Procedures involving animals were approved by the Animal Experimentation Ethics Committee of Ruijin Hospital, and were performed strictly according to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Male wild-type (WT) and SIRT3-knockout (SIRT3-/-) mice were used for the study. The mice were kept on a 12-hour light/dark cycle with free access to food and water.
SAE mouse model
SAE was induced in mice between 6 to 8-week-old by intraperitoneal (i.p.) administration of 10 mg/kg LPS (Escherichia coli, serotype 0111: B4, catalog L2630) from Sigma-Aldrich (St. Louis, MO, USA), similar to previous studies [50, 51, 52]. Animals were divided into 4 groups (n = 8 each): WT Control (WT Con) group, WT LPS group, SIRT3-/- Control (SIRT3-/- Con) group and SIRT3-/- LPS group. Mice were euthanized 24 hours after LPS administration. The hippocampal tissues were isolated and stored at -80 °C for further analysis.
Behavioral tests included the open field test (OFT) and fear conditioning test (FCT). Spontaneous locomotor activity of the mice in OFT was evaluated as previously described . The apparatus is made up of a white polyester resin chamber (40 cm x 40 cm x 40 cm). Mice were placed in the center of the arena and allowed to explore for 300 s, and the total distance moved and the time spent in the center were recorded. The chamber was cleaned with 75% ethanol after each trail.
The FCT was performed following the protocol as previously described [54, 55]. On the day of training, mice were placed into an enclosed training chamber and allowed to explore for 180 s. Mice were then exposed to a tone (30 s, 70 dB, 3 kHz), followed by a 2 s foot shock (0.45 mA). Afterward, mice were left in the chamber for additional 30 s. The training period was repeated 5 times. 24 hours after the training session, mice were re-exposed in the same chamber for 300 s for the contextual fear conditioning test (a hippocampus-dependent task). 4 hours later, mice were placed into a novel chamber which was different in shape, color, and smell from the original chamber for the cued fear conditioning test (a hippocampus-independent task). After 180 s exploratory period, a training tone (30 s, 70 dB, 3 kHz) was applied for another 30 s and repeated 4 times at an interval of 40 s, and the time of freezing was recorded. The chamber was cleaned with 75% ethanol after each trial.
Mice were anesthetized and transcardially perfused with pre-cooled heparinized physiological saline and 4% paraformaldehyde. After dehydration with gradient ethanol elution and cleared in xylene, cerebral tissues were sliced into 10 μm coronal sections using a cryostat (Leica CM1860 UV, Leica) and stored at -20 °C. Nissl staining (Beyotime Institute of Biotechnology, Shanghai, China) was conducted according to the manufacturer's instructions. In brief, the sections were soaked in 1% toluidine blue for 5 min at 50 °C, washed by double distilled water, and dehydrated with gradient ethanol elution. The slice sections were observed under a fluorescent microscope.
The TdT-mediated dUTP Nick-End Labeling (TUNEL) staining was used to evaluate neuronal apoptosis in the hippocampus. In Situ Apoptosis Detection Kit was used with manufacturer’s instruction (Beyotime Institute of Biotechnology, Shanghai, China). The nuclei were stained with DAPI, and the fluorescent microscopy was used to evaluate neuronal apoptosis.
The brain tissues were stored in 4 % paraformaldehyde and sectioned at 30 μm, and immunofluorescence staining for SIRT3, NeuN and DAPI were performed. The sections were incubated with SIRT3 antibodies (catalog D22A3, Cell Signaling Technology, Danvers, MA, USA) and NeuN (catalog 66836-1-Ig, ProteinTech Group, Chicago, IL, USA) overnight at 4 °C. Anti-rabbit (catalog 147626) and anti-mouse (catalog 146552) secondary antibodies from Jackson ImmunoResearch were added and incubated for 2 h at 37 °C, and sections were then washed three times with PBST. After final washing, sections were protected with coverslips, with the nucleus visualized with DAPI (catalog C1005, Beyotime Institute of Biotechnology, Shanghai, China). The brain tissues were observed and analyzed using fluorescence microscope.
HT22 cells were fixed in 4% paraformaldehyde in PBS for 15 min at room temperature. Cells were then permeabilized in 0.1% Triton X-100 in PBS for 15 min and blocked with 10% donkey serum in PBS for 1 h. The culture dishes were then incubated with anti-SHC phosphor S36 (catalog 54518, Abcam, Cambridge, UK) and anti-TIMM44 (catalog HPA043052, Sigma) overnight at 4 °C, and for 1.5 h at room temperature for secondary antibody incubation. After final washing, the nucleus visualized with DAPI (catalog C1005, Beyotime Institute of Biotechnology, Shanghai, China). The cells were observed using confocal microscopy.
NAD+ and NADH measurements
Fresh mouse hippocampal brain tissue (20 mg) was collected and homogenized in 400 μL NAD+/NADH extract buffer on ice. Total NAD+ and NADH were tested using an NAD+/NADH assay kit (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer’s protocol . NAD+/NADH ratio was calculated based on the valve of total NAD+ and NADH (NAD+ = total NAD+ - NADH).
Hippocampal neuronal cells (HT22) and microglia cells (BV2) were respectively cultured in Dulbecco’s modified Eagle’s medium (DMEM), 10 % fetal bovine serum (FBS) and 1 % streptomycin and penicillin in incubator containing 5 % CO2 at 37 °C. To induce LPS-induced hippocampal neuron stress in vitro, BV2 cells were first exposed to LPS (1 μg/ml) for 24 h, and the supernatants of LPS-stimulated microglia (Mi-sup) were collected as previously described . HT22 cells were then cultured in the Mi-sup for 24 h.
Co-immunoprecipitation (Co-IP) assays
HT22 cell lysate was harvested, and Co-IP assay was performed using an Immunoprecipitation kit from Proteintech. Briefly, 2 μg of antibodies against the proteins of interest or a negative control IgG of the same species were added to the cell lysate. Then the IP buffer was mixed with protein A/G beads. The immunoprecipitate was separated by SDS-PAGE and analyzed by western blotting.
Plasmids and transfection
The pDC315-SIRT3 was designed and purchased from Shanghai Gene-Pharma Co. (Shanghai, China). The plasmid was transfected into HEK293 cells and the supernatant was collected. The viral supernatant was identified and amplified to obtain adenovirus-SIRT3. The plasmids were transfected into the HT22 cells to overexpress SIRT3 with Lipofectamine 2000 (Thermo Fisher Scientific, Inc.) according to the manufacturer’s protocol. Null vector transfection was used as the control group.
Total cellular ROS production was determined by detecting the fluorescence intensity of dichlorofluorescein diacetate (DCFH-DA) according to the manufacturer’s protocol  (Reactive oxygen species assay kit, Beyotime, China). Briefly, HT22 cells were cultured in 12-well plates and incubated with freshly prepared DCFH-DA reagent in dark at 37 °C for 30 min. HT22 cells were washed three times with PBS and analyzed using fluorescence microscope.
JC-1 staining was used to evaluate MMP according to the manufacturer’s protocol (Mitochondrial membrane potential assay kit with JC-1, Beyotime, China). Pretreated HT22 cells were washed three times with PBS, followed by the additional 1 ml JC-1 working solution per sample and incubated at 37 °C for 20 min. After incubation, HT22 cells were washed twice with JC-1 staining buffer and detected using fluorescence microscope.
MPTP opening was measured by calcein-AM following the manufacturer’s protocol (Mitochondrial permeability transition pore assay kit, Beyotime, China). HT22 cells were co-stained with 1 μM calcein-AM, 1mM CoCl2, and 200 nM Mitotracker for 20 min at 37 °C. After staining, HT22 cells were washed with PBS and captured by fluorescent microscope. ImageJ software was used for quantification and measurement of fluorescent intensity.
Western blot analysis
Frozen hippocampus sample were homogenized in ice-cold extraction buffer. Homogenates were centrifuged at 12,000 g for 15 min at 4 °C, the final supernatants were obtained. In contrast to frozen hippocampus tissues, HT22 cells were washed twice with PBS and lysed in an extraction buffer. Mitochondrial extracts were obtained with a mitochondrial isolation kit (Beyotime Institute of Biotechnology, Shanghai, China). Protein concentrations were determined by BCA Protein Assay Kit (Beyotime Institute of Biotechnology, Shanghai, China). Equal amounts of protein were separated on a 10% SDS-PAGE and transferred to a PVDF membrane. After being blocked, the membranes were incubated with correspondent primary antibody at 4 °C for overnight. Antibodies against α-tubulin (catalog 11244-1-AP), GAPDH (catalog 10494-1-AP), Bcl-2 (catalog 12789-1-AP), Bax (catalog 50599-2-Ig), Cytochrome C (catalog 66264-1-Ig), SHC (catalog 10054-1-AP) were obtained from Proteintech Group. Antibodies against VDAC (catalog D73D12), SIRT3 (catalog D22A3), PSD95 (catalog D27E11), BDNF (catalog 47808S) were from Cell Signaling Technology. Anti-Cyclophilin 40 (catalog 181983), anti-acetyl Lysine (catalog 80178) and anti-SHC phosphor S36 (catalog 54518) were from Abcam. Anti-JNK (catalog 7345) and anti-p-JNK (catalog 6254) were from Santa Cruz Biotechnology. After washing, the membrane was incubated with secondary antibodies (catalog SA00001-2, Proteintech Group) for 80 min at room temperature. Immunoreactive protein bands were detected using chemiluminescence reagents (Beyotime Institute of Biotechnology, Shanghai, China). In the present study, α-tubulin and GAPDH were used as loading control in hippocampus homogenates and whole-cell extracts, while VDAC was used as loading control in both hippocampus and cell mitochondrial extracts.
Data were analyzed using GraphPad Prism 8 software and the data were presented as mean ± standard error. Statistical significance was evaluated by the Student’s t-test for comparing two experimental groups. For datasets having multiple groups, statistical significance was evaluated using a one-way analysis of variance (ANOVA) followed by Tukey’s test for the post hoc analysis. Results were considered as significant at P < 0.05.