Study Of The Protection And Mechanism Of Insulin-like Growth Factor-1 In PC12 Cells Induced By Aβ_25-35

Alzheimer disease is the most neuro-degenerative disease.The causes and mechanisms of AD are very complicated. At present, Aβcan lead to AD through neurotoxicity caused by mechanism of cell apoptosis and phosphorylation of tau protein .so major scholars deem Aβplays an important role in the pathogenesis of AD.In recent years, the effect of insulin like growth factor-1 on cognition function was being payed close attention. In this paper, Aβ_25-35, as an inductor, acted on PC12 cells to make cell model of AD. Apoptosis and phosphorylation of tau protein of Aβ_25-35were studied. This paper introduced IGF-1 into AD cell model, The aim of this study is to elucidate the p rotective and anti-apop totic effects of IGF-1 againstβ-amyloid (Aβ) and investigate the effect of IGF-1 on Aβ-induced tau phosphorylation.And mechanisms of IGF-1 on anti-apoptosis were investigated through discussing the relationship betweenIGF-1 and expression of NF-κB, bcl-2 mRNA and protein, in order to supply theory basis for exploring pathogenesis and exploiting new drugs of AD.1. Set up AD cell model and the anti-apoptosis effect of IGF-1 on PC12 cells induced by Aβ_25-351.1 set up AD cell modelEffect of Aβdepends on state of peptide aggregation and concentration of protein, and its toxic segment is a peptide of _25-35 amino acid. Therefore, Aβ_25-35 induced PC12 cells to set up cell model of AD.In this study, the relationship between time and effect, the relationship between dosage and effect were analyzed by MTT, Aβ_25-35 at concentration of 30μmol·L^-1 induces PC12 cells for 36 hours to develop an ideal AD cell model.1.2 Study on cell apoptosis in PC12 cells induced by Aβ_25-35 and the effects of IGF^-1By means of HE staining, the flow cytometry, electron microscope and DNA fragment, cell apoptosis form of PC12 cells induced by Aβ_25-35 was observed. in order to observe the effects of inhibitor apoptosis pretreatment with 1.0μg/ml IGF^-1 .Results: Numbers of apoptosis PC12 cells increased, DNA ladder could be seen in PC12 cells in the group of Aβ_25-35; nucleolus concentrate, cataclasm, vacuole inform in electron microscope numbers of apoptosis PC12 cells in the pre-protection group with IGF^-1decreased significantly, DNA ladder couldn't be seen in PC12 cells with IGF^-1 by DNA electrophoresis,Conclusion: Aβ_25-35 induced PC12 cells apoptosis and IGF^-1 can inhibit cell apoptosis.2. The mechanism of Aβwas used to induce the apoptosis of PC12 cell and the protective effects of IGF^-1Western-Blot analysis showed that the level of phospho-GSK-3βSer9 and Akt Ser473 phoryphoslation level in PC12 cells induced by Aβ_25-35. in order to observe whether IGF^-1 can protect neuron through inhibiting the GSK-3βactivity or through PI3-K/Akt signal transduction pathway. western blot was performed on PC12 cells exposed to 30μmol/L Aβ_25-35 in the presence or absence of 36-hour pretreatment with 1.0μg/ml IGF^-1, 10mmol/L LiCI or Ly294002,1.0μg/ml IGF^-1. Results: Akt Ser473 and GSK-3βwere inhibited in group of Aβ_25-35. IGF-1 inhibited Akt and induce GSK-3β, and IGF-1 showed the reverse effect. and more, while IGF-1 activated Akt, the activity of GSK-3βwas inhibited.Conclusion: Aβ_25-35 induced PC12 cells apoptosis through inhibiting Akt, then activating GSK-3β. on the contrast, IGF-1 exerted its protection by activating Akt and inhibiting GSK-3βin turn.3. The mechanism of aggregated Aβ_25-35 on the level of tau protein phosphorylation in PC12 cells, and the protective effects of IGF-1 on Aβ_25-35-induced tau prote in hyperphosporylation.In order to detect the levels of tau phosphorylation, total tau protein and GSK-3β,western blot was performed on PC12 cells exposed to 30μmol/L Aβ_25-35 in the presence or absence of 36-hour pretreatment with 1.0μg/ml IGF-1, 10mmol/L LiCI or Ly294002, a specific inhibitor of PI3-K/Akt.Results: Aβ_25-35 (30μmol/L) exposure of PC12 cells for various periods induced tau protein hyperphorylation. The levels of tau protein phosphorylation in the sites of Ser396, Ser202 and the amount of whole tau increased after 3hr exposure and reached the maximum level after 12hr exposure, then gradually declined after 12 hr exposure. Meanwhile, the expression so ft he amount of whole GSK-3βand activated GSK-3βwere also increased after Aβ_25-35 exposure of PC12 cells for 12h. Pretreatment with 1.0μg/ml IGF-1 or 10mmol/L LiCl, markedly attenuated Aβ_25-35-induced tau hyperphosphorylation and the expression of GSK-3β. on the contrast, Ly294002 reduced the protection of IGF-1 and enhance tau protein phosphoralation, the activity of GSK-3βwas promoted.Conclusion: GSK-3βactivation by Aβ_25-35 may lead to extensive tau phosphorylation. IGF-1 can attenuate Aβ_25-35-induced tau protein hyperphosphorylation by inhibiting the activation of GSK-3β, the protective mechanism of inhibited tau phosphorylation through PI-3K/Akt signal transduction pathway.4. Expression of NF-κB, bcl-2 mRNA and protein in the PC12 cells induced Aβ_25-35 and effects of IGF-1 on themIn this paper, mRNA expression of NF-κB P65 and Bcl-2 were detected by RT-PCR, protein expression of NF-κB P65 were detected by Western blot.Results: Aβ_25-35 could increase the protein and mRNA expressions of NF-κB and decrease the protein and mRNA expressions of Bcl-2; IGF-1 could decrease the protein and mRNA expressions of NF-κB and increase the protein and mRNA expressions of Bcl-2.Conclusion: Aβ_25-35 could induce cell apoptosis of PC12 cells by increase NF-κB and decrease Bcl-2; IGF-1 could inhibit activation of NF-κB and activation of Bcl-2 to protect PC12 cells.