| Objective:To explore the effect and mechanism of Reelin-dab1 signal transduction pathway on the abnormal deposition of A?induced 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 0?M,50?M,100?M,200?M and 400?M 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 A?in 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 0?M Al?mal?3?control group?,200?M Al?mal?3treated group,100?M STZ treated group,100?M STZ+200?M Al?mal?3 treated group,1?M corticosterone treated group and 1?M corticosterone+200?M 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,A?42and A?400 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 A?in 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 0?M Al?mal?3?control group?,200?M Al?mal?3 treated group,100?M PrP treated group,100?M PrP+200?M Al?mal?3treated group,20?M cypermethrin treated group and 20?M cypermethrin+200?M 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,A?422 and A?400 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 400?M 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 400?M Al?mal?3 was too high?69.218%?,so 200?M Al?mal?3 is selected for subsequent experiments.2.The role of expression changes of reelin on the abnomal deposition of A?in PC12cells induced by aluminum?1?The mRNA and 330 kDa protein fragment expression of reelin in PC12 cells exposed to 200?M 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 100?M STZ+200?M Al?mal?3 at 12 h,24 h and 48 h were significantly lower than those in the 200?M Al?mal?3 at 12 h,24 h and 48 h?P<0.05?.PC12 cells were treated with 100?M STZ+200?M Al?mal?3 at 24 h,the 330 kDa protein expression of reelin was lower than those in the 200?M Al?mal?3 treated group?P<0.05?.PC12 cells were treated with100?M STZ+200?M Al?mal?3 at 12 h and 24 h,the 180 kDa protein expression of reelin was lower than those in the 200?M Al?mal?3 treated group?P>0.05?.The protein expression of A?422 in PC12 cells exposed to 200?M 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 A?400 was significantly lower than those in the control group at the same exposure time?P<0.05?.PC12 cells were treated with 100?M STZ+200?M Al?mal?3 at 12 h,24 h and 48 h,the protein expression of A?422 was significantly higher than those in the 200?M Al?mal?3 exposure group?P<0.05?,while the protein expression of A?400 was significantly lower than those in the 200?M Al?mal?3 treated group at the same exposure time?P<0.05?.?2?The mRNA expression of reelin in PC12 cells exposed to 1?M corticosterone+200?M Al?mal?3 at 12 h and 24 h were significantly higher than those in the 200?M Al?mal?3 treated group?P<0.05?,while the mRNA expression of reelin at 48h was significantly lower than those in the 200?M Al?mal?3 treated group?P<0.05?.The180 kDa protein expression of reelin in PC12 cells exposed to 1?M corticosterone+200?M Al?mal?3 at 48 h was significantly lower than those in the200?M Al?mal?3 treated group?P<0.05?.The 330 kDa protein expression of reelin in PC12 cells exposed to 1?M corticosterone+200?M Al?mal?3 at 24 h was significantly higher than those in the 200?M Al?mal?3 treated group?P<0.05?.The protein expression of A?422 in PC12 cells exposed to 1?M corticosterone+200?M Al?mal?3 at 12 h and 24 h were significantly lower than those in the 200?M Al?mal?3treated group?P<0.05?,while the protein expression of A?400 was significantly higher than those in the 200?M Al?mal?3 treated group at the same exposure time?P<0.05?.The protein expression of A?422 in PC12 cells exposed to 1?M corticosterone+200?M Al?mal?3 at 48 h was significantly higher than those in the 200?M Al?mal?3 treated group?P<0.05?,while the protein expression of A?400 was lower than those in the 200?M Al?mal?3 treated group?P>0.05?.3.The role of expression changes of dab1 on the abnomal deposition of A?in PC12cells induced by aluminum?1?The mRNA and protein expression of dab1 in PC12 cells exposed to 200?M 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 100?M PrP+200?M Al?mal?3 at 12 h,24 h and 48 h were significantly lower than those in the200?M Al?mal?3 treated group?P<0.05?.The protein expression of A?422 in PC12 cells exposed to 200?M 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 A?400 in PC12 cells exposed to 200?M 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 A?422 in PC12 cells exposed to 100?M PrP+200?M Al?mal?3 at 24 h and 48 h were significantly higher than those in the 200?M Al?mal?3 treated group?P<0.05?,while the protein expression of A?400 were significantly lower those in the 200?M 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 20?M 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 20?M cypermethrin at 12 h and 48 h were significantly higher than those in the 200?M Al?mal?3 treated group?P<0.05?.The protein expression of A?422 in PC12 cells exposed to 20?M cypermethrin+200?M Al?mal?3 at 12 h and 24 h were significantly lower than those in the 200?M Al?mal?3treated group?P<0.05?,while the protein expression of A?400 were significantly higher than those in the 200?M Al?mal?3 treated group at the same exposure time?P<0.05?.Conclusion:1.The abnormal deposition of A?can 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 A?induced by aluminum in neuron. |
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