2.1 Materials and reagents
Acetonitrile, Methanol, pyridine, n-hexane, methoxylamine hydrochloride, and BSTFA were purchased from CNW Technologies GmbH (Düsseldorf, Germany), L-2-chlorophenylalanine was from Shanghai Hengchuang Biotechnology Co., Ltd. (Shanghai, China). Atorvastatin Calcium Tablets was purchased from Beijing JIALIN pharmaceutical Co., Ltd (Beijing, China). Herbs of SHXZF (Table 1) were purchased from the Xiyuan Hospital, China Academy of Chinese Medical Sciences (Beijing, China).
2.2 Animal handling
Eight-week male ApoE−/− mice and C57BL/6L mice were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd (Beijing, China). 10 C57BL/6L mice were set to blank group and fed with normal diet. 30 ApoE−/− mice were fed with a standard western diet (containing 20% protein, 50% carbohydrate and 21% fat, Beijing HFK bioscience Co., Ltd) to establish AS model. Then they were randomly divided into three groups: model group, Atorvastatin control group and SHXZF-treated group. The mice were fed adaptively for 3 days prior to the experiment. MIce in SHXZF-treated group were administrated with SHXZF once a day in 0.1 mL /10g body weight for 8 weeks and blank group were given equal quantities of water. Mice in atorvastatin group were administrated with atorvastatin once a day in 2 mg /kg body weight.
2.3 Oil red O staining section method and Pulse wave velocity (PWV) measurement
2.3.1 Oil red O staining section
The fixed aortic root tissues was dehydrated with 10%, 20%, and 30% sucrose solutions. Frozen sections (6μm slice) were cut using a cryostat. Then soak the slices in 60% different propanol for 10 minutes, and dye with oil red staining solution for 10 minutes. Put the slices in 60% isopropanol to differentiate for 10 seconds, rinse with water after clarification, and mount a glass slide containing glycerin gelatin.
2.3.2 PWV measurement
The propagation time of the pulse wave from the aortic arch to the abdominal aorta is measured by the time delay between the upstroke (foot) of each pressure peak..Distance between the two measurement points is determined. Then divide the distance by the pulse wave travel time (in meters per second) to calculate the PWV. The travel time were taken by averaging at least 10 normal continuous cardiac cycles.
2.4 Collection and preparation of biosamples
80 μL of each serum sample was added to a 1.5-mL Eppendorf tube with 10 μL of 2-chloro-l-phenylalanine (0.3 mg/mL) dissolved in methanol as internal standard, and the tube was vortexed for 10s. Subsequently, 240 μL of ice-cold mixture of methanol and acetonitrile (2/1, v/v) was added, vortexed for 1 min, ultrasonicated for 5 min, stored at -20 ℃ for 10 min, centrifuged at 12000 rpm, 4 ℃ for 10 min. An aliquot of the 150 μL supernatant was taken for vacuum-dry at room temperature. Then added 80 μL of 15 mg/mL methoxylamine hydrochloride in pyridine. The resultant mixture was vortexed vigorously for 2 min and incubated at 37 ℃ for 90 min. 80 μL of BSTFA (with 1% TMCS) and 20 μL n-hexane was added, then vortexed vigorously for 2 min and derivatized at 70 ℃ for 60 min.
2.5 Metabolic profiling platform
2.5.1 Chromatography and mass spectrometry conditions
Metabolic profiling analysis was performed using an GC-MS system with Agilent 7890B gas chromatography system and Agilent 5977A MSD system (Agilent Technologies Inc., CA, USA). A DB-5MS fused-silica capillary column (30 m × 0.25 mm × 0.25 μm, Agilent J & W Scientific, Folsom, CA, USA) was utilized to separate the derivatives. Helium (> 99.999%) was used as the carrier gas with a flow rate of 1 mL / min. The injector temperature was 260 ℃. Injection volume was 1 μL by splitless mode. The initial oven temperature was 60 ℃, ramped to 125 ℃ at a rate of 8 ℃/min, to 210 ℃ at a rate of 5 ℃/min,to 270 ℃ at a rate of 10 ℃/min, to 305 ℃ at a rate of 20 ℃/min, and finally held at 305 ℃ for 5 min. The temperature of MS quadrupole and ion source was 150 and 230 ℃, respectively. The collision energy was 70 eV.
2.5.2 Data processing
MD-DIAL software were used for data processing. Metabolites were annotated through LUG database (Untarget database of GC-MS from Lumingbio).
2.5.3 Data analysis and biomarker identification
Principle component analysis (PCA) an orthogonal partial least-squares-discriminant analysis ( OPLS-DA ) were performed to visualize the metabolic difference among experimental groups, after mean centering and unit variance scaling. The Hotelling’s T2 region, shown as an ellipse in score plots of the models, defines the 95% confidence interval of the modeled variation. Variable importance in the projection (VIP) ranks the overall contribution of each variable to the OPLS-DA model, and those variables with VIP > 1 are considered relevant for group discrimination. The differential metabolites were selected on the basis of the combination of a statistically significant threshold of VIP values obtained from the OPLS- DA model and p values from a two-tailed Student’s t-test on the normalized peak areas from different groups, where metabolites with VIP values larger than 1.0 and p values less than 0.05 were considered as potential biomarkers.
2.6 TMT-based proteomics analysis
2.6.1 Sample preparation and protein extraction
Frozen samples of aortic arch tissue were transferred into low protein binding tubes (1.5ml Eppendorf) and lysed with 300 µL lysis buffer supplemented with 1mM PMSF. Then the samples were further lysed with sonication. (80W, 3min). After sonication, the samples were centrifuged at 12000g for 10min to collect supernatants. Protein concentration was determined by BCA assay and aliquoted to store at -80°C.
2.6.2 Protein digestion and TMT labeling
Add 25mm DTT of corresponding volume into the protein solution. Then add the corresponding volume of iodoacetamide so that the final concentration is about 10mm. Subsequently, 6 times of the volume of precooled acetone in the above system to precipitate the protein. According to the amount of protein, add the corresponding volume of enzymolysis diluent (protein: enzyme = 50:1 (m/m), 100ug of protein add 2ug of enzyme) to redissolve the protein precipitate. For TMT labelling, 88µL acetonitrile were added to TMT reagent vial. The centrifuged reagents were dissolved for 5 min and mixed for centrifugation for 2 times. Then 41 μL of the TMT label reagent was added to each sample . Then incubated at room temperature for 1 h. After that, 8 µL of 5% hydroxylamine were added and incubated for 15 min. The labeling peptides solutions were lyophilized and stored at -80°C.
2.6.3 Liquid chromatography-mass spectrometry
All analyses were performed by a QE mass spectrometer (Thermo, USA) equipped with an Easyspray source (Thermo, USA). Samples were loaded by a capillary trap column (100μm×2cm, RP-C18, Thermo Fisher) and then separated by a capillary analytical column (15 cm × 75 µm, RP-C18, Thermo Fisher) on an EASY-nLCTM1200 system (Thermo, USA). The flow rate was 300 nL/min. The linear gradient was set as follow: 0~40 min,5-30% B;40~55 min,30-50% B;55~60min,50-100% B. Full MS scans were acquired in the mass range of 300-1600 m/z with a mass resolution of 70000 and the AGC target value was set at 1e6. The 10 most intense peaks in MS were fragmented with higher-energy collisional dissociation (HCD) with collision energy of 28. MS/MS spectra were obtained with a resolution of 35000 with an AGC target of 2e5 and a max injection time of 80 ms. The dynamic exclusion was set for 15.0 s and run under positive mode.
2.6.4 Protein identification and quantitative analysis
The data were analyzed by Proteome Discoverer software 2.4 (Thermo Fisher Scientific, San Jose, CA, USA) and a database including 36,508 sequences (uniprot-reviewed-Mus musculus-2020_08). The search parameters were as follows: enzyme: trypsin; two missed cleavages allowed; fragment mass tolerance, 0.02 Da; mass values: monoisotopic; dynamic modification: Oxidation(M), Acetyl(N-term); peptide mass tolerance: 20 ppm; and fixed modifications: carbamidomethyl of cysteine. In addition, quality control was performed to determine if a reanalysis step was needed. The false discovery rate for both protein and peptide identification was set to be less than 0.01. Fold changes > 1.5 or < 0.75 and p < 0.05 were set as cut-off values to designate significant protein expression changes.
2.6.5 Pathway enrichment and interaction network analysis
Functional analysis of these genes expressed differentially was performed by DAVID (https://david.ncifcrf.gov).. Kyoto Encyclopedia of Genes and Genomes (KEGG) was utilized to analyze canonical pathways involved for those differentially expressed proteins.The protein–protein interaction network (PPI) was constructed using the online STRING database (https://string-db.org).
2.7 Statistical analysis
The experimental results were presented as mean ± SD and were compared using an 2-sample Student’s t-test or a one-way ANOVA. Statistical difference was set at p value < 0.05.