Preparation of CRD
CRD was prepared from a total of 4320 g of Conioselinum anthriscoides 'Chuanxiong' (Chuanxiong), Panax ginseng C.A. (Renshen), Pueraria montana var. lobata (Willd.) Maesen & S.M. Almeida ex Sanjappa & Predeep (Gegen), Ginkgo Biloba L. (Yinxingye), and Reynoutria multiflora (Thunb.) Moldenke (Heshouwu) in a 1:2:2:2:2 ratio. All herbs were obtained from Huadong Medicine Co., Ltd. (Zhejiang, China, Batch No. 20210208) and were fully validated by Associate Professor Ying Yao (Department of Pharmacognosy, School of Pharmacy, Hangzhou Medical College) according to the Chinese Pharmacopoeia 2015. The herbs were slightly crushed and placed in a flask with 8 volumes (W/V) of 60% ethanol and subjected to reflux heating for 2 h, followed by filtering. The alcohol extract was filtered, placed in a refrigerator at 4 °C for 24 h, and filtered again to obtain the drug solution. This solution was concentrated to 1.44 g/mL (14.4 g/kg) by a rotary evaporator and stored at the Zhejiang Institute of Traditional Chinese Medicine. When used in animal experiments, the solution was diluted to 0.72 g/mL and 0.36 g/mL with distilled water, which were equivalent to CRD 7.2 g/kg and 3.6 g/kg, respectively.
Quality control markers of CRD
With reference to the requirements of the Chinese Pharmacopoeia 2020 regarding the above Chinese medicinal materials, we selected ferulic acid, ginsenoside Rg1, puerarin, ginkgolide A and emodin as the quality control markers for Conioselinum anthriscoides 'Chuanxiong' (Chuanxiong), Panax ginseng C.A. (Renshen), Pueraria montana var. lobata (Willd.) Maesen & S.M. Almeida ex Sanjappa & Predeep (Gegen), Ginkgo Biloba L. (Yinxingye), and Reynoutria multiflora (Thunb.) Moldenke (Heshouwu), respectively.
Animals and drug administration
Experimental 3 × Tg-AD transgenic mice (APPSwe, tauP301L, PSEN1dE9, females) and C57BL/6 background mice (25 ± 5 g) were placed separately in plastic cages at 22 ± 1 °C and 55% ± 5% humidity according to the 12-h light–dark cycle. All procedures involving experimental animals were performed according to the guidance on the treatment of the experimental animals issued by the Ministry of Science and Technology of the People's Republic of China and were also approved by the animal experiment ethics committee of Zhejiang University of Traditional Chinese Medicine (Ethics Approval No. ZSLL-2018-045). Five-month-old 3 × Tg-AD transgenic mice were randomly divided into 5 groups with 9 mice in each group: the 3 × Tg-AD group, three CRD groups (3.6 g/kg/d; 7.2 g/kg/day; 14.4 g/kg/d), and the donepezil treatment group (1 mg/kg/d, DNPQ, batch 1705080, Eisai Pharmaceutical Co., Ltd., Benxi, Liaoning, China). Nine C57BL/6 mice were used as the wild-type (WT) group. We used the WT group as the normal control group, the 3 × Tg-AD group as the model control group, and the donepezil group as the positive control group. The CRD groups were intragastrically administered (0.1 mL/10 g) once a day for 4 months. The WT group and 3 × Tg-AD group were given the same amount of normal saline by gavage.
Morris water maze (MWM) tests
At 1, 2, 3 and 4 months after administration, all mice were subjected to the MWM test for 5 days to evaluate their learning and memory ability. The water temperature was kept at 22 ± 0.5 °C during the test.
Place navigation test
The learning and access memory abilities of mice were evaluated by a place navigation test for 5 days. The day before the experiment, the mice were put into the water to swim freely for 60 s to familiarize themselves with the experimental environment. The tank was divided into four quadrants, and four fixed points were chosen as starting points. A mouse was placed in the water at one of the four points. The escape latency and the time to reach the platform were recorded.
Spatial probe test
On the sixth day of the experiment, a spatial probe test was performed. The hidden platform was removed, and a mouse was placed in a tank with its face toward the wall in a randomly selected quadrant. The swimming trajectory and the number of times the mice crossed the original platform were recorded within 60 s.
Brain tissue staining
H&E staining was performed as follows: 30 min after the last administration, the mice were anesthetized with 3% pentobarbital sodium intraperitoneally. Cardiac perfusion was performed before brain tissue staining. The left side of the brain was separated and fixed in 10% neutral formaldehyde fixative for 24 h. After the brain tissue was dehydrated and cleared, it was embedded in paraffin and sectioned. The tissue was dried at 65 ℃, dewaxed with xylene twice for 5 min, and washed with distilled water. Slides were placed in hematoxylin staining solution for 5 min after washing for 5 min. Differentiation was performed using acid alcohol for 30 s, and tap water immersion was performed for 15 min, followed by eosin staining for 2 min. Finally, the dehydration, clearing, rinsing and mounting were performed as follows: 95% ethanol for 1 min, ethanol for 3 min, anhydrous ethanol for 5 min (repeat twice), xylene for 5 min (repeat twice), and neutral balm for mounting [20].
Nissl staining was performed as follows: (1) dewaxing paraffin sections; (2) distilled water washing; (3) soaking in 1% toluidine blue aqueous solution in a constant temperature box at 50–60 °C and for 30 min; (4) distilled water washing; (5) rapid differentiation in 95% ethanol; and (6) anhydrous ethanol dehydration, xylene treatment for transparency, and neutral gum sealing [20].
Immunofluorescent staining
Brain sections were roasted at 60 °C for 2 h and dewaxed. After washing, the sections were repaired with citrate sodium buffer in a microwave oven. Antibodies against Aβ (amyloid 1–16, 1:100, 803001, BioLegend) were added and incubated at 4 °C overnight following BSA blocking. Then, the slices were incubated with goat anti-mouse secondary antibody at room temperature for 1 h. Five different fields were randomly selected for photos under a 10× objective lens. All images were statistically analyzed by ImageJ software, and the degree of immunofluorescence staining was reflected by the cumulative optical density of Aβ plaques. Cumulative optical density (IOD) refers to the sum of fluorescence intensity in an image. The formula is IOD (cumulative optical density) = ∑area (positive expression area) × density (average fluorescence intensity).
Western blot assay
Lysates of the hippocampus in brain tissues were collected, and the protein concentrations were determined by the BCA protein assay (Beyotime, China). Equal amounts of protein were loaded in each lane and separately subjected to SDS‒PAGE before transfer onto PVDF membranes (Millipore, Massachusetts, USA). After blocking with 5% skim milk for 1.5 h at 37 °C, the membrane was incubated with the indicated primary antibodies at 4 °C overnight and subsequently with the respective near-infrared dye-tagged secondary antibodies for 1 h at 37 °C. Antibodies were purchased from Thermo Fisher (anti-tau (phospho-Ser202 Thr205), MN1020), Immunoway (anti-tau, YT4546), and Hangzhou Huaan Biotechnology Co., LTD (GAPDH EM1101). Image acquisition and documentation of the blots were performed by an Odyssey double color infrared laser imaging system (LI-COR, Nebraska, USA). The analysis software of the Odyssey two-color infrared laser imaging system was used for analysis. The equation was as follows: relative gray value = (sample gray value/sample internal reference)/(control gray value/control internal reference). After obtaining the core target through network pharmacological prediction, we verified it through western blotting. The antibodies used were anti-EGFR (ab52894, Abcam), anti-CASP3 (14220S, Cell Signaling Technology) and anti-β-tubulin (AM1031A, Abcepta). An Amersham ImageQuant 800 system (Cytiva, China) and ImageJ software were used to quantify the expression of EGFR and CASP3.
Preparation of homogenates from brain tissue and serum
Ten 3 × Tg-AD mice (female mice in an SPF grade, weight 25 ± 2 g, 5 months old) were randomly assigned to the control group and CRD group (1.44 g/kg/d). Experimental mice were placed separately in plastic cages at 22 ± 1 °C and 55% ± 5% humidity according to the 12-h light–dark cycle (Ethics Approval No. ZJCLA-IACUC-20020056). The CRD group was intragastrically administered CRD once a day for 5 days with a gavage volume of 0.1 mL/10 g. The control group was given the same volume of distilled water in the same way. After the last administration, anesthesia was administered, the brain was removed by craniectomy, the brain tissue was repeatedly rinsed with normal saline until it became colorless, and the water droplets on the surface of the brain tissue were dried with paper. The processed brain tissue was accurately weighed and transferred to EP tubes, 3 volumes of normal saline was added, and homogenate beads were added for homogenization. Thirty minutes after the last drug administration, blood was taken from the abdominal aorta and allowed to stand at room temperature for 30 min, followed by centrifugation at 3000 rpm at 4 °C for 15 min. The supernatant was collected and stored in an EP tube at − 80 °C.
UPLC-Q–TOF–MS analysis
Equipment and materials
The instruments and reagents used for mass spectrometry were as follows: SCIEX X-500R Quadrupole Time of Flight Mass Spectrometer (AB SCIEX, USA); TurboIonSpray ion source (AB SCIEX); Waters ACQUITY I-Class Plus UPLC Ultra-High-Performance Liquid Chromatography System (Waters); Thermo ST40R Low-temperature high-speed centrifuge (Thermo Fisher); IKA Miniature scroll mixing instrument (IKA Germany); AUW220D electronic balance (Shimadzu Company, Japan). Methanol, acetonitrile and formic acid (Merck, Germany), Milli-Q ultra-pure water (Millipore, USA); and other reagents were obtained in analytically pure form.
Preparation of CRD test solution
The preparation method of the original drug solution was performed as previously described. Then, 200 μL of the original drug solution (1.44 g/mL) was diluted in 800 μL of water and vortexed for 1 min to obtain a 0.384 g/mL solution. Then, 0.5 mL of 0.384 g/mL solution was mixed with 0.5 mL of methanol, vortexed for 1 min, and centrifuged at 14,000 rpm for 20 min. A 2 μL sample of the supernatant was obtained.
Preparation of standard curve solution
First, 200 μL of 1 mg/mL ginsenoside Rg1 standard solution, 200 μL of 1 mg/mL ferulic acid standard solution, 100 μL of 1 mg/mL emodin standard solution, 60 μL of 1 mg/mL ginkgolide A standard solution, and 60 μL of 1 mg/mL puerarin standard solution were placed in a centrifuge tube, and 40 μL of methanol was added. The mixture was vortexed to obtain the solution with concentration 1. The solution with concentration 1 was diluted by a factor of two. Then, 500 μL of the solution with concentration 1 was placed in a new centrifuge tube and diluted with 500 μL of methanol, and the same procedure was repeated to obtain standard solutions at seven concentrations. After centrifugation at 12,000 rpm/min for 20 min, the supernatant was collected and injected into the sample.
Preparation of homogenate of brain tissue and serum test solution
Brain tissue homogenate (200 μL) was mixed with methanol (800 μL), vortex shocked for 3 min, and centrifuged at 8000 rpm for 10 min. Next, 600 μL of supernatant was blown dry with nitrogen, dissolved in 200 μL of methanol, vortex shocked for 3 min, and centrifuged at 13,000 rpm for 10 min. A sample of the supernatant was taken for determination.
The serum was thawed at room temperature, 1.0 mL was placed in a centrifuge tube, and 20 μL of phosphoric acid was added accurately. Ultrasonic treatment was carried out for 1 min, vortex mixing was carried out for 30 s, and the samples were transferred to the SPE column, which previously activated and equilibrated with 3 mL of methanol and 3 mL of water in advance. The samples were washed with 3 mL of water, discarded, and eluted with 3 mL of methanol, and the eluent was collected and lyophilized. The residue was redissolved in 150 μL of methanol and centrifuged at 14,000 rpm at 4 ℃ for 15 min. The supernatant was used as the serum test solution.
UPLC-Q–TOF–MS conditions
The chromatographic conditions were as follows: an ACQUITY UPLC BEH C18 (150 × 2.1 mm, 1.7 μm) column with mobile phases of 0.1% formic acid acetonitrile (A) − 0.1% formic acid water (B); gradient elution procedure: 0–15 min, 95–50% B; 15–16.5 min, 50–30% B; 16.5–20 min, 30–20% B; 20–22.5 min, 20–10%; 22.5–23 min, 10–1% B; flow rate: 0.3 mL/min; injection tray temperature: 8 ℃; column temperature: 40 ℃; injection volume: 4 μL.
The mass spectrometry conditions were as follows: ion source gas 1 (Gas1): 55, ion source gas 2 (Gas2): 55, curtain gas (CUR): 35, source temperature: 600 ℃, ion spray voltage floating (ISVF): 5500 V/− 4500 V; TOF MS scan m/z range: 50–1500 Da, production scan m/z range: 25–1000 Da. The data were also decluttered by information-dependent acquisition (IDA) in high-sensitivity mode. Collision energy: 35 ± 15 eV; IDA: exclude isotopes within 4 Da, candidate ions to monitor per cycle: 12.
Identification of the ingredients
SCIEX OS (v2.0.1) software was used to collect and process the data. We first used the TCM MS/MS Library (TCM MS/MS Library contains more than 1000 secondary data of TCM compounds) as a database to identify CRD test solutions. Then, we used the identified ingredients as a self-built database to perform SCIEX OS software analysis of brain tissue homogenate of the CRD group and control group.
Target collection
The potential targets of the ingredients in CRD were searched in SwissTargetPrediction [21], a web server that accurately predicts bioactive molecular targets based on the combination of 2D and 3D similarity measures of known ligands. A probability greater than 0.1 was considered to indicate a possible regulatory target of CRD ingredients. The targets related to AD were selected from the databases Disgenet and Genecards. Then, a Venn diagram was drawn to identify the intersection of ingredient-related targets and disease-related targets, which are potential targets for the treatment of CRD in AD.
Protein‒protein interaction (PPI) network construction
Potential targets of CRD and AD were uploaded to STRING 11.0 (https://string-db.org/). The protein type was set to "Homo sapiens", and the minimum interaction score was 0.4. The results obtained from STRING were imported into Cytoscape V 3.8.2 software, and the core targets of the PPI network were determined by using the centriscape computing degree centrality (DC).
Gene ontology and Kyoto encyclopedia of genes and genomes pathway enrichment analysis
Metascape was used to perform pathway enrichment and biological process annotation. Metascape perfectly makes up for the shortcomings of DAVID while retaining its advantages. The data are updated frequently to ensure timeliness and reliability. We entered the core potential targets of CRD ingredients into Metascape and selected "Homo sapiens" for enrichment analysis to examine the role of potential targets in gene function and signaling pathways.
Molecular docking
In this study, we aimed to identify the interactions between ingredients and their targets and explore their binding patterns. Therefore, we selected the ingredients that could be absorbed and 20 core targets for molecular docking verification. The core ingredient PDB format was obtained from the UniProt database, and the X-ray crystal structures were obtained from the RCSB database. The LibDock module utilizes the molecular docking function of Discovery Studio 2016 (DS) to perform ingredient–target molecular docking. Then, we drew a heatmap of the core ingredients for molecular docking using the OMIC Studio website [22].
Statistical analysis
All data are presented as the mean ± SD and were analyzed using SPSS 24.0 software (IBM Corp., Armonk, NY, USA). The data of each group were tested for normality: if the data were normally distributed, one-way ANOVA was used; if the equations were homogeneous, the LSD test was used; if the equations were not homogeneous, the Games-Howell (A) test was used; otherwise, the Kruskal‒Wallis H test method was used for analysis. Differences at p < 0.05 and p < 0.01 were considered to be significant.
