Effeets Of Glyeosides Of Cistanche On Learning Cognitionand Synaptic Plasticity in Sleep Deprivation Model Rats

ABSTRACT

Objective: To explore whether the glycosides of cistanche can improve the learning and cognitive function of sleep deprivation model rats by regulating synaptic plasticity. 

Methods: Fifty male Wistar rats were randomly divided into a blank control group ( Control), platform control group ( PC), model group ( Model), glycosides of cistanche group(GCs), and estazolam group ( Est ). The sleep deprivation model of rats was prepared by a modified multi-platform water environment method. Morris water maze and open field test were used to detect the spatial cognitive function and emotional changes of rats. Nissl staining was used to observe the changes in morphology and quantity of nerve cells in the hippocampal CAl area of rats. Western blot and R'T - PCR were used to detect the expression levels of synaptophysin(SY'N), postsynaptic membrane dense substance 95 ( PSD -95 ), and brain-derived neurotrophic factor ( BDNF). 

Results; Morris water maze results showed that compared with the Control group, the learning and cognitive ability of rats in the Model group was significantly decreased ( P <0. 05 ), and the learning and memory function of rats was improved GCs treatment ( P <0. 05). The results of the open field test showed that compared with the Control group, the movement distance and standing times of the Model group were significantly increased ( P <0. 05 ), the resting time was significantly shortened( P <0.05 ), and the anxiety was increased, while after GCs treatment, the movement distance and standing times decreased ( P <0. 05 ), the resting time increased ( P <0. 05), and the anxiety was improved. The results of Western blot and R'T - PCR showed that the protein and gene expression levels of BDNF, SYN, and PSD -95in the Mlodel group were significantly decreased ( P <0. 05 ), while after treatment with GCs, the expression levels ofBDNF, SYN and PSD -95 were significantly up-regulated ( P <0.05). Conclusion: Glycosides of cistanche may af. feet synaptic plasticity by regulating the expression of synaptic-related markers, thereby improving the learning and cognitive function of sleep deprivation model rats.

HERBAL CISTANCHE FORMULATION SUPPLEMENTS FOR  IMPROVING LEARNING AND COGNITIVE FUNCTION

KEY WORDS Glycosides of cistanche; Hippocampus; Sleep deprivation; Learning and memory; Anxiety; Synaptic plasticity

Sleep disorder (SD) is a functional disorder of abnormal sleep caused by a series of complex factors such as insomnia, dreaminess, and easy awakening. In recent years, the incidence of SD has been gradually increasing. Related studies have reported that SD can cause cardiovascular diseases, metabolic dysfunction, immune diseases, and central nervous system degenerative diseases such as Alzheimer's disease [1]. SD can cause oxidative damage to hippocampal neurons, cause cognitive dysfunction, and produce mood changes such as anxiety and depression [2]. Cistanche deserticola is a precious medicinal material that can be used as both medicine and food in China. It has the effects of tonifying the kidney and replenishing qi, moistening the intestines promoting bowel movements, and strengthening yang. The main extract of Cistanche deserticola, glycosides of cistanche (GCs), has antioxidant, anti-apoptotic, liver-protecting, and neuroprotective effects [3]. Our previous research found that GCs can improve the learning and cognitive function of SAMP8 mice by playing an antioxidant and anti-apoptotic role [4].

Related studies have shown that cognitive dysfunction caused by SD is closely related to synaptic plasticity [5]. However, there are few reports on the mechanism by which GCs regulate synaptic plasticity and improve learning and memory impairment and cognitive decline in rats induced by sleep deprivation. Therefore, this study aims to explore whether GCs affect synaptic plasticity by regulating the expression of synaptic-related markers, thereby improving the learning and cognitive function of rats with sleep deprivation models, and provide new treatment ideas and plans for GCs to treat learning and cognitive impairment caused by sleep disorders.

1 Materials and methods

1.1 Experimental animals

Male Wistar rats aged 6 to 8 weeks, weighing 280 to 300 g, were purchased from Beijing Sibeifu, with an animal production license [SCXK (Beijing) 2019-0010]. All animals were kept in the Animal Experiment Center of Baotou Medical College under the following conditions: temperature (24 ± 1) °C, humidity 50% to 55%, 12 h light and 12 h dark, and no restrictions on food and water. This experiment was approved by the school ethics committee [Baoyi Lunshen 2022 No. (63)].

1. 2 Main reagents and instruments

Total glycosides of Cistanche deserticola were purchased from Chengdu Xiyu Cistanche Biotechnology Co., Ltd. (batch number: KSM20220609011); Estazolam tablets were purchased from CSPC Pharmaceutical Group Ouyi Pharmaceutical (batch number: 044220343); protein loading buffer (P1015), RIPA lysis buffer (R0010), chemiluminescent solution (PE0010), etc. were all purchased from Beijing Solebao; postsynaptic density substance in 95 (PSD-95) antibody (AB192757) and synaptophysin (SYN) antibody (ab32127) were purchased from Abcam, USA Company; brain-derived neurotrophic factor (BDNF) (48503-2) and β-actin (52901) were purchased from SAB Company, USA; goat anti-rabbit IgG secondary antibody (SA00001-20) was purchased from Proteintech Company, USA; rat melatonin (MT) and ELISA Scientific Research Kit (MM-0657R1) were purchased from Jiangsu Enzyme Immunoassay Industry Co., Ltd. Paraffin slicer (Leica, Germany); optical microscope (Leica, Germany);

Morris water maze, open field test box (Shanghai Xinruan); vertical electrophoresis apparatus (Beijing Liuyi Company); decolorization shaker (Beijing Liuyi Company); protein imaging analysis system (Shanghai Tanon Technology); fluorescence quantitative PCR instrument (Xi'an Tianlong Technology Co., Ltd.).

1.3 Experimental methods

1.3.1 Animal grouping and drug administration

50 male Wistar rats were randomly divided into blank control group (Control group), platform control group (Plat-form control, PC group), model group (Model group), total glycosides of Cistanche deserticola group (GCs group, 50 mg/kg), and estazolin group (Est group, 0.54 mg/kg), with 10 rats in each group. The Control group and PC group were given 0.9% saline by gavage. All groups were gavaged at 7 am every day after the modeling began, and the gavage lasted for 21 days.

1.3. 2. Preparation of sleep deprivation animal model

The sleep deprivation model was established using the modified multi-platform water environment method [6]. The experimental equipment was a 130 cm × 50 cm × 45 cm rectangular polyethylene resin water tank. A small platform with a diameter of 5 cm and a height of 20 cm was placed in the water tank. The platform of the PC group had a diameter of 10 cm and a height of 20 cm. The water tank was filled with water to a depth of about 18 cm and the water temperature was maintained at (20 ± 1) ℃. One week before the start of the experiment, the rats were placed in the sleep deprivation box every day for 60 min to familiarize themselves with the environment. After the model was established, except for the Control group, the rats of each group were placed on the small platform on time from 8 am to 8 pm every day for sleep deprivation for 21 consecutive days.

All rats had free access to food and water and moved freely on the platform. The light and dark cycle was 12 hours every day.

1. 3. 3 Morris water maze experiment

The Morris water maze was divided into 4 areas, filled with water, and the platform was placed in the 4th quadrant. The water surface was about 2 cm above the platform. Food-grade edible melanin was added to the water, and the water temperature was maintained at (25 ± 1) ℃. (1) Positioning navigation experiment: The center of each area was selected and the rats were gently placed in the water. They were allowed to explore freely. The time it took for the rats to find the target platform within 60 seconds was recorded. Rats that did not find the platform were guided to the platform and stayed for 20 seconds. The training was continued for 5 days. (2) Spatial exploration test: On the 6th day, the platform was removed and the rats were placed in the water from the center of the 2nd quadrant against the wall. The number of times the rats passed the platform within 120 s and the time they stayed in the 4th quadrant were recorded.

1. 3. 4 Open field experiment

A 100 cm × 100 cm × 50 cm experimental box was used, and the bottom of the box was divided into 9 grids. Each group of rats was placed in the open field experimental room in advance to adapt to the indoor environment for 60 min. At the beginning of the experiment, the test rats were placed in fixed corners of the open field experimental box in turn, and the number of standing times, movement distance, and static time of the rats within 5 min were recorded using behavioral analysis software. The environment was required to be quiet during the experiment, and the experimental device was wiped with water and alcohol respectively between two experiments to prevent the smell of the first rat from affecting the behavioral judgment of the next rat.

1. 3. 5 Enzyme-linked immunosorbent assay (ELISA)

Detection of serum MT levels in rats Rats were anesthetized by intraperitoneal injection, and 5 mL of blood was collected from the abdominal aorta. After waiting for 30 min at room temperature, the blood was centrifuged to obtain serum. 40 μL of sample diluent was added to each well, and then 10 μL of the sample to be tested was added. After sealing with a sealing film, the plate was incubated at 37 °C for 30 min. Washed 5 times, 50 μL of enzyme labeling solution was added, and the plate was sealed with a sealing film and incubated at 37 °C for 30 min. Washed 5 times, and then the color-developing solution and the reaction termination solution were added in turn. The OD value of each well was measured by an enzyme labeler at a wavelength of 450 nm.

1. 3. 6 Nissl staining

Three rats were selected from each group, and brain tissue was removed by decapitation and fixed with a 4% polyoxyethylene solution. The tissue was dehydrated, immersed in wax solution, embedded, and sliced by conventional methods. The slices were kept with a thickness of 5 μm, rinsed with PBS, stained with 0.1% toluidine blue for 0.5 h, differentiated with 95% alcohol, dehydrated with 100% alcohol, transparentized with xylene, and sealed with neutral gum. The changes in the morphology and number of neurons in the CA1 region of the rat hippocampus were observed under a microscope and images were collected.

1. 3. 7 Western blot experiment

Four rats were selected from each group, and the hippocampus was isolated by decapitation. The hippocampus tissue was mixed with RIP A lysis buffer in an ice bath environment and the tissue was centrifuged to obtain the supernatant. The protein concentration was detected by the BCA method. 20 μg of protein was added with protein loading buffer for electrophoresis. After the protein electrophoresis was completed, the membrane was transferred at 300 mA constant flow, blocked with 5% skim milk powder, the primary antibody was incubated at 4 ℃ overnight, and the secondary antibody was incubated at room temperature for 2 h. After the color development solution was used, the protein bands were statistically analyzed using a chemiluminescence imaging analysis system.

1. 3. 8 RT-PCR experiment

Three rats were selected from each group to prepare hippocampal tissue homogenate. The total RNA in the tissue fluid was extracted by the Trizol method and the concentration was measured. A fastking cDNA synthesis kit was used for reverse transcription, and a fluorescence quantitative amplification reaction was performed using a fluorescence quantitative kit to detect the relative expression level of synaptic-related protein mRNA. The results were analyzed using 2-ΔΔCt. The primer sequences are shown in Table 1.

Table 1 RT-PCR primer sequences

1.4 Statistical methods

The data results that conformed to the normal distribution were expressed as mean ± standard deviation (x ± s), and statistical analysis was performed using GraphPad Prism 9.5 software. One-way analysis of variance was used for comparison between multiple samples, and P < 0.05 indicated that the difference was statistically significant.