| Objective:To investigate the inhibition effect of?7 nAChRs activation on?-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.A?1-42 oligomer was prepared by using chemically synthetic A?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,?7 nAChRs agonists?Nicotine,S24795,PNU282987?,?7nAChRs blocker?MLA?,?7 nAChRs agonists in combination with?7 nAChRs blocker,PI3K/Akt signaling pathway inhibitor LY294002,?7 nAChRs agonists combined with LY294002 groups and A?,agonists+A?,LY294002+A?groups.control group did not any treat.The?7 nAChRs agonist group was treated with 5?M of three different agonists for 6,12,18 and 24 h,and?7 nAChRs blocker group was added with 0.1?M of MLA to treat,then MLA combined with?7 nAChRs agonists?Nicotine,S24795 and PNU282987?treatment groups were pretreated with concentration of 0.1 or 0.05,0.1 and 0.2?M for 2 h,and then incubated with 5?M?7nAChRs 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 A?in 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 with?7nAChRs agonists Nicotine,S24795 and PNU282987 that HSP70 and HSF1 in the?7nAChRs blocker MLA combination agonist group or?7 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 with?7 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 with?7 nAChRs blocker MLA or LY294002,compared with the agonist group?Nicotine,S24795,PNU282987?.Compared with the control group,the expression of A?of?7 nAChRs agonist?Nicotine,S24795,PNU282987?group was higher?P<0.05?;compared with the A?group,the?7 nAChRs agonist?Nicotine,S24795,PNU282987?group of expression of A?was significantly lower?P<0.05?;and the agonist?Nicotine,S24795,PNU282987?group of expression of A?was significantly lower than the blocking agent group?P<0.05?.Conclusion:In the primary astrocyte model,?7 nAChRs agonists Nicotine,S24795 and PNU282987 can inhibit A?accumulation by activating?7 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. |
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