The study was approved by the ethics committees of Tianjin Medical University General Hospital and Tianjin Anding Hospital.
2.1 Grouping, modeling and intervention in mice
Fifty male C57BL/6J mice (four weeks old) were purchased from laboratory animal center of the academy of military medical sciences (Beijing, China). The mice were supplied with normal, balanced and adequate diets, and were kept at room temperature with a light/dark cycle (12h/12h) for seven days before our experiments.
Then, the animals were divided into five groups according to randomization principle: sham group, HFD group, IGF-1 treatment group (IGF group), IGF-1 receptor inhibition group (IGFR_I group) and HO-1 inhibition group (HO_I group). Each group contained 10 mice.
After grouping, the mice in the HFD, IGF, IGFR_I and HO_I groups were treated with HFD (D12492, Research Diets) for 12 weeks. Its energy composition was fats (60%), carbohydrates (20%) and proteins (20%). During the same period (12 weeks), the sham mice were fed with normal diet (D12450B, Research Diets). Their body weights were measured weekly, and their fasting serum glucose and lipids were determined at the end of the modeling process. Briefly, fasting blood was obtained from caudal vein of each mouse, and an automatic biochemical analyzer (Hitachi 7170, Hitachi, Japan) was adopted to measure these biochemical markers.
In the next step, the mice in the IGF, IGFR_I and HO_I groups were treated with polyethylene glycol - IGF-1 (PEG-IGF-1) by intraperitoneal injection for four weeks (1 mg/kg, twice a week) (Sama et al. 2018). Meanwhile, the mice in the IGFR_I and HO_I groups were separately treated with IGF-1 receptor blocker AXL1717 and HO-1 blocker Znpp IX by intraperitoneal injection for four weeks (AXL1717: 20 mg/kg, once a day; Znpp IX: 20 mg/kg, twice a week) (Wang et al. 2018; Xu et al. 2019). The mice in the sham group were treated with 0.9% normal saline by intraperitoneal injection once a day for four weeks. AXL1717 and Znpp IX were separately obtained from GlpBio and Sigma-Aldrich (USA), and were dissolved and diluted using DMSO according to their instructions. In this period, the sham mice were also fed with normal diet, and the other mice were fed with HFD.
2.2 Grouping, modeling and intervention in cells
A human neurons-hippocampal (HN-h) cell line was purchased from ScienCell Research Laboratories (USA), and was cultured according to routine procedure. Briefly, the cells were maintained in poly-L-lysine-coated cell culture flasks with specific neuronal medium (NM, Cat. #1521). The flasks were placed at 37℃ in an atmosphere containing 5% carbon dioxide. The medium was renewed every 48 hours. With the division and growth of the cells, they were transferred to new flasks for more space. Eventually, the cells were seeded into 6-well plates for our experiments.
The prepared cells were divided into control group, palmic acid treatment group (PA group) and pyroptosis inhibition group (Pyr_I group). Each group contained ten wells. The cells in the PA and Pyr_I groups were treated with one kind of saturated fatty acids - palmic acid (800 μmol/l) for 12 hours, and the cells in the Pyr_I group were also treated with pyroptosis blocker necrosulfonamide (4 μmol/l) for 12 hours (Yao et al. 2018). Palmic acid and necrosulfonamide were purchased from Sigma-Aldrich (USA). Palmic acid was dissolved using NaOH (0.1 mol/l) at 70℃ for 5 minutes and then mixed using bovine serum albumin (10%) at 55℃ for 10 minutes. The mixture was added with specific neuronal medium (NM, Cat. #1521) to form the concentration of 800 μmol/l. Necrosulfonamide was dissolved and configured by DMSO (0.1%) to produce the concentration of 4 μmol/l.
2.3 Cognitive function test in mice
After modeling and intervention, cognitive function was evaluated using Morris water maze. Detailed steps were described in our previous literature (Barnhart et al. 2015). During a day of adaptive training, the rats were arranged to familiarize themselves with an experimental pool with a platform in it. Subsequently, navigation test was conducted on day 2 to day 5. The rats were randomly placed any quadrant in the pool, and their trajectories and times were recorded using a video-tracking system (TSE Systems, Bad Homburg, Germany). “Escape latency” was defined as the length of time a rat took from being placed in a pool to climbing the platform. If the escape latency of one rat was longer than 60 seconds, its escape latency was recorded as 60 seconds. This rat were also guided to the platform and placed on it for another 30 seconds. After the navigation test, the platform was removed in order to perform spatial probe test on Day 6. Each rat was placed in the pool again, and let them swim in the pool for 120 seconds. The length of time each rat spent in the target quadrant and the number of times it went through the target quadrant were recorded. The “target quadrant” was defined as the quadrant where the platform was originally located.
2.4 Specimen collection and preparation in mice
After the behavioral evaluation, the mice were humanely euthanized under anesthesia. Cerebrospinal fluid specimen was obtained from cisterna magna using a glass capillary tube (Liu and Duff 2008). Hippocampus tissue was collected and cleaned using double distilled water. The fresh tissue was cut as small as possible and digested with trypsin (0.2%) for about 30 minutes until the tissue was dispersed. The tissue suspension was filtered with a 100 mesh nylon filter. The obtained cell suspension was centrifugated at 1000 rpm for 5 minutes, and was mixed with PBS to form a hippocampus neuron suspension for further experiments.
2.5 Levels of tau proteins in cerebrospinal fluids
Cerebrospinal fluid levels of p-tau (Ser 181) and p-tau (Thr 205) were determined using a Singulex Erenna immunoassay platform (USA) (Hastings et al. 2017). HT7 was adopted as capture antibody. AT270 and AT8 which separately recognized p-tau (Ser 181) and p-tau (Thr 205) served as detection antibodies. In the process, the measurement was performed in the presence of HaltR Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher). Briefly, in 96-well plate, each well contained 10 mg capture beads, 50 μl Singulex assay buffer and 50 μl cerebrospinal fluids (or standard peptide), which was diluted in PBS-T (0.2% Tween 20 in PBS). The plate was incubated at 25℃ for 4 hours, and was washed using a Biotek 405TM TS microplate washer. Then, 20 μl AT270 or AT8 antibody (50 ng/ml) was added into the plate, and was incubated and shaken overnight at 25℃. The cerebrospinal fluid specimen was developed according to the instruction, and the result was analyzed using Sgx link software (Singulex Erenna).
2.6 Protein expression in hippocampus tissue and HN-h cells
Expression of Nrf2, HO-1, p-tau (Ser 181), p-tau (Thr 205), t-tau, NLRP3, cleaved Caspase-1, IL-1β and GAPDH in the hippocampus tissue and HN-h cells were determined using western blotting.
Briefly, RIPA lysis buffer (Thermo Fisher Scientific, MA, USA) was adopted to extract the protein from the specimens, and Pierce™ modified Lowry protein assay kit (Thermo Fisher Scientific, MA, USA) was used to measure the total protein levels. A certain amount of protein (50 μg) was separated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then were transferred onto a Bio-Rad's nitrocellulose membrane (Hercules, CA, USA). After 2-hour blocking with 5% skimmed milk at 25℃, the membrane was incubated anti-Nrf2 (Ser 9) (CST, #12721, 1:1000 dilution), anti-HO-1 (CST, #82206, 1:1000 dilution), anti-p-tau (Ser 181) (CST, #12885, 1:1000 dilution), anti-p-tau (Thr 205) (CST, #49561, 1:1000 dilution), anti-t-tau (CST, #46687, 1:1000 dilution), NLRP3 (CST, #15101, 1:1000 dilution), anti-cleaved Caspase-1 (CST, #89332, 1:1000 dilution), IL-1β (CST, #31202, 1:1000 dilution) and anti-GAPDH (CST, #5174, 1:1000 dilution) at 4℃ overnight. Subsequently, the membrane was incubated with an anti-rabbit IgG, HRP-linked secondary antibody (#7074, CST). Chemiluminescence detection was adopted to visualize the immunoreactive bands (Immolilon Western, USA). Image J software was used to measure the intensities of the bands.
2.7 Pyroptosis rate in hippocampus tissue and HN-h cells
Pyroptosis rate in the specimens was determined using a FAM-FLICA Caspase-1 detection kit (ImmunoChemistry) (Zeng et al. 2019). Briefly, prepared cells were stained with FAM-FLICA (10 μl) and PI (5 μl) at 26℃ for 20 minutes. Then, fluorescence intensities of the specimens were measured by a Coulter Epics XL flow cytometer. Pyroptosis rate was calculated using a formula: number of double-positive cells / number of total cells × 100%.
2.8 Statistical analysis
Continuous variable in the study was expressed using mean ± standard deviation. Variable comparison between two groups was conducted using independent sample t test. Variable comparison among more than two groups was performed using variance analysis (ANOVA) with LSD test. P value less than 0.05 was regarded as statistically significant. All statistical analysis was performed using Statistical Product and Service Solutions (SPSS) version 19.0.