Effect Of α7 Nicotinic Acetylcholine Receptor On β-amyloid Peptide Aggregation In Cultured Astrocytes

Objective: To investigate the effect of activated α7 nicotinic acetylcholine receptor(n ACh R) on the aggregation of β-amyloid peptide(Aβ) in astrocytes. Methods: Primary cultured astrocytes were prepared from the cortex of the brains of newborn Sprague-Dawley(SD) rats. The purity of the passaged cells from the original generation of cultured astrocytes were demonstrated by immunocytochemical stainings with anti-GFAP antibody. Aβ oligomer was prepared by using chemically synthetic Aβ in vitro. The cultured astrocytes were divided into two class, i.e., the first including control, agonist, and agonist+agonist groups; and the second including control, Aβ, agonist+Aβ, and antagonist+agonist+Aβ groups. The agonists were nicotine, PNU and s24795; the antagonist was methyllycaconitine(MLA). The m RNA and protein levels of individual n ACh R subunits in the cultured astrocytes were examined by RT-PCR and Western blotting, respectively; the oligomers of Aβ were identified by western blotting and sliver staining. The astrocytes were treated with different concentrations of Aβ oligomer and the survival rates of the cells detected via Cell Counting Kit-8(CCK-8) assay. The expression of α7 n ACh R protein and the degradation of Aβ after activation of astrocytic α7 were detected by Western blotting. Results: Astrocytes were isolated and cultured successfully, and the purification of the cultured cells was above 95%, then can perform the later experiment. The protein and m RNA expressions of α2, α3, α4, α7 and β2 n ACh R subunits were found in the primary cultured astrocytes whlie the β3 was not detected. The major types of Aβ were dimer, trimer and nonamer in astrocytes besides of small amount of monomers(4 k Da) unaggregated. The neurotoxicity of Aβ oligomer on astroytes was significantly detected with a manner of dose-dependence. The results concerning α7 n ACh R protein expression were shown as follow.(1) As compared with the control group, the protein expression of α7 n ACh R in the nicotine group was obviously increased(P<0.01); in comparison with the nicotine group, the α7 n ACh R protein in the MLA+nicotine group was decreased(P<0.05).(2) In comparison with the control group, the α7 n ACh R protein expression in PNU group enhanced(P<0.05); in comparison with the PNU group, the α7 n ACh R in the MLA+PNU group decreased(P<0.05).(3) Compared with the control group, the α7 n ACh R expression of the s24795 group obviously increased(P<0.01); in comparison with the s24795 group, the α7 n ACh R protein expression in the MLA+s24795 group obviously decreased(P<0.01). The results concerning Aβ were shown as follow.(1) In the culture medium and cell lysis, respectively, as the reference to the control group without adding Aβ, Aβ in the nicotine+Aβ group was significantly lower compared with that in the Aβ group(P<0.01); Aβ in the MLA+nicotine+Aβ was significantly increased compared with that in the nicotine+Aβ group(P<0.01).(2) Collect the culture medium and cell lysis, respectively, for analysis as reference to the control group without adding Aβ, Aβ in the PNU+Aβ group was significantly lower compared with that in the Aβ group(P<0.01); Aβ in the MLA+PNU+Aβ group was significantly increased(P<0.05) compared with that in the PNU+Aβ group.(3) In the culture medium and cell lysis, respectively, as the reference to the control group without adding Aβ, Aβ in the s24795+Aβ group was significantly lower compared with that in the Aβ group(P<0.01); Aβ in the MLA+ s24795+Aβ was significantly increased compared with that in the s24795+Aβ group(P<0.01). Conclusion: Aβ oligomer has a obvious neurotoxic effect on astrocytes. There are expressions of multiple n ACh R subunits in the cells. The agonists used in the study can stimulate and upregulate the expression of α7 n ACh R. The increased expression of α7 n ACh R can degrade Aβ in astrocytes through the phagocytosis and secretion of related enzymes.