| ObjectiveTo investigate the effects of manganese exposure on the PI3K/Akt signaling pathway and the role of this signal pathway in the oxidative stress and apoptosis induced by manganese by in vitro PC12 cell experiments.MethodsPC12 cells were exposed to 0,25,75,125 ?M manganese sulfate(MnSO4)for 24 hours,cell morphology was observed under the inverted phase contrast microscope.The survival rate was determined by CCK8,and the apoptotic rate was detected by Annexin V-FITC apoptosis kit.The levels of SOD,GSH-Px,CAT and MDA were detected by Nanjing Jiancheng kits(Jianchen Biochemical Inc,Nanjing,China).The relative expression of PI3K/Akt signaling pathway related proteins Akt and FoxO3 and their phosphorylation levels were determined by Western Blot,and the mRNA relative expression levels of Bcl-2,Bax and Caspase-3 were measured by fluorescence quantitative PCR.In addition,the PI3K/Akt signal pathway inhibitor LY294002,with a concentration of 50 ?M was treated with PC12 cells in advance for 1 hours,then the dose,time and index of manganese exposure were similar to those mentioned above.Results(1)The activity of SOD,GSH-Px,CAT in PC12 cells decreased and the content of MDA increased with the increase of manganese dosage.The activity of SOD and GSH-Px in high dose group were 219.308±6.602 U/ml and189.383±17.824 U/ml respectively,which were significantly lower than those in control,low dose and medium dose groups(P <0.05).The activity of CAT in medium and high dose groups were 10.832±1.395 U/ml and 9.202±1.046 U/ml respectively,which were significantly lower than the control group and low dose group(P <0.05).The MDA content(11.912±2.734 nmol/L)in high dose group was significantly higher than the control,low,medium,and high dose groups(P<0.05).(2)The expression level of phosphorylated protein of PI3K/Akt signaling pathway in PC12 cells was significantly increased with the increase of manganese dose.The expression levels of p-Akt,p-FoxO3,p-Akt/ Akt and FoxO3/ FoxO3 were significantly higher in each Mn group than that in control group(P <0.05).(3)The apoptosis rate of PC12 cells was increased with the increase of Mn concentration.The apoptosis rate in control,low dose,medium and high dose groups were-0.002±8.049,7.093±2.905,17.614±2.678,and 34.529±1.739,respectively,and there was significant difference among the groups(P <0.05).The mRNA expression level of Bax gene increased,while the mRNA expression levels of Bcl-2 and Caspase-3 decreased.The mRNA relative expression of Bcl-2 gene in the medium and high dose groups were 0.776±0.031 and0.418±0.030 respectively,which were significantly lower than those in the low dose group(0.984±0.034)and control group(P < 0.05).The mRNA relative expression of Bax gene in the high dose group(2.496±0.281)was significantlyhigher than those in the control,low dose(1.103±0.027)and the medium dose groups(1.327±0.043)(P < 0.05).The mRNA relative expression of Caspase-3genein the low,middle and high dose groups were 0.897±0.038,0.547±0.035,and 0.473±0.008,respectively,which were significantly lower than that in the control group(P < 0.01).The morphology of cells also changed in varying degrees with the increase of manganese concentration.(4)The relative expression of p-Akt and p-FoxO3 proteins were significantly correlated with oxidative stress index of SOD,GSH-Px,CAT and MDA.The relative expression of p-Akt protein was negatively correlated with SOD,GSH-Px and CAT,with the correlation coefficients were-0.774,-0.947,and-0.802,respectively(P < 0.01);and there was a positive correlation between p-Akt protein expression and MDA(r=0.766,P < 0.01).The relative expression of p-FoxO3 protein was negatively correlated with SOD,GSH-Px and CAT,with the correlation coefficients were-0.766,-0.946,and-0.826,respectively(P < 0.01);while there was a positive correlation between p-FoxO3 protein expression and MDA(r=0.773,P < 0.01).(5)The relative expression of p-Akt and p-FoxO3 proteins were correlated with apoptosis index of Bcl-2,Caspase-3 and Bax.The relative expression of p-Akt was negatively correlated with Bcl-2 and Caspase-3,with the correlation coefficients were-0.819 and-0.969 respectively(P < 0.01);and there was a positive correlation between p-Akt protein expression and Bax(r=0.732,P <0.01).The relative expression of p-FoxO3 protein was negatively correlated with Bcl-2 and Caspase-3,with the correlation coefficients were-0.831 and-0.879 respectively(P < 0.01);and there was a positive correlation between p-FoxO3 protein expression and Bax(r=0.804,P < 0.01).(6)With the increase of Mn concentration,the activity of SOD,GSH-Px,and CAT in the PC12 cells in the inhibitor + Mn exposed group was lower than that in the Mn exposed group,but the MDA content was higher than that in the Mn exposed group(P < 0.05).The apoptosis rate of PC12 cells was increased significantly after the cell exposure to Mn for 24 h,and the cell apoptosis rate in the inhibitor + Mn exposed group was significantly higher than the Mn exposed group(P < 0.05);The relative expression of Bcl-2 mRNA was significantly lower than that in the Mn exposed group(P < 0.05),and the relative expression of Bax and Caspase-3 was significantly higher than that in the Mn exposed group(P < 0.05).Conclusions(1)MnSO4 exposure can lead to the morphological changes of PC12 cells,reduce the cell survival rate and increase the cell apoptosis rate.(2)MnSO4 exposure can increase the level of oxidative stress and decrease the antioxidant capacity in PC12 cells.(3)MnSO4 exposure can activate the PI3K/Akt signaling pathway.(4)PI3K/Akt signaling pathway may play a protective role in the neurotoxicity of MnSO4. |
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