| Objective Alzheimer's disease (AD) is one of the most common neurodegenerative disorders. It is characterized by cognition obstacle and the cholinergic neuronal degeneration. Amyloidβ-peptide(Aβ) is the major constituent of the senile plaques in the brains of patients with AD. It relates with the neuronal loss by apoptosis pathway. Nerve growth factor (NGF) is a main member of the neurotrophin family for cholinergic neuron, which prevent cholinergic neuron from damage investigated in aminal model and cell culture,and have attracted considerable reseach for potential application in AD therapy. NGF mediate signaling with its high affinity receptor-tyrosin protein kinase TrkA, may relate with PI3K (phosphatidylinostol3'-OH kinase) /Akt and Ras-MAPK (mitogen-activated protein kinase) signaling pathway. The cause of the more AD are still uncertain. Acquired disturbances of cell metabolism in AD are supposed to indicate Aβmay play crucial roles. In this experiment, it is amyloidβ-peptide fragment 25-35(Aβ25-35) that induces apoptosis in the rat pheochromocytoma lines PC12 cells which have been widely used for the cell model of AD. The effects of NGF purificated from cobra venom on Aβ25-35 -induced in PC 12 cells were investigated . The application of inhibitors of extracellular- signal-regulated protein kinase(ERK) and PI3-K, detection of caspase-3 activity and Bcl-2 expression all were to explore the NGF and its signaling pathway of protect PC12 cells from Aβ25-35-induced apoptosis. The main aim of this study was to elicit the molecular mechanism of NGF protect Aβ25-35-induced nerodegeneration through signaling pathway which provides the experimental evidences for the clinical use of treatment in the neurodegenerative disorders.Methods (1)Isolation and purification: NGF was purified from the venom of cobra by successive column chromatography on Sephadex G-50 ,CM-sepharose FF cation exchang column and Sephacryl S-200 High Resolution column.The identific action of NGF fraction was assayed by PC12 cells, which the biological activity is based upon the neurite outgrowth when it is in the presence of active NGF.The molecular weight and PI of NGF were detected with SDS-PAGE and IEF-PAGE respectively. (2) In this study PC 12 cell line was served as an established model system for studying mechanisms of neuroapoptosis induced by Aβ25-35. To explore if NGF from cobra venom could have protective effect on AD cell models induced by Aβ25-35 in PC 12 cells. The PC12 cells were cultured, treated with Aβ25-35 or/and NGF and collected for apoptotic analysis. The cytotoxicity of Aβ25-35 was assessed by the MTT and LDH release assay. Morphological change was screened by fluorescence microscopy and DNA by agarose gel electrophoresis. The apoptosis ratio analyzed by flow cytometry. Compare the difference between each group. (3) PI3-K /Akt and Ras/ERK are the important signal transduction pathways of NGF receptor for the survival and differentiation of cells. To elucidate the regulations role of PI3-K and ERK1/2 in the protection of NGF against Aβ25-35-induced apoptosis in PC 12 cells, LY294002 and PD98059, the PI3-K and ERK1/2 inhibitors, were used. The activity of ERK1/2 and PI3K stimulated by NGF were detected with the phosphorylation of ERK1/2 and Akt respectively by Western Blot. Apoptosis ratio was confirmed by flow cytometric analysis when ERK1/2 or PI3K/ Akt pathway was abolished by the specific inhibitor PD98059 and LY294002 respectively .The catalytic activity of Capase-3 and expression of Bcl-2 were detected with Western Blot.Results (1) Cobra crub venom was applied on a Sephadex G-50 column and four fractions were pooled. Each fraction was tested for NGF activity, and of these, fractionsⅡcontained the NGF and was concentrated and subfractionated by CM-Sepharose FF cation exchange column and six fractions were pooled. Among these, FractionsⅡwas identified as NGF and was then passed through a Sephacryl S-200 High Resolution column. NGF was obtained and showed a single band on SDS-PAGE and IEF-PAGE respectively and with the molecular weight of 24.5 kD, and PI of 8.23. NGF from cobra venom had a nice bioactivity with PC12 cell differentiation test. The effect was in a dose- and time- dependence. (2) The cytotoxic effect of Aβ23-35 and the protection of NGF from cobra venom on PC12 cells was evaluated . MTT and LDH release assay were performed to observe the change of PC 12 cells viability when cells were exposed to the different concentrations of Aβ25-35. Compared with the control, the cell viability was decreasing with the concentration increasing of Aβ23-35, and at the concentration of 20μmol/L Aβ25-35 , with the concentration of NGF increasing, cell viability was increasing. Screened by fluorescence microscopy it was observed that treatment with 20μmol/L Aβ25-35 on PC12 cells for 48h induced cell morphological changes, such as condensed chromatin, ruptured nuclei and apoptotic body ,and DNA ladder by agarose gel electrophoresis was detected . Analyzed by flow cytometry, treatment with Aβ25-35 for 48h, apoptosis ratio in PC12 cells evidenced increased in a dose -dependent manner. The apoptocis ratio increased from 2.1±0.3%(control) to 18.6±3.4%,and when pretreated with 100ng/mLNGF, the apoptocis ratio decreased from 18.6±3.4% to 11.2±3.7%. These rusults indicated that efficiently induced apoptosis in PC12 cells by 20μmol/L Aβ25-35 and 100ng/mL NGF can partly protect PC 12 cells from apoptosis. (3) To confirm that against apoptosis by NGF from cobra venom occurs via the PI3K/Akt dependent pathway, LY294002 (PI3K inhibitor) was used. NGF from cobra venom can stimulate Akt phosphorylation in a dose and time dependent. When PC12 cells pre-incubated with LY294002, the degree of Akt phosphorylation decrease in a dose-dependence . NGF inhibited the effects of decrease Bcl-2 expression and increase the cleavage of caspase-3 by Aβ25-35. LY294002 significantly increase the apoptotic ratio of Aβ25-35 -induced cells protected by NGF and decrease Bcl-2 expression and increase the cleavage of caspase-3 .It seems that once the activation of PI3-k was blocked, the protection of NGF was discharged. (4) To confirm if the role of against apoptosis by NGF occurs via the Ras/ERK dependent pathway, PD98059 (ERK inhibitor) was used. NGF from cobra venom can stimulate ERK1/2 phosphorylation in a dose- and time-dependence. When PC12 cells pre-incubated with PD98059, the degree of ERK1/2 phosphorylation decrease in a dose dependence .The apoptotic rate of the PC12 cells induced by Aβ25-35 pretreatments with PD98059 plus NGF presented the same kind of changes as in the cells pretreated with NGF only. The similar results were obtained with Bcl-2 expression and cleavage of caspase-3 measuration.Conclusions (1)The purified NGF was obtained by simple and efficient method,and with the molecular weight of 24.5 kD,PI of 8.23. The biological activity of NGF is excellent with PC12 cells test. (2) The cytotoxity of Aβ25-35 is in a dose- and time- dependent manner to PC12 cells. Both the morphological changes and the apoptosis ratio analysis suggested that 20μmol/L Aβ25-35 efficiently induced apoptosis in PC12 cells. (3) NGF from cobra venom had a protective effect against Aβ25-35-induced apoptosis in the PC12 cells. . (5)LY294002—the specific inhibitor of PI3-K , could block the protection of NGF from cobra venom against Aβ25-35-induced apoptosis in PC 12 cell. PI3-K/Akt is an effective against apoptosis pathway in NGF-TrkA of PC12 cells induced by Aβ25-35, and inhibition of caspase-3 activity and increase of Bcl-2 expression were involved in. (4)PD98059—the specific inhibitor of ERK , could not block the protection of NGF. It seems that NGF prevents Aβ25-35-induced apoptosis in PC12 cells not through Ras-mediated ERK activation pathway.On the basis of the results obtained, we proposed that apoptosis of PC12 cells by Aβ25-35 occurs partly against by NGF via the PI3-K/Akt- dependent pathway signal cascade's activation, and inhibition of caspase-3 and increase of Bcl-2 expression were involved in. |
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