| Objective: Traumatic brain injury(TBI)is a major cause of human injury and a common type of injury in forensic medicine.Late survivors are often accompanied by cognitive dysfunction and dementia-like symptoms,which are caused by persistent oxidative stress and chronic inflammatory stimulation in the brain.NF-E2-related factor(Nrf2)is a central regulator of antioxidant response in cells.Studies have shown that(including ours study)Nrf2 is dynamically expressed in different cells at the early stage of TBI and is involved in the regulation of antioxidant stress,anti-inflammatory and anti-apoptotic processes.A large number of studies have also shown that decreased Nrf2 content or abnormal expression is closely related to the occurrence and development of neurodegenerative diseases,such as Alzheimer’s disease,Parkinson’s disease,and amyotrophic lateral sclerosis.Alzheimer’s disease(AD)is A degenerative nervous system disease characterized by progressive cognitive dysfunction,which is characterized by deposition of amyloid beta protein(Aβ)and abnormal phosphorylation of tau protein(NFTs).Aβ plaque is formed by over-expression of presenilin 1 and presenilin 2 by triggering gene mutation of precursor protein(APP)in Aβ.The neurotoxicity caused by the deposition of Aβ in the brain leads to the oxidative stress damage and inflammatory response of nerve cells.Studies have shown that the occurrence and development of AD is closely related to factors such as brain injury,environmental damage,and heredity.Meanwhile oxidative stress and inflammation play an important role in the neurodegenerative process of Alzheimer’s disease.ROS produced by oxidative stress causes per-oxidation damage to tissues and cells,thereby inhibiting their normal functions.Nrf2 is an important central regulator of antioxidant stress response,regulating the expression of various antioxidant enzymes,detoxification enzymes and inflammation-related genes.Nrf2 activates the downstream genes regulating antioxidant protein and Ⅱ phase detoxification enzyme through ARE pathway,regulates the expression of antioxidant stress protein and Ⅱ phase detoxification enzyme(NQO1,GCL,HO-1,etc.),and enhances the capacity of the antioxidant response of cells.In addition,the expression of pro-inflammatory factors can be increased by inhibiting the up-regulation of transcription of these genes,or the expression of inflammatory factors can be negatively regulated by direct transcription.Activation of Nrf2 protects the body from damage by up-regulation of the antioxidant defense pathway,suppression of inflammation,and maintenance of protein homeostasis.Studies using brain tissue from Alzheimer’s patients after death and animal models of Alzheimer’s disease have found that Nrf2 levels decrease with age.In this study,we applied APP/PS1 double transgenic mice,a widely used animal model of Alzheimer’s disease.By established Nrf2 knockout APP/PS1 double transgenic mice,we found that Nrf2 knockout in mice exacerbated further declines in spatial learning and memory,accompanied by associated changes in Alzheimer’s disease-like pathological features,which were closely associated with excessive oxidative damage and neuroinflammatory processes.Methods: Genotypes of APP-/PS1-/Nrf2het(WT),APP-/PS1-/Nrf2-/-(Nrf2-KO),APP/PS1/Nrf2het(AT),APP/PS1/Nrf2-/-(AT/Nrf2-KO)were obtained by hybridization of Nrf2-KO C57BL/6 and APP/PS1 mice,and related experiments were conducted from the age of 12 months to 22 months.Firstly,water maze experiment was conducted to test the spatial learning ability and memory function of the genotyped mice.The mice were killed and collected from the hippocampus,part of which was used for morphology and part for molecular detection.Nrf2,Aβ,p-taus404 and 8-oHdg were stained by immunohistochemistry.Microglia and astrocytes were stained by immunofluorescence.Enzyme-linked immunosorbent assay and real-time quantitative PCR were used to detect the expression levels of pro-inflammatory factors IL-1β,IL-6,TNF-α,anti-inflammatory factors IL-4,IL-10,and TGF-β in the APP/PS1+ group.The expression levels of Nrf2 downstream antioxidant enzymes HO1,NQO1,GCLC and GCLM were detected by real-time quantitative PCR.GraphPad Prism 6.0 Software(Graph Pad Software,La Jolla,CA,USA)was used for statistical analysis.Comparisons between different groups were statistically analyzed using the t-test.P <0.05 was considered statistically significant.Results: In the study of 12-month-old mice,immunohistochemical staining showed that Nrf2 was widely expressed in hippocampal neurons and glial cells.There were significant differences between AT mice and AT/Nrf2-KO mice in the swim distance to the platform quadrant,the latency to the platform quadrant,the frequency of crosses to the platform quadrant,and the frequency of crosses to the platform,indicating that Nrf2 deficiency aggravated the damage of spatial learning and memory ability of APP/PS1 double transgenic mice.Immunohistochemical staining showed that Aβ and p-tau S404 were expressed in hippocampal neurons and hippocampal glial cells,and AT/Nrf2-KO mice showed higher expressions of Aβ and p-tau S404 in hippocampal Aβ than in AT mice.Nrf2 gene knockout enhanced the deposition of plaque and the expression of p-tauS404 in hippocampus Aβ of APP/PS1+ mice.AT/Nrf2-KO mice had more Aβ deposition in the hippocampus than AT mice,and the number of microglia and astrocytes surrounding the plaques increased.In both AT/Nrf2-KO and AT groups,M1-type microglia cells around plaque Aβ were dominant,and the number of M2-type microglia cells was significantly higher than that of M2-type microglia cells.It is noteworthy that the number of M1-type and M2-type microglia around plaque Aβ in AT/Nrf2-KO mice was higher than that of AT group.AT/Nrf2-KO mice showed increased levels of pro-inflammatory factor IL-1β,IL-6 and TNF-α mRNA in the hippocampus of AT mice.In contrast,levels of anti-inflammatory factors IL-4,IL-10,and TGF-β protein and mRNA were decreased.This suggested that Nrf2 knockout may enhance the activation of microglia and astrocytes in APP/PS1+ mice,further enhancing the pro-inflammatory response and weakening the anti-inflammatory response.In addition,immunohistochemical staining showed that 8-oHdg was expressed in hippocampal neurons and glial cells.AT/Nrf2-KO mice showed increased expression of 8-oHdg positive cells in the hippocampal CA1 region of AT mice.The mRNA levels of HO1,NQO1,GCLC and GCLM were decreased in AT/Nrf2-KO mice compared with AT mice.This suggested that the oxidative stress damage was more obvious in the hippocampus of APP/PS1+ mice with Nrf2 knockout.Similarly,mice in the 22-month-old group also showed similar results in the 12-month-old group,but their learning and cognitive impairment were more severe,the pathological features of Alzheimer’s disease were more obvious,the inflammatory response was stronger,and the oxidative stress damage was more severe.Conclusion: In the APP/PS1+ mouse model,Nrf2 gene knockout aggravated the decline of spatial learning ability and memory function in the mice.In the hippocampus of AT/Nrf2-KO mice,more serious Aβ deposition,glial cell response and pro-inflammatory response were observed,which also aggravated oxidative stress damage.It is suggested that the absence of Nrf2 is involved in the occurrence and development of early aging lesions in APP/PS1,and is closely related to its regulated anti-inflammatory and antioxidant reactions... |
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