Theresearching Sanatory Infulence And Regulatory Molecular Mechanism Of Tho Kinds Of Phytoestrogens From Drynaria Fortunei On PC-12 Cell Injured By A?_25-35

Objective:Researching the sanatory infulence and regulato-ry molecular mechanism of two kinds of phytoestrogens(kaempherol and luteolin)from Drynaria fortunei on PC-12 cell injured by A?25-35,and exploring their the ER and MAPK signaling pathway regulatory mechanism.Which provide theoretical bassis for the application of Chinese medicine phytoestrogens for treatment of Alzheimer's disease(AD).Methods:Using different concentrations of A?25-35(5?mol/L,10?mol/L,20?mol/L,40?mol/L)damaged PC-12 cells and to observe the PC-12 cell morphology under the inverted microscope,to measure PC-12 cells injury rate using MTT method.Based on the best damage concentration and time,to choose best concentration of A?25-35.Then using different concentrations of kaempherol(10 ?mol/L,1 ?mol/L,10-1 ?mol/L?10-2 ?mol/L,10-3 ?mol/L,10-4 ?mol/L,10-5 ?mol/L,10-6?mol/L)or luteolin(10 ?mol/L,1 ?mol/L,10-1 ?mol/L,10-2 ?mol/L,10-3 ?mol/L,10-4 ?mol/L,10-5 ?mol/L,10-6 ?mol/L)to heal the damaged PC-12 cells and to study the protective effect of kaempferol and luteolin on PC-12 cell injured by A?25-35.Using Western blot method detect the influence of two kinds of phytoestrogens to PC-12 celluar ERa,ER?,ERK1/2,Bcl-2,Bax and Caspase-3 protein expression.Using RT-RCR method dectect the influence of two kinds of phytoestrogens on PC-12 celluar ER?,Bcl-2,Bax and Caspase-3 mRNA expreesion.Further to explore the possible mechanism of the protective effectts of the two kinds of plant estrogen on cell apoptosis.Results:Among the four different concentrations of A?25-35,We found(20?mol/L)A?25-35 were treated on PC-12 cells for 24h can obviously inhibit their growth,and the damages are easy to observe and detect.And the damaged PC-12 Cells can be healed under the treatment of phytoestrogens,so we chose(20 ?mol/L)A?25-35 as the preparation concentration for the in vitro mode of the AD.Comparing to the blank group,the A?25-35 made the expression of ER?,p-ERK1/2 and Bcl-2 protein in PC-12 cells significantly decreased(P<0.01),and the expression of Caspase-3 and Bax protein significantly increased(P<0.01).Luteolin(10-2?mol/L)and kaempferol(10-4?mol/L)can significantly increase the expression of ER?,p-ERK1/2 and Bcl-2 protein in the PC-12 cells that had been damaged by A?25-35(P<0.01),and significantly reduced the expression of Caspase-3 and Bax protein(P<0.01).Given the ER antagonist Fulvestrant,kaempferol(10-2?mol/L)and luteolin(10-4?mol/L)group were not detected the expression of p-ERK1/2 protein;Given ERK blocking inhibitor U0126 or ER antagonist Fulvestrant,kaempferol(1 0-2?mol/L)and luteolin(10-4?mol/L)still promoted the expression of the Bcl-2 protein and inhibit the expression of Bax and caspase-3,but compared with kaempferol(10-2 ?mol/L)and luteolin(10-4?mol/L)group,there is a significant difference(P<0.01).Influences of luteolin(10-2?mol/L)and kaempferol(10-4?mol/L)on cellular ER? Caspase-3,Bax and bcl-2 mRNA expression accord with their protein expression.Conclusion:Two kinds of phytoestrogen including kaempferol and luteolin can selectively promote ER? and p-ERK1/2 protein expression,activate of ER and MAPK signaling pathway,and through estrogen receptor mediated downstream MAPK kinase signaling pathway,inhibit cell apoptosis,and then play a key role in healing the damaged the PC-12 cells.