The Research Of PC-12Cell Apotosis Through Wnt/β-catenin Signal Pathway

[Objective] To investigate if and how alumiun cause pc-12cell apotosis through the Wnt/β-catenin signal pathway[Methods]1.There are two parts of PC-12cells:1)the first part, PC12cells were randomlydivided into four groups, they are control group,100μmol/L Al（mal）₃group,200μmol/L Al（mal）₃group and400μmol/L Al（mal）₃group, all of them were treated for48hours,2) the second part:PC12cells were randomly divided into six groups, they respectively are control group, DMSOgroup, Wnt agonist group,200μmol/L Al（mal）₃group, Wnt agonist+200μmol/L Al（mal）₃groupand DMSO+200μmol/L Al（mal）₃group, also all of them were treated for48hours.2. The cell viability was quantified by Cell Counting Kit-8; and the flow cytometry byAnnexin V FITC-PI double staining quantified the apoptotic rates. The expression of BCL-2,BAX, caspase-3, Wnt3a, Dishevelled, β-catenin and GSK-3β protein and mRNA was detectedby ELISA, qRT-PCR.[Results]1.Cell viability:1) the first part: the cells’ viability was significantly decreased atdifferent dose group（P<0.05）.2) the second part: the cells’viability of control group, DMSOgroup and the Wnt agonist group were nearly the same,they have no differences. the viabilities of200μM Al（mal）₃group, DMSO+200μM Al（mal）₃group and Wnt agonist+200μM Al（mal）₃group were significantly decreased（P<0.05）compared with the control group, and comparedwith200μM Al（mal）₃group cell viability of Wnt agonist+200μM Al（mal）₃group wassignificantly higher（P<0.05）.2. Apoptotic rates:1)the first part: the apoptotic rates were significantly decreased atdifferent dose group（P<0.05）.2)the second part:â—‹1compared with the control group，theapotosis rates of the DMSO group, the Wnt agonist group and the Wnt agonist+200μM Al（mal）₃group were nearly the same, they had no differences（P＜0.05）,and the apotosis rates of the200μM Al（mal）₃group and the DMSO+200μM Al（mal）₃group weresignificantly increased（P＜0.05）.â—‹2compared with the200μM Al（mal）₃group， the apoptosis rates of Wntagonist+200μM Al（mal）₃group decreased significantly （P＜0.05）， and that of theDMSO+200μMAl（mal）₃group didn’t change a lot.3.RT-PCR:1) the first part:â—‹1The expression of BAX and caspase-3mRNA increased withthe exposure dose,and the expression of BCL-2mRNA decreased. Compared with the controlgroup the level of BAX and caspase-3mRNA in200and400μM Al（mal）₃group increasedsignificantly （p<0.05）, while the BCL-2is the opposite.â—‹2The expression of GSK-3β mRNAincreased with the exposure dose, and the expression of Wnt-3a, Dishevelled, β-catenin mRNA decreased.ï¼›Compared with the control group, the level of GSK-3β mRNA in400μM Al（mal）₃group increased significantly （p<0.05）, while the Wnt-3a, Dishevelled and β-catenin are theopposite.（p<0.05）. The expression of Dishevelled in200μM Al（mal）₃group also decreasedsignificantly.2)the second part:â—‹1Compared with the control group, the expression of BCL-2、BAX、caspase-3mRNA in Wnt agonist group and DMSO group didn’t change evidently.The content of BAX and caspase-3mRNA in200μM Al（mal）₃group and DMSO+200μMAl（mal）₃group increased significantly （p<0.05）, that of BCL-2mRNA decreasedevidently（p<0.05）.â—‹2Compared with the200μM Al（mal）₃group, the content of BAX、caspase-3mRNA in Wnt agonist+200μM Al（mal）₃group decreased significantly and that ofBAX mRNA increased significantly（p<0.05）, while the content of all three mRNAs ofDMSO+200μMAl（mal）₃group didn’t change significantly.4ELISA:1)the first part: The expression of BAX、caspase-3protein increased with theexposure dose, and the expression of BCL-2protein decrease. Compared with the control group,the expression of BAX and caspase-3protein in200and400μM Al（mal）₃group increasedsignificantly （p<0.05）, while the BCL-2decreased（p<0.05）.2)the second part：○1Comparedwith the control group, the expression of BCL-2、BAX、caspase-3protein in Wnt agonistgroup and DMSO group didn’t change evidently, while in the Wnt agonist+200μM Al（mal）₃group the expression BAX and caspase-3protein didn’t change significantly, BCL-2leveldecreased significantly（p<0.05）, in200μM Al（mal）₃group and DMSO+200μM Al（mal）₃group，the level of BAX、caspase-3protein increased significantly（p<0.05）,and that of BCL-2proteindecreased significantly（p<0.05）；○2. Compared with the200μM Al（mal）₃group,in Wntagonist+200μM Al（mal）₃group,the expression of BAX、 caspase-3protein decreasedsignificantly, while that of BCL-2protein increased significantly （p<0.05）. In DMSO+200μMAl（mal）₃group the expression of these protein above didn’t change significantly.[Conclusion] The aluminium can cause apoptosis of PC-12cells, and with the specificityactivation of Wnt/β-catenin pathway, the apoptosis rate of PC-12cells can be pulled down.