Effect And Mechanism Of Reelin-dab1 Signaling Transduction Pathway On The Abnormal Metabolism Of β-amloid Protein Induced By Aluminum

Objective:To explore the effect and mechanism of Reelin-dab1 signal transduction pathway on the abnormal deposition of Aβinduced by aluminum,and to provide a theoretical reference for further understanding the mechanism of aluminum neurotoxicity and the possible mechanism of aluminum in the development of AD.Methods:1.Preparation of cell model induced by neurotoxicity of aluminumThe PC12 cells were exposed to the medium,including Al（mal）₃ at the final concentrations of 0μM,50μM,100μM,200μM and 400μM at 12 h,24 h and 48 h,cell morphology were detected by using an inverted microscope,cell activities were detected by using a Cell Counting Kit 8（CCK-8）assay,determining the dose and time of exposure to Al（mal）₃ for the preparation of cell model induced by neurotoxicity of aluminum.2.The role of expression changes of reelin on the abnomal deposition of Aβin PC12cells induced by aluminumCell activities were detected by using a CCK-8 assay,which induced by different concentrations of reelin-specific antagonist streptozotocin（STZ）and agonist corticosterone treated PC12 cells at different times,the optimal concentration and exposure time were determined.PC12 cells were divided into 0μM Al（mal）₃（control group）,200μM Al（mal）₃treated group,100μM STZ treated group,100μM STZ+200μM Al（mal）₃ treated group,1μM corticosterone treated group and 1μM corticosterone+200μM Al（mal）₃ treated group,continuing to cultivate at 12 h,24 h and 48 h,cell morphology were detected by using an inverted microscope,cell activities were detected by using a CCK-8 assay,The mRNA expression of reelin and dab1 were measured by qRT-PCR,the protein expression of reelin and dab1 were detected by Western-blot,the protein expression of pdab1,Aβ₄₂and Aβ₄₀0 in PC12 cells was detected by ELISA,the intensity of intracellular Ca²⁺fluorescence([Ca²⁺]i)were analyzed by using flow cytometry.3.The role of expression changes of dab1 on the abnomal deposition of Aβin PC12cells induced by aluminumCell activities were detected by using a CCK-8 assay,which induced by different concentrations of the dab1-specific antagonist prion peptide PrP（PrP）and agonist cypermethrin at different concentrations,the optimal concentration and exposure time were determined.The PC12 cells were randomly divided into 0μM Al（mal）₃（control group）,200μM Al（mal）₃ treated group,100μM PrP treated group,100μM PrP+200μM Al（mal）₃treated group,20μM cypermethrin treated group and 20μM cypermethrin+200μM Al（mal）₃ treated group,continuing to cultivate at 12 h,24 h and 48 h,cell morphology and activities were observed,the mRNA and protein expression of dab1 in PC12 cells were detected,the protein expression of pdab1,Aβ₄₂2 and Aβ₄₀0 in PC12 cells were detected,the[Ca²⁺]i were analyzed by using flow cytometry.All methods were the same as above.Results:1.The activities of PC12 cells showed a decreasing trend with the increasing of exposure dose and the raise time of Al（mal）₃,the activities of PC12 cells exposed to 200and 400μM Al（mal）₃ were significantly lower than those in the control group（P<0.05）,while the cell inhibition rate of PC12 cells exposed to 400μM Al（mal）₃ was too high（69.218%）,so 200μM Al（mal）₃ is selected for subsequent experiments.2.The role of expression changes of reelin on the abnomal deposition of Aβin PC12cells induced by aluminum（1）The mRNA and 330 kDa protein fragment expression of reelin in PC12 cells exposed to 200μM Al（mal）₃ at 12 h,24 h and 48 h were significantly lower than those in the control group（P<0.05）.The m RNA expression of reelin in PC12 cells exposed to 100μM STZ+200μM Al（mal）₃ at 12 h,24 h and 48 h were significantly lower than those in the 200μM Al（mal）₃ at 12 h,24 h and 48 h（P<0.05）.PC12 cells were treated with 100μM STZ+200μM Al（mal）₃ at 24 h,the 330 kDa protein expression of reelin was lower than those in the 200μM Al（mal）₃ treated group（P<0.05）.PC12 cells were treated with100μM STZ+200μM Al（mal）₃ at 12 h and 24 h,the 180 kDa protein expression of reelin was lower than those in the 200μM Al（mal）₃ treated group（P>0.05）.The protein expression of Aβ₄₂2 in PC12 cells exposed to 200μM Al（mal）₃ at 12 h,24 h and 48 h were significantly higher than those in the control group（P<0.05）,while the protein expression of Aβ₄₀0 was significantly lower than those in the control group at the same exposure time（P<0.05）.PC12 cells were treated with 100μM STZ+200μM Al（mal）₃ at 12 h,24 h and 48 h,the protein expression of Aβ₄₂2 was significantly higher than those in the 200μM Al（mal）₃ exposure group（P<0.05）,while the protein expression of Aβ₄₀0 was significantly lower than those in the 200μM Al（mal）₃ treated group at the same exposure time（P<0.05）.（2）The mRNA expression of reelin in PC12 cells exposed to 1μM corticosterone+200μM Al（mal）₃ at 12 h and 24 h were significantly higher than those in the 200μM Al（mal）₃ treated group（P<0.05）,while the mRNA expression of reelin at 48h was significantly lower than those in the 200μM Al（mal）₃ treated group（P<0.05）.The180 kDa protein expression of reelin in PC12 cells exposed to 1μM corticosterone+200μM Al（mal）₃ at 48 h was significantly lower than those in the200μM Al（mal）₃ treated group（P<0.05）.The 330 kDa protein expression of reelin in PC12 cells exposed to 1μM corticosterone+200μM Al（mal）₃ at 24 h was significantly higher than those in the 200μM Al（mal）₃ treated group（P<0.05）.The protein expression of Aβ₄₂2 in PC12 cells exposed to 1μM corticosterone+200μM Al（mal）₃ at 12 h and 24 h were significantly lower than those in the 200μM Al（mal）₃treated group（P<0.05）,while the protein expression of Aβ₄₀0 was significantly higher than those in the 200μM Al（mal）₃ treated group at the same exposure time（P<0.05）.The protein expression of Aβ₄₂2 in PC12 cells exposed to 1μM corticosterone+200μM Al（mal）₃ at 48 h was significantly higher than those in the 200μM Al（mal）₃ treated group（P<0.05）,while the protein expression of Aβ₄₀0 was lower than those in the 200μM Al（mal）₃ treated group（P>0.05）.3.The role of expression changes of dab1 on the abnomal deposition of Aβin PC12cells induced by aluminum（1）The mRNA and protein expression of dab1 in PC12 cells exposed to 200μM Al（mal）₃ at 24 h and 48 h were significantly lower than those in the control group（P<0.05）.The m RNA and protein expression of dab1 in PC12 cells exposed to 100μM PrP+200μM Al（mal）₃ at 12 h,24 h and 48 h were significantly lower than those in the200μM Al（mal）₃ treated group（P<0.05）.The protein expression of Aβ₄₂2 in PC12 cells exposed to 200μM Al（mal）₃ at 12 h,24 h and 48 h were significantly higher than those in the control group（P<0.05）.The protein expression of Aβ₄₀0 in PC12 cells exposed to 200μM Al（mal）₃ at 24 h and 48 h were significantly lower than those in the control group（P<0.05）.The protein expression of Aβ₄₂2 in PC12 cells exposed to 100μM PrP+200μM Al（mal）₃ at 24 h and 48 h were significantly higher than those in the 200μM Al（mal）₃ treated group（P<0.05）,while the protein expression of Aβ₄₀0 were significantly lower those in the 200μM Al（mal）₃ treated group at the same exposure time（P<0.05）.（2）The mRNA,and protein expression of dab1 in PC12 cells exposed to 20μM cypermethrin at 12 h,24 h and 48 h were significantly higher than those in the control group（P<0.05）.The mRNA and protein expression of dab1 in PC12 cells exposed to 20μM cypermethrin at 12 h and 48 h were significantly higher than those in the 200μM Al（mal）₃ treated group（P<0.05）.The protein expression of Aβ₄₂2 in PC12 cells exposed to 20μM cypermethrin+200μM Al（mal）₃ at 12 h and 24 h were significantly lower than those in the 200μM Al（mal）₃treated group（P<0.05）,while the protein expression of Aβ₄₀0 were significantly higher than those in the 200μM Al（mal）₃ treated group at the same exposure time（P<0.05）.Conclusion:1.The abnormal deposition of Aβcan be induced by aluminum exposure in PC12 cells.2.The expression changes of reelin and dab1 may play a major role in Reelin-dab1signal transduction pathway,these results suggest that the Reelin-dab1 signal transduction pathway may correlate to the abnormal deposition of Aβinduced by aluminum in neuron.