Study On The Antagonistic Mechanism Of Zinc Against Cadmium Induced Cytotoxicity On Macrophage

Cadmium(Cd)is a common toxic metal,which can cause multiple organ and system toxic damage.Macrophages play an important role in the body’s immune system,especially in innate immunity.Cadmium affects the immune function of macrophages by causing toxic damage to macrophages.Zinc can effectively inhibit the toxic effect of cadmium on macrophages,but the effect and mechanism of zinc on the toxic damage of cadmium on macrophages are still unclear.The aim of this study was to investigate the toxic effects of cadmium on macrophages and the mechanism of zinc inhibiting the toxic effects of cadmium on macrophages.In this study,RAW 264.7 macrophage lines were used as the research subjects,and Cd2+(20 μmol/L,50 μmol/L)was added to the cell culture medium to establish the cell cadmium poisoning model,and the regulatory effect of zinc on cadmium poisoning macrophage toxicity was studied by adding Zn2+(20 μmol/L)to the cell cadmium poisoning model.After adding Zn2+ into cadmium challenge cells for 12 h,the effects of cadmium poisoning macrophages and the effects of zinc on cadmium poisoning macrophages were evaluated by detecting changes in cell morphology and ultrastructure,apoptosis,mitochondrial injury,ROS production,and intracellular Cd2+ and Zn2+ contents.In addition,by adding Cd2+(20 μmol/L,50 μmol/L)and Zn2+(20 μmol/L)to the cell culture medium,the changes in the expression of metal binding elements(MTF-1)and metallothionein(MTs)genes and proteins in the process of zinc regulating cadmium toxic macrophages were detected.The mechanism of zinc-based MTF-1 and MTs on the regulation of cadmium-poisoning macrophages was revealed by TPEN chelation of Zn2+ and MTF-1 si RNA.The results are as follows:(1)Cadmium concentration of 20 μmol/L and 50 μmol/L could cause significant contraction of RAW 264.7 cells,increase cell surface processes and decrease pseudopodia.At the same time,the ultrastructure of cells was changed obviously,chromatin was concentrated and marginalized,nuclear membrane and nucleolus disappeared,organelles were reduced and cytoplasmic vacuolation were observed,and the damage of cell surface structure and ultrastructure was more serious with the increase of concentration.(2)Cadmium could induce RAW 264.7 cell apoptosis,decrease of mitochondrial membrane potential,excessive production of ROS and significant increase of Cd2+ in RAW 264.7 cells(P<0.05).After adding Zn2+ with concentration of 20μmol/L in cadmium poisoning RAW 264.7 cells,the apoptosis rate decreased,the mitochondrial depolarization degree decreased,the ROS production decreased,the cell Cd2+ level decreased significantly,and the Zn2+ content increased significantly(P<0.05).(3)Cadmium poisoning can significantly promote the expression of MTs and MTF-1 genes and proteins in RAW 264.7 cells(P<0.05).The expression levels of MTs and MTF-1 genes and proteins in 50 μmol/L Cd2+ cells were higher than 20 μmol/L Cd2+(P<0.05).After adding Zn2+ to cadmium poisoning cells,the expression levels of MTs and MTF-1 genes and proteins were significantly higher than those of Cd2+ alone(P<0.05).(4)After TPEN chelated Zn2+,the apoptotic rate of Cd-treated cells increased significantly(P<0.05),and the expression of MTF-1 and MTs genes and proteins decreased significantly(P<0.05).After MTF-1 si RNA interfered with MTF-1 expression,the apoptotic rate of RAW 264.7 cells treated with Cd2+ increased significantly(P<0.05),while the gene and protein expression of MTs and MTF-1 decreased significantly(P< 0.05),which did not recover effectively with the supplementation of Zn2+.Test results show: cadmium could cause concentration-dependent cell damage in RAW 264.7 cells,and zinc had a significant protective effect on macrophages poisoned by cadmium.Moreover,zinc protects macrophages from cadmium poisoning by regulating MTs,which is mainly mediated by zinc-mediated MTF-1 expression.