1.Establishment of animal model
After one week of adaptive feeding, aged C57 mice were randomized into two groups, namely Control and DMP (n = 10 per group). The DMP group was given 500 mg/kg DMP solution (corn oil as solvent) daily, while the Control group was given an equal dose of corn oil for 12 weeks by continuous intragastric administration. The mice were fed and watered ad libitum.
In the mechanism research, C57 mice were classified into Control, DMP, siRNA + DMP and Cel + DMP groups. In the siRNA + DMP group, siRNA-COX2 (GenePharma, Suzhou, China) was used to silence the brain tissue expression of COX2. Using microneedles, 3 nmol of siRNA-COX2 was injected into the mouse brain once every 3 d. For the inhibition of COX2, mice were intragastrically administered with Cel (celecoxib) once daily. Similarly, DMP was given by continuous intragastric administration for 12 weeks.
2.Network pharmacological analysis
The PubChem database (https://pubchem.ncbi.nlm.nih.gov) was used for obtaining the effective ingredients and their SMILES (Simplified Molecular Input Line Entry System) strings. Then, the SMILES strings were imported into the SwissTargetPrediction database (http://www.swisstargetprediction.ch) to predict the effective targets. Intersections of active drug targets and disease targets were obtained using the Venny (https://bioinfogp.cnb.csic.es/tools/venny/) software, which served as the potential key targets for the disease treatment by drugs. Relevant files were imported into the Cytoscape 3.9.0 software for network topological analysis, and the target pattern, color, transparency and size were adjusted in accordance with the Degree value (number of connections per gene), thereby constructing the "drug-target-disease" network maps. PPI correlations were derived by importing the intersecting genes via the String (https://string-db.org/) platform. The results were imported into the Cytoscape 3.9.0, and "NetworkAnalyzer" was chosen to acquire the network topology parameters. Next, the downloaded TSV file was imported into the Cytoscape to generate PPI network, and the top 10 core targets were screened in accordance with the Degree value. Utilizing Bioconductor (http://www.bioconductor.org/), an open source software for bioinformatics, the screened core targets were subjected to GO and KEGG functional enrichment analyses of biological processes, and the results were visualized via the Bioinformatics platform. The GO analysis was consisted of three parts, where the role of drug target proteins in gene function was explained from the following three aspects: Biological Process (BP), Cellular Component (CC) and Molecular Function (MF). The KEGG analysis primarily aimed to elucidate the major signaling pathway involved in the drug treatment of disease.
3. Morris water maze [10]
The water temperature of the maze was controlled at 25 ± 1 oC. Before the test, the mice were placed in the conditioning chamber for 30 min, and thus they had sufficient time to adapt to the experimental environment. One week before inducing the cognitive impairment, the mice were exposed to adaptive training once daily. During the formal test, orientation navigation and spatial probe experiments were carried out. Orientation navigation experiment: The mice were positioned in the water maze with their heads facing a wall. In case the mice boarded the central platform of the maze within 90s, the video recording ended. Otherwise, the mice were guided to swim to and board the central platform using a wooden stick. The time taken by the mice to find the platform was recorded, as well as the average speed. Spatial probe experiment: After removing the central platform, the mice were positioned into the same area facing a wall, their movement trajectory within 90s was observed, and the number of their platform crossings was recorded.
4. Enzyme-linked immunosorbent assay (ELISA)
In terms of the inflammatory cytokine assays, the brain tissues were pulverized with liquid nitrogen and lysed on ice with 1 ml of NP-40 buffer (Beyotime Biotechnology, Shanghai, China) for 30 min, followed by collection of protein supernatant. In the cellular experiment, the collected medium was cryopreserved at -80 oC, and after obtaining all the samples, they were centrifuged at 3,000 r/min for 20 min, and then assayed as per the instructions of ELISA kits (Jiancheng Bioengineering Institute, Nanjing, China). Calculation was performed by the standard curve method.
5. H&E and Nissl
Cerebral perfusion was performed after killing the mice, and their brain tissues were prepared into 4-µm serial frozen sections, which were then operated as per the instructions of H&E staining kit (Beyotime Biotechnology, Shanghai, China). The sections were stained using hematoxylin for 3 min, washed in tap water for 2 min, treated with 1% hydrochloric acid alcohol for 2s, rinsed in tap water for 2 min, and later treated with 1% ammonia water for 20s. Thereafter, the sections were stained using 0.5% eosin alcohol for 10s, dehydrated with gradient alcohol, permeabilized in xylene and sealed with neutral gum. Finally, the brain pathological changes were observed under optical microscope. During the Nissl staining, the sections were dehydrated, added dropwise with Nissl reagent (Beyotime Biotechnology, Shanghai, China), for 30 min of staining, and then washed in tap water, dehydrated with alcohol, treated with xylene, and finally observed under optical microscope.
6. Immunohistochemical staining (IHC)
The mouse brain tissues were fixed in 4% paraformaldehyde, dehydrated with sucrose solution, embedded in OCT and then sectioned. After washing in PBS, the tissue sections were blocked with 5% serum for 30 min, incubated overnight with IBA-1 monoclonal antibody (1:100 dilution in TBST, Abcam, Massachusetts, USA) at 4oC, and then washed in PBS thrice. Finally, the sections were visualized with peroxidase substrate kit (Abcam, Massachusetts, USA) and observed microscopically.
7. Western-Blotting
After grinding the tissues with liquid nitrogen, NP-40 buffer was used to extract cellular protein. The cells were washed in PBS, and then directly lysed on ice with NP-40 buffer for 30 min. The protein fluid was collected, boiled in 5x loading buffer, electrophoresed at 80–120V voltage, and then transferred onto membranes with a 300mA constant flow for 0.5–2 h. After 2 h of blockade with 5% skim milk powder, the membranes were incubated with monoclonal primary antibodies (1:500 dilutions in TBST; Abcam, Massachusetts, USA), and further with HRP-labeled goat anti-rabbit secondary antibody (Abcam, USA). After incubation, chemiluminescent immunoassay was carried out, and optical density (OD) was explored through Image Pro-Plus 6.0.
8. Cell model establishment
Mouse microglia BV2 cells (Procell Biotechnology, Wuhan, China) were cultured in complete medium at 37oC with 5% CO2 saturated humidity. The cell viability was assayed by Trypan blue staining, and the log-phase cells were collected for experiment, which were classified into Control and DMP groups. The Control group consisted of control cells, while the cells in the DMP group were treated with 20 µM of DMP. In the mechanism research, the cells were categorized into Control, DMP, siRNA-DMP and Cel-DMP groups, which were treated separately with siRNA-COX2 and Cel-COX2 for inhibiting COX2.
9. CCK-8
Microglia were inoculated into 96-well microplates, and incubated in a three-gas incubator for 4 h by supplementing each well with 100 µl of phenol-free red medium and 10 µl of CCK-8 reagent (MCE, Shanghai, China). After replacing the medium, the OD values were measured at 450 nm, the cytotoxicity was calculated, and the results were denoted as %.
10. Immunofluorescence staining (IF)
For the assay of IBA-1 expression, the microglia were washed in PBS twice, fixed in methanol, permeabilized with 0.2% Triton X-100, and incubated on a shaker with TBST-diluted monoclonal antibody (Abcam, Massachusetts, USA) at 4oC. Thereafter, the cells were sealed with 95% glycerin and observed by fluorescence microscopy.
11. Statistical methods
All measurement data were processed using SPSS 17.0 and expressed as x̅±s. After homogeneity of variance test, two groups of data were compared by two-sample t-test, while three or more groups of data were compared through one-way ANOVA. Subsequent pairwise comparisons among groups were all made by LSD method. The above test processes were all two-tailed, and the differences represented statistical significance when P < 0.05.