The Role And Mechanism Of OXR1A Regulating Oxidative Stress Via P53-p21 Signal Pathway In Alzheimer’s Disease

Objective Alzheimer’s disease（AD）is a common progressive neurodegenerative disease in middle-aged and elderly people.The main pathological features were deposition ofβ-amyloid protein（Aβ）in the central nervous system（CNS）to form senile plagues.Hyperphosphorylation of SP and Tau proteins leads to neurofibrillary tangles,synaptic loss and neuron death.At present,the pathogenesis of AD is not very clear,and a large number of evidences show that oxidative stress plays an important role in the pathogenesis and development of early AD.Oxidation resistance gene 1（OXR1）is a conserved gene family with antioxidant function that was discovered in 2000.Studies have found that OXR1 plays an important role in anti-oxidative stress in eukaryotes.It has been reported that OXR1 gene mutation will lead to neurodegenerative changes in children,such as neurological retardation and cognitive dysfunction.In addition,studies have found that the expression subtype of OXR1-OXR1A is only expressed in the brains of humans and rodents,suggesting that OXR1A may play an important role in the pathogenesis of neurodegenerative diseases represented by AD and have a protective effect on cognitive function.At present,there are no studies on the role of OXR1A in AD and related specific mechanisms.This project intends to construct SD rat AD animal model and hippocampal AD cell model of OXR1A overexpression and silencing through in vivo and in vitro experiments,to study the role of OXR1A in AD animal model and cell model and to explore its potential mechanism.Method In vivo experiments:Thirty healthy male Sprague-Dawley rats（SD rats）weighing 250-300 g and aged 9-12 weeks were randomly divided into negative control group（Sham group）and AD model group（AD group）,with 15rats in each group.SD rats in AD group were injected with 5ul Aβ1-42（2.5ug/ul）solution into hippocampal CA1 region of bilateral brain of SD rats,while Sham group was injected with 5ul 0.9%normal saline.After dark avoidance test and Morris Water Maze behavior test,hippocampal tissue of each rat was collected for RT-q PCR,Western blot,immunohistochemical staining and immunofluorescence.The changes of learning and memory ability,the expression of OXR1A protein and m RNA in hippocampus,the damage of nuclear and mitochondrial DNA,the relative copy number of mitochondrial DNA and the expression of Caspase-3 were observed in the hippocampus of rats,to study the gene and protein expression of OXR1A and the differences of oxidative stress response level and apoptosis level in hippocampal area of the two groups of rats.In vitro experiments:1)primary hippocampal neurons from SD rats were extracted and treated with 10 u M Aβ_1-42 to establish AD cell model.OXR1A was overexpressed or silenced by overexpressed lentivirus and si RNA transfection.Cells were divided into NC group:normal control group;Model group:Alzheimer’s disease cell damage Model;Model+OE-OXR1A group:Alzheimer’s disease cell damage Model+transfected with overexpressed OXR1A vector;Model+OE-Ctrl group:Alzheimer’s disease cell damage Model+transfection blank vector;Model+si-OXR1A group:Alzheimer’s disease cell damage Model+transfected with OXR1A interference si RNA;Model+si-Ctrl group:Alzheimer’s disease cell damage Model+transfected with blank interfering si RNA.Then,the proliferation rate of neurons was detected by MTT method to verify the effect of overexpression or silencing of OXR1A on the proliferation ability of AD neurons.The mitochondrial DNA copy number of neurons was detected by q PCR,the DNA damage degree of mitochondria and nucleus was quantitatively determined by RADF assay.The activities of superoxide dismutase（SOD）and glutathione peroxidase（GSH-Px）,the content of malondialdehyde（MDA）and nitric oxide（NO）were detected by ELISA.The effects of overexpression or silencing of OXR1A on the expression of key factors in the oxidation system and antioxidant system were observed to verify the effect of OXR1A on the antioxidant capacity of AD neurons.The expression levels of p53 and p21 were detected by RT-q PCR to explore its possible mechanism.2)The activation of p53-P21 signaling pathway was blocked by P53 inhibitor MDM-2,and hippocampal neurons were divided into NC group:normal control group;Model group:Alzheimer’s disease cell damage Model;Model+MDM-2 group:Alzheimer’s disease cell damage Model+P53 inhibitor MDM-2;Model+OE-OXR1A group:Alzheimer’s disease cell damage Model+transfected with overexpressed OXR1A vector;Model+OE-OXR1A+MDM-2group:Alzheimer’s disease cell damage Model+transfection of OXR1A vector+p53 inhibitor MDM-2.The expression levels of p53 and P21 were detected by RT-q PCR,and the proliferation rate and antioxidant capacity of neurons in the quantitative groups were detected by the above detection method,so as to observe the the effect of p53 inhibitor MDM-2 on the oxidative damage level and antioxidant capacity of OXR1A through the p53-P21 pathway,and to explore whether OXR1A plays an anti-oxidative stress role in AD through the p53-p21 pathway.Results In vivo experiments results:1.Successfully constructed AD model of the SD rat.In the dark avoidance experiment,the incubation period of dark avoidance in AD group was shortened and the number of errors was significantly increased when compared with Sham group.In Morris water maze behavior test,compared with Sham group,the escape latency of SD rats in AD group was prolonged and the number of platform crossing was significantly reduced.The above animal behavior results indicated that the SD rat model of AD was successfully constructed in this experiment.2.The protein and m RNA levels of OXR1A in hippocampal tissue of AD group were significantly higher than those in Sham group.In addition,compared with Sham group,the damage degree of nucleus and mitochondria relative DNA in cerebral cortex of AD group was more serious,the relative copy number of mitochondrial DNA was relatively reduced,and the expression level of caspase-3 was significantly increased.3.Compared with Sham group,the average m RNA levels of p53 and p21 in hippocampus of AD group were decreased.In vitro experiment results:1).With the extension of neuron culture time,the proliferation rate of neurons in Model group decreased significantly compared with NC group;Compared with Model group,the proliferation rate of neurons in Model+OE-OXR1A group was significantly increased,while the proliferation rate of neurons was significantly significantly lower in Model+si-OXR1A group.2).Compared with NC group,the degree of nuclear and mitochondrial relative DNA damage in Model group was significantly increased,and the relative mitochondrial DNA copy number was decreased;Compared with Model group,the relative DNA damage degree of Model+OE-OXR1A group was significantly lower,while the relative mitochondrial DNA copy number was increased.In addition,compared with Model group,the degree of relative DNA damage was significantly increased in Model+si-OXR1A group.3).Compared with NC group,SOD activity in Model group decreased,while GSH-Px activity increased,MDA and NO content increased;Compared with Model group,SOD activity in Model+OE-OXR1A group increased,GSH-Px activity decreased,while MDA and NO contents decreased.In addition,SOD activity decreased,GSH-Px activity increased and MDA and NO content increased in Model+si-OXR1A group when compared with Modelgroup.These results suggest that OXR1A could enhanced SOD activity,reduced MDA and NO content,reduced mitochondrial DNA damage,increased mitochondrial DNA copy,and played an anti-oxidative stress role in primary hippocampal neurons induced by Aβ_1-42.4).Compared with NC group,m RNA expression of pathway regulatory factors p53 and p21 increased in Model group;Compared with Model group,the expression levels of p53 and p21were decreased in Model+OE-OXR1A group,while the expression levels of p53 and p21 pathway regulatory factors increased in Model+si-OXR1A group.In addition,compared with Model group,the expression of p53 and p21 in Model+MDM-2 group was significantly down-regulated.In addition,compared with Model group,the m RNA levels of P53 and P21 in Model+MDM-2 group were significantly down-regulated.The m RNA levels of p53 and p21 in Model+OE-OXR1A+MDM-2 group were significantly lower than those in Model+MDM-2 group5).Compared with Model group,SOD activity in Model+MDM-2 group increased,GSH-Px activity decreased,MDA and NO content decreased;Compared with Model+MDM-2 group and Model+OE-OXR1A group,SOD activity in Model+OE-OXR1A+MDM-2 group was significantly increased,GSH-Px activity was decreased,and MDA and NO contents were significantly decreased.Conclusion In this study,through animal experiments found that the gene transcription level and protein expression of OXR1A in the hippocampus of AD rats were significantly increased,accompanied by the aggravation of the relative DNA damage in the nucleus and mitochondria of rats,the decrease of the relative copy number of mitochondrial DNA and the significant increase of caspase-3 expression level.The results suggest that OXR1A may be correlated with the incidence of AD.In vitro experiments results showed that overexpressing OXR1A increased the survival rate of hippocampal neurons in AD model,while silencing OXR1A increased the inhibition rate of cells.OXR1A can also affect the damage of hippocampal neuron nucleus and mitochondrial DNA,SOD activity,GSH-Px activity,MDA and NO content.At the same time,there were changes in m RNA levels of p53 and p21.Finally,by using MDM2-p53 inhibitor,we proved that MDM2 could inhibit p53-P21 active and the protective effect of OXR1A on hippocampal neurons,as well as the regulation of SOD activity,GSH-Px activity,MDA and NO content.In conclusion,our study preliminarily found that OXR1A has a high expression in the brain of AD rats and a protective effect on hippocampal neurons.The possible mechanism is that OXR1A increases the activities of SOD and GSH-Px by inhibiting the activity of p53-P21 pathway,and reduces the levels of MDA and NO in cells.Thus,it can reduce the damage of nuclear and mitochondrial DNA,protect the perfection of mitochondrial DNA,and then play the role of neuron protection.