Background
Alzheimer’s disease (AD) is the most common type of dementia. AD is a chronic degenerative disease of the primary central nervous system, characterized by progressive cognitive dysfunction and memory impairment. The incidence of AD has increased annually, and it is currently estimated that more than 10% of the elderly are suffering from AD. As such, AD has become the third leading cause of death in the elderly [
1]. Currently, drugs for the treatment of AD consist of cholinesterase inhibitors and N-methyl-D-aspartate receptors (NMDA) antagonists; however, these drugs cannot control or reverse the progression of AD. Moreover, some medicines have certain side effects. Therefore, we need to explore new drugs to slow down cognitive decline.
The deposition of Aβ leads to the destruction of synaptic signaling pathways and the destruction of dendritic spines, which affects synaptic morphology and function, leading to memory and behavioral defects [
2]. Many studies suggest that the protection of synapses can diminish the observed memory deficits in AD [
3]. Long-term potentiation (LTP) is widely considered to be one of the major cellular mechanisms of learning and memory [
4], which has received much attention in Alzheimer’s disease (AD), a neurodegenerative disease that causes obvious cognitive decline and dementia. The accumulation of Aβ is considered to be a main pathogenic feature of AD. Studies have found that Aβ levels increase in the brain of AD patients and AD mouse models. Aβ accumulation may cause multiple neuronal damage and lead to cognitive impairment [
5].
The Huatuo Zaizao pill (HT), a pure natural preparation derived from plants, which is a TCM formulation marketed in China [
6]. HT was listed in the pharmacopoeia of the People’s Republic of China Part I (Pharmacopoeia Commission of the People’s Republic of China, 2015), as HT can be used to promote rehabilitation after stroke. HT was widely used in the treatment of cerebrovascular diseases, stroke and hemiplegia [
7‐
9]. Meta-analysis showed that HT promoted the functional recovery in patients with ischemic stroke [
10,
11]. HT treatment could promote functional recovery by enhancing the expression of brain-derived neurotrophic factor (BDNF) and increasing the level of neurogenesis in cerebral I/R animal [
12]. HT mainly composed of five Chinese herbs:
Ligusticum chuanxiong hort (Umbelliferae), Evodia rutaecarpa (Juss.) Benth, Carthamus tinctorius L. (Safflower), and Angelica sinensis (Oliv.) Diels, Borneol. Researches have shown that HT have many pharmacological effects, which could improve the cognitive decline [
13]. HT combined with donepezil improved the cognitive function in the treatment of subcortical ischemic vascular dementia [
14]. Another study showed that HT improved the degree of cognitive function and memory impairment by improving blood hypercoagulability and microcirculation [
15]. HT contains many ingredients, such as Ferulic acid (FA), Evodin, Evodiamine and Rutaecarpine, especially FA has a therapeutic effect in the treatment of AD. FA reduced the progression of cognitive deficits in animal models [
16‐
18].
Although some clinical reports indicated that HT can be used to treat cognitive decline, but there is no study on its mechanism of action. In our study, we hypothesize that HT alleviates cognitive impairments. As one of the mouse models of AD, APPswe/PS1dE9 (APP/PS1) co-expressing the delta exon 9 variant of presenilin 1 (PS1) and the Swedish mutation of β-amyloid precursor (APP) [
19,
20]. Additionally, APP/PS1 transgenic mice have developed a few Aβ plaques and began to have deficits in learning and memory at 6 months of age compared to wild-type (WT) mice [
21‐
23]. Thus, in this study, we showed whether HT could attenuate memory impairment in the APP/PS1 mice and investigate the molecular mechanisms involved in the protection of synapses.
Methods
Drugs
HT was obtained from GuangZhou BaiYun Shan QiXing Pharmaceutical Co, Ltd. (Guangzhou, China; batch number: 17121), and placed in 4 °C. The dosage for clinical patients is 8–16 g per day. HT quality management data can be found in the pharmacopoeia of the People’s Republic of China Part I (Pharmacopoeia Commission of the People’s Republic of China, 2015). The HT solution was dissolved in 0.9% physiological saline with HT powder (0.0625 g/ml).
Animals
Six-month-old male APP/PS1 transgenic mice which have memory deficits (provided in the Additional file
1: Figure S1) were provided by the Model Animal Research Center of Nanjing University in China. The mean body weight was 36 ± 5.2 g. All animals were housed in standard cages under 12 h/12 h light/dark conditions with free access to food and water. All animal experimental procedures were approved by the Animal Care Committee of Nanjing University and conformed to the Animal Ethical Standards.
Grouping and treatment
Animals were placed into each group using a randomized block design. The selected HT dosages (0.25 g/kg) were based on our previous experiment (provided in the Additional file
2: Figure S2). Mice were divided into the following groups: APP/PS1 group, HT at 0.25 g/kg group, and MEM at 5 mg/kg group. HT and MEM were dissolved in 0.9% saline. The APP/PS1 group was given the same volume of saline. The treatments were administered intragastricly once daily for 4 weeks. At the end of the fourth week, The Morris water maze, Novel Object Recognition, and Open-field tests were used to assess cognitive behavioral changes. The mice were anesthetized depth with sodium pentobarbital (50 mg/kg) and killed by decapitation in an unconscious state [
24]. Then the brains were harvested for electrophysiology, Golgi Staining, histopathological, Immunofluorescence staining, and Western blotting assessments.
Morris water maze
The cognitive function of the animals was evaluated using a Morris water maze as previously described [
25]. The Morris water maze (MWM) test was administered before treatment (to confirm behavioral deficits in APP/PS1 mice) and after the HT, MEM and saline treatments. In brief, mice can escape onto the platform within 60 s and then stay on the platform for at least 5 s during acquisition phase trials (days 1–5). If the mice failed to find the platform within the permitted 60 s, they were manually placed on the platform for 30 s so that they could recollect the location of the platform, and the latency was recorded for 60 s. The escape distance and escape latency were analyzed by Any-maze software (Stoelting, USA). On the 6th day, the platform was removed from the water maze, and the mice were released to swim freely for 60 s. The number of times they crossed the target platform and the time spent in the target quadrant were automatically recorded by the video/computer system.
Novel object recognition
The Novel Object Recognition test is conducted in an open field arena. During habituation, the mice are allowed to explore an empty arena. Three days after habituation, the mice were presented with two similar objects during the first session. Then, one of the two objects was replaced by a new object during a second session. The time spent exploring each object and the discrimination index percentage were recorded. This test can be used to assess the cognitive impairment of transgenic mice and to assess the impact of new objects on their cognition.
Open field test
The open field test (OFT) is primarily used to observe a variety of behaviors after the mouse is released into the open environment and reflects the neuropsychiatric activities of the experimental animal. Spontaneous open field experiments can observe the spontaneous activities of mice. The experimental device was a square Plexiglas box, and the wall was a brown color. A video tracking system was fixed above the experimental platform. The mice were placed in the same position in the box, and the total distance the mice traveled within 5 min was recorded. The field instrument was cleaned up after the experiment to avoid leaving an abnormal odor. The following observations were made: the total distance, central time, corner time, and the trajectories of the mice.
Electrophysiology
Hippocampal CA1 long-term potentiation (LTP) was recorded as described previously [
26]. Transverse slices of the hippocampus (400 mm) were cut from the mouse brain. The brains were rapidly removed after decapitation and maintained in cold oxygenated (95% O2/5% CO2) physiological media. The hippocampal slices were continuously perfused with artificial cerebrospinal fluid (ACSF) composed of the following: NaCl 124 mM, CaCl2 2.0 mM, KCl 4.5 mM, MgCl2 1.0 mM, NaHCO3 26 mM, NaH2PO4 1.2 mM, D-glucose 10 mM, and pH 7.4. The orthorhombic stimulus was used to stimulate the Schaffer collateral/connective pathway using a polished bipolar tungsten electrode. The stimulator (SEN-3301, Nihon Kohden, Japan) provides a constant current pulse (0.1 ms, 0.05 Hz). The excitatory postsynaptic potential (EPSP) was recorded from the radiation layer of the CA1 region and connected to a preamplifier with a high pass pulse of 5 kHz. The signal was amplified using a different AC amplifier (A-M Systems, 1700, Seattle, WA). EPSP was digitized using the pCLAMP system (Axon Instrument Inc, Foster City, CA). The input/output (I/O) function was measured by the average EPSP slope and the stimulus intensity of 0.1–1.1 mA. Long-term enhancement (LTP) is generated by high frequency stimulation (HFS, 100 Hz, pulse 100 Hz) with the same intensity as the previous HFS. Subsequently, the single pulse recording was resumed and continued for 60 min. When the EPSP slope is more than 60 min longer than 20% of the baseline, it is considered to be LTP induced.
Golgi staining for dendritic spines
According to the manufacturer’s instructions, Golgi staining was performed using a fast Golgi staining kit (FD Neurotechnologies, Elliot City, MD, USA). The brain tissues were mixed with solution A and B at room temperature for 2 weeks in the dark and then transferred to solution C at 4 °C for at least 2 days and up to 7 days. Subsequently, the samples were placed in a 200-μm slice by a cryostat and placed on a microscope slide. The sections were rinsed twice for 4 min each with a double distilled water or Milli-Q water. The slices were placed in a mixture containing 1 part of solution D, 1 part of solution E and 2 parts double distilled water or Milli-Q water. Then, Golgi Staining was performed as described previously [
6]. The slides were dehydrated and cleared, and a coverslip was placed.
Western blotting
Proteins were isolated from the hippocampus of brain according to the protein extraction kit instructions (Beyotime Biotechnology, China). Protein concentrations were determined using the BCA protein assay kit (Bioworld, USA). Equal volumes of protein were separated using 10% dodecyl sulfate, sodium salt polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked in 5% non-fat milk for 1 h and incubated overnight at 4 °C with primary antibodies as follows: 6E10 (1:1000; Covance, USA), anti-APP (1:1000, Abcam Technology, UK), anti-PSD95 (1:1000, Cell Signaling Technology, USA), anti-SYN (1:1000, Abcam Technology, UK), anti-IDE (1:1000,Abcam Technology, UK), anti-ADAM10 (1:1000, Millipore Technology, USA), anti-BACE1 (1:1000, Millipore Technology, USA), anti-PS1 (1:1000, Cell Signaling Technology, USA), and anti-GRPDH (1:2000, Cell Signaling Technology, USA). Membranes were rinsed with Tris-buffered saline with Tween 20 (TBST) three times. Then, membranes were incubated with HRP-conjugated anti-rabbit or anti-mouse secondary antibodies for 2 h. Protein signals were visualized with the chemiluminescence reagents provided with the ECL kit (Bioworld, USA), and quantitation of proteins was determined by densitometric analysis using ImageJ software (Bio-Rad, Hercules, USA).
Hematoxylin-eosin staining (HE) staining
HE staining was used to observe the pathologic changes in the hippocampal CA1 and cortex areas in APP/PS1 mice. The brain slices embedded in paraffin were cut into 4-μm slices. The brain slices were dewaxed in xylene and dehydrated by fractional alcohols. Then, sections of each group were stained with HE substance cresyl violet. The neurons in the hippocampal CA1 and cortex were examined using an Olympus optical microscope.
Immunofluorescence staining
Immunofluorescence staining was performed as described previously [
27]. After anesthesia, the mice were perfused with 0.9% saline and 4% paraformaldehyde. The brain was removed and placed in 15 and 25% sucrose solution. Subsequently, the brain was cut into 40 μm slices using a low temperature thermostat (Leica, Germany). Each section was incubated with 10% normal sera, followed by overnight incubation with the primary antibodies, 6E10 (1:500, Covance, Princeton, NJ, USA), at 4 °C. Then, sections were incubated with the appropriate secondary antibodies for 2 h at room temperature in the dark. The Aβ antibody showed dense amyloid deposits in the cortex and hippocampus of APP/PS1 mice with an Olympus microscope and a BX51 digital camera (Olympus, Japan). The images were quantified with IPP 6.0 software. All measurements were performed in a randomized and blinded manner.
Statistical analysis
The results were expressed as the mean ± SEM and analyzed using SPSS 18.0 statistical analysis software (SPSS, USA). Group differences in escape latency were analyzed using two-way analysis of variance (ANOVA) with repeated measures followed by Bonferroni multiple comparison test with day and treatment as the sources of variation. One-way ANOVA and Bonferroni’s post hoc were performed to analyze other data. P values < 0.05 were considered statistically significant.
Discussion
AD is the most common neurodegenerative disorder in aging populations worldwide. Aβ-induced synaptic dysfunction and loss, which is associated with memory deficits, reduction of synapses, and decreases in synaptic markers occurs in the early and late stages of AD [
31]. Aβ is very important in the pathogenesis of AD. Aβ deposition form senile plaques damages synaptic function and eventually leads to neuronal death and cognitive decline. Inhibition of Aβ production is considered to be a potential strategy in AD treatment [
32,
33]. There are three main mechanisms of reducing Aβ deposition, i.e., reducing Aβ production [
34]; increasing Aβ degradation [
35]; and immune clearance of Aβ [
36].
APP/PS1 transgenic mice are a typical AD animal model. This model has abnormal behavior and Aβ production [
37]. The 6-7 month APP/PS1 mice have memory deficits, learning disabilities and Aβ deposition [
38,
39]. Therefore, we choose 6–7-month-old APP/PS1 mice as our experimental animals. The MWM test is a classic behavioral experiment that can test spatial memory. We used escape latency to observe spatial memory and crossing platform frequency and swimming speed to observe working memory. We found that HT can improve spatial memory and working memory in APP/PS1 transgenic mice.
LTP was first observed by Terje Lømo in 1966 in the Oslo, Norway, laboratory of Per Andersen [
40]. In neuroscience, LTP constantly enhances synapses based on the recent pattern of activity. LTP is a well-characterized form of synaptic plasticity that fulfils many of the criteria for a neural correlate of memory [
41]. In a 2003 study, Rowan et al. proposed one model to explain the impact of AD on LTP [
42]. Hippocampal LTP impairment may lead to cognitive decline in early AD. In turn, AD may also damage LTP by a different Aβ mechanism. In our experiments, we found that HT can reverse LTP impairment in the hippocampus of APP/PS1 mice.
Our experiments with APP/PS1 mice showed that pretreatment with HT can reduce Aβ deposition in the brain of APP/PS1 mice. According to the Aβ cascade hypothesis, Aβ is a direct initiation factor that causes a series of pathological processes in AD. The amyloid precursor protein (APP) undergoes constitutive shedding by a protease activity called α-secretase. Three enzymes belong to a disintegrin and metalloproteinase (ADAM) family, ADAM9, ADAM10 and ADAM17. This is considered an important mechanism preventing the generation of the Alzheimer’s disease Aβ peptide (Aβ) [
43,
44]. ADAM10 is a member of the ADAM family, which is thought to be a constitutive α-secretase during amyloid-beta protein precursor (AβPP) cleavage. Studies have demonstrated the beneficial role of ADAM10 in reducing the pathologic damage of Alzheimer’s disease (AD) [
45,
46]. It has been observed that both ADAM10 localization and its activity are reduced in synapses in the brains of AD patients, especially in hippocampus. Therefore, given the synaptotoxic nature of Aβ and the role of ADAM10 in reducing Aβ generation, an appropriate level of ADAM10 in synapses is indispensable in regulating Aβ activity and maintaining normal synaptic function [
47]. Our study showed that HT can increase the expression level of ADAM10. γ-secretase is an aspartyl protease that cleaves a few number of substrates within the membrane environment [
48]. It contains at least four components that are required for enzymatic activity: the catalytic component presenilin (PS1 or PS2), anterior pharynx-defective-1 (Aph-1), presenilin enhancer-2 (Pen-2) and the essential cofactor nicastrin [
49,
50]. Inactivation of PS1 in APP transgenic mice results in a high accumulation of APP-CTFs at the front of the synaptic front, which could lead to impaired synaptic plasticity and long-term memory deficits [
51]. In addition, memory deficiencies in APP/PS1 transgenic mice can be treated by genetic inactivation of PS1, which is one method to effectively reduce Aβ accumulation and prevent plaque deposition [
52]. Therefore, a strategy for AD treatment may include the selective increase of α-secretase or γ secretase activity reduction to reduce Aβ production. The results show that HT can reduce the deposition of Aβ, reduce the formation of APP, and promote the degradation of IDE. HT can regulate the α and γ degradation pathway of APP at the same time. ADAM10 is thought to be a constitutive α-secretase during amyloid-beta protein precursor (AβPP) cleavage. HT can improve dementia through regulating ADAM10 expression. However, the underling molecular mechanisms are still not well established. Study showed that ADAM10 expression can be mediated by AMPK/SIRT1 manner [
20]. Additional, Sirtuin type 1 (SIRT1) upregulated ADAM10 gene expression via coactivation of the retinoic acid receptor (RAR) transcription factor, leading to reduced production of Aβ and plaques in mice model [
53]. In further research, we plan to explore the relationship between HT and SIRT1, in order to illustrate the possible mechanism of HT in treatment of AD.
TCM is the traditional medicine of the Chinese nation that maintains the nation’s reproduction and health; it provides hope for AD treatment. TCM composition is complex, which leads to several significant effects [
54] and may possess multi-target treatment for disease. HT consists of several medicinal herbs, including
Ligusticum chuanxiong hort (Umbelliferae), Evodia rutaecarpa (Juss.) Benth, Carthamus tinctorius L. etc. Clinical reports showed that HT could improve the cognitive function [
14,
15]. Results confirmed that there are mainly three compounds included in the HT, as ferulic acid, onjixanthone I and albiflorin [
7], especially FA has an effect on the treatment of Alzheimer’s disease [
55]. Since from Chinese medicine theory, HT contains many herbs which have function of promoting brain smart, resuscitation and these effects may be could improve cognitive function in AD patients. In this study, we found that HT can improve the memory disorders in APP/PS1 mice via enhancing partially the synaptic plasticity and reducing Aβ deposition.