Effects And Mechanisms Of Ce³⁺ And CeO_2-x Nanoparticles On Mesenchymal Stem Cells And Osteoclasts Through Modulating The Intracellular Reactive Oxygen Species

Background:In recent years,inorganic materials based on Ce have been widely used in industrial production,environmental protection,biomedical and other fields.The reasons are mainly as follows:First,Ce is easy to obtain.Ce is the most abundant rare earth element in the earth’s crust,and its mining and refining are easier than other rare earth elements;Second,Ce atoms have unique chemical properties.Ce atoms have two valence states of Ce³⁺and Ce⁴⁺,and the conversion between the two valence states is easy to achieve,which makes the oxides of Ce have excellent catalytic activity.The application of CeO_2-x nanoparticles（Ceria nanoparticles）in many fields has been rapidly promoted in recent years,among which the research of CNPs in the biomedical field has attracted the most attention.Recent studies have shown that CNPs can simulate the catalytic activity of some natural enzymes,such as Superoxide dismutase（SOD）,catalase,oxidase,phosphatase,etc.,among which SOD enzyme activity plays a key role in promoting the application of CNPs in biomedical field.A large number of studies have shown that CNPs can remove Reactive oxygen species（ROS）of cells through their SOD enzyme activity,thus preventing cells from oxidative stress damage.Bone is a kind of metabolically active tissue.Bone tissue realizes dynamic balance through bone resorption of osteoclasts and bone formation of osteoblasts.If this balance is damaged,bone homeostasis will be seriously damaged.ROS plays an important role in the homeostasis of bone.ROS can not only regulate the differentiation and maturation of osteoclasts,but also regulate the signal transduction of osteoblasts.Therefore,the following studies were carried out in this paper:firstly,the effect of Ce³⁺regulating ROS on osteoclast differentiation was investigated;secondly,the effect of CNPs regulating ROS on osteoclast differentiation was studied;finally,the feasibility of CNPs assisting mesenchymal stem cells（MSCs）to cope with oxidative stress was preliminarily explored.Methods:（1）The effect of ROS on osteoclast differentiation regulated by Ce³⁺ions.Ce（NO₃）₃ and Ce Cl₃ were used as sources of Ce³⁺.The effects of Ce³⁺on osteoclast differentiation were evaluated by TRAP staining,FAK staining and PCR.The effects of Ce³⁺ions on osteoclast function were investigated in vitro and in vivo.DCFH-DA reagent was used to detect the change of fluorescence intensity by fluorescence microscopy and flow cytometry,and the effect of Ce³⁺ions on ROS level of osteoclasts was investigated.The gene and protein expression levels of NOX1 were quantitatively detected by PCR and WB,and the effect of Ce³⁺on the Mitochondrial membrane protein（MMP）of osteoclasts was evaluated by JC-1reagent.（2）CNPs regulate the effect of ROS on osteoclast differentiation.Spherical CNPs with a diameter of about 42.8 nm were synthesized by thermal decomposition.The effect of CNPs on osteoclast differentiation was evaluated by CCK8,TRAP staining,FAK staining and PCR detection.Transmission electron microscopy（TEM）was used to observe the entry of CNPs into cells,and Inductively coupled plasma（ICP）was used to determine the rate and mechanism of CNPs entry into cells.The intracellular total ROS level was detected by DCFH-DA kit.Mito Sox Red was used to detect the change of fluorescence intensity by fluorescence microscopy and flow cytometry,and the effects of CNPs on the mitochondrial ROS level of osteoclasts were qualitatively and quantitatively detected.JC-1 kit was used to test MMP,and Mitotracher Green FM fluorescent dye was used to detect the total number of mitochondria.Through the above studies,the effect of CNPs on the ROS level of osteoclasts through mitochondrial pathway and the effect of ROS level changes on their differentiation were comprehensively evaluated.（3）CNPs regulate the effect of ROS on antioxidant stress of MSCs.CNPs with similar size and different parameters were synthesized by microemulsion method and high temperature thermal decomposition method.CCK8 was used to detect its cytotoxicity.Paraquat was used to construct the oxidative stress model of MSCs,and CCK8 was used to evaluate whether CNPs could protect MSCs from oxidative stress damage by inhibiting ROS.Through the above studies,the protective effect of CNPs on regulating ROS level of MSCs was preliminarily evaluated.Results:（1）Ce³⁺can promote the differentiation of osteoclasts and enhance the bone resorption function of osteoclasts.The anions in cerium salts did not interfere with the differentiation of osteoclasts.The mechanism and structure of the study showed that Ce³⁺induced the high expression of NOX1,and then increased the intracellular ROS level,which was the main reason that Ce³⁺promoted osteoclast differentiation.（2）CNPs can promote osteoclast differentiation.CNPs enter cells through clathrin-mediated and caveolin-mediated endocytosis,but CNPs don’t through its SOD activity inhibition of intracellular ROS generation,in contrast the existence of CNPs make osteoclast ROS levels increased significantly,the reason may be that acid environment of osteoclast causes CNPs to release a large number of Ce³⁺/Ce⁴⁺ions inside the cells,which leads to mitochondrial dysfunction and induces the increase of intracellular ROS level,thus leading to the acceleration of osteoclast differentiation.（3）CNPs can prevent oxidative stress from damaging MSCs.Paraquat can induce the increase of ROS level in MSCs cells,and then produce toxicity to cells.CNPs can reduce the ROS level in MSCs cells through the activity of SOD enzyme,thus promoting the fight against oxidative stress of MSCs.Crystallinity has an important effect on SOD enzyme activity of CNPs,and the lower the crystallinity,the stronger the enzyme activity of CNPs,and the better the protection effect on MSCs.Conclusion:In this paper,the effects of Ce³⁺and CNPs on ROS levels of MSCs and osteoclasts were studied,and the following specific conclusions were drawn:（1）Ce³⁺can induce the high expression of Nox1 in osteoclasts,which increases the intracellular ROS level,thereby promoting the differentiation of osteoclasts,and the corresponding enhancement of osteophagy function.（2）CNPs can enter osteoclasts through endocytosis and release Ce³⁺/Ce⁴⁺ions in their acidic environment,leading to mitochondrial dysfunction,and then increasing intracellular ROS level to promote osteoclast differentiation.（3）CNPs can exert SOD activity in MSCs and reduce intracellular ROS level through SOD activity to protect cells from oxidative stress injury.The above research conclusions showed that CNPs could increase and decrease ROS levels in osteoclasts and osteoblasts,respectively.The reason for this result is closely related to the structure and function of the two types of cells.The acidic environment of osteoclasts easily causes CNPs to release Ce³⁺ions,resulting in the increase of intracellular ROS level.In MSCs,there is no large area of acidic environment similar to the fold edge of osteoclasts,so CNPs exert SOD enzyme activity in MSCs and thus reduce intracellular ROS level.The conclusions obtained in this study can provide some important theoretical and experimental basis for the future application of Ce ions and CNPs in bone repair materials or bone-related diseases.