ABSTＲACT 

Objective: To clarify whether general cistanosides ( GCs) ameliorates cognitive dysfunction in rapidly aging mice ( SAMP8) by mediating specific androgen receptors ( AＲ) in the regulation of expression of synaptic protein． 

Methods: A total of 76 7-month-old male SAMP8 mice were randomly divided into the model group, GCs group, GCs + flutamide ( F) group, GCs + fulvestrant ( ICI) group, F group, and ICI group． The learning and memory functions of mice in each group were tested with the Morris water maze． Western blotting and RT-PCR were used to test the expression level of synaptophysin ( SYN), postsynaptic density － 95 ( PSD － 95), and the brain-derived neurotrophic factor ( BDNF) of mice in each group． 

Results: GCs significantly improved the learning and cognitive function of SAMP8 mice． 

Compared with the GCs group, the learning and memory function of mice in the GCs + F group and GCs + ICI group was significantly lowered． Western blotting and RT-PCR results showed that the expression level of BDNF, SYN, and PSD － 95 were significantly increased at the protein level and gene level in the GCs group compared with the Model group ( P ＜ 0． 05) ． SYN expression level was increased in the GCs + F and GCs + ICI group, and expression levels of BDNF and PSD － 95 were significantly decreased in the GCs + F and GCs + ICI group compared with the GCs group ( P ＜ 0． 05)． 

Conclusion: GCs may specifically mediate AＲ to enhance synaptic plasticity in SAMP8 mice, which in turn improves learning and cognitive function．

KEYWODS:General cistanosides; Androgen receptor; Synaptic plasticity; SAMP8 mice 

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Alzheimer's disease (AD) is a common neurological degenerative disease, clinically characterized by progressive cognitive function decline and changes in mental and psychological status [1]. Studies have found that testosterone levels in male AD patients are significantly lower than in normal men.

The cognitive function of patients has been improved to varying degrees after ketone replacement therapy [2], which indicates that age-related decreases in androgen levels may be a risk factor for AD in older men.

Cistanche deserticola total glycosides (general cistanosides, GCs) are one of the main extracts of Cistanche deserticola, which can improve AD-related learning and cognitive dysfunction [3]. In addition, administration of GCs by gavage significantly increased testosterone levels in animals [4]. GCs also have the function of promoting yang and can exert

Androgen-like pharmacodynamic effects. Previous studies have reported that testosterone can improve the learning and cognitive functions of AD model animals, and this process relies on specific androgen receptor (androgen receptor, AR)-mediated [5].

It is well known that cognitive dysfunction in AD patients is closely related to synaptic plasticity. However, the specific mechanism through which GCs regulate synaptic plasticity and thereby improve learning and cognitive functions is still unclear. This study aimed to explore whether GCs mediate specific ARＲ to affect synaptic plasticity and thereby improve learning and cognitive functions in AD.

1 Materials and methods

1. 1 Experimental animals and equipment

Male 3-month-old SAMP8 mice were purchased from Peking University School of Medicine (SCXK [Beijing] 2016-0010) and were raised until they were 7 months old. Animals were kept in a clean environment at (24 ± 1) ℃ and

The animals were provided with natural lighting, and free access to food and water, and humanitarian care was given in accordance with the 3R principles for the use of experimental animals. This experimental study has been approved by the Experimental Animal Ethics Committee of Baotou Medical College (Baotou Medical College No. 20201102).

The main reagents in the experiment are: Cistanche deserticola total glycosides (GCs, Xi'an Ciyuan Biological Co., Ltd.); androgen receptor antagonist-flutamide (Sigma Company, USA); estrogen receptor antagonist-fulvestrant (ICI 182780, USA) Med Chem Express); Rabbit anti-mouse BDNF antibody,

PSD-95 antibody, SYN antibody, β-actin antibody (Proteintech Company of the United States); protein electrophoresis solution, protein transfer solution, TEMED, acrylamide buffer, ammonium persulfate, Tris (Beijing Solebao Technology Co., Ltd.); cDNA Synthesis kit, Talent fluorescence quantitative detection

Reagent kit (Tiangen Biochemical Technology Co., Ltd.), etc. The main experimental equipment includes a vertical plate electrophoresis tank, electrophoresis apparatus, transfer tank, destaining shaker (Beijing Liuyi Biotechnology Co., Ltd.), protein gel imaging analysis system (Shanghai Tanon Technology Co., Ltd.), real-time fluorescence quantitative PCR instrument (USA Roche) etc.

1. 2 Grouping and administration

Two mice died naturally during the feeding process, and the remaining 78 mice were randomly divided into model group, GCs group, GCs + flutamide (F) group, GCs + fulvestrant (ICI) group, F group, ICI

Groups of 13 animals each. Except for the model group, F group, and ICI group, the other groups were continuously administered GCs at a dose of 50 mg/(kg·d) for 30 days.

From the 24th day after GCs administration, the GCs + flutamide (F) group and the GCs + fulvestrant (ICI) group were given intraperitoneal injection of F (5 mg/kg/d) 30 minutes after GCs were administered by gavage. ), ICI (1 mg/kg/d), continuous injection for 7 days. Adjust the dosing concentration to ensure that the maximum gastric volume of the mice is 0. 5 mL, 1 time/d.

1. 3 Morris water maze test

The mice adapted to the water environment one day in advance. The Morris water maze lasted for 6 days. The first 5 days were the positioning and navigation test. The mice were trained once a day in each of the four quadrants for 1 minute each time. Each training interval was 15 minutes, and records were recorded at the same time. Escape latency of the animal positioning and navigation test; on the 6th day, remove the platform from the water and conduct a space exploration test. Record the number of platform crossings and the percentage of time spent in the target quadrant of the mouse. the target quadrant) and spatial exploration swimming trajectory (Swimming trajectory of spatialprobe test) and other indicators.

1. 4 Western blot detection

Expression of SYN, PSD-95, and BDNF synapse-related proteins. After the Morris water maze test, all mice were anesthetized routinely, their heads were decapitated, their brains were removed, and their hippocampi were stripped off. The hippocampal tissue was protein lysed, disrupted by ultrasound at 4°C, left on ice for 30 min, and centrifuged at 12,000 r/min for 15 min at 4°C. The supernatant was taken, and the protein concentration was measured using the BCA method. After loading the protein, perform stacking gel electrophoresis at a constant voltage of 80 V, separation gel electrophoresis at a constant voltage of 120 V, and wet transfer at a constant flow of 300 mA; take out the PVDF membrane, block it with 5% skim milk for 80 minutes at room temperature, and wash the membrane three times with TBST. 15 min/time, primary antibody was incubated overnight on a shaking table at 4°C; the next day, the membrane was washed 3 times with TBST.

15 min/time, the secondary antibody was incubated at room temperature for 2 h, then ECL color development was performed, and imaging was performed with a chemiluminescence imaging system. β-actin was used as the internal reference, and the optical density value of the band was analyzed using Image J software.

1. 5ＲRT－PCRＲDetection

The mRNA expression of SYN, PSD-95, and BDNF was extracted from hippocampal tissue total RNA according to the Trizol extraction method, and cDNA was generated after reverse transcription. The Talent fluorescence quantitative detection kit was used for relative fluorescence quantitative detection. The experiment was repeated three times, and the results were converted to 2 - ΔΔCT was analyzed. Mouse β-actin, SYN, PSD-95, and BDNF primers were designed by Shanghai Sangon Company, and the primer sequences are shown in Table 1.

1. 6 Statistical analysis

Via SPSS 25. 0 statistical software for processing, Graphpad Prism 8 software was used to draw statistical graphs, and one-way ANOVA was used to compare pairwise means of multiple samples. P < 0. 05 means the difference is statistically significant.