Synapse-protective Effects Of Flavonoid Extract From Leaves Of Diospyros Kaki In APP/PS1 Mice

BackgroundNowadays, with the increasing problems of aging population, the prevalence of various neurodegenerative diseases, such as Alzheimer’s disease (AD) and Pakinsons’ disease (PD), have increased worldwide. AD, the most prevalent cause of dementia, has drawn more and more attention. However, therapeutic effect of drugs in clinical is still not very good. AD is a late-onset disease, asymptomatic in some phase during the progression, and therapeutic effect on late phase is limited. It may be inspiration if drug was used earlier. Synapse plays a key role in combination among neurons, and it was convinced to be an early biomarker of AD. Thus, the importance of synapse has drawn increasing attention of researchers in recent years.APP/presenilin-1(PS1) double transgenic mice is the most widely used mice model for AD, and it was revealed that in APP/PS1 mice brain, plaques and neurofibrillary tangles can be tested in early phase, and exhibit learning and memory impairment as early as 6-month-old.Flavonoids is widely spread in nature, and flavonoids extract from coca, blueberry, ginkgo leaf and green tea have been studied for its free radical scavenging, anti-inflammatory, anti-oxidative, anti-virus and anti-tumor activity in both physical and pathological status. Researches showed that flavonoid intake in daily life may be responsible for the decreased incidence of neurodegenerative disease. Flavonoids extract from leaves of Diospyros kaki (FLDK) was able to alleviate brain injury from ischemia and protect neurons from excitotoxicity with few side-effects. In our experiment, APP/PS1 mice model was used to investigate the neuroprotective effect of FLDK on synapse structure and function.ObjectiveAPP/PS1 double transgenic mice model was used as AD model. Morris water maze test was used to test learning and memory abilities, transmission electron microscope (TEM) was used to observe ultrastructure of synapse in mice hippocampus regions, and western blot and immunohistochemistry were used to investigate the expression levels of related proteins.MethodsTwenty APP/PS1 mice of 4-month-old were randomly divided into 2 groups:AD model group (APP/PS1 group) and treated group (APP/PS1+FLDK group) (n=10 in each group). Twenty 4-month-old C57BL/6 mice were randomly divided into normal control group (NC group) and treated group (FLDK group) (n=10 in each group). According to previous research, the dose of FLDK administration was selected as 80mg/kg/day, and same dose of saline was used for APP/PS1 group and NC group. FLDK and saline were administered intragastrically for six months. Morris water maze assays the escape latency (EL) and times of crossings. TEM observes the ultrastructure of synapse in mice hippocampus. RhoA and Racl activity were detected by Rho GTP activity kit. Western blot detected the expression levels of ERK1/2, CREB, synaptophysin and drebrin. Immunohistochemistry was used for the distribution of RhoA and the expression levels of ERK1/2 and CREB.Results1. Influence of FLDK on APP/PS1 double transgenic mice in a behavioral test: Compared with APP/PS 1 group, EL was significantly shorter (P<0.01) and number of crossings were significantly increased (P<0.05) in APP/PS1+FLDK group. Compared with NC group, EL was significantly shorter (P<0.05) and number of crossings were significantly increased (P<0.05) in FLDK group.2. Ultrastructure of synapse in mice brain observed by TEM: In the APP/PS 1 group, the structure of synapse was unclear, with reduced synaptic vesicles and few PSD. In contrast, FLDK-treated APP/PS1 mice exhibited less severe pathological changes, the synaptic ultrastructure was integrate, with a clear synaptic structure and increased PSD. In the NC and FLDK groups, the integrity of the synapse ultrastructure was clearly seen with synaptic vesicles in the presynaptic membrane and thick postsynaptic densities (PSD) in the postsynaptic membrane.3. The activity assay of RhoA and Rac1: RhoA activity was significantly higher in APP/PS1 mice than in normal mice (P< 0.01), and FLDK treatment significantly decreased RhoA activity in APP/PS1 mice (P<0.05). In contrast, Racl activity was significantly lower in APP/PS1 mice than in normal mice (P<0.01), and FLDK treatment did not significantly reduced Racl activity in APP/PS1 mice.4. Expression levels of ERK1/2, CREB, SYP and Drebrin in each mice brain: Six months of FLDK treatment significantly upregulates the expression levels of ERK1/2, CREB, SYP and Drebrin, in both APP/PS1 mice (P<0.01) and normal mice (P<0.05).5. Distribution of RhoA in cortex and CA1 region of mice brain by immunohistochemistry: In APP/PS1 mice, RhoA-immunopositive puncta appeared more in neurites and less in cell bodies. After FLDK treatment for six months, RhoA-immunopositive puncta decreased in neurites and increased in the cytosol. In normal mice, RhoA was mainly located in the cytosol of the neuron.6. Expression of phospho-ERK1/2 and phospho-CREB in CA1 region of each group detected by immunohistochemistry With specialized anti-Phosphor-ERK1/2 and anti-phosphor-CREB antibodies, nucleus were stained brown. Compared with APP/PS1 group, brown puncta in NC group and FLDK group were obviously increased, and expression of immunopositive puncta of phosphor-ERKl/2 and phosphor-CREB were higher in APP/PS1 mice and normal mice after administration of FLDK.ConclusionFLDK significantly rescued cognition impairment in both APP/PS1 mice and 10-month old C57BL/6 mice, protect the ultrastructure and integrity of synapse, elevated the expression of synapse-related proteins. Thus, FLDK might be a potential agent for Alzheimer’s disease.