Cl^- Channel Blockers Prohibits PC12 Cells From Apoptosis Induced By Aβ_25-35 And The Mechanism Of The Protection

BackgroundAlzheimer's disease (AD) is a neurodegenerative disorder clinically characterized by progressive loss of memory. Common pathological features include senile plaques, neurofibrillary tangles and neuronal loss in brain regions involved in learning and memory.The accumulation of amyloidβpeptides(Aβ) in forming fibrillar deposits is a principal component of senile plaques. Aβ-induced neurotoxicity leads to cell necrosis and apoptosis. The aggregation of Aβplays an important role in the development of AD.But, the molecular mechanisms of Aβ-induced neuronal cell death is unclear.Membrane ion channels are essential for they play an important role in fundamental cellular functions.To be the main ion inside the cell, Cl–play a important role in the regulation of the cell volume and apoptosis.The relationships between chloride channels and apoptosis become a hot spot. Many researches manifested the upregulation of Cl- channels induced the efflux of Cl-, the apoptotic volume decrease,and the activation of apoptosic related protein such as cytochromec and caspase3.The Cl- channels blockers could inhibit the efflux of Cl^-, the activation of cytochromec and caspase3, as a result , The Cl- channels blockers protected the cells from apoptosis.Purpose1. To study the effects of the release of cytochromec and the activation of caspase3 on the Aβ_25-35 cellular toxicity2. To study the influence of DIDS on the apoptosis of PC12 cells induced by Aβ_25-35..MethodsIn this study , the rat adrenal glandin medullosuprarenoma cells(PC12 cells) were investigated. we divided PC12 cells into 3 groups: the control group ,the Aβ_25-35 group﹙PC12 cells were treated with 40μmol/L Aβ_25-35﹚,the protective group with DIDS﹙PC12 cells were treated with both 40μmol/L Aβ_25-35 and 50μmol/L DIDS﹚.MTT assay was used to detect the cellular viability; Hoechst 33258 fluorescence staining was used to observe the cellular morphous ; Flow cytometric analysis was used to detect the early apoptosis; In order to identify the mechanism of DIDS protection, western blot was used to detect the expression of cytochromec and caspase3.Results1. The first part of this study validated Aβ_25-35 induced PC12 cells apoptosis .⑴The cellular viability decreased in a time dependence after PC12 cells were treated with 40μmol/LAβ_25-35.⑵The cell nucleolus in Aβ_25-35 group exhibited typical apoptosis which were strong bluestaining and contained many broken bits formed by DNA after staining with Hoechst 33258.⑶The results of flow cytometric analysis manifested that the early apoptosis of PC12 cells in the Aβgroup was 35.7%±6.1%,and was 1.5%±0.5% in the control group.Compared with control group, Aβ_25-35 could increase the early apoptosis of PC12 cells significantly, P <0.05.2. The second part of this study validated that DIDS had a protective influence on PC12 cells apoptosis induced by Aβ_25-35.⑴PC12 cells were treated with 10μmol/LDIDS+40μmol/LAβ_25-35,30μmol/LDIDS+40μmol/LAβ_25-35 and 50μmol/L DIDS+40μmol/LAβ_25-35. DIDS can protect PC12 cells from apoptosis in a dose dependence by MTT assay.⑵The results of Hoechst 33258 staining manifested DIDS could protect PC12 cells from apoptosis morphologically;The results of flow cytometric analysis manifested that the early apoptosis in the DIDS group was 5.6%±1.0%. Compared with the Aβ_25-35 group,the early apoptosis in the DIDS group shew a significant diffirence, P>0.05.⑶The results of western blot were: the expression of the cytochromec in the Aβ_25-35 group increased after 6h.There was no significant expression of the cytochromec in the control group and the DIDS group; the expression of the caspase3 in the Aβ_25-35 group increased after 12h..There was no significant expression of the caspase3 in the control group and the DIDS group.ConclusionsIn conclusion , our experiments confirmed that :1. Aβ_25-35 induced the apoptosis of PC12 cells by the injury of the mitochondria, in other words, Aβ_25-35 induced the apoptosis of PC12 cells by releasing the cytochromec and activating the caspase3. 2. DIDS attenuated the Aβ_25-35-induced apoptosis of PC12 cells by inhibiting the release of cytochromec and the activation of caspase3.