2.1 Chemicals, reagents, and materials
Z. bungeanum Maxim. (Catalog No. 20180802) was purchased from Hehuachi tradition herbal medicine market (Chengdu, China). D-galactose (D-gal, Catalog No. 2018070901) was provided by Cologne Chemical Co., Ltd (Chengdu, China). Lipopolysaccharides (LPS, Catalog No. 032M4082V) was purchased from Sigma-Aldrich (St. Louis, USA). Adenosine 5′-triphosphate disodium salt (ATP, Catalog No. S0714A) was purchased from Meilun Biotechnology Co., Ltd (Dalian, China). Hydergine (Catalog No. 6G862T) was provided by Huajin Pharmaceutical Co., Ltd (Tianjin, China). Baicalin (Catalog No. 20170319) was obtained from Biological Engineering Co., Ltd (Xi’an, China). The primary antibodies including anti-caspase-1, anti-GSDMD, and anti-NLRP3 (Catalog No. 36458S, 67314S, 15101S) were from Cell Signaling Technology (Beverly, USA); anti-IL-1β (Catalog No. 510681) was from Zhengneng Biotechnology Co. LTD (Chengdu, China); anti-IL-18 (Catalog No. GR3285548-4) was from Abcam (Cambridge, UK); anti-ionized calcium binding adapter molecule 1 (anti-Iba1), anti-Mac-1, alphaM integrin, Cr3, MO-1, C3bi Receptor (anti- CD11b ) (Catalog No. #18m3752, #DF2911) were from Affinity Biosciences, Inc (Cincinnati, OH, USA). Superoxide dismutase kit (SOD, Catalog No. 20180605), malondialdehyde kit (MDA, Catalog No. 20180611) were obtained from Jiancheng Bioengineering Institute (Nanjing, China). Tunel kit (Catalog No. XZ201902) was from Seville Biotechnology Co., Ltd (Wuhan, China). Cell culture medium, double antibody and serum were purchased from Gibco (Grand Island, NY). All organic reagents were purchased from Kelong Chemical Co., Ltd (Chengdu, China).
2.2 Preparation of extraction
Solvents of four different polarities solvents (petroleum ether, dichloromethane, ethyl acetate and n-butanol) were used to fractionate Z. bungeanum extracts. The dried Z. bungeanum (500 g) was soaked in 5000 mL 95% ethanol for 24h and then filtered it. We repeated this operation one more time, and transferred the filtrate to a flask for evaporation. After the liquid was concentrated to 400 ml, we used petroleum ether, dichloromethane, ethyl acetate and n-butanol were used for extraction twice in turn. These filtrates were concentrated by rotary evaporator. Finally, each extract was collected in a screw tube and kept them in a vacuum desiccator for 3–5 days to completely remove each organic solvent. The extracts were away from light stored at 4 °C until use. We referred to the fraction with petroleum ether as PE, the fraction with dichloromethane, as DCM, the fraction with ethyl acetate, as EA, and the fraction with n-butanol, as N-BAI.
2.3 Animals
Male Kunming mice (8 weeks old) were purchased from the Dashuo Experimental Animal Co., Ltd and housed under suitable conditions of temperature (22 ± 2℃) and humidity (65 ± 5%) in the animal observation room of Chengdu University of Traditional Chinese Medicine with free access to water and food. All animal experiments were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the Institute of Material Medica Integration and Transformation for Brain Disorders ethics committee approved the animal protocols. Mice were randomly divided into seven groups (n=6 in each group): solvent plus saline (control group), solvent plus D-gal (aging group), 0.9 mg/kg Hydergine plus D-gal (HG group), PE plus D-gal (PE group), DCM plus D-gal (DCM group), EA plus D-gal (EA group), and N-BAI plus D-gal (N-BAI group). Drugs were dissolved in 0.5% CMC-Na for administration, and the dose of each extract was equal to 450 mg/Kg of raw material. After oral administration of the for 1 h, 500 mg/kg D-gal was subcutaneously injected once a day for 42 days. After that, all the animals were executed for further study.
2.4 Behavioral evaluation
The protocol followed the sequence of passive avoidance test and Morris water maze test. All tests were performed in conditions of dim light and low noise. The passive avoidance test was performed as previously described [18]. The test was performed daily for 2 consecutive days, and the first day was the training trial, and the second day was the test trial. Mice were individually placed into the apparatus and permitted free exploration for 5 min. The mice were individually placed in a lit room and allowed free access to the corresponding dark room when a trap door was opened. The latency time taken to pass through the trap door was recorded as " Escape latency ", and the number of times the mice passed through the trap door was counted as " The number of errors ". If the mouse did not pass through the trap door within 300 s, a latency of 300 s was recorded.
The Morris water maze test was conducted based on earlier methodology [18]. In the orientation navigation experiment, the mice were placed in the opposite quadrant of the platform and allowed to swim until it found the hidden platform within 60 s. The time of finding the platform was recorded as" Escape latency ". If the mice could not find the platform within 60 s, it was gently guided to the platform and kept there 10 s, and the was recorded as 60 s. The continuous period of the hidden platform experiment was 5 days. Next the spatial probe trial was performed by removing the platform on the sixth day. The swimming time in target quadrant and the number of crossing the platform were recorded in the spatial probe trial.
2.5 Hematoxylin and eosin staining
For hematoxylin and eosin (HE) staining, four mice brain samples in each group were collected and postfixed in 4% paraformaldehyde. The hemisphere of each mouse was embedded in paraffin and cut into 4 μm slices, dewaxed, which were then stained successively with hematoxylin and eosin to observe lesions in the hippocampus. And only large intact cells with clearly stained cytoplasm and a distinct nucleus, usually with evident nucleoli, were counted as healthy neurons [19].
2.6 Immunohistochemistry
Immunohistochemistry of the paraffin blocks were performed as previously described [20]. The brain slices were dewaxed, rehydrated, blocked, incubated with primary antibodies (NeuN,1:500, Iba-1,1:200) overnight for 4°C, incubated with secondary antibody and colored with a DAB kit.
2.7 Measurement of the activity of SOD and the content of MDA
The activity of SOD and the content of MDA were determined strictly according to the kit instructions.
2.8 Tunel staining
The paraffin blocks were cut into 4 μm slices, dewaxed, repaired with proteinase K working solution, disrupted cytomembrane, incubated with TdT and FITC-12-dUTP for 1 h. After rinsing with phosphate-buffered solution (PBS), the nuclei were stained with DAPI, then anti-fluorescence quenching agent was added. Then the samples were observed immediately under fluorescence microscope after sealed with neutral balsam on slides. Pay attention to avoid light in the experiment.
2.9 Cell culture and Cell viability measurements
BV-2 microglial cells were purchased from Chinese Academy of Sciences (shanghai, China). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum and 1% streptomycin/penicillin, maintained at 37°C under 5% CO2. Cells in the exponential growth phase were used for all experiments. Cells were plated into 96-well plates at a density of 1×104 or plated into 6-well plates at 1×10 5 densities. After culturing for 24h, drugs were first added for 1h, then 100 ng/ml LPS was added for 12 h, and finally, ATP (2.5 mM) was added for 3h. After the cells were treated as described above, 10μl of MTT solution was added to each well. Finally, after the plates were incubated in the incubator for 4h, remove the supernatant, add dimethyl sulfoxide, incubate in a shaker, and absorbance was measured at 490 nm. Cell experiments were independently performed at least three times.
2.10 Western blotting
Tissues and collected cells were lysed with lysate, centrifuged, collected supernatant, added with sample buffer, and stored at -80℃. SDS-PAGE was used to isolate proteins, then the protein was transferred to PVDF membrane,blocked with 5 % skimmed milk, incubated with primary antibodies (cleaved GasderminD,1:1000; Cleaved Caspase-1,1:1000; NLRP3, 1:500; IL-1β, 1:1000; IL-18, 1:1000; GAPDH, 1:5000) overnight at 4℃,and the second antibody were incubated the next day. Finally, the relative expression of the target protein was analyzed by the Quantity One software.
2.11 Sample preparation and Mass spectrometry analysis
EA fraction was extracted with methanol solution by ultrasonic extraction, and filtered through a 0.22 μm micropore film to yield the sample solution for HPLC-MS/MS. The sample was analyzed by an Agilent Technologies 1290 LC and an Agilent Technologies 6410 triple quadrupole mass spectrometer (Agilent Technologies, CA, USA). And the chromatographic separation was performed on a poroshell 120 EC-C18 column (100 × 4.6 mm, 2.7 μm, Agilent Technologies, CA, USA) at 29°C. The mobile phase consisted of H2O (containing 0.05% formic acid, A) and acetonitrile (B) in the gradient elution program: 0 ⟶ 3 min, 5% ⟶ 18% B; 3 ⟶ 10 min, 18% B; 10⟶ 20 min, 18% ⟶ 63% B. The flow rate was 0.3 mL/min, and the sample injection volume was 10 μL. Mass spectrometric scan was obtained by electrospray ionization (ESI) in positive-ion mode with a scanning interval m/z 100–1300. The main parameters for MS were set as follows: gas temperature, 300 °C; gas flow, 11L·min–1; nebulizer, 35 psig; capillary voltage, 4000 V; and atomizer pressure 15 psi (1 psi = 6.895 Kpa).
2.12 Statistical analysis
All dates are represented as the mean ± standard error of the mean (SEM). The escape latency in the Morris water maze test navigation phase was analyzed by two-way analysis of variance (ANOVA). For other data, date with a normal distribution was analyzed by one-way ANOVA. Student’s test was performed for comparisons between two groups. And Bonferroni post hoc analysis was conducted, and the statistical significance was tested at P < 0.05.