Comparative Study Of AlCl₃ And Al（mal）₃ On Inhibition Of CK-BB And Cytotoxic Effect To C6 Glioma Cells

The situation of aluminum pollution in food is very serious,which has caused the human body’s exposure to aluminum through food-borne routes is inevitable.The absorbed aluminum tends to accumulate in the human body and causes damage to multiple systems.The central nervous system is the main target of aluminum accumulation,and the metal toxicity is largely determined by the metal form.This study investigated the toxicity mechanisms of two different forms of aluminum from two levels:brain-type creatine kinase(CKB)and astrocytes.First,using large yorkshire pigs brain tissue as raw material,electrophoretic pure CKB samples can be prepared through the separation steps of 65%alcohol dissolution,80%alcohol precipitation,70%ammonium sulfate salting out,DEAE-Sepharose CL-6B anion exchange chromatography and Sephacryl S-300 gel filtration chromatography.The enzyme activity was 139300 U/mg,the purification factor was 74.29 times,and the enzyme recovery rate was 1.24%.Subsequently,the effect of inorganic AICl3 and organic Al(mal)3 on the catalytic activity of CKB was studied by inhibition kinetics.The results showed that AlCl3 exhibited a strong inhibitory effect on the catalytic activity of CKB.The half inhibitory concentration of AlCl3 on the catalytic activity of CKB is 0.67 mM,and the suppression type is mixed suppression.The half inhibitory concentration of Al(mal)3 on the catalytic activity of CKB is 3.81 mM.When the substrate is PCr,the inhibition type is hyperbolic non-competitive inhibition,and when the substrate is ADP,the inhibition type is anti-competitive inhibition.Secondly,the effect of AlCl3 and Al(mal)3 on protein structure was analyzed by fluorescence spectroscopy.The results showed that both of them would lead to enhanced hydrophobicity of CKB.Finally,molecular simulation docking was used to study the binding sites of AlCl3,Al(mal)3 and CKB and the amino acid residues interacting with each other.The results showed that AlCl3 was located near the active site of CKB.The experimental results of protein structure and inhibition mechanism show that different forms of aluminum have different inhibition mechanisms on CKB.Based on the above CKB experiment,astrocytes as the main accumulation site of aluminum in the brain tissue,and are rich in CKB characteristics.Using C6 astroglioma cells as experimental models to further study the difference between AlCl3 and Al(mal)3 toxicity level.It was concluded that the half inhibitory concentration of AlCl3 and Al(mal)3 on cell viability was 3.5 mM and 0.1 mM,respectively.AlCl3 will cause obvious changes in cell morphology,mainly caused by ATP depletion by inhibiting CKB activity in the cell.Al(mal)3 because of its lipophilicity is easier to enter the cell than AlCl3,inducing the cell to produce more serious oxidative damage and apoptosis.The above results further reveal that different forms of aluminum-induced cytotoxic effects are different.In conclusion,the experimental results of creatine kinase and C6 cells together proved that different forms of aluminum can induce different toxicity mechanisms,thus exhibiting different toxic effects.The above conclusions provide a theoretical basis for the scientific analysis of the hazards of food-borne aluminum and the mechanism of aluminum-induced neurotoxicity and neurodegenerative diseases.