Aluminum Induced Neuroblastoma Cell Death And Its Intervention

Objective Aluminum is known as a kind of neurological toxin,it is related to kinds of degenerative diseases if being chronically exposed in a long-term lifespan.Processing cell death is one of the most important features of neural cells degeneration,a lot of studies have shown that aluminum might induce neural cells death.To investigate the mode of cell death induced by aluminum and effect of inhibition,the present study focus on the types of cell death in SH-SY5Y cells,a kind of common used model of mixed neural cells,and the effects of inhibitions. Therefore,the first aim of the present study is to investigate the cell death types in SH-SY5Y cells induced by aluminum.Based on this,the next aim is to inhibit apoptosis by RNA interference to apoptotic related genes:bak and caspase-3.Besides,Necroptosis is a type of novel cell death pathway apart from apoptosis and necrosis,which was found in ischemic brain injury by Prof.Jun Yingyuan's group in 2005.To probe whether necroptosis exists in neural cell death induced by aluminum and effect of its specific inhibitor,Nec-1,are the third aim of the study.Concerning interaction of the cell death types and combined effect of the inhibitors,there comes the forth aim of the present work.Finally,to investigate the role of neuroglias on aluminum induced neural cells apoptosis and RNA interference effect on specific bax gene are the fifth aim of the research.Methodsâ‘ To find out the mode of cell death induced by aluminum,SH-SY5Y cells were cultured in vitro and treated with 0-8mM AlCl₃·6H₂O,types of cell death were detected 24h after treatment.The cell status was observed under a phase contrast microscope,apoptosis and necrosis were shown by AO-EB fluorescent double staining and Hoechst sigle staining respectively.Furthermore,flow cytometry by Annexin V-PI double staining was used to quantify the apoptotic and necrotic rates,expressions of apoptotic related proteins NF-κB and Cty-c were detected by immunohistochemistry.Finally,ultrastructure images of transmission electronic microscope were used to confirme the cell death types of aluminum treated SH-SY5Y cells.â‘¡According to the necrotic like morphous and programmed cell death characteristics,neuroptosis was recognized in SH-SY5Y cells,which were incubated with various concentrations of aluminum and/or Nec-1.Morphous of aluminum treated SH-SY5Y cells was observed under a phase contrast microscope,apoptosis and necrosis were shown by Hoechst-PI double staining,cytometry was used to quantify cell viability, mitochondria membrane potential(MMP),content of reactive oxygen species(ROS),and apoptotic rate/necrotic rate.Autophogosomes were imagined in electronic microscope photographs.â‘¢To specific decrease apoptotic rate,3 siRNA sequences were designed for inhibition of bak and caspase-3 genes respectively,which were transfected into SH-SY5Y cells. Cell viabilities on siRNAs,concentrations of transfected reagent,time courses of transfection were assayed by CCK-8 method.Interference rates of genes were detected by QRT-PCR, inhibition on proteins was measured by immunohistochemistry,apoptotic rate and necrostic rate were quantified by flow cytometer.â‘£To inhibite apoptosis and necrosis simultaneously,bak RNAi and caspase-3 RNAi were performed to reduce the apoptotic rate and Nec-1 was used to decline the necrosis rate,LC3 expression was detected by western blot after performation of bak RNAi,caspase-3 RNAi and/or Nec-1 treatment.⑤To investigate the role of neuroglias on aluminum induced apoptosis,bax RNA interference was performed on gliocytoma cells,cell viability was detected by CCK-8 kit on different siRNA sequences,various transfection concentrations,and diverse transfection courses.Transfection efficiency was determined by percentage of CY3 fluorescent staining cells in DAPI numbered cells,and interference efficiency was measured by QRT-PCR,immunohistochemistry was used to quantify protein content. Besides,flow cytometry was used to measure apoptotic rate and necrotic rate in aluminum treated SH-SY5Y cells transfected by the selected bax siRNA.Resultsâ‘ The co-exist status of apoptosis and necrosis was present by AO-EB fluorescent staining and Hoechst staining, expression of apoptotic related protein Cty-c was significantly upregulated with aluminum concentration increasing(P＜0.05),while which of apoptotic preventive protein NF-κB downregulated(P＜0.05,P＜0.01).Apoptotic rate and necrotic rate detected by flow cytometer quantified their significant upregulation(P＜0.05,P＜0.01).Finally,ultrastracture in aluminum treated SH-SY5Y cells confirmed the apoptosis and autophagy features.â‘¡Nec-1 ameliorated the necrotic-like cell morphology,enhanced cell viability(P＜0.05,P＜0.01),increased cell MMP (P＜0.05,P＜0.01),and retained ROS content in aluminum treated SH-SY5Y cells(P＞0.05). Besides,Nec-1 could significantly decrease the necrotic rate(P＜0.05,P＜0.01),while it has no significant effect on apoptotic rate(P＞0.05).â‘¢Based on the viabilities and gene inhibitive rates of bak siRNA sequences,siRNA1 was selected as the optimal siRNA sequence,the optimal transfection concentration was 10nmol/L,and the optimal time course was 24 h after transfection. The transfection efficiency was above 90%,the interference efficiency was 57.76%,significant inhibitive effect on Bak protein was found as well(P＜0.05).Furthermore,the apoptotic rate in aluminum treated SH-SY5Y cells were significantly decreased by bak siRNA1 transfection (P＜0.05),while there was no significant effect on necrotic rate(P＞0.05).Similarly,based on the viabilities and gene inhibitive rates of caspase-3 siRNA sequences,siRNA1 was selected as the effective sequence,the optimal transfect concentration was 10nmol/L,the optimal transfection time was 48h after transfection.Furthermore,the transfection efficiency was 93%,the interference efficiency was 63.02%,and there was also a significant difference in the expression of Caspase-3 protein among the various siRNA transfection concentrations(P＜0.05).â‘£Results of LC3 expression in bak RNAi,caspase-3 RNAi and/or Nec-1 treatment shown that there was interaction among apoptosis,necroptosis and autophagy,bak RNAi and caspase-3 RNAi could inhibite cell apoptosis while LC3 marked autophagy upregulated(P＜0.05).However,Nec-1 could reduce not only necrosis(P＜0.05,P＜0.01) but also LC3 expression of autophagy(P＞0.05), thereafter,it could decrease sequenced apoptotic rate through cell signal transfer.The coeffect of Nec-1 and bak siRNA or caspase-3 siRNA shown that LC3 expression in coeffect cells was lower than that in Nec-1 treated alone cells,but higher than those in bak siRNA or caspase-3 siRNA treatment alone cells(P＜0.05,P＜0.01).There was significant increase in cell viability of aluminum treated cells(P＜0.05,P＜0.01),and significant decrease in apoptotic rate and necrotic rate(P＜0.05,P＜0.01).⑤The optimal bax siRNA sequence was selected based on the cell viability and the gene inhibitive rate.The transfection efficiency was above 90%,the interference efficiency of bax gene was 62.3%,and the optimal transfection time was 72h after transfection, the optimal transfection concentration was 20nmol/L,there was a significant decrease in Bax protein content after transfection(P＜0.05).Besides,apoptotic rate was significantly decreased (P＜0.05),while necrotic rate showed no significant difference after transfection(P＞0.05). Conclusion Apart from apoptosis,necrosis and autophagy,there is necroptosis in aluminum induced cell death.Apoptosis is one of the major cell death pathways in SH-SY5Y cells induced by aluminum,it can be specitifically reduced by transfection of bak and caspase-3 siRNAs. When chemically synthesized siRNAs are inducted to neural cells,it can significantly reduce bak and caspase-3 gene level,decrease Bak and Caspase-3 protein expression.Nec-1,as a specific inhibitor of necroptosis,can effectively block the necroptosis induced by aluminum,it indicates that necroptosis plays an important role in aluminum induced cell death.The cell viability could be enhanced significantly by caspase-3 siRNA combined Nec-1,besides apoptotic rate and necrotic rate decrease simultaneously.The present study also hints that neuroglia cells play a major role in aluminum induced neuron injury.Being mass number in nervous system and extraordinary resistence to aluminum toxicity,the neuroglia cells may be another target in performing neuron protection.