| Alzheimer's disease (AD) is a kind of neurodegenerative disease in central nervous system (CNS) related to age. With its population ageing, there is an increase trend in the incidence of AD. It is such a heavy burden on society and family that none of us can ignore. Therefore, there is intense interest in searching for drugs reliable interventions to AD. The onset of AD exists evident sex differences according to epidemiological study. Postmenpause women are 1.5-3 times more likely to develop AD than men among the people with the same age. The estrogen replace therapy (ERT) has been reported recently that it can not only play a role in the prevention and treatment of AD, but also delay the patients' age of onset. But the protective mechanism hasn't been known.Premarin, purified from pregnant horse urine, is one of water-soluble conjugated equine estrogen (CEE). 17β-estradiol, the essential component in CEE, can be oral administration convenient-ly and has good compliance, which can effectively cure, prevent and control hypoestrogen and osteoporosis resulted in primarily hypovaria. Whether can Premarin be used to treat AD? What is its protective mechanism? Where is its action target in CNS? All of them will be resolved in the study.AD is a consequence coordination with multiple factors due to complicated mechanisms such as cholinergic theory,β-amyloid peptide (Aβ) theory and Tau protein abnormal phosphorylation theory. In the present studies, the effect of Premarin on behavior in the hormonoprivia and dementia model rats and the changes of choline acetyl-transferase (ChAT), brain derived neurotrophic factor (BDNF), Aβ, Tau protein and estrogen receptor (ER) in frontal lobe and hippocampus was observed respectively. The aim of the study is to investigate the protective mechanism of Premarin and to provide theoretical basis for clinical application to AD.Part I Establishment of the hormonoprivia and dementia model rats and effect of Premarin on behavior and histology1. Establishment of AD model rats30 adult female Wistar rats 4 months of age (with body weight of 200-300g) were randomly divided into 3 groups: sham-operated group, ovariectomized (OVX) plus di-hippocampal fimbria-fornix transected group (model group), model plus Premarin group (Premarin group). Each group was 10 respectively. Briefly, rats were anesthetized with 10% chloral hydrate (3.0ml~3.5ml/kg) and hibaterally blocked up hippocampal fimbria-fornix on the first day. A week later, hibateral ovaries were resected. Sham-operated animals were subjected to the same anesthesia without hippocampal fimbria-fornix block-up and OVX. After 2 weeks of recovery, Premarin group were orally injected with Premarin (0.02mg/d) for 8 weeks, while the other groups with saline sodium (1.5ml/d) as control.2. Behavioral testingSpatial learning and memory were examined using the Morris water maze task. During the trials, the time to reach the platform (escape latency) and the frequency to cross the platform (crossing frequency) were recorded. Significant group differences were seen in mean escape latency and crossing frequency wherein the model group did not perform the task as well as the sham-operated group. Compared to the model group, Premarin group rats took the shorter distance and more times to reach the goal. These results suggest that the impairment of learning and memory function can occur after OVX plus di-hippocampal fimbria- fornix transsection, but Premarin may help enhance the recovery of lost function.3. HE stainAnalysis of HE-stained tissue sections revealed that there were no neuronal loss and structural changes in the sham-operated animals. However, the raritas and disorder of pyramidal cells and swelling and pyknosis of the neuronal cells were seen in the model group. The Premarin group was shown to decrease injury in the cortex of frontal lobe and hippocampal CA1 region cells significantly. These results suggest that Premarin can protect CA1 neurons against the damage to which OVX plus dihippocampal fimbria-fornix transsection led.Part II Changes of cholinergic system and BDNF and effect of Premarin on themThe expression levels of ChAT and BDNF protein were detected by immunohistochemistry. Compared with the sham-operated group, the number of ChAT and BDNF positive neurons in the cortex of frontal lobe and hippocampal CA1 region decreased in the model group. Meanwhile, immunoreactive cells showed smaller cell bodies and weaker staining. There were significant differences in changes of ChAT and BDNF between the model group and the Premarin group, suggesting that Premarin can further improve the function of cholinergic system in CNS.Part III Changes of Aβand Tau protein and effect of Premarin on themThe main pathologic changes of AD is the formation of senile plaques (SP) and neurofibrillary tangles (NFTs). Excessive deposition of Aβconstitutes essential component of SP, however abnormal phosphorylation of Tau protein related to NFTs formation. The positive neurons of expressing Aβand Tau protein were measured by immunohistochemistry. The neuronal arrangement in disorder was observed in the model group, while neuron in the sham-operated group was seen no apparent abnormality. There was significant amelioration in neuronal structure in the Premarin group, showing that Premarin can reduce the accumulation of Aβand Tau protein, further lowering their neurotoxicity.Part IV Changes of ER in different encephalic regions and effect of Premarin on themEstrogen mainly play physiological roles by binding to its ER to form E-ER compound. ERαand ERβwere demonstrated to express and have distinct distribution in brain. RT-PCR technique was used to measure the mRNA levels of ERαand ERβin different encephalic regions. The results showed that Premarin up-regulated ERαmRNA level in cortex, basal forebrain and hippocampus, but it had no effect on ERβ.In conclusion, our study indicated that Premarin, binding to ERαreceptor can enhance the expression of ChAT and BDNF activity, inhibit the accumulation of Aβand Tau protein and thus improve the learning and memory ability.
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