| Objective:To explore the protection effect of phytoestrogenic naringin from Rhizoma Drynariae on PC 12 cells induced by Aβ25-35 and regulation mechanism of naringin on ER-PI3K-AKT-GSK-3β-Tau signaling pathway and ER-mediated apoptosis in PC 12 cells induced by Aβ25-35,which provides theoretical basis for the prevention and treatment of phytoestrogen on Alzheimer’s disease.Methods:1.Protective effect of naringin on PC 12 cells induced by Aβ25-35The effect of different concentrations of naringin on cell viability was detected by MTT assay,The expression of estrogen receptor subtype mRNA was detected by RT-qPCR method,and to investigate the effect of estrogen receptor blocker ICI182780 on naringin function,which determines whether the biological effect of naringin is mediated by ER.The AD cell model was established by the treatment of PC 12 cells with 2×10-5mol/L Aβ25-35 for 24h.Morphological changes and cell proliferation rate of PC 12 cells induced by Aβ25-35 were detected by morphological method and MTT assay to investigate the effective concentration of naringin.2.Protective mechanism of naringin on PC 12 cells induced by Aβ25-35The cell proliferation rate and apoptosis rate of AD cell model with the treatment of naringin was detected by MTT assay and Annexin V-FITC/PI double staining flow cytometry,The expression of ERβ,p-AKT(Ser473)/AKT,p-AKT(Thr308)/AKT,p-GSK-3β(Ser9)/GSK-3β,Tau,p-Tau(Thr231)and p-Tau(Ser396)protein in AD model cells were detected by Western blotting and to investigate the effect of estrogen receptor blocker ICI182780,PI3K/AKT signaling pathway blocker LY294002 and GSK-3β inhibitor Licl on naringin function,which determines whether naringin induced a decrease of hyperphorylation of Tau protein was mediated through ER-PI3K-AKT-GSK-3β signaling pathway.3.Effects of naringin on apoptosis factors of PC 12 Cells induced by Aβ25-35The cell apoptosis rate of AD cell model by naringin pretreatment was detected by Annexin V-FITC/PI double staining flow cytometry,The expression of Bax,Bcl-2 and Caspas-3 protein in AD model cells were detected by Western blotting,while the expression of apoptosis factor mRNA in AD model cells was detected by RT-qPCR,which determines whether the anti-apoptotic effect of naringin was mediated by ER signaling pathway.Results:1.Protective effect of naringin on PC 12 cells induced by Aβ25-35MTT results showed that 10-3mol/L naringin could significantly promote cell proliferation(P<0.05),(10-10-10-4mol/L)naringin had no significant effect on the proliferation of PC 12 cells compared with the blank group.RT-qPCR results showed that naringin could significantly promote the expression of ERβ mRNA(P<0.01),the above effects could be reversed by estrogen receptor blocker ICI182780,which indicated that the biological effect of naringin is mediated by ER.Morphological results showed that the cell viability of the Aβ25-35 group decreased,the cell debris increased,the cell connection decreased,some cells shrinked,the cell cytoplasm particles became more and the cell membrane was not translucent compared with the blank group.After pretreatment with naringin,cell viability was increased,cell debris was decreased,intercellular connection was increased,shrinkage cells was decreased,cell membrane was translucent.MTT results showed that the cell proliferation rate of the Aβ25-35 group was significantly decreased compared with the blank group(P<0.01);The cell proliferation rate of(10-8mol/L-10-6mol/L)naringin group was significantly increased compared with the Aβ25-35 group(P<0.01),The protective effect of naringin was dose dependent,and 10-6mol/L naringin had the strongest protective effect.The effect of naringin was consistent with estradiol.2.Protective mechanism of naringin on PC 12 cells induced by Aβ25-35MTT and Annexin V-FITC/PI double staining flow cytometry showed that the cell proliferation rate of the Aβ25-35 group was significantly decreased,while the cell apoptosis rate of the Aβ25-35 group was significantly increased compared the blank group(P<0.01).The cell proliferation rate of naringin group and LiCl group was significantly increased,while the cell apoptosis rate of those was significantly decreased compared with the Aβ25-35 group(P<0.01).The cell proliferation rate of ICI 182780+naringin+Aβ25-35 group and LY294002+naringin+Aβ25-35 group were significantly decreased,while the cell apoptosis rate of that was significantly increased(P<0.01).The effect of the above statements could be reversed by estrogen receptor blocker ICI182780,PI3K/AKT signaling pathway blocker LY294007 and GSK-3β inhibitor Licl,which indicated the protective effect of naringin was mediated by activating the ER signaling pathway,the PI3K/AKT signaling pathway and inhibiting the activity of GSK-3β.The effect of naringin was consistent with estradiol.Western blot analysis showed that the expression of ERβ,p-AKT(Ser473)/AKT,p-A KT(Thr308)/AKT,p-GSK-3β(Ser9)/GSK-3β protein in Aβ25-35 group was significantly decreased,while the expression of p-Tau(Thr231)/Tau and p-Tau(Ser396)/Tau protein was significantly increased compared with the blank group(P<0.01);the expression ofERβ,p-AKT(Ser473)/AKT,p-AKT(Thr308)/AKT,p-GSK-3β(Ser9)/GSK-3β protein in naringin group was significantly increased,while the expression of p-Tau(Thr231)/Tau and p-Tau(Ser396)/Tau protein was significantly decreased compared with the Aβ25-35 group(P<0.01);the expression of ERβ,p-AKT(Ser473)/AKT,p-AKT(Thr308)/AKT,p-GS-3β(Ser9)/GSK-3β protein in ICI182780+naringin+Aβ25-35 group was significantly decreased,while the expression of p-Tau(Thr231)/Tau and p-Tau(Ser396)/Tau protein in ICI182780+naringin+Aβ25-35 group was significantly increased compared with naringin group(P<0.01);the expression of p-AKT(Ser473)/AKT,p-AKT(Thr308)/AKT,p-GSK-3β(Ser9)/GSK-3β protein in LY294002+naringin+Aβ25-35 group was significantly decreased,while the expression of p-Tau(Thr231)/Tau and p-Tau(Ser396)/Tau protein in LY294002+naringin+Aβ25-35 group was significantly increased compared with naringin group(P<0.01);the expression of p-GSK-3β(Ser9)/GSK-3β protein in LiCl group was significantly increased,while the expression of p-Tau(Thr231)/Tau and p-Tau(Ser396)/Ta u protein in Licl group was significantly decreased compared with Aβ25-35 group(P<0.01),which indicated naringin induced a decrease in the hyperphosphorylation of Tau protein was mediated by activating the ER signaling pathway,the PI3K/AKT signaling pathway and inhibiting the activity of GSK-3β.The effect of naringin was consistent with estradiol.3.Effects of naringin on apoptosis factors of PC12 Cells induced by Aβ25-35Annexin V-FITC/PI double staining flow cytometry showed that the cell apoptosis rate of the Aβ25-35 group was significantly increased compared the blank group(P<0.01);the cell apoptosis rate of naringin group was significantly decreased compared with the Aβ25-35 group(P<0.01);the cell apoptosis rate of ICI182780+naringin+Aβ25-35 group was significantly increased compared with naringin group(P<0.01),which indicated the anti-apoptotic effect of naringin was mediated by activating the ER signaling pathway.The effect of naringin was consistent with estradiol.Western blot analysis showed that the expression of Bcl-2 protein in the Aβ25-35 group was significantly decreased,while the expression of Bax and Caspase-3 protein was significantly increased compared with the blank group(P<0.01);the expression of Bcl-2 protein in the naringin group was significantly increased,while the expression of Bax and Caspase-3 protein was significantly decreased compared with the Aβ25-35 group(P<0.01);the expression of Bcl-2 protein in ICI182780+naringin+Aβ25-35 group was significantly decreased,while the expression of Bax and Caspase-3 protein was significantly increased compared with the naringin group(P<0.01).The effect of naringin on the expression of Caspase-3,Bax and Bcl-2 mRNA was consistent with the protein results,which indicated the anti-apoptotic effect of naringin was mediated by activating the ER signaling pathway.The effect of naringin was consistent with estradiol.Conclusion:Phytoestrogenic naringin can increase the activity of PC 12 cells induced by Aβ25-35 and the expression of ERβ,p-AKT and p-GSK-3β,thereby inhibiting the hyperphosphorylation of Tau protein,which was associated with ER and its downstream PI3K/AKT,GSK-3β signaling pathway,and increasing the expression of anti-apoptotic gene Bcl-2 and decreasing the expression of pro-apoptotic gene Bax and Caspase-3 could play an anti-apoptotic effect,whcih was mediated through ER signaling pathway. |
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