Testosterone Deficiency Aggravates Oxidative Damage In The Hippocampus Of Male APP/PS1 Mice

Alzheimer’s disease(AD)is an age-related neurodegenerative disease,which is typically characterized by Aβ deposition and excessive phosphorylation of Tau protein,irreversible loss of neurons and synapses in hippocampus and entorhinal cortex,leading to cognitive impairment.Based on projections of a rapidly aging global population,the number of AD patients will increase proportionally.Therefore,it is very important to prevent/delay the pathological changes of AD.A large number of studies have shown that the decrease of antioxidant capacity plays an important role in aging related neurodegenerative diseases such as AD.Therefore,it is an urgent medical issue to explore the potential risk factors for the decrease of antioxidant capacity induced by neurodegenerative diseases.In age-related neurodegenerative diseases,the balance between oxidants and antioxidants is upset;Excessive Reactive oxygen species(ROS)can cause serious oxidative stress,leading to the lack of antioxidants,and in turn,oxidative damage to lipids,proteins or nucleic acids.Studies have shown that the activity of various antioxidant enzymes in AD brain tissue is decreased,and the increase of ROS and the decrease of antioxidant capacity will lead to and aggravate the neurodegeneration.Studies have found that in older men,levels of circulating Testosterone(T)decrease gradually as they age,which may aggravate the pathological progression of AD and exacerbate symptoms in men with AD.Testosterone is not only an important sex hormone affecting the development of sex organs,but also regulates the functional activities of the brain and protects neurons from oxidative stress damage.Therefore,it is of great significance to explore the effect of testosterone deficiency on hippocampal antioxidant capacity of APP/PS1 mice in AD animal model,which can alleviate/delay the neurodegeneration of AD.Objective : To explore the effect of testosterone deficiency on hippocampal antioxidant capacity in male APP/PS1 mice.Methods:1.Two transgenic male mice overexpressing the APPswe/PSEN1 d E9mutant gene(APP/PS1)and age-matched male wild-type mice(WT)were used in the experiment,which included the following four groups: WT group,APP/PS1 Sham operation group(APP/PS1-sham),APP/PS1 castration group(APP/PS1-GDX);APP/PS1 castration plus testosterone propionate supplementation group(APP/PS1-GDX-TP).Bilateral orticectomy was performed in APP/PS1-GDX and APP/PS1-GDX-TP groups at 3 months of age,and artificial hand treatment was performed in WT and APP/PS1-Sham groups.Mice in APP/PS1-GDX-TP group were injected subcutaneously with1.0mg/kg TP,WT mice,APP/PS1-sham mice and APP/PS1-GDX mice were injected subcutaneously with the same amount of sesame oil,once a day,for 4weeks.2.Exploration object localization memory of animals were detected by novel object localization test,novel object recognition test.And Morris water maze.3.The percentage of karyopyknosis was explored in neurons in the hippocampal CA1,CA3,and DG region stained with hematoxylin-eosin.Aβdeposition and immune response of 3-Nitrotyrosine(3-NT)in the hippocampus in CA1,CA3 and DG regions.4.The levels of serum testosterone,oxidative stress indexes MDA and GSH/GSSG in hippocampus,and the enzyme activities of Cu Zn-SOD and Mn-SOD in hippocampus were determined by spectrophotometry.5.The expression of SYP,PSD-95 and Mn-SOD in the hippocampus were detected by real-time fluorescence quantitative PCR(q PCR)6.The protein expressions of SYP,PSD-95 and Mn-SOD in hippocampus were detected by Western Blot.1.Compared with WT,APP/PS1-Sham and APP/PS1-GDX-TP mice,the serum T level decreased in APP/PS1-GDX mice.(P<0.01).2.Testosterone deficiency induced impairment of object localization memory in APP/PS1 mice2.1 novel object localization The exploration time of APP/PS1-GDX mice in the new location was less than that in the old location(P<0.05).APP/PS1-GDX mice showed significantly low recognition index compared with WT mice(P<0.05).The APP/PS1-GDX mice spent less time in their new position than the other three groups.2.2 novel object recognitionThe mice in WT,APP/PSl-Sham and APP/PS1-GDX-TP mice exhibited more time exploring the novel object and APP/PS1-GDX mice spent similar time exploring either the new or the old object,with no statistical difference.The APP/PS1-GDX mice showed a lower preference for new objects than the other groups.3.Testosterone Deficiency Exacerbated the Spatial Memory Deficits in Male APP/PS1 MiceThe escape latency of WT mice progressively decreased,while APP/PS1-Sham mice exhibited longer escape latency than WT mice in the 2ed,4th and 5th day(day 2: P<0.05;Day 4: P<0.05;Day 5: P<0.01).The escape latency of APP/PS1-GDX-TP mice was significantly shortened.Compared with WT mice,the time of APP/PS1-Sham mice in the target quadrant was shortened(P<0.01).Compared with APP/PS1-Sham mice,the time of APP/PS1-GDX mice in the target quadrant was significantly shortened(P<0.01).Relative to WT mice,APP/PS1-GDX mice showed the reduced number of platform crossing(P<0.05).4.Testosterone Deficiency Induced Pathological Alterations of Hippocampus in Male APP/PS1 Mice4.1 The percentage of karyopyknosis in hippocampusMost neurons in WT mice,APP/PS1-Sham mice and APP/PS1-GDX-TP mice had clear and obvious nuclei and nucleoli.We observed that there wereResults:evidently more indistinct kernels and nuclear pyknosis in the CA1,CA3,and DG regions in hippocampal tissue of APP/PS1-GDX mice.Compared with WT,APP/PS1-Sham and APP/PS1-GDX-TP mice,the percentage of nuclear pyknosis in hippocampal CA1,CA3 and DG regions were markedly increased in APP/PS1-GDX mice.(P<0.01).4.2 The effect of testosterone deficiency on Aβ42 deposition in APP/PS1 mice was observed by immunohistochemical stainingIn WT,APP/PS1-Sham,APP/PS1-GDX and APP/PS1-GDX-TP mice,Aβ42-immunoreactive profiles was only observed in the hippocampus of APP/PS1-GDX mice.4.3 Expression of SYP and PSD-95The results of q PCR and Western blot showed that compared with WT mice,m RNA and protein levels of SYP and PSD-95 in hippocampus of APP/PS1-Sham male mice were decreased(m RNA: SYP: P<0.01,PSD-95:P<0.05;Protein: P<0.01).Compared with APP/PS1-Sham group,the expressions of SYP and PSD-95 in hippocampus of APP/PS1-GDX mice were further decreased(m RNA: P<0.01;Protein: P<0.01).After TP supplementation,m RNA and protein levels of SYP and PSD-95 in APP/PS1-GDX mic were increased(m RNA: P<0.01;Protein: SYP: P<0.01,PSD-95: P<0.05).5.Effect of castration on oxidative homeostasis in the hippocampus of APP/PS1 mice5.1 Immunohistochemical results of 3-NTNo significant differences in 3-NT expression in the CA1,CA3 and DG regions of the hippocampus were found among the WT,APP/PS1-Sham and APP/PS1-GDX-TP mice.The average optical density(AOD)of 3-NT in the hippocampus of APP/PS1-GDX mice in CA1,CA3 and DG regions was significantly increased(P<0.01).5.2 The results of MDAThe MDA content in the hippocampus was significantly higher in male APP/PSl-Sham mice than in WT mice(P<0.01).The MDA content in APP/PS1-GDX mice was further increased than in APP/PS1-Sham mice(P<0.01).5.3 GSH/GSSG ratioThe ratio of GSH/GSSG in the hippocampus was less in APP/PSl-Sham mice than WT mice(P<0.01).the ratio of GSH/GSSG in APP/PS1-GDX mice further lower than in APP/PS1-Sham mice(P<0.01).5.4 Antioxidant enzyme activityThe activities of Mn-SOD and Cu Zn-SOD enzymes were less in APP/PS1-Sham than WT.The activity of Mn-SOD enzymes was less in APP/PSl-GDX mice than APP/PSl-Sham(P<0.05),there was no obvious difference in Cu Zn-SOD activity.The activity in APP/PS1-GDX mice was lower than that in APP/PS1-Sham mice(P<0.05).Compared with APP/PS1-GDX mice,the Mn-SOD activity of APP/PS1-GDX-TP mice was increased(P<0.05),and recovered to the level of APP/PS1-Sham mice.5.5 m RNA and protein expression of Mn-SODMn-SOD m RNA and protein levels of APP/PS1-Sham mice were less than WT mice(P<0.01).The m RNA and protein levels of Mn-SOD in APP/PS1-GDX group were less than APP/PS1-Sham mice(P<0.01).The m RNA level of Mn-SOD in APP/PS1-GDX-TP mice was significantly higher than that in APP/PS1-GDX mice(P<0.01).Conclusions:1.4-month-old male APP/PS1 mice showed cognitive deficits and oxidative damage in hippocampus.2.Testosterone deficiency aggravated cognitive deficits and induces neuropathic changes in AD in male APP/PS1 mice.3.Testosterone deficiency enhances hippocampal oxidative damage in male APP/PS1 mice.4.Cognitive deficits and induced neuropathic changes in AD in male APP/PS1 mice aggravated by testosterone deficiency may be related to the enhancement of oxidative damage in hippocampus by testosterone deficiency.