CK (Chengdu Desite Biotech Co., Ltd., China, Purity: ≥90%) solution was dissolved in Aβ42 (Jill Biochemical (Shanghai) Co., Ltd., China, Purity: 95.20%) monomer solution to obtain final concentrations of 0 μM, 50 μM, 100 μM, and 200 μM, the final concentration of Aβ42 was 50 μM. Aβ42 monomer solution was incubated alone or with CK for 5 days at 37°C. The Aβ42 monomer and treated samples were dropwise to glow-discharged carbon-coated 300-mesh copper grids, were adsorbed for 10 min and blotted with filter paper, and air-dried. Images were examined with a JEM-1230 electron microscope operated at 80 keV.

Two hundred microliters (200 μL) of different concentrations of CK (1 mg/mL, 2 mg/mL, 4 mg/mL, 8 mg/mL) were mixed with 200 μL H₂O₂ (Xilong Chemical Co., Ltd., China) (6 mM) and 100 μL FeSO₄ (Liaoning Quan Rui Reagent Co., Ltd., China) (6 mM), The mixture was stirred and mixed for 10 min. Next, the resultant mixture was added 200 μL salicylic acid (Beijing Yongding Chemical Factory, China) (6 mM) maintained at 37°C for 25 min. The absorbance of the resultant mixture was measured at 510 nm and the following equation was used to determine radical scavenging rate: S c a v e n g i n g   r a t e = 1 − A 1 − A 2   /   A 0 × 100 % . A₁ is the absorbance of CK mixed with the reaction solution. A₂ is the absorbance measured with distilled water instead of H₂O₂. A₀ is the absorbance of CK replaced by distilled water.

The 3D structure SDF file of ginsenoside CK was first obtained from the PubChem database. File format conversion to PDB was peformed using Discovery Studio 2019 software. The protein structures of Aβ, Nrf2-Keap1, and the receptor protein were downloaded from the RCSB PBD database (https://www1.rcsb.org/). The https://swift.cmbi.umcn.nl/servers/html/prepdock.html website was used to manipulate receptor proteins, including correcting side chains and water molecules. The receptor protein and ligand drug molecules were de-watered and non-polar hydrogen atoms were added using AutoDock Tools 1.5.6 software to construct a docking site cavity. After docking was complete, the best docking conformation was selected for verification based on the principles of conformational rationality and low energy, and the docking results were visualised using PyMOL 2.2.0 software.

ICR mice (weighing 25–30 g) were purchased from Changchun Yisi Experimental Animal Co., Ltd. (China). SCOP was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (China). Memantine Hydrochloride (MH) was purchased from Adamas Reagent Co., Ltd. (China). Mice were housed in a sterile environment with controlled room temperature at 22°C–25°C and allowed food and water. All experiments were approbed by the Changchun University of Chinese Medicine Animal Ethics Committee. Mice were randomly divided into three groups, with 10 mice in each group: Control group (0.9% Saline), SCOP group (SCOP 2 mg/kg), MH group (Memantine Hydrochloride, MH 3 mg/kg + SCOP 2 mg/kg), CK1 group (CK 20 mg/kg + SCOP 2 mg/kg), CK2 group (CK 40 mg/kg + SCOP 2 mg/kg). The Control group, MH group, CK group were administered saline (i.g), MH (i.g), CK (i.g) once daily for 14 consecutive days. Starting from the 7th day, except in the case of control group mice, SCOP was injected intraperitoneally, 30 min after treatment with the corresponding drug.

A three day NOR paradigm was used with an open field measuring 40 cm (w) × 40 cm (L) × 40 cm (h). On day 1, all mice were habituated to the empty opaque plastic chamber for 10 min. After 24 h of familiarization, mice were placed in the chamber with two identical objects and allowed to explore for 10 min. A testing trial occurred on day 5 and one object was replaced with a randomly chosen novel object, and the mice allowed to explore for 10 min. Object exploration was defined when the nose of the mice directed towards the object at a distance less than 2 cm and measured by the discrimination index which indicates the difference of time spent between a novel (TN) and familiar object (TF). It was calculated using the total amount of time spent with both objects in the test phase [Discrimination Index = TF/(TN + TF)].

The Step-Down Passive-Avoidance (SDPA) test was detected the learning and memory ability of mice by passively avoiding electrical stimulation. An insulated platform was placed in the reaction chamber where the bottom grid floor can be energized. During the training, the mice was subjected to an electric shock on the grid floor, and mice were eventually trained to stay on the platform. Retention tests were carried out 24 h after the traning session. Placed the mice on the platform and recorded the latency and number of errors within 5 min. After behavioral testing, the mice were anesthetized and sacrificed. The brain tissue was quickly removed and placed on ice, washed with PBS buffer solution, and some brain tissues were stored at −80°C for subsequent RNA extraction and microarray analysis, and some brain tissues were placed in 4% paraformaldehyde for subsequent immunohistochemistry.

Mouse brain tissue is dehydrated and embedded in paraffin, paraffin sections of brain tissue were dewaxed and hydrated, and incubated in 3% H₂O₂ to eliminate endogenous peroxidase activity. Slides were washed in PBS and sections were incubated overnight for rabbit anti-beta Amyloid Antibody (BOSTER, China), rabbit anti-IDE (Proteintech, China), rabbit anti-BACE1 (Proteintech, China), APP (Cell Signaling Technology, United States ), rabbit anti-PS1 (Bioss, China) at 4°C. Sections were incubated with biotinylated goat anti-rabbit lg-G for 30 min at room temperature. Slices were incubated in DAB (ZSGB-Bio, China) stain for 5–8 min until a brown precipitate is produced. Image observation and analysis using ImageScope software.

Control group, SCOP group, and CK2 group of mice were sacrificed, and brains were isolated as described earlier. And total RNA was purified using Trizol as per manufacturer’s protocol. Evaluate RNA integrity was using the Agilent 2,100 Bioanalyzer with an RNA LabChip Kit (Agilent Technologies). cDNA was prepared from total RNA using a random priming method followed by fragmentation of double-stranded cDNA, labelling and hybridization onto the GeneChip WT Terminal Labeling and Controls Kit. Microarrays were scanned with the Affymetrix GeneChip Scanner 3,000 7G. Raw data using Affymetrix GeneChip Operating Software. The obtained data were screened, and the differentially expressed genes were analyzed by unsupervised hierarchical clustering and displayed as heatmap. The GO Enrichment Analysis method uses the differential gene to perform gene function annotation based on the GO database (http://www.geneontology.org). Obtain the molecular function of the gene, and then screen out the significant function of the gene. Calculate the significance level (p-value) and false positive rate (FDR) of each function using Fisher’s exact test and multiple comparison test. The standard of significant screening: p-value < 0.01.

Lyophilized Aβ42 was reconstituted in 100% 1,1,1,3,3,3 hexafluoro-2-propanol (HFIP) (Shanghai Macklin Biochemical Technology Co., Ltd., China) to make a 1 mM dispersion. The mixture was incubated at 25°C for 60 min, placed on ice for between 5 and 10 min, moved to a fume hood to evaporate the HFIP, and subsequently formation of an Aβ peptide film occurred after air-drying. The film was resuspended in 5 mM anhydrous DMSO, and PBS was added to dilute the mixture to a 1 mM concentration.

Mouse hippocampal HT22 cells were obtained from Shanghai Enzyme Research Biotechnology Co., Ltd. (Shanghai, China). Cultured in high glucose DMEM supplemented with 10% FBS at 37°C in a water-saturated atmosphere of 5% CO2 incubator (Thermo, United States). Cells were subcultured 3 times a week. All experiments were performed after the cells were attached for 24 h, Cells treated with different concentrations of CK (2.5, 5, 10 μM) for 24 h, In addition to Control, cells treated with 10 μM Aβ42 for 24 h.

Detection of cell viability using CCK-8 (Dojindo Molecular Technologies, INC., Japan) assay. HT22 cells were cultured in 96-well plates at 1 × 10⁵ cells/mL concentration. Treated cells were added 10 μL CCK-8 per well. Determination of absorbance at 450 nm using microplate reader (TECAN, Switzerland) and calculated cell viability.

Treated cells were added DCFH-DA diluted in serum-free medium at a ratio of 1:1,000, Incubate at 37°C for 20 min, Observing the fluorescence expression of ROS under fluorescence microscope (Leica, Germany). Fluorescence intensity was measured by fluorescence microplate reader (TECAN, Switzerland) at 485 nm excitation and 525 nm emission.

Treated cells were rinsed in PBS and permeabilized with 4% paraformaldehyde for 15 min. The cells were added 0.5% Triton X-100 at room temperature for 20 min after rinsing with PBS. The cells were incubated with primary antibody anti-rabbit Nrf2 (Proteintech, China) overnight at 4°C. After incubation, HT22 cells were rinsed three times with PBS and incubated with specific secondary antibody for 1 h at room temperature. The cells were incubated with DAPI for 5 min at room temperature to stain nucleus. Photographing and observing with a laser confocal microscope (Olympus, Japan).

Cell lysed protein were eletrophoretically separated on 15% SDS-PAGE and transferred to PVDF membranes (Millipore, United States). The membranes were incubated with 5% skim milk for 1 h to block non-specific binding protein, and reacted with primary antibody at 4°C overnight. Afterwards, the membranes were incubated with secondary antibody conjugated with goat anti-rabbit (Proteintech, China) or goat anti-mouse HPR (Proteintech, China) at room temperature for 2 h. The protein bands was detected using the ECL kit (Proteintech, China), and image was scanned using an imaging system (Aplegen, United States). Protein gray value was detected by ImageJ software, and then calculated the relative expression levels of proteins.

Data are presented as mean ± SE. All data were analyzed using the Prism 6 program (GraphPad Software, Inc.). Statistical significance was assessed using Student’s t-test. p-value< 0.05 was considered to be statistically significant

Aβ-treated HT22 cells showed a significant decrease in cell viability (79.15 ± 4.02) %, and increased cell viability after CK treatment. However, 2.5 μM CK was insignificant in increasing cell viability of HT22 cells, with the cell survival rate gradually increasing with an increase in the CK dose (Figure 1).

Mice with SCOP-induced cognitive dysfunction showed similar preference for familiar and novel objects, while mice treated with CK had significantly more exposure time to new objects than familiar objects and an increased recognition index (Figures 2A,B). The latency of mice was significantly decreased after intraperitoneal injection of SCOP, with an increased error number being observed. There was no significant increase in the latency after pre-administration of memantine hydrochloride; however, the number of errors was reduced in these mice. The latency increased after pre-administration of CK, and the number of errors also reduced (Figures 2C,D). These results indicate that CK can improve the memory of mice, with an improvement better than memantine hydrochloride being recorded.

Through molecular docking, ginsenoside CK was shown to bind to Aβ by interacting with Lys16 and Glu3 of Aβ42 (Figure 3A). Unincubated Aβ monomer showed no obvious structure (Figure 3B). Aβ produced more fibrous filaments and a small amount of cluster-like structures after incubation; 50 μm CK and Aβ co-incubation did not significantly reduce the formation of filaments and cluster structures. Non-etheless, fiberous filaments and cluster structures gradually decreased after co-incubation of 100 μM or 200 μM CK with Aβ. These findings indicate that CK can inhibit the formation of Aβ aggregates.

Increased Aβ deposition plaques was observed in the hippocampus of mice after SCOP injection; however, these plaques were significantly reduced and the gray value was reduced in mice pretreated with CK. Increased expression of APP, BACE1, PS1, and Aβ, and reduced expression of IDE were observed in Aβ-injured HT22 cells and mice with SCOP-induced cognitive dysfuction. Contrarily, CK treatment decreased the expression of APP, BACE1, PS1, and Aβ, and increased that of IDE (Figure 4). These findings suggest that ginseng CK can inhibit the production of APP and further inhibit the cleavage of APP by BACE1 and PS1, produce a large number of IDE, and thus regulate the deposition of Aβ in neurons.

The expression levels of SYP and PSD95 in HT22 cells after Aβ injury were significantly decreased, significantly increased SYP expression levels after pretreatment with different doses of CK, high dose of CK increases the expression level of PSD95, CK of 2.5 uM and 5 uM had no significant effect on the expression level of PSD95 in HT22 cells. The expression levels of SYP and PSD95 in the brain of mice were significantly decreased after intraperitoneal injection, pretreatment of memantine hydrochloride and CK showed a significant increase in the expression levels of SYP and PSD95 in mouse brain (Figure 5). Through in vivo and in vitro experiments, it is speculated that CK can improve synaptic function and structure of injured neurons.

The GO database can provide functional classification for genomic data, including categories of biological processes (BP), cellular component (CC), and molecular function (MF). Herein, we mainly performed MF analysis. The p-value is presented as a logarithm and has a negative value; therefore, the larger the value of (-LgP), the smaller the p-value, indicating a higher GO significance level. We screened out the molecular functions of 21 significant GO enrichments and, as previously reported, molecular functions such as oxygen binding, peroxidase activity, oxidoreductase activity, hemoglobin (Hb) binding, hemoglobin beta (Hb β) binding, and hemoglobin alpha (Hb α) binding had a direct or indirect relationship with the regulation of ROS (Richter et al., 2009; Al Ghouleh et al., 2011; Barman and Fulton, 2017; Altinoz et al., 2019) (Figures 6A,B). Hb and oxygen combine to form oxyhemoglobin, which increases the level of oxidative stress in neurons and increases the production of ROS (Agyemang et al., 2021). Both ROS production and scavenging are determined by oxidoreductase activity. Peroxidase, a type of oxidoreductase, is also involved in the production and clearance of ROS (Vlasova, 2018). To confirm that CK can affect the regulation of ROS, we determined its abilities to scavenge hydroxyl radicals (Figure 6C), and a fluorescent probe displayed that a large amount of ROS was produced in Aβ-injured HT22 cells. Reduction of ROS in HT22 cells was observed after pretreatment with CK (Figures 6D,E). These results further indicate that CK is involved in the regulation of ROS and reduces its production.

The overproduction of ROS generates oxidative stress, Nrf2 plays an important role in antioxidant stress (Deshmukh et al., 2017). CK molecularly bound to the Nrf2/Keap1 complex through interacting with ILE-559 and VAL 606 of Nrf2/Keap1 complex (Figure 7). Nrf2 fluorescence intensity was reduced in Aβ-injured HT22 cells, and the expression of nuclei and total Nrf2 were reduced in Aβ-injured HT22 cells and mice with SCOP-induced cognitive impairment. Non-etheless, after pretreatment with CK, the fluorescence intensity of Nrf2 in HT22 cells increased and the expression of total and nuclei Nrf2 increased. Keap1 and HO-1 act as downstream factors of Nrf2 and participate in the regulation of ROS and oxidative stress (Abed et al., 2015; Guo et al., 2015). Aβ induced the increased expression of Keap1 and decreased expression of HO-1 in HT22 cells (Figure 8). CK reversed the expression of these two factors, findings similar to a previously reported in vivo study (Yang et al., 2019). Our findings indicate that CK activates the Nrf2/Keap1 signaling pathway and inhibits oxidative damage in HT22 cells.

An increase in the levels of proapoptotic proteins Cytochrome C (Cyt C), Caspase-3, and cleaved Caspase-3 was observed in HT22 cells after Aβ injury as well in brain tissue of mice with SCOP-induced cognitive impairment; however, these levels decreased after treatment with CK (Figure 9). To this end, CK can alleviate Aβ-damage in HT22 cells and mice with SCOP-induced cognitive impairment by inhibiting apoptosis. Further, as the dose of CK increased, the protective effect of HT22 cells was enhanced.