| Alzheimer’s disease(AD) as a neurodegenerative brain disorder is the most common cause of dementia, it characterized by amyloid-β plaque accumulation, intracellular tangles and neuronal loss in selective brain regions. It is the most common cause of dementia, affecting 35 million people today, and also, it is a typical age-dependent neurodegenerative disease that affects 5 % of individuals >65 years, 20 % of those >85 years, and more than one-third of those >90 years. It will cause heavy burden to the family and society, therefore, it particularly urgent the research about the pathogenesis and prevention for AD. The increased brain iron deposition has been found in Alzheimer disease(AD) patients, which might be the result of misregulation of brain metal transporters. Misregulation of brain iron homeostasis is central to early neuropathological events in AD, including brain iron deposition has been proposed to play an important role in the pathophysiology of AD. Thus, the concept of iron chelation holds considerable promise as a therapeutic atrategy for AD pathogenesis. A single-blind, 2-year clinical study with the prototype iron chelator desferrioxamine(DFO) showed that sustained intramuscular administration slowed the chinical progression of the dementia associated with AD. Another metal ligand, clioquinol, also ameliorated cognitive decline in AD patients. However, clioquinol is highly toxic, while the hydrophilic nature of DFO and its large molecular size limit absorption across the gastrointestinal tract and prevent it from penetrating the blood-brain barrier(BBB). Oppositely, traditional chinese medicines are characterized by security, nontoxic, brain-permeable, and multifunction, which can overcome the pharmaco-clinical limitations of iron chelator. The beneficial effects of epimedium, astragalus and radix puerariaehave been attributed to the presence of active compounds that are powerful anti-oxidants, anti-inflammatory, and regulation of each organ function. Therefore, it is believed that active compounds of epimedium, astragalus and radix puerariaemay protect against brain iron overload and attenuate memory deficits. Therefore, the present study will evaluate the therapeutic effect of the active compounds of epimedium, astragalus and radix puerariaeon the neuropathology, deficits of spatial learning and memory, brain iron deposition in amyloid in amyloid precursor protein(APP)-transgenic(Tg) AD mice, and further experiments will be performed to study the molecuiar mechanisms. The aim of our study is to reveal novel therapeutic targets for neurodenerative disorders. Part 1 Effects of effective fraction of Epimedium, Astragalus, Radix Puerariae on behavioral changes in a transgenic mouse model of Alzheimer’s diseaseObjective: To investigated the effects of Epimedium, Astragalus, Radix Puerariae on behavioral changes in the APP/PS1 double transgenic mouse model of AD.Methods: A total of 108 specific-pathogen-free male APP/PS1 double transgenic mice aged 9 months were equally and randomly assigned to model, epimedium, astragalus, radix puerariae, compound and DFO groups. An additional 18 9-month-old C57BL/6J mice served as negative controls group. Mice in the epimedium, astragalus, radix puerariae and compound groups were given 120-mg/kg epimedium herb, 80-mg/kg milkvetch root, 80-mg/kg kudzuvine root, 120-mg/kg epimedium herb + 80-mg/kg milkvetch root + 80-mg/kg kudzuvine root via intragastric administration, once a day, for 3 consecutive months. Mice in the deferoxamine group were intraperitoneally injected with 30-mg/kg deferoxamine, once a day, for 3 consecutive months. Mice in the model and control groups were intragastrically administered 1 m L of normal saline. Using Morris water maze to investigated effects of Epimedium, Astragalus, Radix Puerariae on behavioral changes in the APP/PS1 double transgenic mouse model of AD.Results: The analysis of the place navigation trial showed that the escape latencies decreased from Day 1 to Day 5 in both the groups(p<0.05). The APP/PS1 mice displayed longer escape latencies than C57 mice(p<0.05), compared with AD model group and epimedium, astragalus, radix puerariae group, the escape latencies of compound group and DFO group were shorter(p<0.05), there was no significant difference between compound group and DFO group(P>0.05). there was no significant difference of the average swimming velocity in all groups(P>0.05). The spatial probe trial showed, compared with C57 group, the platformcrossing times, the time spent in the target quadrant and the score of search strategies of the mice in APP/PS1 AD model group were significantly decreased, compared with the AD model group and epimedium, astragalus, radix puerariae group, the mice in compound group and DFO group were increased, there was no significant difference between compound group and DFO group.Conclusions: The APP/PS1 double transgenic mice were cognitive deficits on learning and memory performance, and the compound of epimedium, astragalus, radix puerariae can improve the learning and memory ability of AD model mice. Part 2 Effects of effective fraction of Epimedium, Astragalus, Radix Puerariae on pathological changes in a transgenic mouse model of Alzheimer’s diseaseObjective: To investigated the effects of Epimedium, Astragalus, Radix Puerariae on pathological changes in the APP/PS1 double transgenic mouse model of AD.Methods: A total of 36 specific-pathogen-free male APP/PS1 double transgenic mice aged 9 months were equally and randomly assigned to model, epimedium, astragalus, radix puerariae, compound and DFO groups. An additional 18 9-month-old C57 mice served as negative controls group. Mice in the epimedium, astragalus, radix puerariae and compound groups were given 120-mg/kg epimedium herb, 80-mg/kg milkvetch root, 80-mg/kg kudzuvine root, 120-mg/kg epimedium herb + 80-mg/kg milkvetch root + 80-mg/kg kudzuvine root via intragastric administration, once a day, for 3 consecutive months. Mice in the deferoxamine group were intraperitoneally injected with 30-mg/kg deferoxamine, once a day, for 3 consecutive months. Mice in the model and control groups were intragastrically administered 1 m L of normal saline. Using Nissl’s staining, Bielschowsky staining, and transmission electron microscope to investigated the effects of Epimedium, Astragalus, Radix Puerariae on pathological changes in the APP/PS1 double transgenic mouse model of AD.Results: Nissl staining demonstrated that in the control group, nerve cells were arranged neatly and densely, abundant Nissl bodies were visible in the cytoplasm, Nissl bodies were present as dark blue staining, and nuclei were light blue in the cerebral cortex of mice. In the model group, neuronal swelling was observed and the number of cells were decreased. Cells were arranged sparsely and the intercellular space was increased. Intracytoplasmic Nissl bodies were reduced with unclear boundaries, and were stained light blue. Neuronal swelling was relieved, and the number of cells slightly increased in the epimedium herb, milkvetch root and kudzuvine root groups compared with the model group. In the compound and deferoxamine groups, nerve cells were arranged regularly and densely in the cerebral cortex, with the presence of abundant Nissl bodies in the cytoplasm, improving swelling and increasing the number of cells. The modified Bielschowsky staining showed that the neuro-fibers of the cerebral cortex of APP/PS1 double transgenic mice were enlarged, swollen, and dense. There were also senile plaques and nerve fiber tangles in the cerebral cortex of C57 mice. The mice in compound group and DFO group take a turn for the better than AD model group and epimedium herb, milkvetch root and kudzuvine root groups.The ultrastructure of cerebral cortex neurons in mice of each group by transmission electron microscopy, the neurons of the C57 group were normomorph, the neurons of the mice in APP/PS1 AD model group Severe degeneration and necrosis, the neurons of the mice in compound group and DFO group take a turn for the better than AD model group and epimedium herb, milkvetch root and kudzuvine root groups.Conclusions: The compound of epimedium, astragalus, radix puerariae can repair the cortex neurons in APP/PS1 AD model mice. Part 3 Effects of effective fraction of Epimedium, Astragalus, Radix Puerariae on brain iron load changes in a transgenic mouse model of Alzheimer’s diseaseObjective: To investigated the effects of Epimedium, Astragalus, Radix Puerariae on brain iron load changes in the APP/PS1 double transgenic mouse model of AD.Methods: A total of 36 specific-pathogen-free male APP/PS1 double transgenic mice aged 9 months were equally and randomly assigned to model, epimedium, astragalus, radix puerariae, compound and DFO groups. An additional 10 9-month-old C57 mice served as negative controls group. Mice in the epimedium, astragalus, radix puerariae and compound groups were given 120-mg/kg epimedium herb, 80-mg/kg milkvetch root, 80-mg/kg kudzuvine root, 120-mg/kg epimedium herb + 80-mg/kg milkvetch root + 80-mg/kg kudzuvine root via intragastric administration, once a day, for 3 consecutive months. Mice in the deferoxamine group were intraperitoneally injected with 30-mg/kg deferoxamine, once a day, for 3 consecutive months. Mice in the model and control groups were intragastrically administered 1 m L of normal saline. Using Perls’ staining and Graphite furnace atomic absorption spectrometry method to investigated the effects of Epimedium, Astragalus, Radix Puerariae on brain iron load changes in the APP/PS1 double transgenic mouse model of AD.Results: Brain iron deposition was assessed using Perls’ staining, the result reveal that the iron load in the mice brain. Compared with the C57 group, there were more blue spots in AD group(P<0.05). compared with the AD model group, the blue spots of epimedium, astragalus, radix puerariae groups are lower(P<0.05), Compared with epimedium, astragalus, radix puerariae groups, compound of epimedium, astragalus and radix puerariae and DFO group can reduce the blue spots of AD model mice(P<0.05). There was no difference between compound group and DFO group(P>0.05).Using a Graphite furnace atomic absorption spectrometry method, total iron concentrations in the cortex was detected. The results show that Compared with the C57 group, the iron concentration is higher in AD group(P<0.05). compared with the AD model group, the iron concentration of epimedium, astragalus, radix puerariae groups are lower(P<0.05), Compared with epimedium, astragalus, radix puerariae groups, compound of epimedium, astragalus and radix puerariae and DFO group can reduce the the iron concentration of AD model mice(P<0.05). There was no difference between compound group and DFO group(P>0.05).Conclusions: The iron concentration of AD model group is much higher, the compound of epimedium, astragalus and radix puerariae can reduce the iron concentration of AD model mice. Part 4 Effects of effective fraction of Epimedium, Astragalus, Radix Puerariae on the expression of DMT1、FPN1 in a transgenic mouse model of Alzheimer’s diseaseObjective: To investigate the effects of Epimedium, Astragalus, Radix Puerariae on DMT1 and FPN1 expression in the cerebral cortex of APP/PS1 double transgenic mouse model of AD.Methods: A total of 36 specific-pathogen-free male APP/PS1 double transgenic mice aged 9 months were equally and randomly assigned to model, epimedium, astragalus, radix puerariae, compound and DFO groups. An additional 10 9-month-old C57 mice served as negative controls group. Mice in the epimedium, astragalus, radix puerariae and compound groups were given 120-mg/kg epimedium herb, 80-mg/kg milkvetch root, 80-mg/kg kudzuvine root, 120-mg/kg epimedium herb + 80-mg/kg milkvetch root + 80-mg/kg kudzuvine root via intragastric administration, once a day, for 3 consecutive months. Mice in the deferoxamine group were intraperitoneally injected with 30-mg/kg deferoxamine, once a day, for 3 consecutive months. Mice in the model and control groups were intragastrically administered 1 m L of normal saline. Using immunohistochemistry and molecular biology methods to investigated the effects of a compound combining the effective components of epimedium, astragalus, radix puerariae, on DMT1 and FPN1 expression in the cerebral cortex of APP/PS1 double transgenic mouse model of AD.Results: Immunohistochemical staining results revealed that DMT1-with/without IRE expression was higher(P<0.05), and FPN1 expression was lower(P<0.05) in the cerebral cortex of the model group compared with the control group. DMT1-with/without IRE expression was lower in the epimedium herb, milkvetch root, kudzuvine root, compound and deferoxamine groups than in the model group(P<0.05). FPN1 expression was higher in the epimedium herb, milkvetch root, kudzuvine root, compound and deferoxamine groups than in the model group(P<0.05). DMT1-with/without IRE expression was higher(P<0.05), but FPN1 expression was lower(P<0.05) in the epimedium herb, milkvetch root and kudzuvine root groups compared with the deferoxamine group. No significant difference was detected in DMT1-with/without IRE and FPN1 expression in mouse cerebral cortex between deferoxamine and compound groups(P>0.05). Real time-PCR results indicated that DMT1-with IRE m RNA and DMT1-without IRE m RNA expression was higher(P<0.05), and that FPN1 m RNA expression was lower(P<0.05) in the cerebral cortex of the model group cmpared with the control group. DMT1-with IRE m RNA and DMT1-without IRE m RNA expression was lower(P<0.05) and FPN1 m RNA expression was higher(P<0.05) in the epimedium herb, milkvetch root, kudzuvine root, compound and deferoxamine groups compared with the model group. DMT1-with IRE m RNA and DMT1-without IRE m RNA expression was higher(P<0.05), and FPN1 m RNA expression was lower(P<0.05) in the epimedium herb, milkvetch root, and kudzuvine root groups compared with the deferoxamine group. No significant difference in DMT1-with/without IRE m RNA and FPN1 m RNA expression was detected between compound and deferoxamine groups(P>0.05). Western blot assay indicated that DMT1-with IRE and DMT1-without IRE expression was higher(P<0.05) and that FPN1 expression was lower(P<0.05) in the model group compared with the control group. DMT1-with IRE and DMT1-without IRE expression was lower(P<0.05) and FPN1 expression was higher(P<0.05) in the epimedium herb, milkvetch root, kudzuvine root, compound and deferoxamine groups compared with the model group. DMT1-with IRE and DMT1-without IRE expression was higher(P<0.05) and FPN1 expression was lower(P<0.05) in the epimedium herb, milkvetch root and kudzuvine root groups compared with the deferoxamine group. No significant difference in DMT1-with IRE and DMT1-without IRE and FPN1 expression was observed between the compound and deferoxamine groups(P>0.05). Western blot assay results were consistent with the results of real time-PCR.Conclusions: downregulated divalent metal transporter 1 expression, upregulated ferroportin 1 expression, and inhibited iron overload in the cerebral cortex of mice with Alzheimer’s disease. These compounds reduced iron overload-induced impairment of the central nervous system, indicating a new strategy for developing novel drugs for the treatment of Alzheimer’s disease. |
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