The Role And Molecular Mechanism Of Oxidative Stress In The Experimentally Pathological Injury Of Central Nerve System Induced By ?-amyloid Protein And Fluoride

Objective:Oxidative stress plays an important role in the pathogenesis of a variety of diseases.In previous studies,the research group found that?-amyloid peptide?A??,which is one key protein in the pathogenesis of Alzheimer's disease?AD?can elevate levels of oxidative stress;chronic fluorosis induced by long term intake of excessive fluoride can result in the damages of whole body,the main damage mechanism of which is the high level of free radicals caused by fluoride.In order to understand the pathogenesis of OS,this research selected brain tissues of AD patients,the transgenic mice with overexpression of?-amyloid precursor protein?APP?,the rats with chronic fluorosis and the primary cultured neurons in vitro exposed to A?to investigate the role and molecular mechanism of OS in the injury in central nerve system of both diseases.Methods:1.Study of AD:?1?Research object:the autopsy samples of hippocampus and temporal cortex from AD patients and control group?without nervous system disease?brains,APPswe/PSEN1dE9 double transgenic mice model?APP overexpression?and neurons cultured in vitro or SH-SY5Y cells.?2?Treatment methods:starting at the ages of 4 and 8 months of transgenic animals.APP/PS1 and WT mice received physiological saline,RSV?a silent information regulators,SIRTs 20 mg/kg?,suramin?SIRTs reaction inhibitor,20 mg/kg?,while control group received physiological saline;both groups were treated for two months.The primary cultured neurons or SH-SY5Y cells were exposed to A?Os?0.5mmol/L?,RSV?10 or 50mmol/L?,Suramin?200 or 300?g/mL?,ZLN005?20mmol/L?and the transfection plasmid of peroxisome proliferator-activated receptor-?coactivator 1??PGC-1??in individual or combination treatment for 24⁴8 h.?3?Determination method:Observing the histological structure in human or mouse brains with HE,Nissl staining,transmission electron microscope and immunohistochemistry;the expression and location of SIRTs?SITR1,SIRT3,SIRT4 and SIRT5?in brains were detected by immunofluorescence or immunohistochemistry;the cell survival rate was detected with CCK-8 test;the vitro culture cell apoptosis and reactive oxygen species?ROS?levels were detected by flow cytometry or confocal;the content of?APPs in serum and CSF,and A?1-42 in brains were detected by ELISA kit;the proteins expressions,mRNA levels and nicotinamide adenine dinucleotide?NAD+?level of various brain areas of mouse and cultured cell were measured by Western-blotting,Real-time PCR and NAD+kit;then the behavioral changes of learning and memory capacity of mice were measured by Morris water maze.2.Study of chronic fluorosis:?1?Research object:Sprague-Dawley?SD?rats.?2?Treatment methods:this experiment replicated the rat models with chronic fluorosis and antioxidant treatment by feeding the drinking water containing sodium fluoride?50 mg/L F?and vit E?50 mg/kg/day?gavage treatment for 10 months.?3?Determination method:replicating examination of animal model of chronic fluorosis,including the weight of brains,dental fluorosis levels,fluoride content in urine and bone tissues determined by fluoride ion selective electrode;the levels of O^2·-and malondialdehyde?MDA?were detected by biochemical methods;the Muscarinic acetylcholine receptors?mAChRs?protein levels were detected by immunohistochemistry and the behavioral changes were measured by Morris water maze.Results:1.AD:?1?Human brains sample:compared with healthy people,the results of immunohistochemistry showed that the protein levels expressions of SIRT1,SIRT3,SIRT4 and SIRT5 in AD human brains were significantly lower,and a large number of senile plaques were found.Interestingly,the reduced protein levels of SIRTs were negatively correlated with the number of senile plaques in AD brains.?2?AD animal model:after extensive breeding and genotyping,AD animal model was established successfully;the learning and memory abilities of APP/PS1 mice in different age were significantly lower than those in wild-control mice,and the brain weights of APP/PS1 mice were also decreased;compared with wild-control group,the number of Nissl's Body in neurons obviously reduced,while different number of senile plaques were found in cortex and hippocampal area of the APP/PS1 mouse brains,and the number and distribution increased with age.Furthermore,under the electron microscope,there is partial congenital absence in nuclear envelope of neurons of APP/PS1 mouse of different ages,even fusion,fracture and disappearance in magination,cristae mitochondriales and membrane.Treated with RSV for APP/PS1 mice,the learning and memory ability and the pathological changes of brain were obviously improved,while Suramin could further aggravate these changes.For APP/PS1 group,the protein levels and the mRNA expressions of SIRT1,SIRT3,SIRT4,SIRT5 were lowered with different degrees,and distributed in hippocampus and cortex;meanwhile,PGC-1?,OGG1,?7 nAChR,Syn,SNAP25 in mouse brains were significantly lowered,and levels of protein expression of Apo E and Caspase3were obviously increased.Protein expression of ADAM10 and BACE1,BACE2showed increase and decrease of different levels.The previous stated changes could be reversed by RSV,and animal senile plagues obviously reduced.While the content of?APPs in the serum and CSF,and NAD+in the mouse brains were decreased,the level of insoluble A?1-42 in the mouse brains increased.While the RSV treated APP/PS1 mice,content of?APPs and NAD+obviously increased,level of insoluble A?1-42 obviously decreased,applying Suramin would yield opposite reaction.Levels of OS and antioxidant enzymes of serum,brain tissues and mitochondrion of APP/PS1 mice obviously increased or decreased;applying RSV,which would decrease OS and increase activities of antioxidative enzymes,wchile Suramin would yield opposite reaction.?3?Cell model:The results of immunostaining showed that the purity of primary cultured neurons was about 83%;the protein levels of PGC-1?silencing neurons or SH-SY5Y cells with PGC-1?were obviously lower than the neurons or SH-SY5Y of wild type cells.It was observed that after treated with AbOs,expressions of SIRT1,SIRT3,SIRT4,SIRT5 proteins and mRNA were decreased with various degree;after pretreated with RSV,the expressions of SIRT1,SIRT3,SIRT4,SIRT5 obviously increased,and RSV could obviously eliminate AbOs which was already inside the cell,while Suramin could further enhance the toxic effect of AbOs.After AbOs treated neurons,protein expression of PGC-1?,OGG1 and?7 nAChR obviously decreased;Apo E and Caspase3 obviously increased.Protein expression of ADAM10 and BACE1,BACE2 showed different degrees of increase or decrease,after pretreated by RSV,which could inhibit previously mentioned changes.AbOs could decrease NAD+levels significantly,increased apoptosis,ROS and OS level increase,reducing the antioxidant enzymes significantly.RSV treated with the cells,the changes could be reversed and Suramin could enhance the previously mentioned changes.In addition,inhibited PGC-1?and up-regulated SIRT1 could not inhibit the expression of Apo E,on the contrary,inhibited SIRT1 and up-regulated PGC-1?could regulate Apo E expression.2.Chronic fluorosis:in the rats of the fluoride group,the dental fluorosis with different degrees were observed,the contents in urine and bone tissues were significantly higher than control,and the weight of brains were insignificance;the ability of learning and memory was significantly lower than the control and the protein levels of M₁ and M₃ receptors in the rat brains significantly reduced as compared to controls;while the levels of O^2·-and MDA were increased significantly.The chronic fluorosis rats that treated with the Vit E could obviously reduced these change.The reduced protein levels of M₁ and M₃ were positively correlated with the decreased ability of learning and memory of the rats with fluorosis.Conclusions:?1?Increased OS level of AD patients brain tissue,APP high expression in experiment mice brain tissue and the neurons treated by AbOs,and SIRTs expression decreased appeared to be linked to the numbers of senile plagues.?2?RSV could increase SIRTs expression to reduce toxic effect induced by high level of AbOs.The mechanism might related with activated SIRTs regulated the proteins,reduced the mitochondrial free radical and ROS production,reduced lipid peroxidation levels and enhanced antioxidant enzyme activity,decreased the expression of Apo E,inhibited mitochondrial DNA damage,which could reduce apoptosis to reduce the changes in abilities of learning and memory caused by aging cells.?3?It also enhanced the hydrolysis of APP with the non-amyloid metabolism process,which resulted in stimulating expression of?7 nAChR and up-regulates ADAM 10 by stimulating SIRTs activation,thereby decreased the production of neurotoxic A?.?4?The expression levels of mAChRs at protein were detected in the brains with excess fluoride and increased levels of oxidative stress,levels of MDA and O^2·-increased,the reduced protein levels of receptors were negatively correlated with high levels of OS,these may be the main mechanism of brain injury of learning and memory ability in fluoride rats.Antioxidants can play an important antagonistic effect in the brain damage induced by fluoride toxicity.?5?OS plays an important role in AD and chronic fluorosis with central nervous system damage.Antioxidant treatment or improved antioxidant level may counteract excessive A?or fluoride induced nerve injury.