Aluminum Induced Dementia Model Of Caspase-3 Gene Intervention Study

Objective Through reducing the expression of Caspase-3 gene in aluminum-induced apoptosis by RNA interference,study the effect of RNAi on the neurotoxicity of aluminum,and effects on other modes of cell death.Methods 48 maleness KunMing mice, 3 months old,were divided into 6 groups randomly by weight,8 in each batch:blank control group,Sham group,Normal saline group(NS 4μl),aluminum exposed group(0.5%AlCl3 3μl+NS 1μl),Al plus empty vector group(0.5% AlCl3 3μl plus control siRNA expression vector)and Al plus RNAi group(0.5% AlCl3 3μl plus targeted siRNA expression vector). Every group was treated by lateral cerebral ventricle micro-injection for 5 days. On the 15th day after exposure,the step-down test,open-field test,Morris water maze test were performed to test the learning and memory abilities of mice. Then on the 20th day after exposure,mice were sacrificed. Half of the brain is soaked in 10% formalin 24h which morphological changes were observed by optical microscope,and observed the transfection efficiency,further to do HE staining and thionin staining. The cerebral cortex and hippocampus was removed from the other half. The hippocampus was for observation by electron microscope,and apoptosis rate by flow cytometry and mitochondrial membrane potential detection. The cerebral cortex was placed in EP tube at -80℃to save for the Western-blot to detect the protein expression :AD-related protein:Tau,APP,Aβ,apoptosis-related protein:Bax,Bcl-2,NF-κB,Caspase-3 and activated Caspase-3,autophagy-related protein:LC3-Ⅱ,necroptosis -related protein:RIP1,and the gene expression of APP,Bax,bcl-Ⅱ,NF-κB,LC3-Ⅱ,Caspase-3 and RIP1 by QRT-PCR.Results: 1,learning and memory capability test in mice:Compared with the control group, water maze escape latency of mice in sham group and the normal saline group increased(P<0.05), the other indexes were not significantly different(P>0.05); Compared with saline group,the indicators of mice in aluminum exposed group and the Al+negative control group were significantly different(P<0.05), Al+RNAi group were no significant changes(P>0.05), but enhanced the learning and memory ability was significantly higher than mice in aluminum exposed group(P<0.05). 2,Transfection efficiency and the efficiency of gene silencing:observation by fluorescence microscope and calculate the number of neural cells transfected by caspase-3 siRNA,and the transfection efficiency was more than 90%.The inhibition efficiency of caspase-3 gene was 64.92%.3,Results of apoptosis rate of hippocampus in each group and the mitochondrial membrane potential show: apoptosis of the sham group and blank control group were no significant difference(P>0.05),apoptosis of normal saline group was more than blank control group(P<0.05); compared with normal saline group,apoptosis of aluminum exposed group and the Al+negative control group increased significantly (P<0.05), while apoptosis of the Al+RNAi group was no significant difference(P>0.05), and significantly less than aluminum exposed group(P<0.05).4,HE staining, histological thionin staining, electron microscopy results show that: hippocampal CA3 area of sham group was close to the blank control group, and cells had no obvious apoptosis; the region of normal saline group had slight pathological changes, and results of electron microscopy showed no significant differences; the pathological changes in aluminum exposed group and the Al+negative control group was the typical apoptotic morphology; while the pathological changes of the hippocampus cells in Al+RNAi group had been restored, results of electron microscopy was close to the normal saline group.5,Associated proteins: apoptosis-related genes,proteins showed:aluminum exposed group and the Al+negative control group were significantly different from normal saline group(P<0.05), while the Al+RNAi group had no significant change(P>0.05), and significantly different from aluminum exposed group(P<0.05); AD-related proteins,genes results:aluminum led to significant increase with protein of APP, Aβ, Tau and the corresponding gene expression, but caspase-3 siRNA can significantly reduce their expression(P<0.05);expression levels of LC3-Ⅱand RIP1 in aluminum exposed group and Al+negative control group were significantly higher than normal saline group(P<0.05),while Al+RNAi group were no significantly different(P>0.05),and significantly less than aluminum exposed group(P<0.05).Conclusion: 1 ) Aluminum can induce neuronal apoptosis in vivo, and have neurotoxicity.Caspase-3 siRNA can effectively block the apoptotic process in animal models, to some extent which can reduce the toxic effects of aluminum,and this is important to the treatment and intervention of AD and other neurodegenerative diseases;2)Lentivirus as vector, and labeled with GFP, neural cells can be successful transfected of caspase-3 siRNA by intracerebroventricular injection method;3)Apoptosis,necrosis,phagocytosis and other forms of death have the interaction.Results show that the reduction of apoptosis can inhibit the other cell death forms.