The Effects And Mechanisms Of EGCG In Corticosterone-Injured PC12 Cells

Under chronic mild stress,the abnormal activation of hypothalamic-pituitary-adrenal axis induced the hypersecretion of glucocorticoids in brain,but exposure to too much high levels of glucocorticoids would cause damages to the brain,thereby inducing the occurrence and development of depression and other stress related neuronal disorders.Studies have showed that green tea extrats have neuroprotective effects on stress related neuronal disorders.EGCG,the main ingredient in green tea,is thought to be responsible for the majority of the biological activity of green tea extracts.It had been reported that EGCG also has neuroprotective effects.But there is no clear researches about the effects of EGCG on stress related neuronal disorders.In this study,we used corticosterone-injured PC 12 cells as the in vitro model to investigate the effects of EGCG.Sonic hedgehog signaling and PI3K/Akt signaling,which are the important signaling pathways regulating cell growth,proliferation and survival,are also closely related to some stress related neuronal disorders.So in this study,we explore the association between EGCG ’s effects in CORT-injured neuronal cells and Sonic hedgehog signalling or PI3K/Akt signalling.It will be helpful to the future study on the development or treatment on stress-related neuronal disorders,such as depression.Part 1:Effects of EGCG on Corticosterone-injured PC12 cellsObjective:Firstly,to choose the effective concentrations of CORT to injure PC12 cells and the protective concentrations of EGCG against the injury,then investigate the effects of EGCG on CORT-injured neuronal cells.Methods:Control group and 100,150,250,400,500 μmol/L CORT group were setting to choose the injured concentrations of CORT by MTT assay;the Control group and 1,10,20,30,50 μmol/L EGCG were setting to screen out the appropriate concentration range of EGCG by MTT assay;then the Control group,CORT-injured group and CORT+EGCG(in the appropriate concentration range)protective group were setting to screen out the suitable protective concentrations of EGCG by MTT assay;finally the morphological changes of cells was observed under the microscope,the cell apoptosis was measured via Hoechst 33342 staining and the cell growth and proliferation were detected by CCK8 assay to investigate the protective effects of EGCG on CORT-injured PC 12 cells.Results:After exposed to a range of different concentrations of CORT for 24 h,there was a dose-dependent decrease of cell viability.Compared with the Control group,the cell viability rate of PC 12 cells treated with 400 μmol/L CORT for 24 h decreased significantly.After treatment the normal cells with low concentrations(0-20 μmol/L)of EGCG,the cells were not affected,but after treatment the normal cells with high concentrations(>30 μmol/L)of EGCG,the cell survival decreased.While the cells were treated with the low concentrations of EGCG 1 h before CORT,the cell viability rate all increased,and the cell viability of the group treated with 20μmol/L EGCG was the highest.Besides,compared with 400 μmol/L CORT group,20 p,mol/L EGCG restored the cell morphological changes,decreased the cell apoptosis and alleviated the inhibition of the cell growth and proliferation.Conclusion:CORT caused damage to PC 12 cells,and there was a dose-dependent effects of CORT on PC 12 cells.While high concentration of EGCG had damaging effects on normal PC12 cell,low concentration of EGCG had a protective effect on CORT-injured cells.It can promote the cell survival,growth and proliferation and reduce the cell apoptosis in the CORT-injured neuronal cells.Part 2:Effects of Sonic hedgehog Signalling on the Protection of EGCG in Corticosterone-injured PC12 cellsObjective:To investigate the effects of Sonic hedgehog signalling on the protection of EGCG in corticosterone-injured PC 12 cells.Methods:The mRNA expression of Shh,Glil and N-myc gene were detected by RT-PCR;the proteins expression of Glil and N-myc were detected by Western blotting;use the Shh signal pathway inhibitor Cyclopamine and investigate the changes of cell survival,growth,proliferation and apoptosis in EGCG-protected PC 12 by MTT method,optical microscope,CCK8 method and Hoechst 33342 staining.Results:Compared with the Control group,the mRNA expression of Shh gene was not affected,but the mRNA and protein expressions of Glil and N-myc decreased in 400 μmol/L CORT group;after exposure to 20 μmol/L EGCG,the mRNA expressions of Shh,Glil and N-myc gene increased significantly,and the protein level of N-myc also increased significantly.Finally,while we using the Shh signalling inhibitor Cyclopamine to inhibit the expressions of Glil and N-myc,compared with the EGCG-protective group,the cell viability was decreased,cell growth and cell proliferation were inhibited,cell apoptosis was increased by Cyclopamine;but compared with the CORT group,the cell viability rate was higher and the cell apoptosis was less in the Cyclopamine intervention group.Conclusion:The protective effects of EGCG on CORT-injured PC 12 cells was partly dependent on the Shh signalling pathway.Part 3:Effects of PI3K/Akt Signalling on Protection of EGCG in Corticosterone-injured PC12 cellsObjective:PI3K/Akt signalling is essential for the cell survival and cell proliferation in most situations,but the role of it in the neuroprotection of EGCG is not clear,so we need to investigate the effects of PI3K/Akt signalling on the protection of EGCG in corticosterone-injured PC 12 cells..Methods:The proteins expression of Akt and p-Akt by were detected by Western blotting;use the PI3K/Akt pathway inhibitor LY294002 and investigate the changes of cell survival,growth,proliferation and apoptosis in EGCG-protected PC 12 by MTT method,optical microscope,CCK8 method and Hoechst 33342 staining.Results:Compared with the Control group,the relative protein levels of p-Akt/Akt was significantly decreased in 400 μmol/L CORT group;compared with 400 μmol/L CORT group,the relative protein levels of p-Akt/Akt was increased in 20μmol/L EGCG group;finally,we used the PI3K/Akt signalling inhibitor LY294002 to inhibit the expressions of p-Akt/Akt,and compared with the EGCG-protective group,the cell viability was decreased,the cell growth and cell proliferation were inhibited,the cell apoptosis was increased by LY294002.Conclusion:PI3K/Akt signaling pathway was also involved in the protective effect of EGCG on CORT-injured PC 12 cells.