Effect Of ?7 Nicotinic Acetylcholine Receptor On ?-amyloid Peptide Aggregation In Cultured Astrocytes

Objective: To investigate the effect of activated ?7 nicotinic acetylcholine receptor(n ACh R) on the aggregation of ?-amyloid peptide(A?) 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. A? oligomer was prepared by using chemically synthetic A? 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, A?, agonist+A?, and antagonist+agonist+A? 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 A? were identified by western blotting and sliver staining. The astrocytes were treated with different concentrations of A? oligomer and the survival rates of the cells detected via Cell Counting Kit-8(CCK-8) assay. The expression of ?7 n ACh R protein and the degradation of A? after activation of astrocytic ?7 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 ?2, ?3, ?4, ?7 and ?2 n ACh R subunits were found in the primary cultured astrocytes whlie the ?3 was not detected. The major types of A? were dimer, trimer and nonamer in astrocytes besides of small amount of monomers(4 k Da) unaggregated. The neurotoxicity of A? oligomer on astroytes was significantly detected with a manner of dose-dependence. The results concerning ?7 n ACh R protein expression were shown as follow.(1) As compared with the control group, the protein expression of ?7 n ACh R in the nicotine group was obviously increased(P<0.01); in comparison with the nicotine group, the ?7 n ACh R protein in the MLA+nicotine group was decreased(P<0.05).(2) In comparison with the control group, the ?7 n ACh R protein expression in PNU group enhanced(P<0.05); in comparison with the PNU group, the ?7 n ACh R in the MLA+PNU group decreased(P<0.05).(3) Compared with the control group, the ?7 n ACh R expression of the s24795 group obviously increased(P<0.01); in comparison with the s24795 group, the ?7 n ACh R protein expression in the MLA+s24795 group obviously decreased(P<0.01). The results concerning A? were shown as follow.(1) In the culture medium and cell lysis, respectively, as the reference to the control group without adding A?, A? in the nicotine+A? group was significantly lower compared with that in the A? group(P<0.01); A? in the MLA+nicotine+A? was significantly increased compared with that in the nicotine+A? group(P<0.01).(2) Collect the culture medium and cell lysis, respectively, for analysis as reference to the control group without adding A?, A? in the PNU+A? group was significantly lower compared with that in the A? group(P<0.01); A? in the MLA+PNU+A? group was significantly increased(P<0.05) compared with that in the PNU+A? group.(3) In the culture medium and cell lysis, respectively, as the reference to the control group without adding A?, A? in the s24795+A? group was significantly lower compared with that in the A? group(P<0.01); A? in the MLA+ s24795+A? was significantly increased compared with that in the s24795+A? group(P<0.01). Conclusion: A? 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 ?7 n ACh R. The increased expression of ?7 n ACh R can degrade A? in astrocytes through the phagocytosis and secretion of related enzymes.