| Background and purposeCobalt is the main element of the hard metal industry and one of the main components of human metal implants.Cobalt-containing implants,especially joint prostheses used for artificial joint replacement,will be corroded,resulting in a large number of nanoscale cobalt particles(CoNPs).CoNPs can cause adverse local reactions around the implant and systemic toxic symptoms of multiorgan system.So far,there are no reports of safe and effective therapeutic drugs for the treatment of systemic and local toxicity symptoms.Cobalt nanoparticles enter cells through endocytosis and generate a large number of reactive oxygen species(ROS)through Fenton-like reactions,thereby inducing cells to produce oxidative stress(Oxidative Stress,OS).However,the molecular mechanism and regulation pathways of cell death induced by oxidative stress are still unclear,and there is a lack of reliable detoxification drugs confirmed by in vivo experiments or clinical experiments.From the perspective of ferroptosis,the novel mechanism of CoNPs toxicology is revealed,which provides a theoretical basis for exploring effective new methods of clinical detoxification and has potential broad application prospects.Methods1.Analysis of CoNPs toxicity mechanismA variety of cells such as mouse fibroblasts,vascular endothelial cells,hematopoietic stem,and hematopoietic cells were selected to be treated with different concentrations of CoNPs to detect cell activity and ROS levels,Apoptosis and necrosis,Fe2+ ions and total iron concentration;transmission electron microscopy detects cell ultrastructure changes,and detects the expression of molecules related to oxidative stress and ferroptosis,and analyzes the relationship between cobalt toxicity and ferroptosis.2.In vitro tests to explore the most effective detoxification drugsPrepare CoNPs-treated cell toxicity models,select a variety of antioxidants L-Ascorbic Acid(LAA),alpha-tocopherol(Vit E),Reduced glutathione(GSH),Melatonin(Mel),Alpha Lipoid Acid(Alpha Lipoid Acid,ALA),ferroptosis inhibitors(selenium compourds)and antagonists(ferrous lactate and zinc chloride)were incubated with CoNPs-infected cells 24 Detect cell activity,screen and compare the detoxification drugs with the best detoxification effect,and analyze the detoxification mechanism of some of them.3.In vivo verification and comparative analysis of the detoxification effects of selected detoxification drugs.(3)Verify the effectiveness of the selected detoxification drugs through in vivo animal experiments.Six-eight weeks old ICR mice were involved and divide into 5 groups(including normal control group,cobalt nano group,and three detoxification drug groups),and use 30μl of 1/16 LD50 CoNPs suspension to inject into hip joint every other week.Then add detoxification drugs after continuous injection CoNPs for 4 weeks,measure the changes in the body weight of the mice every week,sacrifice the mice at 17th weeks,check the blood routine and blood biochemical changes,observe the physical changes of liver,kidney,and heart pathology characteristics by HE stains slides.The liver tissue expression of GPX4,a key molecule of ferroptosis,was detected by Western blotting and immunohistochemistry.Compare the differences between the groups,evaluate the detoxification efficacy and detoxification mechanism of each detoxification reagent.Results1.The mechanism of CoNPsCoNPs inhibit cell proliferation in a dose-and time-dependent manner.CoNPs can induce the increase of intracellular ROS and MDA levels.Oxidative stress participates in cobalt-mediated cytotoxicity and increases the level of cell apoptosis.The results of CCK-8 cell activity and Annexin V-FITC apoptosis test showed that the apoptosis rate induced by CoNPs(9.78%)was significantly lower than the corresponding cell death rate(45%).CoNPs induced a significant increase in Fe2+and total iron concentrations in mouse embryonic fibroblasts(Balb/3T3),and decreased the intracellular GSH and GSG/GSSH ratios,and significantly inhibited glutathione peroxidase 4(GPX4)activity.The cells treated with CoNPs showed typical ferroptosis ultrastructure changes under the transmission electron microscope.CoNPs induced hypoxia-inducible factor 1α(HIF-1α)and downstream gene expression in vascular endothelial cells.Silencing HIF-1α can alleviate the oxidative stress induced by CoNPs and weaken the toxic effect of CoNPs on vascular endothelial cells.In addition,CoNPs also significantly increased DNA damage of hematopoietic stem cells(HSC)and hematopoietic progenitor cells(HPC),induced cell cycle arrest and reduced the level of cyclin B protein to a certain extent.Ubiquitination has a moderate effect,thereby reducing the cell’s ability to repair DNA.2.The cell viability test showed that α-lipoic acid(ALA),ferrous reagent(Ferrous Lactate/FL),and selenium substances(selenomethionine)have clear detoxification effects after testing a variety of detoxification drugs.ALA greatly inhibited CoNPsinduced cytotoxicity in a dose-dependent manner and slightly reduced CoNPs-induced apoptosis.By increasing the concentration of GSH and enhancing the activity of GPX4,significantly inhibited the increase in CoNPs-induced ROS levels and lipid hyperlipidemia.Oxidation reduces the intracellular and extracellular cobalt and iron concentrations,showing that it alleviates the cytotoxicity of cobalt nanoparticles by inhibiting cell ferroptosis-like cell death.Ferrous reagent reduces the toxic effect of CoNPs by reducing the HIF-1αactivity induced by cobalt nanoparticles.SeMet prevents the increase in ROS levels and DNA damage induced by CoNPs by enhancing the antioxidant capacity of the GSH-GPX system,showing the efficacy of iron death inhibitors.In addition,SeMet significantly inhibited the increase in HIF-α levels induced by CoNPs,and increased the level of UBE2K protein,enhanced the repair ability of cell DNA damage,thereby alleviating the cellular and genotoxicity of CoNPs.3.The results of in vivo studies have shown that after cobalt nanoparticles are injected around the hip joint,they accumulate in the liver with the blood flow.The toxicity of CoNPs gradually increases with time.The toxicity symptoms are mainly manifested as weight loss,dry hair,malnutrition,etc.There are rapid heart rate and myocardial hypertrophy-like changes.Pathological slices and blood biochemical tests show liver,heart,and kidney dysfunction,liver tissue GSH levels,and GPX4 activity decrease suggesting that the ferroptosis mechanism is involved in the toxicity.Alpha-lipoic acid,ferrous lactate,and selenomethionine all have different degrees of antagonistic effects of cobalt nano-toxicity,and the common mechanism is that all three have antioxidant capacity.The difference is that α-lipoic acid is both fat-soluble and water-soluble,and is more easily absorbed by the body;ferrous lactate has the effect of stimulating hematopoiesis and can improve the tolerance of mice to hypoxia,while selenomethionine enhances the activity of GPX4 more Significantly.Conclusions1.CoNPs has a dose and time-dependent time toxicity to cells,and their toxicity is related to cellular oxidative stress.Cobalt nanoparticles can induce ferroptosis-like cell death,and their toxicity is regulated by the HIF-1α signaling pathway.It activates the HIF-1αsignaling pathway and inhibits the expression of BRCA1 genes related to DNA repair,decreases the DNA repair ability of cells,and then causes cell death.2.Through the comparative analysis of various detoxification reagents,the anti-oxidation function is still the common feature to solve the toxicity of CoNPs.A variety of antioxidants have the effect of antagonizing the toxicity of CoNPs.The results of in vitro studies indicate that α-lipoic acid,ferrous lactate,and SeMet have better detoxification effects than other reagents.3.The results of in vivo studies also confirmed that α-lipoic acid,ferrous lactate,and SeMet all have different degrees of detoxification functions,and antioxidant properties are the common mechanism of the three.Due to its ability to stimulate hematopoiesis,ferrous lactate shows a slightly stronger detoxification effect than the other two drugs in vivo research. |
