Animals and Ethics Statement
Adult male Sprague-Dawley rats (220±20g; No. 00010751; Experimental Animal Center, Wuhan First Hospital, Wuhan, Hubei Province, China; Laboratory animal facility license number: 00014834) were kept in the laboratory with temperature of 22±2 ℃ and humidity of 65±2%, 4 rats per cage. Animals were fed in a 12 h light/dark cycle and were given food and water at will. All operations were performed in accordance with the guidelines approved by the Animal Care Committee of Inner Mongolia People’s Hospital. We made every effort to relieve pain and discomfort.
Surgery procedure
The animals were weighed accurately and anesthetized by intraperitoneal injection of 10% chloral hydrate according to 0.3ml/100g. After intraperitoneal injection of chloral hydrate for 3-5 minutes, the righting reflex disappeared in animals. The animals were fixed on the operating table in supine position to ensure smooth breathing and disappeared with normal neck skin. The skin and subcutaneous tissue were cut through the middle of the neck, then the digastric muscle and sternocleidomastoid muscle were separated bluntly by tweezers. The carotid sheath was exposed. The bilateral common carotid artery and vagus nerve were separated carefully by glass needle, and the operation line 0 was inserted under the common carotid artery for reserve. Bilateral silk thread was drawn and bilateral common carotid artery of rats was clamped with non-invasive arterioles to block blood flow for 10 minutes, then removed and restored perfusion for 10 minutes for twice. Finally, the skin was sutured and the surgical site was disinfected. The gentamicin sulfate (0.2 ml) was injected intraperitoneally for anti-inflammation.
Groups
Animals were randomized into three groups (N=12 rats/group): (1) sham-operate control, SC, (2) the VD samples, VD, (3) the VD rats treated with NGF, VDN. A total 9 samples (n=3/each group) were performed the quantitative proteomics analysis and western bolt detection.
Preparation and detection of pathological specimens
Fresh brain tissue was fixed in 4 % paraformaldehyde and sectioned routinely. Mitochondria in hippocampus were labeled by Mito-Tracker GreenFM490/560, and morphological structure of mitochondria was observed by confocal laser microscopy.
2.5 Mitochondrial preparation
The isolated hippocampus was homogenated with glass homogenizer in the medium with ice residue. The mitochondria were extracted by differential centrifugation and gradient density centrifugation 10 times. The supernatant was centrifuged at 2000g for 3 minutes and repeated once; the supernatant was centrifuged at 12500g for 8 minutes and the supernatant was discarded; the precipitate was dissolved in 0.8ml 3% Ficoll medium and carefully spread on 3.2ml 6% Ficoll medium and centrifuged at 11050g for 30 minutes and the supernatant was discarded; the precipitate was suspended in the separation medium and centrifuged at 12500g for 8 minutes; finally, the precipitate was suspended with an appropriate amount of separation medium and the protein concentration was adjusted to a certain level About 10-20mg / ml. The protein concentration was determined by Lowry ` s method with bovine serum albumin as the standard protein. Fresh mitochondrial suspension was taken to measure respiratory activity. All the above operations were completed at 0-4 ℃.
Hippocampal neuron staining
The hippocampal neuron of the sham-operate control, VD and VD rats treated with NGF groups were taken for 5 mm and fixed with 10 % neutral buffer at 4 ℃, then embedded with conventional wax blocks. Slice thickness was 5um. The slices were immersed in dimethylbenzene I, II and III of 90%, 80% and 70% concentration for 5 minutes. The slices were then immersed in 5g Nissl solution and soaked at 37 ℃ for 20 minutes. After incubation, the slices were washed with distilled water and then separated with 95% ethanol. Microscopically, there are obvious hippocampal neurons. The slices were dehydrated with anhydrous ethanol, transparent with xylene, and fixed with neutral rubber. Image analysis was carried out by using Image-Pro plus 4.5 software of Silver Spring Company in the United States to determine the cumulative integral optical density (IOD sum) and area value (area sum) of the positive product and calculate the average density (IOD / area)[14].
Extraction of total protein from mitochondrial cells in hippocampus
Total mitochondrial cell protein extraction and TMT quantitative proteomic experiment in hippocampus were commissioned by Majorbio Company in Shanghai. The instrument used was Triple TOF 5600 (AB Sciex, USA), and the analysis software was Proteinpilot (Applied Biosystems MDS Sciex, USA), Q-Exactive, EASY-nLC 1200, Orbitrap Fusion Tribrid (Thermo Fisher, USA) and Proteome Discoverer (Thermo Fisher, USA).
Total mitochondrial cell protein extraction
Samples were taken out under freezing condition and frozen on ice. The samples were added 10% SDS (Final concentration 1 %) and protease inhibitor PMSF (7.5 mM) to lyses for 30 minutes, and mixed every 5 minutes for 10 seconds. Protein supernatant was centrifuged at 12000g for 8 mins. BCA quantitative analysis was used, and then detected by SDS-PAGE. Protein was measured using the BCA method.
TMT Sample Preparation
Take 100 mg of above protein sample and supplement the volume with lysate to 100 µl. The final concentration of 10 mM TCEP was added and the reaction time was 60 min at 37 ℃. Add the iodoacetamide (40 mM) at room temperature for 40 minutes. Pre-cooled acetone (6:1 w/w) was added to each tube, and precipitated at -20℃ for 4 hours. The Protein samples were centrifuged at 10000g for 20 mins and then the precipitations were collected. The precipitations were dissolved with 100 µl TEAB (100 mM). Finally, the trypsin solution (1:50 w/w) was added for enzymatic hydrolysis at 37 ℃ overnight.
The above samples were labeled using an TMT reagent 10-plex protein quantitation kit (Thermo Fisher, USA) as follows: The sham-operate control was labeled with TMT10-130N, TMT10-130C and TMT10-131, and the VD samples were labeled with TMT10-128C, TMT10-129N and TMT10-129C, respectively; the VD rats treated with NGF samples were labeled with TMT10-126, TMT10-127N and TMT10-128N, respectively.
High pH Reverse Phase Separation and Nano-LC−MS/MS Analysis
The Sep-Pack columns were used for desalination of TMT-labeled peptide mixtures. The high-pH reversed-phase column peptide fractionation kit (Thermo Fisher, USA) was used to classify the peptide fractions. A total of 10 distillates were charged according to the time. The distillates were concentrated by vacuum centrifugation and dissolved in a mass spectrometry sample buffer for the second dimensional analysis. Mass spectrometry parameters were set as follows: ion spray voltage of the inlet, 2.3 kV; mass spectra range, 350-1300 m/z; accumulation time per spectrum, 100 ms; and dynamic exclusion time, 18 s [15].
Proteomic Data Analysis and Bioinformatics
Proteomic data analysis and bioinformatics methods were described previously [15]. The Proteome Discoverer Software 2.1 (Thermo Fisher, USA), Protein pilot software (Applied Biosystems MDS Sciex) were used. All reported data were based on 95% confidence interval for protein and peptide identification as determined by Protein Pilot (Prot Score ≥1.3) [15]. Meanwhile, a cut off of 1% FDR (false discovery rate) and 40% isolation interference for peptide spectrum matches (PSMs) were required for all reported proteins. The Gene Ontology (GO) annotation was using the database:(http://www.uniprot.org/uniprot/?query=organism:10116).
Verification of Differentially Expressed Proteins Using Western Blotting
Western blot was used to verify the quantitative proteomic results of TMT. The test method is described previously [16]. All the antibodies were diluted with 1:1000, and the secondary antibodies were diluted with 1:10000. The proteins were quantified by Image-J software. Each Western blot strip was measured three times and the average optical density was calculated.
Statistical Analysis
The data were expressed as mean ± SD. A Student’ s t test in R language was used to calculate the P value of significant difference between samples. In this project, the screening criteria for significant difference expression proteins were: P < 0.05 & (FC<0.83 or FC>1.20).