| Objective:To study the protective effects of extracts from waternut Herb on Alzheimer's disease(AD)and its molecular mechanisms in rats.Methods:Waternut Herb extracts were prepared into aqueous extract and alcohol extract in our lab. The screened rats were randomly divided into eight groups:negative group, sham operated group, Alzheimer's disease (AD) model group, positive therapy group, two alcohol extract groups with high or low dose and two aqueous extract groups with high or low dose. Alzheimer's disease (AD) model in rats was established by subdural injection with β-amyloid protein (Aβ,10μg/rat). Morris water maze was used to test the learning and memory abilities of rats; Pathological changes were observed under light microscope (HE staining) and transmission electron microscope; The activities of GSH-Px, T-SOD and the content of MDA in the serum were individually determined by spectrophotometric method. Also ELISA assays were performed to measure the levels of IL-1, IL-6and TNF-α in the serum. The expressions of NOS (including nNOS and iNOS), bcl-2/bax and GFAP proteins in the tissue of cormu ammon (CA) were detected by immunohistochemistry technique. And the transcriptional expressions of NF-κB/IκB, HSP70, APP and BACE1genes in CA tissue were measured by reverse-transcription PCR (RT-PCR) method.Results1. AD model in rats was successfully established and qualified by the results of Morris water maze test and pathological evidences. Escape latency period was significantly prolonged in the AD group (45.5±6.25s), compared with negative group (24.5±5.75s). Similarly, numbers of times across platform were largely decreased in the AD group (1.7±0.89) comparing with (3.2±1.27) in the negative group. Under the transmission electron microscope, slides of AD group showed that cell compartment among the nerve cells were extremely broadened with much less synapse and confluence of synaptic vesicle, as well as severe swelling. And also cell membrane and nuclear membrane were shrinked and partly confluenced with abnormal karyomorphism, heterochromatin aggregation as well as the evident damages of organelles such as mitochondria, ribosome, lysoome and lipofuscin accumulation.2. Waternut Herb extracts obviously improved the capabilities of learning and memory in AD rats. Low and high alcohol extract extremely shortened the escape latency period in dose-dependent way (34.5±7.25s and24.0±6.5s respectively), significant with AD model (45.5±7.3s). Especially, similar effect was observed in both high alcohol extract group and positive therapy group. Meanwhile, the numbers of times crossing platform were increased by the individual Waternut Herb extracts in the different extent with the most evident effect in high alcolhol extract group.3. Pathological examinations by HE staining showed that the quantities of nerve cells in the tissue of CA were significantly increased by the treatment with high alcohol extract, with improved karyomorphism and tight arrange. Also the evidence from transmission electron microscope exhibited that clear cell limit, tighter conjunction and relative normal of nuclear karyomorphism, ribosome, mitochrondria and liposuscin, accompanying with swelling micrangium and astrocyte (AS) in the positive therapy group. High aqueous and alcohol extracts improved, in some extent, pathological appearances such as cell shrinkage, numbers of synapses, nuclear karyomorphism. It should be noted that a large number of autophagosome were observed in the low dose of alcohol extract.4. All Waternut Herb extracts significantly inhibited Aβ1-40level compared with its level (316.8±28.2pg/mL) in AD model (p<0.05, p<0.01), with the most evident in alcohol extracts. Alcohol extracts (both low and high doses) and high aqueous extract decreased the contents of MDA, meanwhile increased the activity of GSH-Px in the serum of rats. All the extracts enhanced the activities of T-SOD compared with AD model (p<0.01,p<0.05). The secretion of inflammatory factors such as IL-1, IL-6and TNF-α in the serum were also obviously suppressed by all extracts, with the most inhibitory effect in the high alcohol extract group.5. Waternut Herb extracts inhibited the expression of GFAP protein, induced the anti-apoptotic protein bcl-2expression, and meanwhile reduced bax apoptotic protein expression which lead to the increase of bcl-2/bax ratio in CA. In addition, both aqueous and alcohol extracts with high doses lessened the expression of induced-NOS(iNOS), while enhanced the expression of neron-type NOS(nNOS).6. Waternut Herb extracts down-regulated APP mRNA expression, the precursor of Aβ and BACE1mRNA, β-site App cleaving enzyme of APP. Additionally, all extracts (especially for alcohol extract) largely inhibited the expression of NF-κB mRNA, while increased IκB mRNA expression, an inhibitor of NF-κB. There is no observed difference in the expression of HSP70between negative group and AD model group (p>0.05).Conclusions1. Alzheimer's disease model in rat was successfully by subdural injection with β-amyloid protein (Aβ,10μg/rat).2. Waternut Herb extracts present the therapeutic effect on AD rats.3. The molecular mechanisms were involved in the inhibition of neuron cell apoptosis and inflammatory factors secretion, as well as in the improvement of anti-oxidant capabilities.
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