Pd@CeO₂ Nanozymes Scavenging ROS And Regulating Macrophage Polarization For Studies Of Osteoarthritis In Vitro

Objective:Osteoarthritis（OA）is a chronic degenerative joint disease that affects the entire joint,including articular cartilage,subchondral bone,synovium,and periarticular tissues.Previous studies have shown that oxidative stress is the key factor of OA.Oxidative stress can lead to the imbalance of cell catabolism,damage DNA and protein,promote the apoptosis of chondrocytes,and promote the degradation of extracellular matrix.Eliminating ROS and regulating the polarization of pro-inflammatory M1-type macrophages to anti-inflammatory M2-type macrophages may be effective measures for the treatment of OA.In this project,a nanocomposite material with a core-shell structure was synthesized.Cerium dioxide（CeO₂）was used as a shell to wrap the core palladium（Pd）particles to synthesize Pd@CeO₂ nanomaterials.To explore the effect of Pd@CeO₂ nanomaterials on ROS and whether it can be used for the treatment of OA in vitro by regulating the polarization of macrophages.Methods:（1）Preparation and characterization of Pd@CeO₂ nanozymes.We used biomolecule-assisted methods to synthesize Pd@CeO₂ nanozymes.The morphology of Pd@CeO₂ nanozymes was detected by transmission electron microscopy（TEM）,High-angle circular dark-field scanning TEM（HAADF-STEM）and EDX-mapping was used to analyze the structure and elements of Pd@CeO₂,the diameter and potential of Pd nanozymes were detected by Malvin particle size analyzer.XRD was used to detect the crystal structure of nanometer,X-ray photoelectron spectroscopy（XPS）was used to analyze the valence states of Ce and Pd,ultraviolet-visible spectrophotometer was used to characterize it.The contents of Pd and Ce were analyzed by inductively coupled plasma atomic emission spectrometer（ICP-OES）.The oxygen production was measured by the dissolved oxygen instrument to evaluate the catalase（CAT）activity.（2）To explore the regulation effect of Pd@CeO₂ nanozymes on the polarization of macrophages.LPS+IFN-γwas used to induce the phenotype of M1 macrophages,and the cells were divided into RAW group,M1 group,M1+CeO₂ group,M1+Pd group and M1+Pd@CeO₂group.MTT was used to evaluate the toxicity of nanozymes to macrophages,Calcein-AM/PI staining was used to assess the viability of macrophages.DCFH-DA fluorescent probe was used to detect the content of ROS in macrophages.The expressions of IL-1β,IL-6,i NOS,CD86,TNF-αand IL-10genes were detected by q RT-PCR,and the expressions of i NOS and CD206proteins were detected by immunofluorescence assay.（3）To investigate the effect of Pd@CeO₂ on inflammation of chondrocytes,macrophage conditional medium（CM）was co-incubated with chondrocytes.The DCFH-DA fluorescent probe was used to detect the content of ROS in chondrocytes,and the JC-1probe was used to detect the mitochondrial membrane potential.The cells were divided into five groups:RAW-CM group,M1-CM group,M1+CeO₂ group,M1+Pd group and M1+Pd@CeO₂ group.The expressions of inflammatory genes IL-1,IL-6,i NOS,MMP-3 and MMP-13 in chondrocytes were detected by q RT-PCR assay.HE staining,safranine O staining,MMP-13immunofluorescence staining and Col-2a1 immunohistochemical staining were used to assess the effect of Pd@CeO₂ on the chondrocyte inflammation.Results:（1）Preparation and Characterization of Pd@CeO₂ nanozymes.TEM results showed that Pd@CeO₂ nanozymes were flower clusters.Pd@CeO₂nanomaterials had a core-shell structure,the results of Malvin particle size analyzer showed that the Pd@CeO₂ hydrodynamic diameter was 81 nm,and the potential Pd@CeO₂ was 12.8±0.5 with positive charge.XRD,XPS and UV-Vis spectrophotometer were used to confirme the successful synthesis of Pd@CeO₂.The antioxidant capacity of Pd@CeO₂was evaluated by the determination of O₂production.（2）Regulation of Pd@CeO₂ Nanozymes on Macrophage Polarization.MTT results showed that Pd@CeO₂ had no toxicity to macrophages when the concentration was 0～200μg/m L（P<0.05）,so 200μg/m L was selected as its working concentration,and the staining results of living and dead cells were consistent with the results of MTT.The results of ROS fluorescence probe detection showed that Pd@CeO₂ could reduce the intracellular ROS level.PCR results showed that Pd@CeO₂ could down-regulate the expression of pro-inflammatory related genes IL-1β,IL-6,TNF-α,M1-type markers i NOS and CD86,and up-regulate the expression of anti-inflammatory related genes IL-10（P<0.05）.Immunofluorescence staining showed that the expression of type M1 marker i NOS was decreased and the expression of type M2 marker CD206 was increased in the Pd@CeO₂ group.（3）Evaluation of Pd@CeO₂ on the anti-inflammatory effect of OA in vitro.DCFH-DA fluorescent probe detection results showed that Pd@CeO₂ could reduce the level of ROS in chondrocytes.JC-1 fluorescent probe detection results showed that Pd@CeO₂ could inhibit early chondrocyte apoptosis.Compared with M1-CM group,PCR results of M1+CeO₂-CM group,M1+Pd-CM group and M1+Pd@CeO₂-CM group showed inflammation-related genes IL-1β,IL-6,i NOS,MMP-3 and MMP-13 was down-regulated（P<0.05）.HE staining and saffine O staining showed that Pd@CeO₂ could maintain chondrocyte phenotype,increase the secretion of proteoglycan,inhibit the secretion of MMP-13 and promote the expression of Col-2a1.Conclusion:Pd@CeO₂ nanozymes may have the function of scavenging ROS and regulating the polarization of M1 macrophages to M2,reducing the effect of OA inflammation in vitro,and providing new ideas for the treatment of osteoarthritis.