Animals, experimental design, experimental grouping
4-month-old male Sprague-Dawley rats were purchased from Changsha Tianqin Biotechnology Co., Ltd. Besides, all the experiments were approved by the Experimental Animal Committee of Zunyi Medical University and in accordance with the guidelines for the nursing and use of experimental animals of the National Research Committee. Four rats in each cage were randomly fed under 12-hour light / dark cycle (23–25°C) to provide enough food and water. In addition, the rats were randomly divided into groups and experiments in an unbiased, double-blind manner.
D-galactose (Solarbio, D8310, Beijing, China) was diluted with normal saline into a liquid with a concentration of 125mg/kg and continuously injected subcutaneously into the neck and back of rats for 42 days to establish an aging rat model. The rats in the experimental group were placed in a thermostatically sealed anesthetic box after starving for 12 hours, which was connected to an anesthetic machine (Drager, Julian, Germany) and an exhaust absorption device. The rats were induced with 3:1 pure oxygen and air mixed with 6% sevoflurane (Lunambett Pharmaceuticals Co., Ltd, Shandong, China), and maintained with 3:1 pure oxygen and air mixed with 3.2% sevoflurane for 6 hours. The anesthetic gas monitor (Drager Medical GmbH, Canada) was continuously monitored during anesthesia to prevent the retention of CO2 and deep anesthesia. The rats in the control group were treated with 3:1 pure oxygen and air mixture for 6 hours, and the rats in the positive control group were intraperitoneally injected with MCC950 (MCE, HY-12815A, New Jersey, USA) for 30 minutes, and then inhaled with 3:1 pure oxygen and air for 6 hours. 30 minutes after intraperitoneal injection of MCC950, the rats in the treatment group were mixed with pure oxygen and air at 3:1 and 3.2% sevoflurane for 6 hours, and the fixed concentration of MCC950 was 10mg/kg.
Rats were randomly divided into four groups in experiment 1 with 15 rats in each group: (1) Ctrl, blank group; (2) MCC950, MCC950 negative control group; (3) Sev, sevoflurane group; (4) MCC950 + Sev, MCC950 pretreatment group; In experiment 2, rats were divided into three groups: (1) Ctrl, blank group; (2) 3-MA, 3 methyladenine group; (3) Sev, sevoflurane group; (4) Rapa, rapamycin group.
Morris water maze test (MWM)
The water maze device consists of a round black water tank (120 cm in diameter and 60 cm in depth). In addition, the water maze was divided into four quadrants, one of which was selected to place a 15 cm diameter escape platform 1 cm below the surface of the water. 24 hours after D-gal injection, the rats in each group continued to carry out the positioning navigation experiment for 5 days. The specific operation was that the rats were put into the water facing the pelvic wall from a fixed point in each quadrant, the time it took for each rat to reach the escape platform was observed, recorded and the rats were guided to the platform for 30 seconds if they did not find the platform within 120 seconds. In addition, the experiment in the next quadrant was carried on.
The spatial exploration test was carried out 24 hours after the intervention of sevoflurane. The specific operation was to withdraw the platform from the original quadrant. In addition, the rats entered the water from any quadrant facing the pelvic wall. The percentage and times of rats crossing the original platform quadrant within 120 seconds, and the total distance and speed of swimming in the water were observed and recorded.
The specific operation of the working memory test was to put the platform into the diagonal quadrant of the original platform quadrant and put the rats down in any quadrant. In addition, the operators observed and recorded the escape platform time again after the rat escape platform rest for 30 seconds, and the second time was recorded as the escape latency time.
The above test results were recorded by a video tracking system (Taimeng Technology Co., Ltd, Chengdu, China).
Nissl staining
The prefrontal cortex of rats was fixed with 4% paraformaldehyde, dehydrated and embedded in paraffin for sections. Besides, the slices were dewaxed in xylene, hydrated with gradient ethanol, washed with pure water and dripped with Nissl staining solution (Solarbio, G1436, Beijing, China) to evaluate the neuronal damage of the prefrontal cortex. Finally, Nissl staining was observed by microscope (Olympus Corporation, BX63, Tokyo, Japan).
Immunohistochemical staining
The prefrontal cortex of the rat brain was fixed with 4% paraformaldehyde, embedded in paraffin, cut into 4–5µm thick sections, dewaxed with xylene, gradient alcohol dehydration, Iba-1 antibody (1:100, Abcam, ab178846, Cambridge, UK) were incubated overnight in refrigerator at 4 ℃, and then incubated with biotin-labeled second antibody (Abcam, ab205718, Cambridge, UK) for 40 minutes. Moreover, the operators conducted diaminobenzidine (DAB, ZLI-9017, Beijing, China) staining, hematoxylin re-staining, and sealing tablets.
Enzyme-Linked Immunosorbent Assay (ELISA)
The concentrations of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in supernatant and serum of brain prefrontal lobe were detected by ELISA kit (Jianglaibio Co., Ltd, CK-E31063; CK-E30219, Shanghai, China).
Real-Time PCR (RT-PCR)
The total RNA of rat prefrontal cortex was extracted by RNAiso Plus Trizol RNA extraction kit (TaKaRa Bio, Japan). The Prime Script RT kit was adopted to reverse transcribe cDNA. Besides, SYBR Green PCR kit (Sangon Biotech, Shanghai, China) was adopted for real-time fluorescence quantitative reverse transcription PCR in the detection system (BIO-RAD Cot., USA). The primer sequences (Sangon Biotech, Shanghai, China) were listed in Table 1.
Table 1 List of primer sequences adopted for RT-PCR.
Accession Number
|
The name of the gene
|
Primer Sequences (F: Forward; R: Reverse)
|
NM_001191642.1
|
NLRP3
|
F. TGT TGT CAG GAT CTC GCA
R. AGT GAA GTA AGG CCG GAA T
|
NM_012762.3
|
Caspase-1
|
F. GAC AAG ATC CTG AGG GCA AA
R. GGT CTC GTG CCT TTT CCA TA
|
NM_031512.2
|
IL-1β
|
F. CTG GAC TCG TGG GAT GAT G
R. GGG ATT TTG TCG TTG CTT GT
|
NM_019165.1
|
IL-18
|
F. AAC GAA TCC CAG ACC AGA C
R. AGA GGG TAG ACA TCC TTC CAT
|
NM_012589.2
|
IL-6
|
F. CAG TTGCCT TCT TGG GGA CT
R. GGT CTG TTG TGG GGT GGT ATC
|
NM_012675.3
|
TNF-α
|
F. CCA CCA CGC TCT TCT GTC
R. GCT ACG GGC TTG TCA CTC
|
NM_031144.3
|
β-actin
|
F. TGT CAC CAA CTG GGA CGA TA
R. GGG GTG TTG AAG GTC TCA AA
|
Western blot
Proteins were quantified with BCA kits (Solarbio, PC0020, Beijing, China), then separated by polyacrylamide gel electrophoresis (BIO-RAD Cot., USA) and transferred to PVDF membrane (Millipore, Cot., USA) after cleavage of rat prefrontal cortex with a mixture of protease inhibitors (RIPA : PMSF=100:1). 5% skimmed milk powder was adopted for sealing after electroporation and with an antibody NLRP3 (Novus, NBP2-12446, Colorado, USA), Caspase-1 (Novus, NB100-56564, Cambridge, UK), IL-1β (BIOSS, bs-20449R, Beijing, China), IL-18 (Proteintech, 10663-1-AP, Wuhan, China), IL-6 (BIOSS, bs-0782R, Beijing, China), TNF-α (Abcam, ab178846, Cambridge, UK), β-actin (Proteintech, 20536-1-AP, Wuhan, China), GAPDH (Proteintech, 10494-1-AP, Wuhan, China), LC3B (HuaBio, ET1701-65, Hangzhou, China), SQSTM1/p62 (HuaBio, R1309-8, Hangzhou, China).
Separation of mitochondria
Mitochondria isolation from prefrontal cortex: Intact cortical mitochondria were isolated from fresh brain tissue using a tissue mitochondrial isolation kit. In brief, 50mg of prefrontal cortex tissue was homogenized in 2mL of pre-ice-cold protein solution, and centrifuging the homogenate at 6000×g for 5min at 4°C, the collected supernatant containing cytosolic protein was then destroyed by adding 1.5mL of pre-ice-cold 1.5mL of protein solution to the supernatant to re-suspend and destroy the particles. After further centrifugation at 11000×g for 10min at 4°C, the supernatant was collected and centrifuged at 6000×g for 10min. The pellet (containing mitochondria) was re-suspended in 750μL mitochondrial purification buffer, centrifuged at 14,000×g for 15min, and a pellet or band containing mitochondria was formed in the lower part of the tube and transferred to a new tube. The suspension was washed three times with 1.5 mL of mitochondrial storage buffer by centrifugation at 8000×g for 10min. Highly purified mitochondria were re-suspended in mitochondrial storage buffer and stored at -80 °C until further use.
Transmission electron microscopy (SEM)
After the anesthetized rats were fixed on the operating table, the brain tissue was quickly removed, and the prefrontal cortex of the rats was stripped. The trimmed tissue was placed in 3% glutaraldehyde fixation solution, fixed at room temperature for 2 days, and then placed at 4°C for later use. The steps were repeated twice: 70% acetone for 15min→ 80% acetone for 15min→ 90% acetone for 15min→ 100% acetone for 1min. Embedding with epoxy resin embedding agent → dodecane succinate hardening agent → diethyl amphetamine accelerator → butyldiester phthalate plasticizer→45°C drying for 12h→ 60°C drying for 36h, special slicer slices into 40nm→ mesh supporting film → drop dye solution (uranyl acetate + lead nitrate) for 15min, transmission electron microscope (Hitachi Limited, Japan).
Statistical analyses
The experimental data were analyzed and processed by SPSS18.0 statistical software. In addition, the measurement data were expressed by mean±SD. The repeated measurements of the water maze were measured by ANOVA after the spherical test. Pairwise comparison between groups was analyzed by LSD test, and the difference was statistically significant when P < 0.05.