Effects Of Stress On MEK/ERK1/2 And PI3K/Akt Signaling Pathways In Hippocampal Neurons And EGCG's Modulation On It

ObjectiveEGCG can improve cognitive function,protect central nervous system against stress-induced injuries.But its protective role of molecular mechanisms and cellular mechanisms are not yet clear.Thereby,to explore the detailed molecular mechanisms of EGCG,this study was conducted to examine the effects of EGCG on signal transduction in stress model of cultured neurons in vitro.These study will pave the way for further research of EGCG as protective agents against stress-damages.Methods1 Newborn wistar rats within 24h were used to culture primary hippocampal neurons,with immunofluorescence method to identify neurons purity.2 The expressions of GR in primary cultured neurons were detected by Western blot.3 Using CCK8,the survival rate of CORT-induced primary cultured hippocampal neurons was observed.4 The neurons were divided into control group,CORT group and EGCG group.Using CCK8,the survival rate was detected.The morphology change was observed by morphological method.Hoechst33342 method was used to detect the changes of neuron apoptosis.5 U0126/LY294002 were added to the cultured neurons in vitro,then the survival rate of hipocmpal neurons were detected.6 Western blot was used to detect the expressions of ERK1/2,PI3K/Akt in the cultured neurons in vitro.7 RT-PCR was used to detect the changes of ERK1/2mRNA and Akt mRNA in the cultured neurons in vitro.Results1 The neurons' purity of primary cultured hippocampal neurons in serum-free media was many more than 90%.2 The GR of neurons in 13d of the primary cultured neurons were matured,which could be used for the next experiments.3 CORT neurotoxicity has a time-concentration dependent manner in the range of 1-100?mol/L.Compared with the normal group,the survival rate of hippocampal neurons treated with 10?mol/L CORT for 24h decreased significantly(P<0.05),both the necrosis rate and apoptosis rate was increased.4 Pretreatment with 0.1-1?mol/L EGCG could significantly reduce stress-induced apoptosis of hippocampal neurons,and improve the survival rate as well as cell morphology.Pretreatment with 5-10?mol/L EGCG reduced the survival rate of hippocampal neurons compared with the normal group(P<0.05),and increased the rate of apoptosis and the necrosis rate.5 Intervention with LY294002 and U0126 could reduce the survival rate of hippocampal neurons of EGCG+CORT group 66.90%,62.54%respectively.6 In CORT group,p-ERK1/2 was reduced within lh,but in EGCG modulation group the p-ERK1/2 increased obviously compared with CORT group.In the whole process,the changes of total ERK1/2,ERK mRNA level were not significant.7 There was decrease of p-Akt at lh following the treatment with CORT.EGCG modulation enhanced the activation of Akt at a range of lower concentrations(0.1-5?mol/L),whereas inhibited Akt activation at higher concentration.In addition,10?mol/L EGCG modulation improved the expression of Akt mRNA expression in contrast to CORT group.ConclusionCORT decreased the survival rate of hippocampal neurons in vitro,and resulted in cell apoptosis.Intervention with EGCG could improve these changes,and the results suggest that the neuroprotective effects of EGCG against stress-induced neural injuries may be involved with the modulations of some signaling transduction pathways,such as MEK/ERK1/2 and PI3K/Akt.Furthermore,the regulation of EGCG on stress neurons is in a protect neurons at lower concentrations,but promote neuron apoptosis at higher concentration.