Effects Of Acute Stress On The Amyloidosis Of Mice Brain

Bovine spongiform encephalopathy (BSE), Creuzfeldt Jakob Disease, Alzheimer's disease (AD) and Scrapie, are the common degenerative diseases of central nervous system in animals and human. The main pathological feature of these diseases is amyloidosis in the brain. The hypothesis of amyloid-β(Aβ) suggests that Accumulation of Aβis an invariant character and primary cause of amyloidosis of brain. Aβproduction is the result of amyloid precursor protein ( APP) which was sequencely shearing ofβ-secretase andγ-secretase. Moreover, the activity ofγ-secretase is essential. The amyloidosis of brain has different types, such as AD, the type of AD can be sporadic or familial in nature. Most of AD cases are sporadic and result from heterogeneous causes, including genetic and environmental factors. At present, lots of studies in AD research focus on the effect of genetic factors on AD pathogenesis, and growing evidence reveals that mutation can affect the activity ofγ-secretase, a pivotal enzyme for Aβproduction, and therefore associate with most of familial AD cases. However, it is largely unknown whether and howγ-secretase activity and Aβproductioq are regulated by environment. Therefore, in this study, we set up an animal model of acute stress and study how stress factor affects the cause and development of brain amyloidosis. The main contents of research for instance,1. Setting up an animal model of acute stress. We designed the primer of C-FOS gene according to the announced data of Gene Bank. C57 mice were immobilized in small cages. After 2 hours, we found that the features of mice being acute stressed, such as increase of bristle pelsge quantity, sensitive, agitated, fussiness, fluffiness of pelage and deficiency of luster. After killed the mice, we gained liver of mice and extracted the total RNA of liver in order to RT-PCR and fluorescent quantity PCR. So after calculating and comparing the data of stress mice to the control ones, our result shows that the mRNA level of C-FOS gene in the liver of acute stress mice is 4 times of the control ones.2. Detecting the level of Aβin the hippocampus of C57 mice with sandwich enzyme linked immunosorbent assay. We collected the hippocampus of the acute stress mice on ice and extracted the protein of hippocampus. And that detected the level of Aβin the hippocampus of C57 mice through ELISA assay, including the level of Aβin the positive control and the native control. At the wave length of 495 nm, we obtained the data with M5 enzyme mark meter that the fluorescent substrate was aroused and produced the fluorescence value. According to the positive protein standard curve, we calculated that the concentration of Aβ₄₀ in the hippocampus of acute stress mice was 1.68nmol/L and the concentration of Aβ₄₂ is 1.7nmol/L. However, the concentration of Aβ₄₀ and Aβ₄₂ in the hippocampus of control mice was 1.01 and 1.0 nmol/L. Our results suggest that both the level of Aβ₄₀ and the level of Aβ₄₂ in the hippocampus of acute stress mice are significantly higher than that of the control mice (p<0.01) . However, we detected the concentrations of Aβ₄₀ and Aβ₄₂ in the cortex of acute mice and the control, the results indicated that both of them are 1.0 nmol/L and that there is no significant difference.3. Examining the activity ofγ-secretase in the hippocampus of C57 mice with fluorescence assay. After acute stress mice, we collected the hippocampus of mice on ice and tissue homogenate, brachytely centrifuged, quantitated the protein, aspirated 6 to 8 times with bullet for insulin injecting in order to mechanically disrupt the membrane of tissue cell, centrifuged at 15,000rpm for 15 min, got out the supernatant fluid, added into the buffer with fluorescence substrates and water-bathed at 37℃for 2 hours. For the wave length of 355 and 440 nm, we obtained the data with M5 enzyme mark meter that the fluorescent substrate was aroused and produced the fluorescence value. We got the fluorescence value ofγ-secretase in the hippocampus of acute stress mice, the value is 95.4 and the value of the control is 65.2 and the value of blank is 53.7. So the data indicated that the activity ofγ-secretase in the hippocampus of acute stress mice enhances 60% more than the control ones. The fluorescence value ofγ-secretase in the cortex of acute stress mice and its control was detected through the same assay, the result displayed that the acute stress had no obvious effect on the activity of cortex in mice since their values were about 60.3. For verifying whether acute stress did influence the production of amyloid-8 only through altering the activity ofγ-secretase, in this study, we detected the activity ofα-secretase andβ-secretase with the above stated assay, and founded that they both of them have no obvious change in the hippocampus and cortex of acute stress mice and their control.4. Determining the phosphorylation level of protein tau in the hippocampus of acute stress mice with Western blot assay. After acute stress, we collected the hipppcampus of mice on ice and extracted the tissue protein with the kit of protein extraction. The protein ran 10% polypropylene acid amide gel, transferred to nitrocellulose membrane and western blot with the antibody of anti-phosphorylation. Through Adobe Photo shop soft work statistic the results of western blot, the results shows that the gray scale value of the phosphorylation of tau protein in acute stress mice is 4.56 and that of the control is 1.48. So the phosphorylation level of protein tau in the hippocampus of acute stress mice is 3 times of the control ones. In summary, our results indicated acute stress can obviously raise the phosphorylation level of protein tau in the hippocampus of mice.