Dysfunction Of Insulin Signal Pathway Contributes To Upregulation Of BACE1 Mediated By Diabetic Metabolism Disorder

ObjectiveTo measure praxiological and pathological change progress of diabetes mellitus of Wistar rats,to investigate expression of APP,BACE1 and Aβin brain for diabetes mellitus of Wistar rats,to investigate expression of BACE1 and molecules of signal pathway in brain for diabetes mellitus of Wistar rats,to study pathophysiological mechanism of Alzheimer's disease from diabetic metabolic disorder.MethodsAnimal model of diabetes mellitus was established by high fat and suger and streptozocin with intraperitoneal injection.Wistar rats were randomly divided into control group(S),4 week diabetes mellitus model group(M4),6 week diabetes mellitus model group(M6) and 8 week diabetes mellitus model group(M8).Behaviour was tested with Morris water maze task and shuttle box.Congo red detected deposition of beta-amyloid in the brain tissues.Bielschowsky stained silver determined senile plaques and neurofibillary tangles in the brain tissues,biochemical assay for blood glucose,blood cholesterol and glycosylated hemoglobin, serum concentration of insulin by radioimmunoassay detection,Expression of Aβwas measured by enzyme linked immunosorbent assay and APP by enzyme linked immunosorbent assay,Western blotting and RT-PCR, BACE1 by immunohistochemistry,enzyme linked immunosorbent assay, Western blotting and RT-PCR,p38MAPK,PKC,PKA,JAK-2,PI-3K,Akt and ERK1/2 by Western blotting and RT-PCR.The light density value was measured by imaging analysis.ResultsAnimal model and praxiologicai disorderThe model group in weighting significantly decreased compared with the control group(482.2±16.5) g(P＜0.05).Blood glucose concentration increased from the control group(6.23±0.62) mmol/ dl to the model group (23.21±3.46) mmol/dl(P＜0.01).Glycosylated hemoglobin levels increased from the control group to 3.44±0.26%of the model group 12.56±0.67% (P＜0.01).Cholesterol concentration increased from the control group (121.6±26.2) mg/dl the model group(291.±27.5) mg/dl(P＜0.01).Serum insulin concentration increased from the control group(365.3±26.5) pmol/l to the model group(406.2±24.5) pmol/l(P＜0.01).Weight,blood glucose, serum glycosylated hemoglobin,cholesterol concentration and serum insulin concentration is no difference in model groups.Weight loss,higher than 16mmol/dl blood sugar,insulin resistance and elevated cholesterol stand for success modeling.Navigation experiments in rats to identify the spatial learning ability:in the morris water maze test,the sample rats' latency of searching for the platform is obviously longer than the control rats(P＜0.01),model group mice no significant difference(P＞0.05).Space exploration test of spatial memory in rats:The times of crossing platform within 120 seconds of sample rats are obviously less than the control ones (P＜0.01),model groups no significant difference(P＞0.05).Shuttle box to test the learning ability for condition reflex:the number of rats subjected to electric shocks increased in model groups than the control group significantly(P＜0.01) from 7.43±2.01 times in control group to 15.46±3.62 times,the model rats no significant differences(P＞0.05).The time of electric stimulation in model groups was significantly longer than the control group from 7.43±2.01 second in control group to 15.46±3.62 second in model.These results show that the model group capacity of learning and memory significantly decreasd in diabetes mellitus model groups and has a clear learning and memory impairment.Histopathological changesCongo Red staining forms non-polar hydrogen bonds with amyloid when viewed by polarised light due to parallel alignment of the dye molecules on the linearly arranged amyloid fibrils.The Alkaline Congo Red Technique uses high concentrations of sodium chloride which act as ionic competitors for the dye thus eliminating background electrochemical (polar) staining while enhancing the hydrogen(non-polar) binding of Congo Red and amyloid.The results by Congo Red staining showed that deposition of amyloid in model group is more significant than control group,but amyloid plaque.Bielschowsky's silver staining shows that nerve fibers in the control group of rats were arranged regularly and closely. Compared with the control group,model groups of nerve fibers is thickening,swelling and disorder,but SP and NFT.Congo red staining and Bielschowsky's silver staining method does not appear senile plaques and neurofibrillary tangles.Diabetic rat brain has no characteristic pathological changes of Alzheimer's disease.Measurement of APP,Aβand BACE1The expression of APP,Aβand BACE1 in MT groups is higher than that of in S group(P＜0.01).The level of Aβand BACE1 has positive correlation with cognitive impairment.The level of APP has negative correlation with cognitive impairment.Three model groups have no significant difference in the expression of APP,Aβand BACE1.The expression of Aβ1-40 increases from(64.13±6.76) pg/mg in normal group to(86.43±7.03) pg/mg in model group by ELISA(P＜0.001) and Aβ1-42 from(67.43±5.12) pg/mg in normal group to(89.45±5.28) pg/mg(P＜0.001) in model group.ELISA assay showed that the expression of APP increases from(116.65±9.21) pg/mg in normal group to(156.73± 8.24) pg/mg in model group(P＜0.001),Western blotting method from 0.63±0.12 in normal group to 1.56±0.19 in model group(P＜0.001),RT-PCR method from 1.68±0.21 in normal group to 3.54±0.22 in model group (P＜0.001).The expression of BACE1 increases from(116.46±8.10)pg/mg in normal group to(158.73±6.24)pg/mg in model group by ELISA and from 0.61±0.11 in normal group to 1.52±0.16 by Western blotting OD valule and from 1.62±0.26 in normal group to 3.61±0.32 by RT-PCR OD valule and from 0.81±0.21 in normal group to 2.01±0.36 by immunohistochemistry OD valule(P＜0.001)。The expression of BACE1 and Aβin MT group is higher than that of in S group(P＜0.01).The level of BACE1 and Aβhas positive correlation with cognitive impairment.The major molecules in insulin signal pathwayThe results of PKC,PKA and JAK-2 by Western blotting show that: the level of protein expression of p38MAPK in model groups was no significant difference,compared with the control group(P＞0.05);RT-PCR results showed that the mRNA level of PKC,PKA and JAK-2 in the model group has been slightly increased,but no statistical significance(P＞0.05).Optical density by Werstern blot analysis revealed that protein level of PI-3K/Akt in model groups has lower than the control group,from the control group 0.914±0.065 to the model group 0.511±0.051(P=0.000); RT-PCR for molecular PI-3K/Akt show the same level of detection of PI-3K mRNA expression than in the control group significantly decreased from the control group 1.289±0.072 to the model group 0.821±0.066 (P=0.000),there was no significant difference in model groups(P=0.352). Optical density by Werstern blot analysis revealed that protein expression p38MAPK/ERK in model groups has lower than the control group,from the control group 0.926±0.072 to the model group 0.631±0.056(P = 0.000);RT-PCR for the detection of the molecular level p38MAPK/ERK mRNA expression showed a more significant downward adjustment from the control group 1.350±0.071 to the model group 0.96±0.069(P = 0.000),there was no significant difference in model groups(P = 0.367). Compared with the control group,,the expression of PI-3K/Akt, p38MAPK/ERK in the hippocampal tissue of diabetes mellitus model group decreased significantly(P=0.000),while the expression of BACE1 increased than the control group(P=0.000).BACE1 and PI-3K negatively correlated(P＜0.05),BACE1 and Akt is also negatively correlated(P＜0.05);BACE1 and p38MAPK was no correlation(P＞0.05),BACE1 and ERK1/2 was no correlation(P＞0.05),Aβ40/42 and p38MAPK negative correlation(P＜0.05),Aβ40/42 and ERK1/2 negative correlation(P＜0.05).Conclusions1.The expression of Aβ,APP increased in diabetes mellitus rats. Diabetes mellitus contributes to the pathogenesis of Alzheimer's disease that diabetic metabolic disorder increases expression of Aβbut APP.APP has a dual mechanism,both the role of cerebral protection, and increased expression of the role of Aβ.2.The expression of BACE1 and Aβincreased in diabetes mellitus rats. Diabetes mellitus contributes to Alzheimer's disease that Diabetic metabolic disorder increases expression of BACE1 and Aβ.3.Signaling pathway MAPK/ERK affect Aβmetabolism and signaling pathway regulates PI-3K/Akt involved in BACE1 expression in the pathogenesis of Alzheimer's disease.PI-3K/Akt signaling pathway might effect the expression of BACE1,which demonstrates that impaired signaling pathway shoud make amyloid precursor protein easy to be processed by BACE1 to involve the pathology of Alzheimer's disease.