Screening of DSS chemical components and prediction of action targets

Utilizing the Traditional Chinese Medicine Systematic Pharmacology Database and Analysis Platform (TCMSP), a search was conducted for the chemical constituents of DSS with “Angelica sinensis,” “Paeonia lactiflora,” “Ligusticum chuanxiong,” “Atractylodes macrocephala,” “Poria cocos,” and “Alisma plantago-aquatica” as keywords. Preliminary screening for active compounds was performed based on two ADME properties: oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18. Furthermore, as supported by the available literature, additional compounds presented in DSS that potentially possessed therapeutic properties, but did not meet the aforementioned criteria of OB and DL, were included for comprehensive analysis. Structural data files for the aforementioned active compounds were obtained from the PubChem database (http://pubchem.ncbi.nlm.nih.gov/). The Swiss Target Prediction analysis platform (http://www.swisstargetprediction.ch/) was employed to predict the target proteins of the active compounds, and the target names were corrected by Uniport database (https://www.uniprot.org).

AD-related target screening

Using “Alzheimer’s disease” as the keyword, potential targets associated with AD were retrieved from the OMIM database (http://www.omim.org), GeneCards database (http://www.genecards.org/), DRUGBANK database (https://go.drugbank.com/), and DisGeNET database (https://www.disgenet.org/). The disease targets were then entered into the UniProt database (https://www.uniprot.org) to standardize the gene nomenclature. After eliminating duplicates and targets that could not be standardized, the Alzheimer’s disease targets were identified. The targets of the active components of the medicine were combined with the disease targets and input into Venny 2.1 (https://bioinfogp.cnb.csic.es/tools/venny/index.html). The intersection of the two sets was taken as the key targets, that is, the potential therapeutic targets of DSS for the treatment of AD, and a Venn diagram was drawn.

Construction and core target prediction of the DSS-AD PPI network

The key targets of DSS for the treatment of AD were uploaded to the STRING platform (https://string-db.org/) for Protein-Protein Interaction (PPI) analysis. The species is set to Homo Sapiens, the minimum required interaction score is set to > 0.7, and the other parameters are left unchanged by default, Isolated nodes in the network were removed to obtain the PPI network of DSS in the treatment of AD. The PPI network data were imported into Cytoscape 3.9.1 and further analyzed using the plugin CytoNCA. The connectivity (Degree), Betweenness Centrality (BC), and Closeness Centrality (CC) of the nodes in the PPI network were calculated, and the average values for Degree, BC, and CC were determined. Nodes and interactions that were simultaneously greater than the average values of Degree, BC, and CC were retained to construct the core target network of DSS in relation to AD.

GO and KEGG pathway enrichment analysis

The key targets of DSS for the treatment of AD were imported into the DAVID database for analysis, which included the Gene Ontology (GO) analysis of biological processes (BP), molecular functions (MF), and cellular components (CC), as well as the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Ten species related to autophagy from each enrichment analysis result were taken for visualization.

Molecular docking

The mol2 format file of the active ingredient was downloaded via TCMSP, and the 3D crystal structures of AMPK and mTOR were obtained on the PDB Protein Data Bank (https://www.rcsb.org/). Subsequently, the proteins and the active ingredients were imported into the AutoDock platform for docking analysis.

Antibodies and reagents

LC3B (ab192890), Beclin1 (ab207612),PSD-95 (ab238135) and IBA1 (ab178846) were purchased from Abcam (Cambridge, MA, USA). P62 (T55546S), p-AMPK (T55608S), and p-mTOR (T565571S) were purchased from Abmart Shanghai Co., Ltd. (Shanghai, China). APP (AF6084), GADPH (T0004) was purchased from Affinity Biosciences LTD (Jiangsu, China), p-Tau (WL03540) was purchased from WanLeiBio (Shenyang, China), IL-1β, IL-6, and TNF-α ELISA (enzyme-linked immunosorbent assay) kits were purchased from UpingBio (Shenzhen, China). EZ-press RNA Purification Kit, Color Reverse Transcription Kit, and 2Color SYBR Green qPCR Master Mix (ROX2 plus) were purchased from EZE Science Biotechnology (Roseville, NM, USA). Human Aβ1–42 peptide (Catalog: AG968) was purchased from Sigma-Aldrich Chemical Co. (St Louis, MO, USA).

Preparation of DSS, SG, and XG

DSS is composed of six raw herbs, including Angelica Sinensis (Oliv.) Diels. (Batch numbers:221,103,461), Paeonia lactiflora Pall. (Batch numbers:221,100,229), Atractyolodes macrocephala Koidz. (Batch numbers:221,260,491), Poria cocos (Schw.) Wolf. (Batch numbers:221,202,451), Alisma orientale (Sam.) Juzep. (Batch numbers:221,000,671), and Ligusticum Chuanxiong Hort. (Batch numbers:221,101,331) were sourced from Kangmei Pharmaceutical Co., Ltd. (Guangdong, P.R. China) and they were combined in a ratio of 3: 16: 4: 4: 8: 8. The Suangan (SG) is composed of Paeonia lactiflora Pall., Atractyolodes macrocephala Koidz., Poria cocos (Schw.) Wolf., Alisma orientale (Sam.) Juzep. and they were combined in a ratio of 16: 4: 4: 8. The Xingan (XG) is composed of Angelica Sinensis (Oliv.) Diels., Ligusticum Chuanxiong Hort., Atractyolodes macrocephala Koidz., Poria cocos (Schw.) Wolf., Alisma orientale (Sam.) Juzep. and they were combined in a ratio of 3: 8: 4: 4: 8. Subsequently, the mixture of dried herbs was briefly immersed in distilled water at a volume-to-weight ratio of 8:1 for one hour, followed by decoction for another hour. The resulting filtrate was collected, and the residue was subjected to a second round of decoction using distilled water at a volume-to-weight ratio of 6:1 for one hour. The filtrates obtained from both decoction rounds were combined and then concentrated.

Animals and drug treatment

Eight-week-old male and female C57BL/6J mice were purchased from Guangzhou Ruige Biological Technology Co., Ltd. The experimental animals were housed in a specific pathogen-free (SPF) room with a controlled temperature of 25 ℃, a relative humidity ranging from 50 to 70%, and a 12-hour light/dark cycle. They were provided with free access to water and food. The experimental protocols were approved by the Animal Experimentation Committee at Guangzhou University of Chinese Medicine and conducted following the National Research Council Guide for Care and Use of Laboratory Animals (approval number: ZYD-2022-267).

Groups and drug administration

After one week of acclimation, all animals were randomly divided into six groups, each consisting of eight mice (four males and four females): the sham group, model group, DSS group, SG group, XG group, and Donepezil group. The Donepezil group was used as a positive control and administered at a dosage of 3 mg/kg/day.

Before the surgical procedure, Aβ1−42 was dissolved in the sterile saline solution to achieve a concentration of 1 µg/1µl. Subsequently, the solution underwent aggregation by incubating it at 37 °C for 7 days [20]. Based on the mouse brain atlas, after anesthetizing with 0.25 g/kg tribromoethanol, all mice except those in the sham group were intracerebroventricularly (ICV) injected with aggregated Aβ1−42 peptide on each side, totaling 5 µl per injection site. The injection coordinates were as follows: AP (anteroposterior): -1.94 mm; ML (mediolateral): ±l.4 mm; DL (dorsoventral): -2.2 mm. In contrast, the sham group received intracerebroventricular injections of physiological saline at the same coordinates. To minimize backflow, the needle was kept in the injection site for 5 min [21].

On the third day after the injection, the treatment regimen was administered based on previous studies [22]. The experimental groups were given DSS (6.4 g/kg/day), SG (4.8 g/kg/day), XG (4.0 g/kg/day), and Donepezil (3 mg/kg/day) daily for 28 consecutive days through oral gavage. In contrast, the control and model groups received the same volume of saline solution. The dosages for the SG and XG groups were adjusted proportionally, referring to the recommended amounts in the book " Synopsis of the Golden Chamber “.

Morris water maze test (MWM test)

The MWM test was performed three weeks following the administration of the drug. It involves a circular pool measuring 1600 cm in diameter and 50 cm in height. The pool is divided into four equal quadrants. The pool is filled with opaque water, and in the designated quadrant, there is a platform with a diameter of 10 cm that is not easily noticeable underwater. The platform is situated 1.0 cm below the water surface.

Animals were randomly released from different quadrants of the pool to train them to locate the hidden platform after a day of acclimatizing to the pool. They underwent this training three times a day for five days, with a 20-minute interval between trials. If the mice failed to find the platform within 60 s, their latency was recorded as 60 s, and they were then guided to the platform location for 10 s. The average escape latency of each animal was recorded daily.

On the sixth day, the mice underwent a probe trial session in which the platform was removed from the pool. The mice were released at the opposite point of the platform’s original position and allowed to swim for 60 s to search for the platform. The time spent in the target quadrant and the number of times each mouse crossed the platform were recorded and analyzed. The experimental design of the conducted studies is delineated in Fig. 1.

Fig. 1

Experimental flow chart for the Aβ1−42 (ICV) model, behavioral studies, and biochemical and molecular analysis

Specimen collection and storage

On the following day after the MWM experiment, mice were euthanized by cervical dislocation after being deeply anesthetized with 0.25 g/kg of tribromoethanol, and then their brains were dissected and separated into left and right hemispheres. The left hemisphere was histologically examined and preserved in 4% paraformaldehyde. The cortex and hippocampus of the right hemisphere were isolated and rapidly frozen in liquid nitrogen before being transferred to a refrigerator set at -80 ℃ for molecular biological analysis.

Real-time quantitative PCR (RT-qPCR)

Tissues were homogenized following the instructions of the manufacturer. Total RNA was extracted from the tissues using the EZ-press RNA Purification Kit reagent (EZBioscience, Roseville, MN, USA). mRNA was reverse transcribed into Complementary DNA (cDNA) using a cDNA synthesis kit (EZBioscience, Roseville, NM, USA). Subsequently, the cDNA was then combined with specific primers according to the 2x Color SYBR Green qPCR Master Mix (EZBioscience, Roseville, NM, USA). The relative expression of the target genes was determined by qPCR, with GADPH serving as the internal control. The relative expression levels of mRNA were calculated using the 2^-ΔΔCT^ method.

The primer sequences can be found in the Table 1.

Table 1 The primer sequences for each geneEnzyme-linked immunosorbent assay (ELISA)

The levels of IL1β, IL6, and TNF-α were measured utilizing the ELISA assay kits (UpingBio, Shenzhen, China) according to the manufacturer’s directions with a multifunctional microplate reader.

Western blot

Brain tissues were briefly lysed in RIPA lysis buffer supplemented with protease and phosphatase inhibitors cocktail. The supernatant was collected, and BCA assays determined the protein concentration. Protein (40 µg) was resolved by 10-12% SDS-PAGE gel electrophoresis and subsequently transferred to polyvinylidene difluoride membranes. After blocking with 5% skim milk for 1 h at room temperature, the membranes were incubated with corresponding primary antibodies: PSD-95, APP, p-mTOR, p-AMPK, P62, Beclin1, LC3B, and GADPH overnight at 4 ℃. On the following day, after three incubations in TBST of 5 min each, the membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-mouse (1: 5000) or Goat anti-rabbit IgG (1: 5000) for 1 h at room temperature. Finally, an ultrasensitive ECL chemiluminescent solution (BL520A, Biosharp, China) was applied, and images were captured by the Chemiluminescence image analysis system (Shanghai Tanon, China). The densitometry of the bands was visualized using ImageJ software.

Hematoxylin-eosin (HE) staining

After being placed at a constant temperature of 65 °C for 1.5 h, brain paraffin sections were washed and dehydrated in alcohol, deparaffinized in xylene, and stained with hematoxylin-eosin for observation of cell morphology.

Immunohistochemistry

Brain paraffin sections were kept at a constant temperature of 65 °C for 1.5 h. Subsequently, the brain sections were dewaxed and rehydrated using various concentrations of ethanol. Antigenic repair was performed using citric acid. Endogenous peroxidase activity was blocked using blocking agents (ZSB-BIO, Beijing, China). The sections were then blocked with 3% bovine serum albumin for 1 h before being incubated overnight at 4 ℃ with IBA1 (diluted 1:1000). The following day, secondary antibodies (Enhanced enzyme-labeled goat anti-rabbit IgG polymer, ZSB-BIO, Beijing, China) were added. The sections were covered with the DAB Detection Kit (ZSB-BIO, Beijing, China) and counterstained with hematoxylin. Finally, the sections were photographed under a microscope, and the IOD value was analyzed using Image-Pro Plus.

Statistical analysis

The data are presented as the mean ± SEM. Statistic Package for Social Science (SPSS, v.20.0) statistical software was used for the data analysis. To analyze the escape latency data in the Morris water maze, repeated measures analysis of variance (ANOVA) was employed. For the analysis of other data, a one-way ANOVA followed by post hoc LSD or Dunnett’s T3 test was used. A statistical significance level of P < 0.05 was considered.