GBE50DP was provided by Zhejiang Shangyao Jiuxu Pharmaceutical Co., Ltd. Donepezil hydrochloride tablets were provided by Chongqing Zein Biotechnology Co., Ltd. Pentobarbital sodium was purchased from Merck KGaA. Superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GSH-Px), catalase (CAT), IL-1β, IL-6 and TNF-α kits were provided by Jiangsu Jingmei Biotechnology Co., Ltd. Terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) kit was provided by Roche Diagnostics GmbH. Rabbit two-step immunohistochemical kit (PV-6001) and 3,3'-diaminobenzidine (DAB) were provided by Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. Caspase-3, Bcl-2, Bax, cytochrome c (Cyto-C), PARP-1, β-actin monoclonal antibody, HRP-labeled anti-rat and HRP-labeled anti-rabbit secondary antibody were provided by Beijing Bioss Biotechnology Co., Ltd. Fas monoclonal antibody was provided by Thermo Fisher Scientific, Inc. PVDF membrane was acquired from Beijing Biotopped Technology Co., Ltd. Enhanced chemiluminescence (ECL) reagent was purchased from Dalian Meilun Biotechnology Co., Ltd. Tissue lysate and SDS-PAGE kits were provided by Beyotime Biotechnology. A Bradford protein quantification kit was provided by Beijing Lanjieke Technology Co., Ltd. MS-grade formic acid and acetonitrile were acquired from Thermo Fisher Scientific, Inc. All other chemical reagents used were of analytical grade.

Open field box (100x100x40 cm), Morris water maze (diameter 150 cm, height 50 cm) and eight-arm maze (radial arm 29x6x15 cm) were provided by Anhui Zhenghua Biologic Apparatus Facilities Co., Ltd. Centrifuges (5810R and 5424R) were acquired from Eppendorf SE, a microplate reader (RT-6000) from Rayto Life and Analytical Sciences Co., Ltd., a homogenizer (FA25) from Fluko Shanghai Equipment Co., Ltd., an embedding machine (BM-VII) from Xiaogan Hongye Medical Instrument Co., Ltd., a microtome (CM3050s) from Leica Microsystems GmbH, an optical microscope (BX-53) from Olympus Corporation, a fluorescence microscope (DM4000B) from Leica Microsystems GmbH and a gel imaging system (C300) from Azure Biosystems.

A total of 120 male Wistar rats (weighing 300-340 g; 9-11 weeks) were supplied by the Changchun Yisi Experimental Animal Technology Co., Ltd. [license no., SCXK(Ji)-2023-0002], housed in a temperature- and humidity-controlled environment (25˚C and 60%) and given free access to water and a standard diet.

After 1 week of adaptation, the rats were randomly divided into the control, model, positive (donepezil 0.45 mg/kg) and GBE50DP low-, medium- and high-dose groups (50, 100 and 200 mg/kg, respectively), with 20 rats in each group. Anesthesia was administered by intraperitoneal injection of 40 mg/kg sodium pentobarbital. An incision was then made at the midline position in the neck, and the bilateral common carotid arteries were isolated and exposed. The control group was exposed without ligation, while the other groups were permanently ligated (16,17) and sutured. The drugs were prepared into suspension with 0.5% sodium carboxymethyl cellulose aqueous solution for intragastric administration once a day for 8 weeks, while the control and model group were given the same volume of pure water.

The experiment was carried out in a dark and quiet environment within the open field box. The rats were placed individually in the box for 5 min twice a day for 2 days, for the adaptive training. At 1 h before the experiment, the tested rat was isolated in a single cage to stimulate its social behavior. The test was performed for 5 min, and the number and duration of contacts were recorded.

The inner wall of the Morris water maze was made black, and four quadrants were distinguished by different markers. A platform was placed in the center of one of the quadrants, 2 cm below the water level, and the water temperature was controlled at 23.0±2.0˚C. Adaptive training: 1 Day before the experiment, the rats were adapted to the environment in a non-platform pool and swam freely for 1.5 min. Positioning navigation experiment: The rats facing the pool wall were randomly placed in one of the three quadrants without the platform. The time frame from their entering the water to boarding the platform (the escape latency) was recorded. The time frame was recorded as 1.5 min if the platform was not found within 1.5 min, and the rats were then guided to the platform and stayed for 10 sec so that they could perform spatial learning and memory according to the markers. The experiments were performed twice a day, in intervals of 4 h, for 5 days total. The 6th day was set as the test period. Space exploration experiment: The platform was removed on the 6th day, and the residence time of the rats in the target quadrant and the number of times they crossed the original platform location within 1.5 min were recorded.

The experiment was carried out in a quiet and odorless environment. There was a removable door at the entrance of each arm, and the arms 1, 2, 6 and 7 were set as the food arms. There were 4 adaption tests before the test, 10 min for each per day. On the first day, the bait was placed in the central area; on the second day, it was placed in the central area and the entrance of each arm; on the third day, it was placed on the top of each arm; from the first day to the third day, three rats were placed in the maze at the same time for exploration; on the fourth day, the bait was placed only at the top of the food arm, was placed in the maze individually for exploration. The rats were then trained for 5 days and fasted for 24 h before the training, with no restriction on drinking water. During the training, only bait was placed in the food arms. After the rats were placed in the central area for 15 sec, the doors of each arm were opened, and the foraging of all arms was free for 10 min or completed in advance. Restricted feed was given after each adaptation and training. The times that the rats repeatedly entered the food arm that had been visited (the working memory errors) and the times that the rats entered the food-free arm (the reference memory errors) were recorded.

After the rats were anesthetized with 3% sodium pentobarbital (40 mg/kg, i.p.), they were fixed in the supine position, and the chest cavity was quickly opened to expose the heart. The perfusion needle was inserted into the aorta through the apex of the left ventricle, and the right atrial appendage was cut open simultaneously. First, 4˚C pre-cooled normal saline (~150 ml) was rapidly perfused until the effluent was clear and the liver turned white; then, 4˚C pre-cooled 4% paraformaldehyde (~200 ml) was perfused for fixation. When the muscles of the rat's limbs and tail became stiff, the rats were immediately decapitated to obtain the brain. The hippocampal tissues were quickly separated on ice and placed in 4% paraformaldehyde for fixation (the volume ratio of fixative solution to tissue was >10:1), fixed at 4˚C for >24 h for subsequent experiments.

After the rats were anesthetized with 3% sodium pentobarbital (40 mg/kg, i.p.), ~1.0 ml of blood was collected from the orbital venous plexus and placed in a heparin sodium anticoagulant tube. After confirming that the rats were in a deep state of anesthesia (with disappearance of the toe clonus reflex), the rats were immediately decapitated to obtain the brain, which was quickly separated into hippocampal tissue on ice, placed in a pre-frozen storage tube, rapidly frozen in liquid nitrogen, and then transferred to -80˚C for storage. The blood samples were centrifuged at 13,523 x g for 10 min at 4˚C to separate the supernatant, which was obtained as the drug-containing plasma. The plasma was frozen at -80˚C for storage. The animal experiment in this study was approved by the Harbin University of Commerce Ethics Committees (approval no. HSDYXY-2023035).

The white transparent ‘herringbone’ hippocampus was collected from the rat brain and quickly transferred to the ice. The tissue was homogenized with pre-cooled PBS at a low temperature until it was completely broken. Following centrifugation at 4˚C, 1,157 x g for 20 min, the supernatant was collected. The contents of SOD (cat. no. JM-01793R1), MDA (cat. no. JM-10323R1), GSH-Px (cat. no. JM-02173R1), CAT (cat. no. JM-10334R1), IL-1β (cat. no. JM-01454R1), IL-6 (cat. no. JM-01597R1) and TNF-α (cat. no. JM-01587R1) were detected by ELISA kits (Jiangsu Jingmei Biotechnology Co., Ltd.) using the microplate reader.

The tissues were fixed using 4% paraformaldehyde (4˚C for >24 h), dehydrated through a graded ethanol series (60% for 4 h, 80% for 4 h, 90% for 4 h, 95% for 4 h twice and 100% for 2 h twice), cleared in xylene (5 min twice), embedded in paraffin (soft wax at 52-54˚C for 30 min, twice; hard wax at 54-56˚C for 30 min, twice), sectioned at a thickness of 4.5 µm, dewaxed by xylene and stained by H&E (19), dehydrated by gradient ethanol and sealed. The damage of nerve tissue in the CA1 region of the hippocampus was observed by optical microscopy (BX-53; Olympus Corporation). For each sample, three random fields of view were captured from this region.

Following the TUNEL kit instructions (20), the slices were dewaxed with xylene and rehydrated through a graded ethanol series. The working solution of protease K was added and incubated at 37˚C for 20 min. The slices were rinsed with PBS, TUNEL reaction solution was added and it was incubated in the dark for 60 min at 37˚C. The sections were rinsed with PBS and sealed with an anti-fluorescence quencher. Apoptosis was observed and calculated using a fluorescence microscope (DM4000B; Leica Microsystems GmbH). The apoptosis rate was calculated as: (number of TUNEL-positive cells/total number of cells) x100.

The tissues were fixed with 4% paraformaldehyde, dehydrated, embedded in paraffin and then sectioned (4-µm thickness), repaired with antigen (0.01 M citrate buffer, pH 6.0, boiling water bath for 15 min). Endogenous peroxidase was eliminated with 3% H₂O₂ (reagent 1 of PV-6001 kit; Zhongshan Jinqiao Biotechnology) at room temperature for 10 min, followed by three washes with PBS (3 min each). Sections were incubated with the primary antibody (Caspase-3, bsm-61071R, dilution 1:200; Bcl-2, bs-0032R, dilution 1:200; Bax, bsm-52316R, dilution 1:200; Cyto-C, bsm-52050R, dilution 1:200; PARP-1, bsm-52408R, dilution 1:200; β-actin, bsm-63325R, dilution 1:1,000; Bioss Biotechnology; Fas, MA5-35308, dilution 1:200; Thermo Fisher Scientific, Inc.) overnight at 4˚C, and then incubated with the secondary antibody (HRP-conjugated goat anti-rabbit IgG; reagent 2 of PV-6001 kit) at 37˚C for 20 min. The slices were stained with DAB (Zhongshan Jinqiao Biotechnology) for 5 min at room temperature, counterstained with hematoxylin (room temperature, 20 sec), differentiated with hydrochloric acid alcohol, dehydrated with gradient ethanol, returned to blue with tap water and sealed with neutral gum. Images were captured using an optical microscopy (BX-53; Olympus Corporation). The visual field was randomly selected, and the average optical density in each visual field was measured using ImageJ software (version 1.53a; National Institutes of Health).

Hippocampal tissue was collected, cut and ground in liquid nitrogen. Lysis solution (RIPA lysis buffer; Beyotime Biotechnology) was added to the tissue overnight at 4˚C. Following centrifugation at 4˚C, 13,523 x g for 10 min, the supernatant was collected and stored at -80˚C. The protein content was determined and quantified (Bradford protein quantification kit, Lanjieke Technology). The sample was mixed with the loading buffer and denatured at 95˚C for 10 min (30 µg/lane). Gel electrophoresis was performed (SDS-PAGE kit; Beyotime Biotechnology) at a voltage of 50 V. The protein bands were transferred to the PVDF membrane, sealed with 5% skimmed milk powder for 1 h (room temperature), then incubated with a primary antibody (Caspase-3, bsm-61071R, dilution 1:1,000; Bcl-2, bs-0032R, dilution 1:1,000; Bax, bsm-52316R, dilution 1:1,000; Cyto-C, bsm-33193M, dilution 1:2,000; PARP-1, bsm-52408R, dilution 1:500; β-actin, bsm-63325R, dilution 1:50,000; Bioss Biotechnology; Fas, MA5-35308, dilution 1:1,000; Thermo Fisher Scientific, Inc.) at 4˚C overnight, and incubated with HRP-labeled secondary antibody (HRP-conjugated goat anti-rat, bs-0293G-HRP, dilution 1:5,000; HRP-conjugated goat anti-rabbit, bs-0295G-HRP, dilution 1:5,000; Bioss Biotechnology) at 37˚C for 1 h. The membranes were exposed with ECL (ECL reagent; Meilun Biotechnology) and then images captured and analyzed using a gel imaging system (C300; Azure Biosystems).

After 8 weeks of intragastric administration of GBE50DP, the orbital blood was collected, and the samples were processed. A total of 200 µl plasma was taken, and 400 µl pre-cooled acetonitrile was added, vortexed and mixed for 5 min, centrifuged at 9,391 x g for 10 min at 4˚C, and the supernatant was filtered using a 0.45 µm microporous membrane for injection analysis. The chemical components of GBE50DP were analyzed respectively in positive and negative ion modes by LC-MS/MS technology, and the chemical components of blank plasma and drug-containing plasma were compared.

The high-performance liquid chromatography (HPLC) analysis was conducted on an e2695 system (Waters Corporation) with an HSS T3 column (2.1x100 mm, 1.7 µm, Waters Corporation). The mobile phase consisted of 0.1% formic acid-water and 0.1% formic acid-acetonitrile with a flow rate of 0.3 ml/min according to the gradient elution program: 5-20% B (0-3 min), 20-100% B (3-15 min). The temperature of the column was 30˚C. The injection volume was set at 5 µl.

Mass spectrometry (MS) detection was performed on a Triple TOF 5600 system (Shanghai AB SCIEX Analytical Instrument Trading Co.) equipped with an electrospray ionization. MS data acquisition was achieved by the positive and negative ion modes, and the ion source voltages were 5.5 and -4.5 kV, respectively. The ion source temperature was 550˚C. The declustering potential was 40/-40 V, and the collision energy to 20/-20 V. Nitrogen was used as an atomizer and auxiliary gas. The sprayer gas (Gas 1) and heater gas (Gas 2) were both 55 psi, and the curtain gas was 35 psi. Information-dependent acquisition set eight peaks with response values exceeding 100 cps for secondary mass spectrometry scanning. Time-of-flight MS and tandem MS (MS/MS) were scanned within the mass range of m/z 80-1,600 Da.

SPSS 22.0 statistical software (IBM Corp.) was used for statistical analysis. Data are expressed as the mean ± standard deviation. One-way analysis of variance followed by Tukey's post hoc test was used for difference analysis between groups if the data conformed to normal distribution. The rank sum test was used for difference analysis between groups if the data did not conform to normal distribution. P<0.05 was considered to indicate statistical significance. Graphs were prepared using GraphPad Prism 6.0 software (Dotmatics).

As shown in Table I, the number of contacts in the model group rats was (8±8) times and the contact duration was 67.21±45.61 sec, both significantly lower than those in the control group (16±4 times, 151.57±30.59 sec; P<0.001). Compared with the model group, the number of contacts (12±6) times and contact duration (111.61±48.75) sec in the GBE50DP high-dose group rats were significantly increased (P<0.05).

The results of the positioning navigation experiment are shown in Fig. 1. Compared with the control group, the escape latency of rats in the model group was significantly prolonged at the 9, 10 and 11th trials (P<0.05 or P<0.01). Compared with the model group, the escape latency of rats in the donepezil group was significantly shortened at the 11th trial (P<0.05), that of rats in the GBE50DP-L group was significantly shortened at the 9 and 11th trials (P<0.05 or P<0.01), that of rats in the GBE50DP-M group was significantly shortened at the 5, 9, 10 and 11th trials (P<0.05), and that of rats in the GBE50DP-H group was significantly shortened at the 10 and 11th trials (P<0.05).

The results of space exploration experiments are shown in Table II. Compared with the control group, the number of platform crossings of rats in the model group was significantly decreased (P<0.05). Compared with the model group, the number of platform crossings in the positive, GBE50DP-M and GBE50DP-H groups was significantly increased (P<0.05 or P<0.01).

As shown in Table III, compared with the control group, the average number of working memory errors and reference memory errors of rats in the model group was significantly increased (P<0.05), and the foraging completion time was significantly prolonged (P<0.01). Compared with the model group, the mean number of working memory errors of rats in the positive, GBE50DP-L and -M groups was significantly decreased (P<0.05), that of reference memory errors of rats in the positive, GBE50DP-M and -H groups was significantly decreased (P<0.05), and the foraging completion time of the positive, GBE50DP-L and -H groups was significantly decreased (P<0.05 or P<0.01).

As shown in Fig. 2A, compared with the control group, the SOD content in the hippocampal tissue of the model group rats (30.31±3.67 ng/l) was significantly decreased (P<0.01), the GSH-Px content (37.51±6.70 nmol/l) was significantly decreased (P<0.01), the CAT content (22.59±1.72 ng/l) was significantly decreased (P<0.001), while the MDA content (5.39±0.67 nmol/l) was significantly increased (P<0.05). Compared with the model group, the SOD content (40.32±7.88 ng/l) in the GBE50DP high-dose group was significantly increased (P<0.01), the GSH-Px content (42.62±4.77 nmol/l) was significantly increased (P<0.05), the CAT content (24.33±1.96 ng/l) was significantly increased (P<0.05), while the MDA content (4.60±0.57 nmol/l) was significantly decreased (P<0.05).

As shown in Fig. 2B, compared with the control group, the IL-1β content (31.08±3.03 ng/l) in the hippocampal tissue of the model group rats was significantly increased (P<0.001), the IL-6 content (128.50±12.07 ng/l) was significantly increased (P<0.01), and the TNF-α content (306.16±21.47 ng/l) was significantly increased (P<0.001). Compared with the model group, the IL-1β content (28.21±1.37 ng/l) in the GBE50DP high-dose group was significantly decreased (P<0.05), the IL-6 content (94.03±22.27 ng/l) was significantly decreased (P<0.01) and the TNF-α content (277.42±23.28 ng/l) was significantly decreased (P<0.01).

As shown in Fig. 3, in the control group, the nerve cells were arranged neatly, the cytoplasm and nucleus were stained clearly, and no inflammatory cell infiltration, capillary dilation or congestion was observed. Compared with the control group, the structure of the hippocampal CA1 region of rats in the model group was loose, the arrangement of nerve cells was irregular and the morphology was incomplete, the cytoplasm was deeply stained, the nuclear condensation, apoptotic bodies and glial cells were improved increased, the inflammatory cells were scattered, and the capillaries were dilated. Compared with the model group, the aforementioned pathological changes in each administration group were improved to various degrees, showing a tighter structure, a decrease in the number of apoptotic bodies and glial cells, and an improvement in telangiectasia. Among them, the improvement in the GBE50DP-H group was the most obvious, followed by the positive group.

As shown in Fig. 4, the number of apoptotic neurons in the CA1 region of the hippocampus in the control group rats was 5±2, while in the model group it was 33±4. Compared with the control group, the number of apoptotic neurons in the model group was significantly increased (P<0.001). Compared with the model group, the number of apoptotic neurons in all treatment groups was significantly decreased (P<0.001), with the GBE50DP high-dose group showing the most pronounced change, having 11±2 apoptotic neurons.

As shown in Fig. 5, compared with the control group, the expression of Bcl-2 in the hippocampal CA1 region of rats in the model group was significantly decreased (P<0.001), while that of Cyto-C, Bax, Caspase-3, Fas and PARP-1 was significantly increased (P<0.001). Compared with the model group, the expression of Bcl-2 in the hippocampal CA1 region of rats in each administration group was significantly increased (P<0.001), while that of Cyto-C, Bax, Caspase-3, Fas and PARP-1 was significantly decreased (P<0.001), particularly in the GBE50DP-H group.

As shown in Fig. 6, compared with the control group, the expression of Bcl-2 in the hippocampal CA1 region of rats in the model group was significantly decreased (P<0.05), while the expression of Cyto-C, Bax, Caspase-3, Fas and PARP-1 were significantly increased (P<0.05 or P<0.01 or P<0.001). Compared with the model group, the expression of Bcl-2 in the hippocampal CA1 region of rats in the positive group and the GBE50DP-H group significantly was increased (P<0.05), while that of Cyto-C, Bax, Caspase-3, Fas and PARP-1 in the positive and GBE50DP-H groups was significantly decreased (P<0.05 or P<0.01).

The molecular formula of the compound in the GBE50DP-containing plasma was speculated by ion peak and cleavage law (see Fig. 7). The main VCI-related compounds were speculated as the ginkgolide A, B, C, K, bilobalide, rutin and quercetin.