Caspase-3 Gene Expression In Aluminum - Induced Apoptosis And Intervention Research

Objective Through in vivo and in vitro experiment to study the neurotoxicity of aluminum,and explore the Caspase-3 gene in aluminum-induced apoptosis in the expression and intervention effect to inhibit the neurotoxicity of aluminum.Methods 1 Aluminum double-transgenic mice exposed to in vivo studies:1) The APP gene was amplified by PCR from the mice genome DNA; 2) The C57BL mice and APP/PS1 double transgenic mice were divided into 3 groups at random,i.e. C57BL control group APP/PSlnormal saline group and APP/PS1 Aluminum exposed group (5 mg/kg),8 for each group. AlC13 was injected into intraperitoneal injection for two monthes. The Morris water maze test was performed to test the learning and memory abilities of mice.3) The necrosis and apoptosis of hippocampus cells was measured by method of Annexin V-PI fluorescent double staining and terrminal-deoxynu-transferase mediated nickend labeling.4) The expression of Caspase-3 was detected by QRT-PCR.2 Aluminum neurotoxicity in vitro:1) Primary cultured nerve cells exposed to using AlC13·6H2O, at final concentrations:0 mM/L (control group),0.5 mM/L(low-dose group),1.0 mM/L(medium-dose group), and 2.0 mM/L(high-dose group) sepatately, then incubated for 24 hr.l) Morphologic characteristics were observed under light microscope, measured with AO-EB fluorescent staining.2) The cell viability of neurons was quantified by method of Cell Counting Kit-8.3) Annexin V-PI fluorescent double staining methods, and the early stage apoptosis rate, late stage apoptosis rate and the total one were detected with flow cytometry.4) The expression of Caspase-3 was detected by QRT-PCR.3 In the role of Caspase-3 RNAi in the aluminum-induced nerve cells:1) Ordered three Caspase-3 RNAi virus reagents,by detecting cell viability and inhibition efficency of Caspase-3 gene selection of effective Caspase-3 RNAi virus reagents intervention studies.2) The expression of Caspase-3 was detected by QRT-PCR.3) Morphologic characteristics were observed under light microscope, measured with AO-EB fluorescent staining and Annexin V-PI fluorescent double staining after RNA interference. The cell viability of neurons was quantified by method of Cell Counting Kit-8.Results:1 Aluminum double-transgenic mice exposed to in vivo studies:1) The APP/PS1 double transgenic mice were identified.2) The latent period of Morris water maze test were significantly increased(P<0.01) as compared with the control group.3) Using Annexin V-PI fluorescent double staining method and terrminal-deoxynu-transferase mediated nickend labeling method,compared with control group, the apoptosis ratios of neuron were measured, apoptosis ratio increased in the APP/PS1 Aluminum exposed group (P<0.01).4) With the dose of aluminum increasing, Caspase-3 increased, compared with normal saline, there existed sig-nificantly difference in the middle,high-dose group (P<0.01).2 Aluminum neurotoxicity in vitro:1) Nuclei shrinkage or decreament of neurocytes volume were noticed under light microscope, quantity of neurite and dendritic reduced with the rising aluminum concentration. Under fluorescence light, alive neurocytes showed a bright green color in the cell body, while did red in the dead, so that we can distinguish living conditions of the neurocytes.2) With the dose of aluminum increasing, the cell viability of neurons decreased. Compared with normal saline, there existed significantly difference in all the groups (P<0.01).3) Apoptosis rate examination showed the higher early stage, late stage and total apoptosis rates of neurocytes after aluminum treatment, which were confirmed to be dose-dependent (P<0.01).4) With the dose of aluminum increasing, Caspase-3 increased, compared with normal saline,there existed difference in the middle and high-dose group (P<0.05).3 In the role of Caspase-3 RNAi in the aluminum-induced nerve cells:1) Compared with the normal living cells,Al-exposed group cell viability deceased siginificantly (P<0.01),and cell viability of three Caspase-3 RNAi virus reagents group siginificantly higher (P<0.01),close to the normal living cells group. The transfection efficiency was above 91%, the interference efficiency was 66.478%.2) Compared with the normal living cell group,the expression of Caspase-3 was significantly higher (P< 0.01) in the 1mM Al group and 1mM Al+empty virus group,and the two sets of them similar. Compared with the 1mM A1 group, the expression of Caspase-3 was markly reduced (P<0.01) in the 1mM Al+Caspase-3 RNAi virus group.3) Compared with the normal living cells,Nuclei shrinkage or decreament of neurocytes volume were noticed under light microscope, quantity of neurite and dendritic reduced in the 1mM A1 group and 1mM Al+empty virus group. Under fluorescence light, most of cell bodies showed a bright green color in the cell normal living group and the 1mM Al+Caspase-3 RNAi virus group. In the 1mM A1 group and 1mM Al+empty virus group the nuclei stained bright red or orange and pink stained cytoplasm also showed that some cell structure unclear or dissolved,the apoptosis rate was significantly higher than the normal living cell group (P<0.01). The necrosis rate increased in 1mM Al group 1mM Al+empty virus group and 1mM A1+Caspase-3 RNAi virus group (P<0.01).Conclusion:1 To identify the genetype of the APP/PS1 double transgenic mice.Genetic mutations in APP and PS1 gene alone can lead to a decline in learning and memory in mice,but genetic factors and environmental factors under the action of aluminum,learning and memory in mice capacity of more obvious barriers,suggesting that aluminum may increase the role of genetic factors,and aluminum can induce neuronal apoptosis.2 Aluminum can induce neuronal apoptosis,decreased cell viability,increased apoptosis.The degree of apoptosis is closely related with the dose of aluminum.3 Aluminum-induced neuronal apoptosis mechanisms is related with the Caspase-3 gene expression.4 By cell viability and the Caspase-3 gene expression level, three Caspase-3 RNAi virus reagents can effectively interference Caspase-3 especially Caspase-3 RNAi virus 1 reagents.5 By Caspase-3 RNAi virus 1 infecting cells,the cell viability was significantly increased,the number of apoptosis cells was significantly decreased,which may be related to inhibition of Caspase-3 gene expression. So Caspase-3 gene plays an important role in aluminum-induced apoptosis.