ABSTRACT
Objective:To investigate the role of down-regulating serine racemase (SRR) in alleviating the β-amyloid peptide (Aβ) induced neurotoxicity and synaptic damage and possible mechanism in PC12 cells.Methods:(1) PC12 cells cultured in vitro were divided into 0, 20, 40 and 80 μmol/L Aβ 25-35 treatment groups; they were treated with 0, 20, 40 and 80 μmol/L Aβ 25-35 for 24 h, respectively; cell counting kit (CCK)-8 was used to detect the survival rate of cells in each group, and Western blotting was used to detect the SRR protein expression. PC12 cells were treated with 40 μmol/L Aβ 25-35 for 0, 12, 24 and 48 h, respectively; cell survival and SRR protein expression were detected by CCK-8 and Western blotting, respectively. (2) PC12 cells were divided into control group, nonsense sequence group, SRR small interfering RNA (siRNA) group 1, SRR siRNA group 2, and SRR siRNA group 3; cells in the later three groups were transfected with SRR nonsense sequence or different SRR siRNA sequences, respectively; 48 h after that, Western blotting was used to detect the SRR protein expression of cells in each group, and SRR siRNA with best effect was selected for subsequent experiments. (3) PC12 cells were divided into control group, AD group, AD+nonsense sequence group, and AD+SRR siRNA group; cells in the latter two groups were transfected with nonsense sequence or SRR siRNA for 48 h, respectively; cells in the latter three groups were added 40 μmol/L Aβ 25-35, and cells in the control group were added same amount of solvent; 24 h after treatment, the SRR protein expression was detected by Western blotting, cell survival was detected by CCK-8, cell apoptosis was detected by Hoechst 33258 fluorescent staining, Caspase 3 activity was detected by enzyme linked immunosorbent assay, and the expressions of activated Caspase 3, N-methyl- D aspartate (NMDA) receptor-associated proteins and postsynaptic dense protein 95 (PSD95) were detected by Western blotting. Results:(1) The survival rate of cells in 0, 20, 40 and 80 μmol/L Aβ 25-35 treatment groups was successively decreased and the SRR protein expression was successively increased, with significant differences ( P<0.05); PC12 cells treated with 40 μmol/L Aβ 25-35 for 0, 12, 24 and 48 h had successively decreased survival rate and successively increased SRR protein expression, with significant differences ( P<0.05). (2) The SRR protein expressions in the SRR siRNA group 1, SRR siRNA group 2 and SRR siRNA3 group 3 were significantly decreased as compared with those in the control group and nonsense sequence group ( P<0.05), and the decrease in the SRR siRNA group 2 was the most obvious. (3) As compared with the control group, the cells in the AD group had significantly increased SRR protein expression and apoptosis rate, statistically decreased cell survival rate, significantly increased Caspase 3 activity and activated Caspase 3 protein expression, significantly increased protein expressions of NMDA receptor 2A (NMDAR2A) and NMDA receptor 2B(NMDAR2B), and statistically decreased PSD95 protein expression ( P<0.05); as compared with cells in the AD group, cells in the AD+SRR siRNA group had significantly decreased SRR protein expression and apoptosis rate, statistically increased cell survival rate, significantly decreased Caspase 3 activity and activated Caspase 3 protein expression, significantly decreased NMDAR2A protein expression, and statistically increased PSD95 protein expression ( P<0.05). Conclusion:Down-regulation of SRR expression can reduce the NMDAR2A protein expression, alleviate the over-activation of NMDA receptor, reduce the cell apoptosis, improve cell survival rate, protect nerve cells, increase PSD95 protein expression, and alleviate synaptic damage in PC12 cells.
ABSTRACT
Objective To study the role ofTAK-242,a Toll-like receptor 4 (TLR4) specific inhibitor,in β-amyloid peptide (Aβ)25-35 inducing PC12 cytotoxicity and its potential mechanism.Methods PC12 cells were cultured with different concentrations of Aβ25-35 (0,10,20 and 30 μmol/L) for 24 h,and then,the cell survival rate was detected by CCK-8 kit to choose the specific concentration of Aβ25-35 to establish cell AD models.The survival rate of Aβ25-35 inducing PC12 cells was further detected one h after TAK-242 intervention.The PC12 cells were divided into four groups:control group,Aβ treatment group,Aβ+TAK-242 pretreatment group and TAK-242 group.The apoptosis of cells was observed with Hoechst 33258 kit.The secretions of interleukin (IL)-1β and tumor necrosis factor-α (TNF-α) were detected with ELISA.The protein expression levels of TLR4,myeloid differentiation factor 88 (MyD88),IκB kinase complexus α/β (IKKα/β) and nuclear factor (NF)-κB were detected by Western blotting.Results The cell survival rate decreased gradually with the increase of Aβ25-35 concentrations after PC12 cells cultured with Aβ25-35 for 1 h.Twentyμmol/L Aβ25-35 was used to establish the AD models,with which the cell survival rate was closely half of the control group.As compared with Aβ treatment group,Aβ+TAK-242 pretreatment group had significantly increased cell survival rate and significantly decreased apoptosis (P＜0.05).The secretions of IL-1β and TNF-α in Aβ treatment group were significantly increased than those in the control group (P＜0.05),and Aβ+TAK-242 pretreatment group had significantly decreased secretions of IL-1β and TNF-α (P＜0.05).As compared with those in the control group,the protein expressions of TLR4,MyD88,IKKα/β and NF-κB in the Aβ treatment group were significantly increased (P＜0.05);as compared with Aβ treatment group,the protein expressions of TLR4,MyD88,IKKα/β and NF-κB in the Aβ+TAK-242 pretreatment group were degraded obviously,with significant differences (P＜0.05).Conclusions Aβ25-35 could reduce the cell survival rate and apoptosis in PC12 cells by up-regulating the expressions of TLR4/MyD88 signal pathway related proteins and increasing the secretions of IL-1β and TNF-α,and the phenomenon is concentration-dependent.TAK-242 could resist Aβ25-35-induced PC12 cytotoxicity through down-regulating the TLR4/MyD88 signal pathway related proteins levels and decreasing the secretions of TNF-α and IL-1β.