GAPT regulates cholinergic dysfunction and oxidative stress in the brains of learning and memory impairment mice induced by scopolamine

Abstract Background Cholinergic dysfunction and oxidative stress are the crucial mechanisms of Alzheimer's disease (AD). GAPT, also called GEPT (a combination of several active components extracted from the Chinese herbs ginseng, epimedium, polygala and tuber curcumae) or Jinsiwei, is a patented Chinese herbal compound, has been clinically widely used to improve learning and memory impairment, but whether it can play a neuroprotective role by protecting cholinergic neurons and reducing oxidative stress injury remains unclear. Methods Male ICR mice were intraperitoneally injected with scopolamine (3 mg/kg) to establish a learning and memory disordered model. An LC‐MS method was established to study the chemical compounds and in vivo metabolites of GAPT. After scopolamine injection, a step‐down passive‐avoidance test (SDPA) and a Y maze test were used to estimate learning ability and cognitive function. In addition, ELISA detected the enzymatic activities of acetylcholinesterase (AChE), acetylcholine (ACh), choline acetyltransferase (ChAT), malondialdehyde (MDA), glutathione peroxidase (GPX), and total superoxide dismutase (T‐SOD). The protein expressions of AChE, ChAT, SOD1, and GPX1 were observed by western blot, and the distribution of ChAT, SOD1, and GPX1 was observed by immunohistochemical staining. Results After one‐half or 1 month of intragastric administration, GAPT can ameliorate scopolamine‐induced behavioral changes in learning and memory impaired mice. It can also decrease the activity of MDA and protein expression level of AChE, increase the activity of Ach, and increase activity and protein expression level of ChAT, SOD, and GPX in scopolamine‐treated mice. After one and a half month of intragastric administration of GAPT, echinacoside, salvianolic acid A, ginsenoside Rb1, ginsenoside Rg2, pachymic acid, and beta asarone could be absorbed into mice blood and pass through BBB. Conclusions GAPT can improve the learning and memory ability of scopolamine‐induced mice, and its mechanism may be related to protecting cholinergic neurons and reducing oxidative stress injury.


| INTRODUC TI ON
AD is thought to be a neurodegenerative disease. The prominent features of AD are the impairment of memory and cognition (Lane, Hardy, & Schott, 2018) and neuropathological hallmarks such as loss of neurons and synapses (Goedert & Spillantini, 2006), the agglomeration of neuritic plaques in the cortex (Grösgen, Grimm, Friess, & Hartmann, 2010), and the presence of intracellular neurofibrillary tangles (NFTs). Several hypotheses have been proposed in earlier studies. However, the mechanisms underlying AD are quite complicated and still uncertain. Some studies have noted that oxidative stress and cholinergic dysfunction play a critical role in the progression of AD (Kim et al., 2019).
To improve the research about cognitive impairments in AD, many mouse models and behavioral tests have been developed.
Scopolamine (a nonselective muscarinic blocker) has been widely used to establish an AD-like model since it can induce cognitive and memory deficits by promoting acetylcholinesterase (AChE) and upregulating brain iron (Wang, Zhong, Gao, & Li, 2017). Acetylcholine (ACh) and cholinergic nerves existing in the hippocampus and cortex are essential for regulation of learning and memory processes (Orta-Salazar, Cuellar-Lemus, Díaz-Cintra, & Feria-Velasco, 2014). The neurotransmitter ACh is employed by all cholinergic neurons, and its normal physiological function guarantees the storage and elicitation of memories (Papandreou et al., 2011). The degeneration of neurons, which is closely related to a loss of cholinergic markers, resulting in cholinergic system dysfunction has been thought to be the most consistent changes in AD cases (Hampel et al., 2018). Choline acetyltransferase (ChAT) catalyzes the synthesis of Ach from choline acetyl-CoA and choline, which is decomposed by AChE (Nalivaeva & Turner, 2016). Some evidence suggests that the increase of AChE is one of the important causes of AD (Lahiri et al., 2002;Racchi, Sironi, Caprera, König, & Govoni, 2001), and currently, clinically available AChE inhibitors for AD, such as donepezil, galantamine, and rivastigmine, can ameliorate the cognitive symptoms and enhance the living quality for AD patients.
Oxidative imbalance and neuronal damage also play a key role in the initiation and progression of AD. In patients with AD, oxidative stress is a state of imbalance between ROS production and antioxidant defense, leading to excessive accumulation of ROS (Tönnies & Trushina, 2017). Excessive ROS in the body can peroxidate unsaturated fatty acids on the cell membrane of neurons in the brain and form lipid peroxidation products. MDA is one of the products of lipid peroxidation. The accumulation of MDA will destroy the cell membrane structure and eventually lead to cell damage and even death. SOD and GPX are the main antioxidant enzymes in the body, which can reduce the memory impairment and cell damage induced by oxidative stress, and jointly remove excessive ROS to maintain the homeostasis between oxidation and antioxidant in the body (Ferreira et al., 2015).
In the treatment of AD, multitarget and multichannel combined treatment has attracted great attention. It is worth mentioning that there are some remarkable traditional Chinese medicine compounds that have been gradually applied in clinical treatment of learning and memory impairment for their multitarget therapeutic effects.
Among them, GAPT, also called as GEPT or GETO in our previous papers, is a combination of herbal extracts and a patented Chinese herbal compound for AD. GAPT, composed of 4.4% ginsenoside from ginseng, 17.3% cistanche, 17.3% prepared Radix Rehmanniae, 13% processed Polygala tenuifolia, 13% Acorus gramineus, 13% Wide Radix Curcumae, 13% Poria cocos, and 9% Salvia officinalis (Shi et al., 2016;Tian et al., 2009), has been clinically widely used to improve learning and memory impairment. A single-blind, randomized controlled clinical trial shows that 75 patients with suspected dementia were treated with GAPT for 3 months, and 1-year follow-up showed that GAPT can significantly improve the memory test scores compared with placebo group (Tian, Zhu, & Zhong, 2003). In another study, GAPT can effectively reduce GSK-3β expression level in the brain cortex of APPV7171 transgenic mice, thus playing a neuroprotective role (Shi et al., 2013). It also regulates the expression of CDK5 and PP2A in hippocampal neurons, thereby inhibiting abnormal tau phosphorylation (Ni et al., 2017). GAPT can increase APP/PS1 transgenic mice's brain glucose uptake and glucose transport and improve the insulin signaling pathway (Mana et al., 2019). Moreover, synapse damage ameliorated by GAPT via regulating bcl-2/Bax balance (Shi et al., 2018).
While the mechanisms behind protecting cholinergic neurons and reducing oxidative stress of GAPT remain unclear, we hypothesized that GAPT can improve the cognitive ability of the scopolamine-induced AD-like mice. We also studied the pharmacodynamics of different doses of GAPT. This study will investigate the optimal dose of GAPT for preventing and treating learning and memory disorder and further explore the neuroprotective mechanism of GAPT from cholinergic system and oxidative stress, thus providing the theoretical basis for the better application of GAPT in clinical practice.
Conclusions: GAPT can improve the learning and memory ability of scopolamineinduced mice, and its mechanism may be related to protecting cholinergic neurons and reducing oxidative stress injury.

K E Y W O R D S
Alzheimer's disease, behavior, cholinergic system, GAPT, memory impairment, oxidative stress, scopolamine 2 | MATERIAL S AND ME THODS

| Animals and drug administration
This research used 6-month-old male ICR mice 28-30 g in weight that purchased from Beijing Huafukang Biotechnology Co., Ltd (SCXK (Beijing) 2014-0004). The animals are kept in SPF grade animal laboratories in Dongzhimen Hospital affiliated to Beijing university of Chinese medicine (Certificate SYXK2015-0001, Beijing, China).
Animals are given regular gavage in the morning and free food and water during feeding. All experiments were performed in compliance with Beijing's regulations and guidelines for the use of animals in research, and the study was approved by the Animal Research Ethics Board of Dongzhimen Hospital .
Animal experiments were divided into two stages. In the first stage, animals were randomly distributed into six groups containing the control group, the model group, the donepezil group, and the low, medium, and high dosage GAPT groups. In the second stage, animals were randomly distributed into four groups containing the control group, the model group, the donepezil group, and the medium dosage GAPT groups. The control group and model group were administered 0.5% CMC, donepezil group mice were treated with donepezil (0.92 mg kg −1 day −1 ), and the GAPT groups were administered a small dose (0.405 g kg −1 day −1 ), a medium dose (0.81 g kg −1 day −1 ), and a large dose (1.62 g kg −1 day −1 ) of GAPT for one-half (first stage) or 1 month (second stage). One and a half hours after intragastric administration, mice were intraperitoneally injected with scopolamine (3 mg/kg, 0.1 ml/10 g). Control groups were administered a 0.9% normal saline injection of the same volume and via the same route.

| LC-MS analysis
Half a month of intragastric administration of GAPT (medium dose), mice plasma, and brain were collected for LC-MS analysis. The UHPLC separation was carried out using Ultimate 3000 ultra-high-performance liquid chromatograph, equipped with a XSelect HSS T3 C18 column (2.1 mm × 75 mm, 2.5 µm). The mobile phase A was acetonitrile, and the mobile phase B was 0.1% formic acid aqueous solution.
The elution gradient was shown as follows: (a) 0-15 min, 5%-50% A, (b) 15-22 min, 50%-95% A, and (c) 22-26 min, 95% A. The column temperature was set at 25°C, and the flow rate was set at 0.40 ml/min. LTQ-Orbitrap velos pro mass spectrometer (ESI) was applied for chemical analysis. Typical ion source parameters were as follows: capillary voltage = 35 V, nozzle voltage = 3.4 kV, gas (N 2 ) flow = 10 arb, sheath gas (N 2 ) flow = 35 arb, capillary temperature = 350°C, and heater temperature = 350°C. Mass standard calibration using external standards method (mass error is less than 5 ppm), the first-order mass spectrum is scanned in FT mode (resolution R is 30,000, scan range is from 50 to 1,500), and the MS 2 and MS 3 are data-dependent scan. Data acquisition and analysis were performed using Xcalibur 2.1 workstation (Thermo-Fisher), Metaworks, Mass Frontier 7.0 software.

| Step-down passive-avoidance test (SDPA)
The step-down passive-avoidance test was carried out according to previous experimental procedures (Figueiró et al., 2011;Mana et al., 2019). The experiment was divided into 2 days (1 day for training and another for testing). During the training (the first day), the mice were put into training apparatus (Shanghai Transfer Information Technology CO., LTD) with five connected 150 × 300 × 300 rooms (had copper grids at the bottom of each room), and then, a 36 V current was passed through the bottom of the room. Under normal conditions, the mice were shocked and jumped onto a shock freezone (SFZ) (an insulating platform 4.5 cm high in the middle of each room) to avoid the shock. 24 hr later, the mice were placed on the SFZ. The step-down latency (SDL) was the time when the mice first jumped off the SFZ, and the number of times the mice jumped off the SFZ within 5 min was the error times (ET).

| Y maze test
The Y-maze is an apparatus with three equal identical black Plexiglas arms (40 × 4.5 × 12 cm, 120° apart), and each arm has a movable partition at the center of the maze. Three arms are randomly distributed into novel arm, a start arm, and another arm. The test contains two phases. First, 10 min after scopolamine injection, mice were placed in the start arm with 3 min free exploring time and one arm blocked.
One hour later, mice were free to explore the entire maze for 3 min with the baffle removed. The time and distance in novel arm were recorded and analyzed (Shanghai transfer information technology CO., LTD).
2.6 | Enzymatic activities of MDA, ACh, AChE, ChAT, SOD, and GPX were determined by ELISA Mice were anesthetized with 20 mg/ml tribromoethanol and sacrificed by cervical dislocation. We immediately stripped brain tissues (removal of the cerebellum) on ice, rinsed with precooled saline, and dried on filter paper. The tissues were weighed and homogenized with ultrasound in saline, centrifuged at 4°C at 3,000-4,000 r/min, and then, supernatant was taken for detecting the activities of ACh, AChE, ChAT, MDA, GPX, and T-SOD by employing mouse-specific ELISA kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's protocols.

| Western blot analysis
The EP tube containing hippocampal tissue was added with RIPA tissue/cell lysate solution (R0010, Solarbio), homogenized by ultrasonic grinder, and centrifuged to obtain supernatant. The concentration of hippocampal tissue was measured with BCA method. Separation of protein by SDS-PAGE, electro transfer, antigen and antibody reaction, color rendering, and exposure were performed respectively. Importantly, in immunodetection, the membranes were probed with primary antibodies: AChE (1:2,000, Abcam), ChAT (1:1,000, Abcam), SOD1 (1:2,000, Abcam), GPX1 (1:1,000, Abcam), and β-actin (1:5,000, Abcam) at 4°C overnight. Finally, the protein bands were analyzed by Image J software, and the gray value of internal reference β-actin was compared to analyze the results and calculate the relative percentage.

| Immunohistochemical Staining
The immunohistochemical staining was performed following the protocol of our previous study . Embedded paraffin tissue from the brain of mice were made into continuous coronal sections with a thickness of about 4 μm, and then, deparaffinize and rehydrate tissue sections, antigen retrieval (microwave), quenching of endogenous peroxides, blocking, primary antibody incubation, and detection were performed. Finally, three fields of vision per section were selected to observe the CA1 region of the hippocampus under 20× objective lens and the number of positive cells was counted. Simultaneously, The ChAT-positive cells in basal forebrain were counted under 5× objective lens. Six paraffin sections were taken from each group and analyzed by Image-Pro Plus image analysis software.

| Statistical analysis
The data were expressed as the mean ± SD. Statistical analysis and data plotting were performed via one-way analysis of variance (ANOVA) using SPSS20.0 software and GraphPad Prism 6. Values of p < .05 were considered to be significant.

| GAPT-medicated plasma and brain contained Ginsenoside Rb1, Beta asarone, and other components by LC-MS analysis
First, to confirm the real effects of GAPT in neurons and brain regions, we chose LC-MS for qualitatively analyzing the components in the GAPT-medicated plasma and brain. As shown in Figure 1 and Table 1, by LC-MS analysis, a total of 83 compounds were identified from GAPT, a total of 42 compounds were identified from GAPTmedicated plasma, and a total of 43 compounds were identified from GAPT-medicated brain. Among 42 compounds in GAPT-medicated plasma, 5 compounds (10, 50, 64, 82, and 83) were identified by comparing with reference standards, while the other 37 compounds were characterized based on literatures. At the same time, among 43 compounds in GAPT-medicated brain, 6 compounds (32, 50, 64, 67, 79, and 82) were identified by comparing with reference standards, while the other 37 compounds were characterized based on literatures.

| GAPT can ameliorate scopolamine-induced behavioral changes in learning and memory impaired mice
Step-down passive-avoidance and Y maze tests were used to study the cognitive changes in mice after the intervention. The step-down passive-avoidance test is aimed to measure the ability to learn new information and to remember spatial location of laboratory animals by regularly offering a passive electrical stimulation. The results from the step-down passive-avoidance tests are shown in Figure 2.
After half of a month of intragastric administration, the number of errors in the model group was increased compared to the control group (p < .01). The latency was shorter in the model group compared to the control group (p < .01). However, GAPT in any dose and donepezil significantly decreased error times and prolonged the step-down latency (p < .01). These results show that GAPT can take effect after half a month of intragastric administration. To further observe the curative effect of GAPT, we choose a clinically effective dose of GAPT (the medium dose) and extended the time of administration to 1 month. As expected, the number of errors in the model group was increased compared to the control group (p < .01) and the latency was shorter in the model group (p < .01, Figure 3).
Again, GAPT in medium dose and donepezil significantly decreased error times and prolonged the step-down latency (p < .01 or p < .05, Figure 3).
The Y maze test is used to study the spatial recognition and memory ability of rodents, which are characterized by the natural habits of exploring new and different environments. The Y maze test can effectively reflect the ability of animals to recognize the new environment. Memory impaired mice usually spend less time and travel shorter distances while exploring the new arm. As shown in Figure 4, the total novel arm distance reordered in the Y maze was markedly shortened in the model group compared with the control group (p < .01). In contrast, the distance of the GAPT medium-dose group and the donepezil group were longer than that of the model group (p < .05). The time spent in the novel arm was also shortened in the model group compared with the control (p < .01).
The mice of GAPT medium-dose group and the donepezil group also spent longer time in novel arm than the model group (p < .01 or p < .05). Although the distance and time in the new arm were extended in the high-dose and low-dose groups, there was no statistical significance. When administration for 1 month, In Figure 5, the total novel arm distance reordered and the time spent in the Y maze were markedly shortened in the model group compared to the control group (p < .01). Interesting, the GAPT medium-dose group and the donepezil group significantly extend the distance and the time in the novel arm than the model group (p < .01 or p < .05).

| GAPT can decrease the activity of MDA and AChE and increase the activity of Ach, ChAT, T-SOD, and GPX in the brain of scopolaminetreated mice
As shown in Figures 6a and 7a

| GAPT can decrease expression of AChE and increase expression of ChAT, GPX1, and SOD1 in hippocampus and basal forebrain of scopolaminetreated mice
As shown in Figure 8 (Figure 9(a,b)). confirm that exposure to scopolamine (1-3 mM) can significantly decrease human neuroblastoma SH-SY5Y cell viability (Puangmalai et al., 2017) and induce PC12 cell mitochondrial and plasma membrane damage (Pandareesh & Anand, 2013). To investigate the particular structures related to memory and learning, scopolamine can also be used (Newman et al., 2017). Improved acetylcholine function recognized as an important way to improve memory. Therefore, in this study, scopolamine was employed to observe whether GAPT can enhance or protect the stability of cognitive activity.
The early research indicates that GAPT can decrease the expression level of endogenous Aβ peptide by inhibiting the PS1 activity in APPV717I transgenic mice (Tian et al., 2009). Eight months and three months after administration with GAPT, spatial learning function and memory abilities were significantly enhanced, suggesting that GAPT could prevent cognitive impairments and protect learning and memory function in an AD-like rat model (Tian et al., 2006).  Ach plays an important role in the central nervous system. Ach is the central neurotransmitter that most closely relates to learning and memory processes. Acetyl-CoA and choline participate in Ach synthesis because of the catalysis of acetyltransferase (ChAT). AD patients usually suffer the loss of cholinergic neurons in the cerebral hippocampus and cortex (Schliebs & Arendt, 2006) along with decreased cholinergic activity, which is possibly due to increased activity of AChE (Khan, 2009). Severely diminished ChAT expression was also observed (Orta-Salazar, Aguilar-Vázquez, et al., 2014) in this process. Some studies also suggested that oxidative stress was closely related to the increase of AChE activity (Inestrosa, Dinamarca, & Alvarez, 2008). In this study, donepezil and GAPT can decrease AChE activity and protein expression level and increase the activity of ChAT and ACh in scopolamine-treated mice. Scopolamineinduced learning and memory disorder was effectively mitigated by GAPT, thereby protecting the cholinergic system and maintaining the normal activity of ChAT and AChE. However, we cannot rule out the possibility of AchR receptor agonist in GAPT compound at present, and we hope to do this work at next step.
The imbalance between pro-oxidant stress and antioxidants frequently leads to oxidative stress. High metabolic rates make the brain the most sensitive organ to hypoxia, and the brain is particu- Fortunately, antioxidant enzymes, including GPX and SOD, can protect tissues against reactive oxygen species (ROS) (Pohanka, 2014).
Our results show that donepezil and GAPT markedly increased SOD activity and GPX activity in the brains of scopolamine-treated mice, indicating that GAPT could improve oxidative stress impairment.
In our previous studies, we report that GAPT has a neuroprotective effect over 3 months of intragastric administration. In this study, we first found that both short-term (half a month) and longterm (1 month) of intragastric administration of GAPT could relieve the effects induced by scopolamine. To confirm the real effects of GAPT in neurons and brain regions after half a month of intragastric administration of GAPT, an LC-MS method was established to study the chemical compounds and in vivo metabolites of GAPT. Of the 83 compounds identified in GAPT, 42 compounds were able to enter the blood, and, surprisingly, 43 compounds might pass through BBB and reach the specific brain areas or neurons. At least, we can sure that echinacoside, salvianolic acid A, ginsenoside Rb1, ginsenoside Rg2, pachymic acid, and beta asarone which identified by comparing with reference standards could be absorbed into mice brain. These findings provided informative groundwork for further pharmacokinetic studies of GAPT prescription.
In conclusion, GAPT can ameliorate the scopolamine-induced behavioral changes in learning-and memory-impaired mice. GAPT reduced the hydrolysis of ACh by reducing the activity and protein expression of AChE. At the same time, it increased the synthesis of ACh by increasing the activity, protein expression, and distribution of ChAT, thus improving the cholinergic nerve function. Meanwhile, GAPT increases the activity, protein distribution, and expression of SOD1 and GPX1, reduces the damage of ROS to cells, improves the damage caused by oxidative stress, and plays a neuroprotective role. Both short term and long term of intragastric administration of GAPT can improve the learning and memory ability of scopolamine-induced memory impairment model mice, and its mechanism is related to protecting cholinergic neurons and reducing oxidative stress injury.

ACK N OWLED G M ENT
This study was supported by the scientific research and graduate training project of Beijing municipal commission of education (2016,2017), the National Natural Science Foundation of China (No. 81573927).

CO N FLI C T O F I NTE R E S T
The authors declare that there are no competing interests associated with the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request. Evidence of oxidative damage in Alzheimer's disease brain: Central