Protein kinase inhibitor was purchased from Calbiochem; trypsin was purchased from Promega; acetonitrile, Ultra-pure water (H2O) from Fisher Chemical; trifluoroacetic acid , formic acid , Fluka, iodoacetamide, dithiothreitol , urea , Triethylammonium bicarbonate（TEAB）from Sigma. Zhishi Rhubarb Soup (ZRS), Chinese herbal regimen was Purchased from the Department of Pharmacy, Nanjing University of Traditional Chinese Medicine Affiliated to Nanjing University of Traditional Chinese Medicine, decocted according to the conventional method, concentrated to 2.5g/ml and stored at 4℃ for later use. Zhishi Rhubarb Soup is consisted of cooked rhubarb 10g, citrus aurantium 10g, magnolia root 10g, scutellaria 10g, woody 3g, licorice 3g.
All animal procedures and protocols were performed in accordance with The Guide for the Care and Use of Laboratory Animals (NIH publication, 85–23, revised 1996) and were reviewed and approved by the Animal Research Committee at National Research Institute of Chinese Medicine. IACUC protocol no: P-99-11; IACUC Approval No: A-99-1. All surgery was performed under anesthesia, and all efforts were made to minimize suffering.
The animal Model building:
The MCAO (middle cerebral artery occlusion) model was built using the thread bolt method24. The method refers to the modified Longa suture method. Briefly, the experimental animals were anesthetized with 10% chloral hydrate (0.3ml/100g) in the abdominal cavity, fixed on the operating table supine (the rectal temperature was controlled at 37.3±0.5℃), and the common carotid artery and vagus nerve were quickly exposed, separated and the proximal end of the common carotid artery and the external carotid artery were connected, thread the internal carotid artery for use, and cut a small opening at the upper end of the common carotid artery ligation from the bifurcation of the common carotid artery, Insert the prepared fishing line into the internal carotid artery along the common carotid artery, and ligate the internal carotid artery when it reaches the specified length (about 18 mm). The tether of different diameters was chosen according to the animal's weight and nutrition, then the incision sutured. After the operation, the body temperature was maintained at 37±0.5℃ with an irradiation lamp, and the rectal temperature, respiration and heart rate were monitored. The animal was kept in a cage until awake for further use.
Nerve function score: After the animal is awake for 2 hours, the rat's behavior and neurological symptoms were observed, and a score was given. According to Longa's 5-level standard scoring method: 0: normal, without any neurological deficits; 1: The front paw cannot be straight when lifted vertically; 2: Lean to the right and rotate to the right when walking; 3: The body falls to the right side while walking; 4: not walk spontaneously or have a consciousness disorder. According to the first score, animals with no neurological deficit, 4 points, dyspnea, early death, and subarachnoid hemorrhage found at the time of execution were discarded. Animals excluded from the group will be supplemented in subsequent experiments.
Animal grouping and administration
Group A (sham group): normal feeding, free drinking water.
Group B (MCAO group): Normally reared after modeling, with free drinking water.
Group C (ZRS group) (effective dose was screened and set at 10g crude drug/kg): start gavage 3h after model building, once a day for 7 days. And the hippocampus tissue was collected after treatment at day 7.
Protein extraction and trypsin treatment
An appropriate amount of tissue（rat hippocampus）was weighed into a mortar pre-cooled with liquid nitrogen, and liquid nitrogen is added to fully grind the tissue to powder. Then samples of each group were added with 4 times the volume of powdered lysis buffer (8 M urea, 1% protease inhibitor, 3 μM TSA, 50 mM NAM and 2 mM EDTA) and lysed by ultrasound. The cell debris is removed after centrifuge, the supernatant was transferred to a new centrifuge tube, and the protein concentration is determined using the BCA kit.
Dithiothreitol was added to the protein supernatant to a final concentration of 5 mM, and reduced at 56 °C for 30 min. Then iodoacetamide was added to make the final concentration 11 mM, and supernatant was then incubated for 15 min at room temperature in the dark. The urea concentration of the sample is diluted to less than 2 M. pancreatin was added at a mass ratio of 1:50 (pancreatin: protein), and protein was digested overnight at 37°C. Finally, the protein was subject to second enzymatic hydrolysis for 4h after with pancreatin added at a mass ratio of 1:100 (pancreatin: protein).
The peptides digested by trypsin were desalted with Strata X C18 (Phenomenex), freeze-dried in vacuo, and then dissolved with 0.5 M TEAB and labelled according to the TMT kit operating instructions. briefly: thaw the labeling reagent and dissolve it with acetonitrile, mix it with the peptide and incubate at room temperature for 2 hours, mix the labeled peptide, remove the salt, and freeze-dry in vacuum.
The peptides were fractionated by high pH reverse HPLC, and the column was Agilent 300Extend C18 (5μm particle size, 4.6 mm inner diameter, 250 mm length). The peptides were subject to a step gradient of 8%-32% acetonitrile, pH 9, and 60 components are separated in 60 minutes, and then the peptides are combined into 9 components, and the combined components are vacuum freeze-dried for subsequent operations.
The peptides were dissolved in the mobile phase A of liquid chromatography (0.1% (v/v) formic acid aqueous solution) and separated using the EASY-nLC 1000 ultra-high-performance liquid-system. Mobile phase A is an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B is an aqueous solution containing 0.1% formic acid and 90% acetonitrile. Liquid gradient setting: 0-30 min, 12%~26%B; 30-52 min, 26%~40%B; 52-56 min, 40%~80%B; 56-60 min, 80%B. The flow rate was maintained at 320 nL/min.
The peptides are separated by the ultra-high-performance liquid system and injected into the NSI ion source for ionization and then analyzed by Orbitrap Fusion Lumos mass spectrometry. The ion source voltage is set to 2.0 kV, and the peptide precursor ions and their secondary fragments are detected and analyzed by high-resolution Orbitrap. The scanning range of the primary mass spectrum is set to 350-1550 m/z, and the scanning resolution is set to 60,000; the scanning range of the secondary mass spectrum is set to a fixed starting point of 100 m/z, and the secondary scanning resolution is set to 15,000. The data acquisition mode uses the data-dependent scanning (DDA) program, that is, the first 20 peptide precursor ions with the highest signal intensity are selected to enter the HCD collision cell and use 32% fragmentation energy for fragmentation after the first scan. Grade mass spectrometry analysis. In order to improve the effective utilization of the mass spectrometer, the automatic gain control (AGC) is set to 5E4, the signal threshold is set to 50000 ions/s, the maximum injection time is set to 70 ms, and the dynamic rejection time of the tandem mass spectrometry scan is set to 30 seconds to avoid precursor ions.
The secondary mass spectrum data was searched using Maxquant (v126.96.36.199). Search parameter settings: The database is Rattus_Uniprot_10116 (29955 sequences), an anti-database is added to calculate the false positive rate (FDR) caused by random matching, and a common contamination library is added to the database to eliminate the contaminating protein in the identification results Impact; set the restriction enzyme digestion method to Trypsin/P; set the number of missed cleavage sites to 2; set the minimum peptide length to 7 amino acid residues; set the maximum modification number of peptides to 5; first search and main search primary precursor ion The mass error tolerance is set to 20 ppm and 5 ppm, respectively, and the mass error tolerance of the secondary fragment ion is 0.02 Da. The cysteine alkylation is set as a fixed modification, and the variable modification is the oxidation of methionine, the acetylation of the N-terminus of the protein, and the deamidation (NQ). The quantitative method is set to TMT-6plex, and the FDR for protein identification and PSM identification is set to 1%.
Twenty micrograms of protein/well was loaded onto 10% gels for separation using sodium dodecyl sulfate-polyacrylamide gels electrophoresis (SDS-PAGE). The gels were electrophoretically transferred onto polyvinylidene fluoride (PVDF) membranes (0.45 or 0.20 μm pore size; Millipore, Billerica, MA, USA). The blotted membranes were blocked with 5% nonfat dry milk in a Tris-buffered saline solution (25 mM Tris, pH 7.5, and 150 mM NaCl) containing 0.05% Tween 20 (TBST) for 2 h at room temperature, followed by incubation with the diluted primary antibody against target protein for 4 h at room temperature. After washing for 10 min in TBST solution, the membranes were incubated with properly diluted secondary antibody conjugated with horseradish peroxidase for 2 h at room temperature. Western signals were developed using ECL chemiluminescent reagents from Thermo Scientific (Waltham, MA, USA). The β-actin levels were used as loading controls.
The two-sample two-tailed The T test method were employed to calculate the p-value. When p-value<0.05, the change of differential expression exceeding 1.2 is used as the change threshold for significant up-regulation, and the change threshold for significant down-regulation is less than 1/1.2. For biological or technical replicate samples, we use principal component analysis (PCA), relative standard deviation (RSD) and Pearson’s Correlation Coefficient to evaluate protein quantitative repeatability.