Neurotoxicity Effect Of β-Amyloid And Microglia In Vitro

[Objectives] To observe the effect of the inflammatory interleukine-lp (IL-1P) on the cytotoxity of Ap and gene expression of amyloid precursor protein(APP). Investigated the relationship between Ap and microglia activation. We also examined the inhibitory effects of indomethacin on the cytotoxity and microglia activation of AP in vitro. To explore the role of p-amyloid (Ap) and microglia (MG) in the pathogenesis of Alzheimer's disease (AD), and that anti-inflammatory drugs might be an effective form therapy.[Materials and methods] Incubation of differentiated rat pheochromocytoma(PC12)cells with Ap with or without IL-lp. PC12 cell survival rate was assayed by MTT assay. APP mRNA expression was detected by RT-PCR. We also observed the morphological changes of cultured murine microglia BV-2 cells after treatment with Ap. The survival rate of BV-2 cells was assayed by MTT assay. In this study, we examined IL-lp mRNA expression and release of proinflammatory cytokines IL-lp and IL-6 by BV-2 cells in response to Ap. IL-lp and IL-6 was examined by radioimmunoassay, IL-lp mRNA expression was analyzed by RT-PCR. The production of nitric oxide (NO) and the activity of inducible nitric oxide synthase (iNOS) were also studied in AP stimulated BV-2 microglia. iNOS mRNA expression was detected by RT-PCR, iNOS protein expression was examined by Western Blot, flow cytometry and immunocytochemistry. We also explored the inhibitory effects of indomethacin on the cytotoxity of Ap and the production of IL-lp and NO in Ap-stimulated BV-2 microglia.[Results]1. Apl-42 and AP25-35 showed similar direct cytotoxity in PC12 cells. The cytotoxity of AP is in a dose and time dependent manner, and correlated with aggregation.2. Coincubation with IL-lp reduced viability of baseline, IL-lp can increase APP mRNA expression and this enhancement is time and dose dependent.3. At the doses studied, neither peptide (Apl-42 and AP25-35) had toxicity in BV-2 cells. BV-2 cells changed their morphology after treatment with Apl-42 in low doses, in correlation with the aggregation of peptides. AP25-35 did not have any effect on the morphology of BV-2 cells.4. Both peptides (Apl-42 and AP25-35) can induce the gene expression and secretion of JL-lp in a dose and time dependent manner, but do not trigger the production of IL-6. The aggregation of peptides was not necessary for the induction.5. NO production was also induced by Apl-42 in a dose and time dependent manner, but not AP25-35. iNOS maybe the main source of NO production.6. Apl-42 can increased NO production, iNOS activity, iNOS mRNA and protein expression in a dose and time dependent manner in BV-2 microglia, but not Ap25-35.7. Indomethacin can inhibit the toxicity of Ap and block IL-1 enhancement of APP gene expression in PC12 cells. Moreover, Indomethacin suppressed IL-lp, iNOS mRNA and protein expression in Apl-42-stimulated BV-2 microglia. [Conclusions]1. Ap have direct and indirect neurotoxic effect in vitro. On exposure to AP microglia exhibit morphological changes, secrete proinflammatory cytokines IL-lpand NO, which may trigger pathways of inflammatory activation and neuronal degeneration in Alzheimer's disease.2. As conventional non-steroidal anti-inflammatory drugs (NSAIDs), indomethacin can inhibit the toxicity of A P , suppress the secretion of IL-lp and NO in Ap-stimulated microglia, thus block the inflammatory cascade reaction and reduce injury of neuron.3. As an effective model in vitro, BV-2 microglia has important value in the study of Alzheimer's disease.