Mechanism Study Of Primary Astrocyte α7 NAChRs Activating PI3K/Akt Signaling Pathway To Regulate HSP70 And Inhibit Aβ Oligomer Aggregation

Objective:To investigate the inhibition effect ofα7 nAChRs activation onβ-amyloid aggregation and its underlying molecular mechanisms in Alzheimer’s disease.Methods:The rat astrocytes were cultured from the cortex and hippocampus of the brains of newborn Sprague-Dawley（SD）rats.The cells were demonstrated purity by immunocytochemi-cal staining with anti–GFAP antibody after passaging for three or four times.Aβ_1-42 oligomer was prepared by using chemically synthetic Aβ_1-42 polypeptide in vitro and then identified by western blotting.Astrocytes were substantially matured when the cells were cultured for 21-29 days,at which time divided into control,α7 nAChRs agonists（Nicotine,S24795,PNU282987）,α7nAChRs blocker（MLA）,α7 nAChRs agonists in combination withα7 nAChRs blocker,PI3K/Akt signaling pathway inhibitor LY294002,α7 nAChRs agonists combined with LY294002 groups and Aβ,agonists+Aβ,LY294002+Aβgroups.control group did not any treat.Theα7 nAChRs agonist group was treated with 5μM of three different agonists for 6,12,18 and 24 h,andα7 nAChRs blocker group was added with 0.1μM of MLA to treat,then MLA combined withα7 nAChRs agonists（Nicotine,S24795 and PNU282987）treatment groups were pretreated with concentration of 0.1 or 0.05,0.1 and 0.2μM for 2 h,and then incubated with 5μMα7nAChRs agonist for 18 hours.The total protein of the cells was collected and measured,and there were proteins of phosphorylated Akt,heat shock protein 70（HSP70）,heat shock transcription factor 1（HSF1）and Aβin the astrocytes of the above groups were detected by Western blotting（WB）.Results:The astrocytes were identified by Immunofluorescence and the results showed that astrocytes accounted for more than 98%of the total cells.Our results suggested that HSP70 and HSF1 in the agonist treatment group showed an increasing trend（P<0.05）after treating with Nicotine,S24795 and PNU282987.However,there was no significant difference from control group in the MLA group or LY294002 group.Compared withα7nAChRs agonists Nicotine,S24795 and PNU282987 that HSP70 and HSF1 in theα7nAChRs blocker MLA combination agonist group orα7 nAChRs agonist combined with LY294002 showed a downregulated trend（P<0.05）.The phosphorylated Akt protein expression demonstrated that the expression of phosphorylated Akt increased over time,which was most significant at 20 min after treated withα7 nAChRs agonist（Nicotine,S24795,PNU282987）for 0,5,10,20 and 30 min compared with the blank control group.Meanwhile,We found that phosphorylated Akt expression was decreased in the blocker group（MLA,LY294002）（P<0.05）treated withα7 nAChRs blocker MLA or LY294002,compared with the agonist group（Nicotine,S24795,PNU282987）.Compared with the control group,the expression of Aβofα7 nAChRs agonist（Nicotine,S24795,PNU282987）group was higher（P<0.05）;compared with the Aβgroup,theα7 nAChRs agonist（Nicotine,S24795,PNU282987）group of expression of Aβwas significantly lower（P<0.05）;and the agonist（Nicotine,S24795,PNU282987）group of expression of Aβwas significantly lower than the blocking agent group（P<0.05）.Conclusion:In the primary astrocyte model,α7 nAChRs agonists Nicotine,S24795 and PNU282987 can inhibit Aβaccumulation by activatingα7 nAChRs to up-regulate HSP70 and HSF1.PI3K/Akt signaling pathway is probably crucial participatory factors in this process.Therefore,to further research into HSP70and HSF1 in the heat shock protein family may provide a fundamental study as treatment of a potential target for AD.