Application of DDX3X inhibitor in the preparation of drugs for treating aseptic loosening of artificial joints

Abstract

The present invention relates to the use of a DDX3X inhibitor in the preparation of a drug for treating aseptic loosening of artificial joints. The present invention clarifies that DDX3X is a potential core regulatory gene in the inflammatory response of macrophages activated by wear particles; inhibiting DDX3X can significantly inhibit the production of macrophage inflammatory factors and the activation of inflammasomes, inhibit the oligomerization level of ASC protein in macrophages, reduce the imbalance of mitochondrial homeostasis of macrophages, reduce the expression level of mitochondrial stress-related proteins in macrophages, promote the expression level of key transcription factors for antioxidant activity in macrophages, reduce the expression level of phosphorylation of endoplasmic reticulum stress-related proteins in macrophages, inhibit the nuclear entry level of endoplasmic reticulum stress-related proteins in macrophages, and significantly inhibit the skull bone dissolution effect. The present invention provides a new idea and theoretical basis for the research and development of drugs for treating aseptic loosening of artificial joints, and has important clinical value and social significance.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to the application of a DDX3X inhibitor in the preparation of a medicine for treating aseptic loosening of artificial joints. Background Art

[0002] Total joint arthroplasty (TJA) can not only reconstruct the patient's joint function, but also significantly improve their quality of life. It is one of the most effective methods for treating end-stage bone and joint diseases. However, the average service life of artificial joints is 15-20 years. Within 10 years after surgery, 5-10% of patients require high-risk and complex joint revision surgery. Factors such as aseptic loosening of the prosthesis, infection, and prosthesis instability limit the service life of artificial joints, among which aseptic loosening of the prosthesis is the main reason. At present, except for revision surgery, there is no effective drug to prevent and treat aseptic loosening of the prosthesis. Therefore, clarifying the occurrence and development mechanism of aseptic loosening of the prosthesis and developing relevant preventive and therapeutic drugs to improve the long-term survival rate of the prosthesis after joint replacement are issues that need to be urgently addressed in the fields of orthopedics and materials science.

[0003] Domestic and foreign studies have shown that the main cause of aseptic loosening of artificial joints is wear particle-mediated macrophage activation. Factors leading to this problem include biological factors (periprosthetic bone dissolution caused by wear particles), mechanical factors (micromotion, stress shielding and high hydraulic pressure, etc.), and individual factors of patients (such as osteoporosis and genetic factors). Although the service life, safety and biocompatibility of artificial joint prostheses have been improved with the continuous improvement of artificial joint prosthesis design and materials, the inflammatory response and bone dissolution caused by wear particles have not been fundamentally solved. Studies have shown that the inflammatory response produced by wear particles activating macrophages is the main cause of aseptic loosening of artificial joints. Wear particles can stimulate the pattern recognition receptors PRRs of macrophages, regulate downstream inflammatory pathways, and promote the production of multiple inflammatory factors through macrophage M1 polarization, activate osteoclasts, and ultimately lead to periprosthetic bone dissolution and aseptic loosening of artificial joints.

[0004] In recent years, studies have found that endoplasmic reticulum stress (ERS) and mitochondrial stress (MS) play an important role in the inflammatory response and M1 polarization of macrophages. Some scholars have found that after LPS stimulates macrophages, ERS is triggered and the expression of its sensor proteins IRE1, PERK and ATF6 is regulated, thereby activating downstream inflammatory responses and aggravating the inflammatory response of macrophages. In addition, after LPS stimulates macrophages, mitochondrial stress leads to the release of a series of mitochondrial signaling molecules, such as ROS and mtDNA, which induce inflammatory responses by activating inflammatory pathways such as NLRP3 inflammasomes, NF-κB and MAPK, as well as macrophage M1 polarization. In addition, endoplasmic reticulum stress can increase the concentration of calcium ions in intracellular mitochondria, causing mitochondrial internal environment disorders and oxidative stress; while mitochondrial stress can lead to a decrease in intracellular ATP and the release of ROS, inducing endoplasmic reticulum stress, and the interaction between the two exacerbates the inflammatory response of macrophages.

[0005] The latest research shows that the close connection between the endoplasmic reticulum and mitochondria creates a dynamic contact point platform between the two, called mitochondrial associated membranes (MAMs), which are mainly composed of the endoplasmic reticulum membrane, the outer mitochondrial membrane and the intermembrane space. Through this structure, signal transmission and material exchange can be carried out between the endoplasmic reticulum and mitochondria, regulating biological processes such as calcium ion transport, inflammasome signal transduction, lipid metabolism and transport, and endoplasmic reticulum-mitochondrial stress interaction. The latest research results show that wear particles can induce endoplasmic reticulum stress and mitochondrial stress in macrophages, promote the formation of MAMs structure and endoplasmic reticulum-mitochondrial stress interaction. It suggests that endoplasmic reticulum-mitochondrial stress is involved in the process of wear particles activating macrophage PRRs to induce inflammatory response, but its specific mechanism of action in this process still needs further exploration. Summary of the invention

[0006] The purpose of the present invention is to solve the problems existing in the prior art, thereby providing the use of DDX3X inhibitors in the preparation of drugs for treating aseptic loosening of artificial joints. The present invention discovers and identifies the significant high expression of DDX3X in wear particle-induced mouse bone marrow-derived macrophages through high-throughput sequencing of mRNA. And detect its expression in Ti particle-induced macrophages and around the prosthesis of patients with aseptic loosening, proving that it is correlated with the occurrence of aseptic loosening. Then, by targeted knockdown / overexpression of DDX3X, the expression of downstream inflammatory factors and inflammatory pathways, as well as the effect of DDX3X on the endoplasmic reticulum-mitochondrial stress of macrophages in this process, are detected. In addition, through in vivo animal experiments, the results of the mouse skull osteolysis model show that the injection of lentivirus that knocks down DDX3X can reduce Ti particle-induced osteolysis, providing a new idea and theoretical basis for the development of drugs for treating aseptic loosening of artificial joints.

[0007] To achieve the above object, the present invention is achieved by the following means:

[0008] In a first aspect, the present invention provides the use of a DDX3X inhibitor in the preparation of a drug for treating aseptic loosening of artificial joints and / or improving physiological indicators related to aseptic loosening of artificial joints.

[0009] Preferably, the physiological indicators related to aseptic loosening of artificial joints are selected from one or more of inflammasome activation, inflammatory factor levels, inflammatory inducing factor levels, inflammasome key protein oligomerization levels, macrophage mitochondrial homeostasis, mitochondrial stress-related protein levels in macrophages, levels of key antioxidant transcription factors in macrophages, phosphorylation levels of endoplasmic reticulum stress-related proteins in macrophages, nuclear import levels of endoplasmic reticulum stress-related proteins in macrophages, and skull bone dissolution effect.

[0010] Preferably, the inflammasome is selected from NLRP3.

[0011] Preferably, the inflammatory factor is selected from IL-1β.

[0012] Preferably, the inflammation inducing factor is selected from Caspase1.

[0013] Preferably, the inflammasome key protein is selected from ASC.

[0014] Preferably, the mitochondrial stress-related proteins in the macrophages are selected from one or more of DRP1 and FIS1.

[0015] Preferably, the key transcription factor for antioxidant activity in macrophages is selected from NRF2.

[0016] Preferably, the endoplasmic reticulum stress-related proteins in the macrophages are selected from one or more of PERK and IRE1.

[0017] Preferably, the endoplasmic reticulum stress-related protein in the macrophages is selected from ATF6.

[0018] Preferably, the DDX3X inhibitor is selected from one or more of siRNA, shRNA, and sgRNA designed based on the DDX3X gene.

[0019] Preferably, the DDX3X inhibitor is selected from siRNA designed based on the DDX3X gene.

[0020] Preferably, the sequence of the siRNA is selected from one or more of SEQ ID NO: 1 and SEQ ID NO: 2.

[0021] A second aspect of the present invention provides a pharmaceutical composition for treating aseptic loosening of artificial joints and / or improving physiological indicators related to aseptic loosening of artificial joints, comprising a DDX3X inhibitor and a pharmaceutically acceptable carrier.

[0022] Preferably, the physiological indicators related to aseptic loosening of artificial joints are selected from one or more of inflammasome activation, inflammatory factor levels, inflammatory inducing factor levels, inflammasome key protein oligomerization levels, macrophage mitochondrial homeostasis, mitochondrial stress-related protein levels in macrophages, levels of key antioxidant transcription factors in macrophages, phosphorylation levels of endoplasmic reticulum stress-related proteins in macrophages, nuclear import levels of endoplasmic reticulum stress-related proteins in macrophages, and skull bone dissolution effect.

[0023] Preferably, the inflammasome is selected from NLRP3.

[0024] Preferably, the inflammatory factor is selected from IL-1β.

[0025] Preferably, the inflammation inducing factor is selected from Caspase1.

[0026] Preferably, the inflammasome key protein is selected from ASC.

[0027] Preferably, the mitochondrial stress-related proteins in the macrophages are selected from one or more of DRP1 and FIS1.

[0028] Preferably, the key transcription factor for antioxidant activity in macrophages is selected from NRF2.

[0029] Preferably, the endoplasmic reticulum stress-related proteins in the macrophages are selected from one or more of PERK and IRE1.

[0030] Preferably, the endoplasmic reticulum stress-related protein in the macrophages is selected from ATF6.

[0031] Preferably, the DDX3X inhibitor is selected from one or more of siRNA, shRNA, and sgRNA designed based on the DDX3X gene.

[0032] Preferably, the DDX3X inhibitor is selected from siRNA designed based on the DDX3X gene.

[0033] Preferably, the sequence of the siRNA is selected from one or more of SEQ ID NO: 1 and SEQ ID NO: 2.

[0034] Preferably, the pharmaceutically acceptable carrier includes one or more of a filler, a binder, a disintegrant, a solvent, a preservative, a lubricant, and a flavoring agent.

[0035] Unless otherwise specified, in the context of the present invention, the DDX3X includes components such as DDX3X nucleotides and DDX3X proteins encoded by DDX3X nucleotides. The DDX3X inhibitor refers to a substance that can specifically downregulate the expression level of DDX3X and / or the transcription level of its mature mRNA and / or the expression level or activity of DDX3X protein, for example, antisense oligonucleotides, siRNA, shRNA, sgRNA, antagomiRs, miRNA sponges, miRNA Erasers, Target Masking and / or multi-target methods are used to downregulate the expression level and / or activity of DDX3X, as long as the level and / or activity of DDX3X can be reduced. The primers and / or primer pairs refer to PCR primers used to synthesize the cDNA chain of the DDX3X gene in PCR, so as to detect the expression level of the DDX3X gene mRNA. In addition to the primers and / or primer pairs listed in the present invention, those skilled in the art are fully capable of designing corresponding primers and / or primer pairs based on the gene sequence of DDX3X using conventional methods and means in the field including but not limited to molecular biology, and screening the designed primers and / or primer pairs by conventional experimental means, as long as they can achieve specific detection of DDX3X expression levels; conventional reagents and methods in the field can also be used to detect DDX3X protein expression levels.

[0036] The inventors of the present invention conducted mRNA sequencing and bioinformatics analysis on mouse bone marrow-derived macrophages (BMDMs) after Ti particle stimulation and found that DDX3X (DEAD-box helicase 3 X-linked) may have a significant association with immunity, inflammation and endoplasmic reticulum stress in macrophages. DDX3X is a highly conserved DEAD-box family protein that plays a role in life processes such as translation control, WNT signaling, viral RNA sensing and interferon-mediated gene expression. Previous studies have shown that DDX3X can interact with the NLRP3 (NACHT) domain through its helicase (Heli) domain to drive NLRP3 inflammasome activation. In addition, DDX3X can interact with IKKα, enhance its autophosphorylation and activation, thereby regulating the NF-κB pathway, and can also mediate macrophage M1 polarization and jointly regulate the expression of downstream inflammatory factors. However, it is still unclear whether DDX3X positively regulates downstream inflammatory pathways and the release of inflammatory factors by promoting endoplasmic reticulum-mitochondrial stress.

[0037] In order to further study the mechanism of DDX3X and aseptic loosening of artificial joints, develop drugs targeting DDX3X to prevent and treat joint loosening, and improve the long-term efficacy of artificial joint replacement, the present invention has found through a large number of studies that DDX3X is a potential core regulatory gene in the inflammatory response of macrophages activated by wear particles, and is closely related to immunity, inflammation and endoplasmic reticulum stress in macrophages; in the inflammatory response of macrophages activated by wear particles, DDX3X positively regulates the downstream inflammatory pathway and inflammatory factor release by promoting endoplasmic reticulum-mitochondrial stress. DDX3X is highly expressed in bone marrow-derived macrophages induced by wear particles, and is also highly expressed in the in vitro model of macrophages stimulated by wear particles and the synovium around the prosthesis of patients with aseptic loosening, indicating that DDX3X has a significant correlation with the occurrence of aseptic loosening. When DDX3X is overexpressed in vivo, it can promote macrophages to produce inflammatory factors. After inhibiting the expression of DDX3X, it can significantly inhibit the production of macrophage inflammatory factors and the activation of inflammasomes, inhibit the oligomerization level of ASC protein in macrophages, reduce the imbalance of mitochondrial homeostasis in macrophages, reduce the expression levels of mitochondrial stress-related proteins DRP1 and FIS1 in macrophages, and promote the expression level of NRF2, a key transcription factor for antioxidant in macrophages. At the same time, inhibiting DDX3X can also reduce the phosphorylation expression level of endoplasmic reticulum stress-related proteins PERK and IRE1 in macrophages, inhibit the nuclear entry level of endoplasmic reticulum stress-related protein ATF6 in macrophages, and significantly inhibit the skull bone dissolution effect. In general, the present invention provides a new idea and theoretical basis for the development of drugs for the treatment of aseptic loosening of artificial joints, and has important clinical value and social significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The volcano plot and heat map are the results of gene expression in wear particle-induced mouse bone marrow-derived macrophages detected using RNA high-throughput sequencing.

[0039] Figure 2 This is a schematic diagram of the results of using WB to detect the expression of DDX3X in Ti particle-induced macrophages and grayscale analysis of the results of each group.

[0040] Figure 3 Schematic diagram of the immunohistochemical detection of DDX3X expression in the periprosthetic synovium of patients with aseptic loosening and the analysis of the immunohistochemical results of sections from the two groups of patients.

[0041] Figure 4 Schematic diagram of the results of PCR detection of the expression of inflammatory factor IL-1β produced by Ti particles in macrophages after targeted overexpression / knockdown of DDX3X.

[0042] Figure 5Schematic diagram of the results of using ELISA to detect the expression of inflammatory factor IL-1β in macrophages induced by Ti particles after targeted overexpression / knockdown of DDX3X.

[0043] Figure 6 Schematic diagram of the results of immunofluorescence detection of the expression of inflammatory factor IL-1β in macrophages induced by Ti particles after targeted overexpression / knockdown of DDX3X.

[0044] Figure 7 Schematic diagram of the results of immunofluorescence detection of the effect of Ti particles on the NLRP3 inflammatory pathway in macrophages after targeted overexpression / knockdown of DDX3X.

[0045] Figure 8 Schematic diagram of the results of using WB to detect the effect of Ti particles on the NLRP3 inflammatory pathway in macrophages after targeted overexpression / knockdown of DDX3X.

[0046] Fig. 9 Schematic diagram of the results of immunofluorescence detection of mitochondrial homeostasis in macrophages after Ti particles induced targeted knockdown of DDX3X.

[0047] Fig.10 Schematic diagram of the results of using WB to detect the expression of mitochondrial stress-related proteins in macrophages induced by Ti particles after targeted knockdown of DDX3X.

[0048] Fig.11 Schematic diagram of the results of using WB to detect the expression of NRF2, a key transcription factor that regulates oxidative stress response in Ti particle-induced macrophages after targeted knockdown of DDX3X.

[0049] Fig.12 Schematic diagram of the results of using WB to detect the phosphorylation expression of endoplasmic reticulum stress-related proteins PERK and IRE1 in macrophages induced by Ti particles after targeted knockdown of DDX3X.

[0050] Fig.13 This is a schematic diagram of the results of immunofluorescence detection of the expression of endoplasmic reticulum stress-related protein ATF6.

[0051] Fig.14 Schematic diagram of the micro-CT detection results of mouse skull. DETAILED DESCRIPTION

[0052] In order to make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0053] Unless otherwise specified, the cells used in the context of the present invention are cultured according to the ATCC guidelines. All cell lines were identified by short tandem repeat analysis of the China Center for Type Culture Collection (Wuhan), and the presence of mycoplasma contamination was verified using a PCR detection kit (Shanghai Biothrive Sci), and were frozen in liquid nitrogen and used for subsequent experiments. The reagents, consumables, etc. used in the present invention are all commercially available or configured according to conventional methods. The experimental methods used in the present invention, such as molecular biology experiments, cell biology experiments, etc., are all conventional methods and techniques in the art.

[0054] Representative results from repeated biological experiments are presented in the context figures, and data are presented as mean ± SD as specified in the figure. All experiments were repeated at least three times. Data were analyzed using GraphPad Prism 8.0. The differences in mean values ​​between two or more groups were compared using t-test or analysis of variance. p A value < 0.05 was considered a significant difference.

[0055] Example 1

[0056] First, an in vitro model of wear particle-stimulated macrophages was established. The specific steps are as follows:

[0057] (1) Under sterile conditions, the femur and tibia of 8-week-old C57BL / 6J male mice were separated and bone marrow cells were extracted. 10 ng / mL macrophage colony stimulating factor was added to DMEM high-glucose medium containing 1% double antibody and 10% FBS, and cultured at a constant temperature of 5% CO2 and 37°C for 5-7 days to obtain mature mouse bone marrow-derived macrophages.

[0058] (2) The titanium particle solution (Alfa Aesar) was diluted with sterile water and filtered using Isopore filters with pore sizes of 10 μm, 1.2 μm, and 0.2 μm to obtain titanium particles in the range of 0.82 ± 0.12 μm. The particles were then washed with 75% ethanol for 48 hours.

[0059] (3) After the ethanol has completely evaporated, weigh the titanium particles and sterilize them with ethylene oxide. Confirm that the titanium particles have been sterilized by using the horseshoe crab amebocyte lysate assay and dilute them with PBS to 9×10 -3 g / mL titanium particle solution.

[0060] (4) At different time points, the titanium particles prepared in step (3) were used to stimulate mouse bone marrow-derived macrophages to establish an in vitro model of wear particle-stimulated macrophages.

[0061] The macrophages derived from the in vitro model of wear particle-stimulated macrophages were analyzed using RNA high-throughput sequencing technology. The specific steps are as follows:

[0062] (1) Mouse bone marrow-derived macrophages stimulated with titanium particles for 4 hours were lysed using a Trizol kit, and total RNA was extracted. The quality of the RNA samples was assessed using an Agilent 2100 bioanalyzer and then further examined by RNase-free agarose gel electrophoresis.

[0063] (2) After rRNA is removed, the remaining mRNA is enriched and fragmented using a fragmentation buffer. Primers are provided for the RNA fragments and reverse transcribed into cDNA. The cDNA is end-repaired, poly(A) is added, and the cDNA is ligated to an Illumina sequencing adapter.

[0064] (3) The cDNA was digested with uracil-N-glycosylase (UNG), size-selected by agarose gel electrophoresis, and amplified by PCR. It was then sequenced using an Illumina HiSeq 4000, excluding reads with adapters or low quality (Q value ≤ 20).

[0065] (4) Gene-level read counts were performed on the mouse reference genome (GRCm38) using MapSplice and HTSeq, and significantly differentially expressed genes (DEGs) were screened using the R package with FDR < 0.05 and log2 (fold change) > 1 as the criteria. Volcano plots and heat map analysis were used to identify mRNAs with significantly different expressions. The volcano plots and heat maps of gene expression results are shown in the following table. Figure 1 The results showed that the volcano plot and heat map showed that DDX3X was highly expressed in bone marrow-derived macrophages of mice induced by wear particles.

[0066] Subsequently, WB (Western Blot) technology was used to detect the expression of DDX3X in Ti particle-induced macrophages. The specific steps are as follows:

[0067] (1) Add 5×10 5 BMDM cells were cultured for 24 h and then lysed with RIPA buffer (containing 1% PMSF, a protease inhibitor, and 1% phosphatase inhibitor).

[0068] (2) Scrape the cells with a cell scraper and transfer them to a 1.5 mL EP tube. Detect the protein concentration using a BCA protein quantification kit and take 75 μL of the protein supernatant for Western blot analysis.

[0069] (3) Prepare the stacking gel and separation gel, add the sample to be tested to a 12-well polyacrylamide gel, and add 30 μg of sample to each well. After electrophoresis and transfer, block the PVDF membrane with 5% BSA solution and incubate the primary antibody on a slow shaker at 4°C overnight. After washing, incubate the secondary antibody solution on a slow shaker at room temperature for 1 hour. Finally, add the ECL chemiluminescent colorimetric reagent and set appropriate exposure conditions to detect protein expression.

[0070] Test results such as Figure 2 The results showed that DDX3X was highly expressed in bone marrow-derived macrophages of mice induced by titanium particles at different time points, and the grayscale analysis of the bands also showed this trend.

[0071] Furthermore, immunohistochemistry was used to detect the expression of DDX3X in the periprosthetic synovium of patients with aseptic loosening. The specific steps were as follows:

[0072] (1) The synovial tissue around the prosthesis of patients with aseptic loosening and the control group were fixed with 4% paraformaldehyde for 12 hours, dehydrated with xylene, embedded in paraffin and sectioned.

[0073] (2) Heat, dewax, and rehydrate the synovial tissue sections. Treat the sections with proteinase K for 15 minutes to permeabilize at room temperature. Add 5% BSA for antigen blocking and place in a 37°C incubator for 30 minutes.

[0074] (3) Add diluted primary antibody to the slices and incubate in a 37°C constant temperature incubator for 2 hours. After washing, add HRP-labeled secondary antibody and incubate in a constant temperature incubator for 30 minutes. After 5 minutes of color development using the DAB color development kit, rinse thoroughly with ultrapure water, stain the cell nuclei in hematoxylin for 20 seconds, and rinse again.

[0075] (4) After drying the slices, seal them with neutral resin and observe them under a microscope. The immunohistochemical staining results of the slices were scored using Bresaliers' semi-quantitative scoring method, which was divided into four levels according to the intensity of cytoplasmic staining: 0 (no staining), 1 (mild staining), 2 (moderate staining), and 3 (deep staining). Ten fields of view were randomly selected from the slices, and the proportion of cells with each staining intensity in the total number of cells in the field of view was calculated and marked as F0, F1, F2, and F3. Staining score (IS) = 0×F0+1×F1+2×F2+3×F3. The average staining score of the ten fields of view was taken as the quantitative result of the whole group.

[0076] The experimental results are as follows Figure 3As shown, the results showed that the expression of DDX3X in the periprosthetic tissue of patients with aseptic loosening (AL) was significantly upregulated compared with that in the control group. The above results collectively indicate that DDX3X is highly expressed in the in vitro model of wear particle-stimulated macrophages and in the periprosthetic synovium of patients with aseptic loosening, indicating that DDX3X is associated with the occurrence of aseptic loosening.

[0077] Example 2

[0078] In order to study the effect of DDX3X on the expression of inflammatory factors in Ti particle-induced macrophages, qPCR, ELISA and immunofluorescence techniques were first used to detect the expression of inflammatory factors in Ti particle-induced macrophages. The specific steps of qPCR detection are as follows:

[0079] Total RNA was extracted from Ti particle-stimulated macrophages using RNAiso Plus kit, and RNA concentration was measured. RNA was reverse transcribed into cDNA using TaKaRa reverse transcription kit, and qPCR was used to detect the expression of IL-1β in Ti particle-stimulated mouse bone marrow-derived macrophages at various time points, using GAPDH as an internal reference. The plasmid targeting overexpression of mouse DDX3X and the siRNA targeting knockdown of mouse DDX3X (si-DDX3X#1, sequence as shown in SEQ ID NO: 1, GGTTAGACTTCTGCAAATA; si-DDX3X#2, sequence as shown in SEQ ID NO: 2, GAAAGAGGTGGAAATAGTC) were designed and constructed by GenePharma, and mock vector (Mock) and negative control siRNA (NC) were used as controls, respectively. In the verification of siRNA knockdown effect, the inhibitory effect of si-DDX3X#1 was relatively better, so si-DDX3X#1 was used for verification in subsequent experiments.

[0080] The specific steps of ELISA detection are as follows:

[0081] (1) 5×10 5 Mouse bone marrow-derived macrophages were seeded into six-well plates and cultured for 24 hours and then cultured in Opti-MEMI reduced serum medium for 12 hours.

[0082] (2) Add 7.5 μL of Lipofectamine RNAiMAX transfection reagent to each well and transfect 200 pmol siRNA. Collect the culture supernatant of mouse bone marrow-derived macrophages that have been transfected with each group of plasmids after Ti particle stimulation, and discard the debris after centrifugation.

[0083] (3) The expression of inflammatory factor IL-1β was determined using an instant enzyme-linked immunosorbent assay (ELISA) kit.

[0084] The specific steps of immunofluorescence detection are as follows:

[0085] (1) Take 1×10 5 BMDM macrophages were seeded into confocal dishes and cultured in a constant temperature incubator for 24 h. Then, DDX3X siRNA (si-DDX3X#1), overexpression plasmids and their controls, and 1.8×10 −3 g / mL Ti particle solution.

[0086] (2) After stimulation, discard the supernatant at the corresponding time point and add paraformaldehyde for fixation. After washing, add 0.1% Triton X-100 to break the membrane and shake slowly for 15 minutes. After washing three times with PBS again, add sheep serum to the dish for blocking for 30 minutes.

[0087] (3) Add the primary antibody to the confocal dish and incubate overnight at 4°C. The next day, discard the primary antibody, wash with PBS, add 100 μL of the corresponding fluorescent secondary antibody and incubate at room temperature for 1 hour in the dark, then discard the secondary antibody and wash three times with PBS. Finally, add anti-fluorescence quenching sealing medium containing DAPI and observe under a confocal microscope.

[0088] Test results such as Figure 4-6 The results showed that compared with the control group, targeted overexpression of DDX3X promoted the production of inflammatory factors by macrophages induced by Ti particles, while targeted knockdown of DDX3X with si-DDX3X inhibited the production of inflammatory factors by macrophages induced by Ti particles.

[0089] Subsequently, immunofluorescence was used to detect the oligomerization degree of the marker protein ASC in the NLRP3 inflammasome. The test results were as follows: Figure 7 The results showed that compared with the control group, targeted knockdown of DDX3X inhibited the oligomerization level of ASC protein in macrophages induced by Ti particles, while overexpression of DDX3X promoted the oligomerization of ASC protein.

[0090] Furthermore, WB technology was used to detect the activation of the NLRP3 inflammatory pathway in macrophages induced by Ti particles after targeted knockdown of DDX3X. The specific steps are as follows:

[0091] (1) Add 5×10 5BMDM cells were cultured for 24 hours, RIPA buffer lysis solution was added, cells were scraped from each well with a cell scraper and transferred to a 1.5 mL EP tube. The protein concentration was then detected using a BCA protein quantification kit, and 75 μL of protein supernatant was taken for Western blot analysis.

[0092] (2) Prepare a stacking gel and a separation gel, add the sample to be tested into a 12-well polyacrylamide gel, add 30 μg to each well, and add 1× SDS-PAGE electrophoresis buffer into the electrophoresis tank, using a protein marker as a reference.

[0093] (3) After the protein was electrophoresed and transferred to the membrane, the PVDF membrane was blocked with 5% BSA solution and incubated with the primary antibody solution overnight on a slow shaker at 4°C. The strips were washed with TBST and then incubated with the secondary antibody solution for 1 hour on a slow shaker at room temperature. Finally, the ECL chemiluminescent ultrasensitive colorimetric reagent was added to the strips and placed in an exposure machine to set appropriate exposure conditions to detect protein expression.

[0094] Test results such as Figure 8 The results showed that compared with the control group, targeted knockdown of DDX3X inhibited Ti particle-induced NLRP3 inflammasome activation in macrophages, and the expression of Caspase1 and IL-1β was significantly decreased.

[0095] Example 3

[0096] First, immunofluorescence technology was used to analyze the mitochondrial homeostasis of macrophages induced by Ti particles after targeted knockdown of DDX3X. The specific steps are as follows:

[0097] (1) Take 1×10 5 BMDM macrophages were seeded into confocal dishes and cultured in a constant temperature incubator for 24 h. Then, DDX3X siRNA (si-DDX3X#1) and its control, as well as siRNA with a concentration of 1.8 × 10 −3 g / mL Ti particle solution.

[0098] (2) After stimulation, discard the supernatant at the corresponding time point and add paraformaldehyde for fixation. After washing, add 0.1% TritonX-100 to break the membrane and shake slowly for 15 minutes. After washing three times with PBS again, add blocking sheep serum to the dish for 30 minutes.

[0099] (3) Add the primary antibody to the confocal dish and incubate overnight at 4°C. The next day, discard the primary antibody, wash with PBS, add 100 μL of the corresponding fluorescent secondary antibody and incubate at room temperature for 1 hour in the dark, then discard the secondary antibody and wash three times with PBS. Finally, add anti-fluorescence quenching sealing medium containing DAPI and observe under a confocal microscope.

[0100] Test results such as Fig. 9 As shown. The results showed that after Ti particle stimulation, MitoTracker in macrophages was significantly aggregated, indicating that homeostasis imbalance occurred after mitochondrial stress. Compared with the control group, targeted knockdown of DDX3X can alleviate Ti particle-induced mitochondrial homeostasis imbalance in macrophages.

[0101] Subsequently, WB technology was used to detect the activation of mitochondrial-endoplasmic reticulum stress marker proteins in macrophages induced by Ti particles after targeted knockdown of DDX3X. The specific steps are as follows:

[0102] (1) Add 5×10 5 BMDM cells were cultured for 24 hours, RIPA buffer lysis solution was added, cells were scraped from each well with a cell scraper and transferred to a 1.5 mL EP tube. The protein concentration was then detected using a BCA protein quantification kit, and 75 μL of protein supernatant was taken for Western blot analysis.

[0103] (2) Prepare a stacking gel and a separation gel, add the sample to be tested into a 12-well polyacrylamide gel, add 30 μg to each well, and add 1× SDS-PAGE electrophoresis buffer into the electrophoresis tank, using a protein marker as a reference.

[0104] (3) After the protein was electrophoresed and transferred to the membrane, the PVDF membrane was blocked with 5% BSA solution and incubated with the primary antibody solution overnight on a slow shaker at 4°C. The strips were washed with TBST and then incubated with the secondary antibody solution for 1 hour on a slow shaker at room temperature. Finally, the ECL chemiluminescent ultrasensitive colorimetric reagent was added to the strips and placed in an exposure machine to set appropriate exposure conditions to detect protein expression.

[0105] Test results such as Fig.10 The results showed that compared with the control group, targeted knockdown of DDX3X inhibited the expression levels of mitochondrial stress-related proteins DRP1 and FIS1 in Ti particle-induced macrophages.

[0106] Furthermore, WB technology was used to detect the expression of NRF2, a key transcription factor regulating oxidative stress response, in Ti particle-induced macrophages after targeted knockdown of DDX3X, and grayscale analysis was performed. Fig.11The results showed that after Ti particle stimulation, the expression of NRF2, a key transcription factor required for the antioxidant response of macrophages, was increased, and compared with the control group, targeted knockdown of DDX3X significantly promoted the expression level of NRF2, a key transcription factor for antioxidant response in Ti particle-induced macrophages.

[0107] In addition, WB technology was used to detect the phosphorylation expression of PERK and IRE1, the endoplasmic reticulum stress-related proteins induced by Ti particles in macrophages after targeted knockdown of DDX3X; and immunofluorescence was used to detect the expression of ATF6, the endoplasmic reticulum stress-related protein. Fig.12 As shown in the figure, compared with the control group, targeted knockdown of DDX3X inhibited the expression level of phosphorylation of endoplasmic reticulum stress-related proteins PERK and IRE1 in macrophages induced by Ti particles. Fig.13 As shown in the figure, the results showed that after Ti particle stimulation, the nuclear entry of endoplasmic reticulum stress-related protein ATF6 in macrophages increased. Compared with the control group, targeted knockdown of DDX3X inhibited the nuclear entry level of endoplasmic reticulum stress-related protein ATF6 in macrophages induced by Ti particles.

[0108] Example 4

[0109] An in vivo model of mouse skull osteolysis was constructed to analyze the mitigating effect of in vivo inhibition of DDX3X on Ti particle-induced osteolysis. The specific method for constructing the in vivo model of mouse skull osteolysis is as follows:

[0110] (1) An 8-week-old male mouse was anesthetized with 10% chloral hydrate by intraperitoneal injection. After successful anesthesia, a 15 mm sagittal incision was made on the midline of the top of the skull to expose the skull. The periosteum in the top area of ​​the skull was scraped with a nerve stripper and a 0.5 × 0.5 × 0.3 cm 3 The size of gelatin sponge was placed on the skull for the next specific injection. The Control group was injected with 100 μL PBS alone; the Ti group was locally injected with 3 mg titanium particles suspended in 100 μL PBS; the NC group was locally injected with 30 μL PBS, 3 mg titanium particles and 70 μL of negative control lentivirus; the si-DDX3X group was locally injected with 30 μL PBS, 3 mg titanium particles and 70 μL si-DDX3X (si-DDX3X#1) lentivirus.

[0111] (2) All mice were observed to have no mortality or complications within 7 days after surgery. The mice were euthanized 7 days later, and the skulls were removed and all soft tissues were cleared for further analysis.

[0112] The mouse skull was collected and fixed thoroughly with 4% paraformaldehyde for 48 hours, and then scanned and analyzed using a high-resolution small animal micro-CT and related software (ZZKS-MicroCT4.1). The relevant micro-CT instrument setting parameters were 60kV and 667μA, and 3D image reconstruction was completed using reconstruction software. The analysis results are shown in Fig.14 As shown, the results showed that titanium particles induced obvious mouse skull osteolysis after micro-CT scanning and 3D reconstruction, and compared with the control group, the injection of lentivirus targeting the inhibition of DDX3X could significantly inhibit the skull osteolysis effect induced by titanium particles.

[0113] From the above, it is clear that DDX3X is a potential core regulatory gene in the inflammatory response of wear particle-activated macrophages, and is closely related to immunity, inflammation and endoplasmic reticulum stress in macrophages; in the inflammatory response of wear particle-activated macrophages, DDX3X positively regulates the downstream inflammatory pathway and the release of inflammatory factors by promoting endoplasmic reticulum-mitochondrial stress. DDX3X is highly expressed in bone marrow-derived macrophages of mice induced by wear particles, and is also highly expressed in the in vitro model of wear particle-stimulated macrophages and the synovium around the prosthesis of patients with aseptic loosening, indicating that DDX3X has a significant correlation with the occurrence of aseptic loosening. When DDX3X is overexpressed in vivo, it can promote the production of inflammatory factors in macrophages, while inhibiting the expression of DDX3X can significantly inhibit the production of inflammatory factors and the activation of inflammasomes in macrophages, inhibit the oligomerization level of ASC protein in macrophages, reduce the imbalance of mitochondrial homeostasis in macrophages, reduce the expression levels of mitochondrial stress-related proteins DRP1 and FIS1 in macrophages, and promote the expression level of NRF2, a key transcription factor for antioxidant in macrophages. At the same time, inhibition of DDX3X can also reduce the phosphorylation expression levels of endoplasmic reticulum stress-related proteins PERK and IRE1 in macrophages, inhibit the nuclear entry level of endoplasmic reticulum stress-related protein ATF6 in macrophages, and significantly inhibit the skull bone dissolution effect. In general, the present invention provides a new idea and theoretical basis for the development of drugs for the treatment of aseptic loosening of artificial joints, which has important clinical value and social significance.

[0114] The above specific implementation method part specifically introduces the analytical method involved in the present invention. It should be noted that the above introduction is only to help those skilled in the art better understand the method and ideas of the present invention, rather than limiting the relevant content. Without departing from the principle of the present invention, those skilled in the art may also make appropriate adjustments or modifications to the present invention, and the above adjustments and modifications shall also fall within the scope of protection of the present invention.

Claims

1. Use of a DDX3X inhibitor in the preparation of a drug for treating aseptic loosening of an artificial joint, characterized in that: The DDX3X inhibitor is selected from siRNA designed based on the DDX3X gene, and the sequence of the siRNA is shown in SEQ ID NO:

1.

2. A pharmaceutical composition for treating aseptic loosening of an artificial joint, characterized in that: It comprises a DDX3X inhibitor and a pharmaceutically acceptable carrier; the DDX3X inhibitor is selected from siRNA designed based on the DDX3X gene, and the sequence of the siRNA is shown in SEQ ID NO: 1.

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