Cerebral Ischemia Increases The Level Of Beta-amyloid Protein In Brain And Its Mechanism

Cerebral ischemia increases the level of beta-amyloid protein in brain and its mechanismBackground;Alzheimer disease(AD),the most common type of dementia,ravages the health of the elderly severely.However,one of the biggest stumbling blocks in developing effective prevention and treatment for AD has been the lack of a comprehensive hypothesis that explains the mechanism behind all of the histopathological changes seen in patients suffering from AD.Up to now,the major theories are the Amyloid Hypothesis and Vascular Etiological Hypothesis.Objective;To explore whether cerebral ischemia promote the generation of beta amyloid protein(Aβ)in the brain and its underlying mechanism.Methods;1.Serum Aβand advanced glycosylation endproducts(AGEs)of patients with acute cerebral infarction(ACI)were detected by radioimmunoassay and by enzyme linked immunosorbent assay.And healthy persons were regarded as control group(CONT).2.Permanent bilateral common carotid artery occlusion(2VO)model of rat was established to mimic the chronic cerebrohypoperfusion.The spatial memory function was tested by Y-maze before operation and execution.Rats were sacrificed in each time point to harvest the brain.The expressions of Aβ1-40 and apoptosis markers bax,bcl-2 in the cortex and hippocampus of rats were measured by immunohistochemistry.The biochemical indicators related with cholinergic function including choline acetyltransferase(CHAT)and acetylcholinesterase(AChE)in brain were determined.3.Realtime PCR and fluorescence detection were applied respectively to assay the mRNA expression and the activities ofα,β-secretase of aged rat brain in 2VO models.4.Primary culture of hippocampal neurons from newborn wistar rat had been done for 7 days.Then,the hippocampal neurons were treated with hypoxic and hypoglycemic culture to mimic the chronic ischemic damage of neurons. The neurons were divided into 4 groups;12h group(neurons were treated with hypoxia and hypoglycemia for 12 hours),24h group(treated with hypoxia and hypoglycemia for 24 hours),36h group(treated with hypoxia and hypoglycemia for 36 hours)and CONT group(without hypoxia and hypoglycemia).Neuron vitality was tested by MTT assay.The mRNA and protein expression and the activities ofα,β-secretase in neurons were detected by realtime PCR,fluorescence detection and Western Blot.Results;1.The serum Aβand AGEs levels in ACI group were significantly higher than those in CONT group,that is Aβ(1.43+0.24ng/mL versus 1.02+0.13ng/mL)and AGEs(13.55+1.00μg/mL versus 9.94+1.24μg/mL),P＜0.05 respectively.2.The spatial learning and memory impairment in 2VO model rats aggravated after cerebral hypoperfusion.The number of Aβ1-40 positive cells in cortex(37.30±4.19)and hippocampus(26.00±3.94)of 2VO group was more than that(22.30±6.00 and 18.30±3.53)of sham-operated group,P＜0.01.Bax(38.50±5.85 in cortex,18.30±4.03 in hippocampus)and Bcl-2(25.30±4.45 in cortex,15.00±2.49 in hippocampus)of 2VO group were higher than Bax(14.60±3.27 in cortex,9.20±2.62 in hippocampus) and Bcl-2(14.60±2.46 in cortex,8.20±2.53 in hippocampus)of sham-operated group, P＜0.01.The Bax/Bcl-2 ratio was higher in 2VO group.The number of ChAT positive cell(82.52±5.10 in cortex,69.13±6.04 in hippocampus)and activity of hippocampal AChE(264.40±18.53 mol.g^-1)of 2VO group was obvious lower than ChAT (153.31±4.29 in cortex,138.43±5.12 in hippocampus)and AChE activity (514.21±22.35 mol.g^-1),P＜0.01.3.The mRNA expressions of hippocampalα-secretase ADAM17 in 2VO group at 2 week,4 week and 16 week post-operation (0.78±0.030,0.78±0.018 and 0.54±0.034,respectively)were decreased significantly (p＜0.01)compared with sham-operated group(1.12±0.049,0.99±0.035 and 1.01±0.037).And the longer of the hypoperfusion time the lower of the hippocampalα-secretase activity in 2VO group.Compared with sham-operated group,the mRNA expression ofβ-secretase BACE1 was decreased at the first week(2.799±0.070 versus 3.306±0.106,P＜0.05)but reached the lowest level at the 2nd week after operation(2.239±0.068 versus 3.318±0.195,P＜0.01).At the followed time points, their mRNA expression ofβ-secretase was no considerable different(3.182±0.220 versus 3.272±0.206,P＞0.05 at 4week,3.292±0.107 versus 3.289±0.112,P＞0.05 at 16week).The activity of hippocampalα-secretase at 1 week,2 week,4 week and 16 week following hypoperfusion had been decreased persistently,there were significant differences(p＜0.01)between 2VO group(33880±1086,37496±817,32295±864 and 30069±1111,respectively)and sham-operated group(39497±838,39802±1052, 40137±776 and 39894±1076).The activity of hippocampalβ-secretase at 1 week,2 week,4 week and 16 week following hypoperfusion had been increased persistently, there were significant differences(p＜0.01)between 2VO group(43490±3355, 35319±3880,31819±3676 and 30932±2845,respectively)and sham-operated group (27406±2209,26543±2880,27261±3637 and 27027±2837).4.The activities of neuron in 12h group(0.1628±0.0759),in 24h group(0.1825±0.0458)and in 36h group(0.2024±0.06784)were lower than in CONT group(0.3685±0.0832),P＜0.01.α-secretase activity in 12h group(5160.3±403.3)was higher than in CONT group (2160.0±343.0),P＜0.01,but there were no differences in 24h group(2534.0±421.9), in 36h group(1922.2±288.8)and in CONT group,P＞0.05.β-secretase activities were higher in 12h group(28130.0±3410.3),24h group(26364.4±2047.7)and 36h group(28237.4±1475.3)than in CONT group(21278.6±1390.3),P＜0.01.There were no considerable different in mRNA expression ofα-secretase among in 12h group (1.00±0.16),24h group(1.01±0.16),36h group(0.95±0.13)and CONT group (1.00±0.14).The mRNA expression ofβ-secretase was obvious higher in 12h group (1.37±0.24),24h group(2.15±0.20)and 36h group(1.88±0.03)than in CONT group (1.00±0.14),P＜0.01.The protein expression ofα-secretase was lower in 12h group (0.85±0.06),24h(0.81±0.02)and 36h(0.74±0.08)than in CONT group(1.00±0.00), P＜0.01.The protein expression ofβ-secretase was higher in 12h group(2.47±0.29), 24h(1.98±0.08)and 36h(2.27±0.11)than in CONT group(1.00±0.08),P＜0.01.Conclusions;Aβ,a heterogeneity peptide with definite neurotoxicity,is derived from cleavage of APP.Cerebral ischemic injure produces expression and activity alterations of theα-secretase andβ-secretase,promoting the amyloidogenic pathway, but inhibiting non-amyloidogenic pathway,finally results in the increased Aβproduction.It is inferred that cerebral hypoperfusin would cause Aβaccumulation in brain,which could participate in an induction of apoptosis in brain cells.Neuronal loss,especially the cholinergic neuron loss,contributes to cholinergic dysfunction and reduced learning and memory ability,ultimately resulting in dementia.