiment after 5 days of culture, and quantitative analysis was carried out. It can be seen that SLC7A11 and GPx4 were significantly reduced after titanium particle treatment, which was consistent with the expression level of the positive control, that is, the erastin group. Moreover, after adding Fer-1, the expression levels of SLC7A11 and GPx4 were significantly increased. Thus, it can be seen that ferroptosis is involved in the process of titanium particle-induced cell death and is related to PPO.

[0050] Please refer to Figure 8, the cell viability of the Control group, Ti group, Ti+L-CA group, and Ti+H-CA group cultured for 48 h was detected by the CCK-8 method. It can be seen that catalpol can improve the decrease in cell viability induced by titanium particles, and it shows a concentration-dependent manner. Erastin was co-treated alone or with low-dose and high-dose catalpol to form the Erastin group, Erastin+L-CA group, and Erastin+H-CA group respectively. The cell viability of each group was detected by the CCK-8 method. It can be seen that catalpol can improve the ferroptosis of cells induced by erastin, and it shows a concentration-dependent manner. BMSCc cells were treated with titanium particles, low-dose catalpol, and high-dose catalpol respectively to obtain the untreated Control group, Ti group, L-CA group, and H-CA group. The intracellular reactive oxygen species (ROS) were labeled with DCFH-DA probe. The fluorescence spectra of cells in each group were detected by the reactive oxygen fluorescence experiment, and the average fluorescence intensity of ROS (ROS Fluorescence Mean Intensity) was quantitatively analyzed. Then, the average fluorescence intensity of ROS in the Control group, Ti group, Ti+L-CA group, and Ti+H-CA group cells was detected and analyzed by the same method. Combining the results of the two experiments, it can be known that catalpol can remove excessive ROS in cells. The intracellular lipid peroxides and ROS were labeled with fluorescence probes respectively, and the relative cell number (Count) was detected by flow cytometry. It can be seen that catalpol significantly reduced the intracellular ROS level and lipid peroxidation production. Then, the average fluorescence intensity of ROS in the Control group, Ti group, Ti+L-CA group, and Ti+H-CA group cells was detected by the reactive oxygen fluorescence experiment, and the average fluorescence intensity of lipid oxidation fluorescence (Lipid oxidation Fluorescence Mean Intensity) and MDA level (MDAlevel) of each group of cells were detected by flow cytometry after labeling. It can be seen that catalpol reduced the intracellular lipid peroxidation production, and at the same time, catalpol also reduced the MDA level.

[0051] Please refer to Figure 9, with GAPDH as the internal reference, the protein expression levels of Nrf2, HO-1, NQO1, SLC7A11, and GPx4 in the cells of the Control group, Ti group, Ti+L-CA group, and Ti+H-CA group cultured for 5 days were detected by WB experiment. The Nrf2 signaling pathway is the direct downstream of ROS. When the intracellular ROS level increases, it will trigger the activation of this pathway. After Nrf2 enters the nucleus, it promotes the expression of antioxidant proteins by regulating the transcription of antioxidant response element (ARE)-dependent genes, thereby balancing oxidative mediators and maintaining cellular redox homeostasis. This process is crucial for protecting cells from oxidative stress damage. According to the results in the figure, it can be seen that the expression of antioxidant proteins Nrf2, NQO1, and HO-1 in the cells was inhibited after Ti particle intervention. On the contrary, after treatment with catalpol at different concentrations, the expression levels of these proteins increased significantly, and catalpol significantly reversed the inhibitory effect of Ti particles on antioxidant proteins.

[0052] Please refer to Figure 10 , the expression and localization of Nrf2 in BMSCc cells treated with Ti particles and catalpol were detected by immunofluorescence, and the integrated density in the area of Nrf2 fluorescence was quantitatively analyzed. It can be seen that the nuclear expression of Nrf2 decreased after Ti particle treatment, while the nuclear expression of Nrf2 recovered after catalpol intervention.

[0053] Please refer to Figure 11 , and then the mRNA expression levels of Nrf2, HO-1, NQO1, SLC7A11, and GPx4 were quantitatively analyzed by RT-PCR. It can be seen that the expression of the above various mRNAs was up-regulated. This indicates that catalpol inhibits Ti particle-induced ferroptosis by activating the Nrf2 / HO-1 pathway.

[0054] Please refer to Figure 12, the expression of Nrf2 in BMSCc cells was knocked down by small interfering RNA (siRNA), and its knockout efficiency was verified by Western blot (WB) assay. It could be seen that the expression of Nrf2 was significantly decreased after transfection. The cells with knocked-down Nrf2 were co-cultured with high-dose catalpol in the presence of titanium particles to form samples of the Ti+H-CA+Nrf2siRNA group. Then, the WB assay was used to detect the expression of downstream antioxidant proteins Nrf2, HO-1, NQO1, SLC7A11, and GPx4, as well as the corresponding mRNAs, in the samples of the Ti+H-CA+Nrf2siRNA group, the Control group, the Ti group, and the Ti+H-CA group. According to the experimental results, compared with the Ti+H-CA group, the protein expressions of Nrf2, NQO1, and HO-1 in the Ti+H-CA+Nrf2siRNA group were significantly decreased, indicating that the therapeutic effect of catalpol was significantly inhibited after knocking out Nrf2. Compared with the Ti group, the expressions of ferroptosis-related proteins SLC7a11 and GPx4 were significantly increased in the Ti+H-CA group, while there was no significant difference in the expressions of SLC7a11 and GPx4 between the Ti+H-CA+Nrf2siRNA group and the Ti group. This indicates that the active expression of the Nrf2 / HO-1 pathway is an important prerequisite for catalpol to inhibit ferroptosis.

[0055] Please refer to Figure 13 , the expressions of osteogenesis-related proteins Runx2 and osteocalcin (OCN), as well as the corresponding mRNAs, in the samples of the Control group, the Ti group, the Ti+H-CA group, and the Ti+H-CA+Nrf2siRNA group were detected by WB assay and reverse transcription polymerase chain reaction (RT-PCR) assay, respectively. It could be seen that compared with the Ti group, the protein and corresponding mRNA levels of Runx2 and OCN were significantly upregulated in the Ti+H-CA group, while there was no significant difference in the protein and corresponding mRNA levels of Runx2 and OCN between the Ti+H-CA+Nrf2siRNA group and the Ti group. Runx2 is an important osteogenic transcription factor and is crucial for promoting bone formation in the early stage. OCN appears at the end stage of osteoblast differentiation and is a late osteoblast marker that promotes bone formation and is crucial for bone mineralization. The above experimental results demonstrated that under the intervention of titanium particles, the therapeutic effect of catalpol on cell differentiation and mineralization may be mediated by the Nrf2 / HO-1 signaling pathway.

[0056] Please refer to Figure 14, samples of the Control group, Ti group, Ti+H-CA group, and Ti+H-CA+Nrf2siRNA group were subjected to ALP staining and ARS staining, and quantitative analysis was performed on the staining conditions. The staining images showed that catalpol significantly increased the ALP activity and the ability of BMSCc cells to differentiate and mineralize after treatment, while the protective effect of catalpol was reversed after co-culture with Nrf2siRNA. The results of the quantitative analysis were consistent with those shown in the staining images, further demonstrating the relationship between the Nrf2 / HO-1 signaling pathway and the therapeutic effect of catalpol on cell differentiation and mineralization.

[0057] Please refer to Figure 15 , the expression levels of Runx2 and OCN in cells of the Control group, Ti group, Ti+H-CA group, and Ti+H-CA+Nrf2siRNA group were further detected by immunofluorescence staining and quantitative analysis. It could be seen from the images that the nuclear expression of Nrf2 in cells was significantly decreased after knocking out Nrf2. Combining with the results of quantitative analysis, it could be seen that the fluorescence intensities of Runx2 and OCN were significantly decreased after Ti particle treatment, while catalpol treatment inhibited this change, and the therapeutic effect decreased after knocking down Nrf2. The research results showed that under the intervention of Ti particles, the therapeutic effect of catalpol on cell differentiation and mineralization was mediated by the Nrf2 / HO-1 signaling pathway.

[0058] Please refer to Figure 16 , the expression levels of Nrf2, GPx4, OCN, and 4-HNE in cells of the Control group, Ti group, Ti+H-CA group, and Ti+H-CA+Nrf2siRNA group were detected by immunohistochemical staining and quantitative analysis, and quantitative analysis of the number of positive cells was performed. Combining the staining images and quantitative analysis graphs, it could be seen that the protein expression levels of Nrf2, GPx4, and OCN in the Ti group were significantly lower than those in the Control group, and the expression levels of GPx4, Nrf2, and OCN in the Ti+H-CA group were higher than those in the Ti+H-CA+Nrf2siRNA group. The protein expression level of 4-HNE in the Ti group was significantly higher than that in the Control group, and the expression level of 4-HNE in the Ti+H-CA group was lower than that in the Ti+H-CA+Nrf2siRNA group. These research results showed that catalpol reduced ferroptosis of osteoblasts in vivo and improved the viability and osteogenic ability of osteoblasts by activating the Nrf2 / HO-1 signaling pathway.

[0059] In this example, the inhibitory effect of catalpol on ferroptosis of osteoblasts induced by wear particles and other factors generated during the long-term wear of artificial joint prostheses was verified. The therapeutic effect of catalpol on the weakened osteogenic differentiation and mineralization ability of bone marrow mesenchymal stem cells caused by wear particles was also verified. It was also demonstrated that this therapeutic effect is mediated by the Nrf2 / HO-1 signaling pathway, which helps to inhibit the aseptic loosening of the prosthesis and plays an enlightening role in the development of joint replacement surgery.

[0060] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0061] The above-described embodiments only represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the invention patent should be subject to the appended claims.

Claims

1. Application of catalpol in the preparation of periprosthetic osteolysis inhibitors.

2. The use according to claim 1, characterized in that The catalpol is used for inhibiting the ferroptosis of osteoblasts induced by titanium particles.

3. The use according to claim 1, characterized in that The catalpol is used for inhibiting the ferroptosis of osteoblasts induced by erastin, a ferroptosis inducer.

4. The use according to claim 1, characterized in that The catalpol inhibits the decrease of osteogenic differentiation ability of bone marrow mesenchymal stem cells.

5. The use according to claim 1, characterized in that The catalpol inhibits the decrease in bone mineralization ability of bone marrow mesenchymal stem cells induced by titanium particles.

6. The use according to any one of claims 2 to 5, characterized in that: The catalpol exerts a therapeutic effect by activating the Nrf2 / HO-1 signaling pathway.

7. The use according to claim 1, characterized in that The periprosthetic bone dissolution is the periprosthetic bone dissolution that occurs after artificial joint replacement surgery.

8. The use according to claim 1, characterized in that The inhibitors were administered by intraperitoneal injection.

9. The use according to claim 7, characterized in that The content of catalpol in the inhibitor is any value between 10 mg / kg and 30 mg / kg.

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