Animal care and drug treatment
Adult male CD1 Elite mice (22-28 g, 10-12 weeks; Jining, China) were used in this study. Nrf2 knockout (Nrf2−/−) CD1 male mice were obtained from Dr. Chunyan Li (Second Hospital of Hebei Medical Univercity). The Institutional Animal Care and Use Committee of Jining Medical University approved all the experiments, which were performed according to the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No.80-23). The mice were housed under standard conditions and were randomly assigned among one of 5 groups: sham, TBI, TBI + low dose (0.5 µg/kg) of calcitriol, TBI + medium dose (1 µg/kg) of calcitriol, and TBI + high dose (3 µg/kg) of calcitriol. The animals received daily gavage of vehicle or different doses of calcitriol for 14 days. Moreover, to confirm the neuroprotective mechanisms of VitD, CQ (30 mg/kg/day) was co-treated with calcitriol by intraperitoneal injection to block autophagy flux.
Mouse model of TBI
A previously described (Cui，Song 2017), a 10-mm-long midline scalp incision was made to expose the skull. TBI was induced by hitting the brain surface at the center of the craniotomy with a 2.5-mm-diameter rounded metal tip at a velocity of 4 m/s and a deformation depth of 2 mm using a controlled cortical impact (CCI) device (CCI Model 6.3; Custom Design, USA). The bone flap was immediately replaced and sealed, and the scalp was sutured.
Neurological score evaluation
At 1–14 days following TBI, the neurological scores were determined as Neurological Severity Scores, a composite of motor, sensory, reflex, and balance tests (normal score: 2–3; maximal deficit score: 18; Additional file 1).
The Morris water maze (MWM) test
The apparatus consisted of a circular black water tank (180 cm in diameter, 50 cm high) filled with water (26 °C) to a depth of 30 cm. An escape platform (diameter 12 cm, height 28 cm, painted opaque) submerged 2 cm below the water surface was placed in the middle of one of the quadrants equidistant from the tank wall and the center of the pool. All the mice were trained to find the platform before the sham operation or the induction of TBI. For each trial, each mouse was randomly placed into a quadrant start point (N, S, E, or W) facing the wall of the pool and was allowed a maximum of 60 s to find the escape platform. The mice that failed to escape within 60 s were placed on the platform for a maximum of 20 s and returned to their cage to await a new trial (intertrial interval, 10 min). Probe trials were conducted at 11–14 days following the induction of TBI or the sham operation. The time spent in the target quadrant and the swim speeds were evaluated on the last day of the test after the platform was removed.
After fixation, the brains were embedded in paraffin and sliced into 4-µm coronal sections at the level of the bregma and stained with hematoxylin and eosin (H&E). Apoptosis was assessed using terminal deoxynucleotidyl transferase-mediated cyanine–dUTP nick-end labeling (TUNEL) following the manufacturer’s protocol. Nuclei were counterstained with DAPI (Beyotime Biotechnology, China). For each group, sections from three different mice were used for quantification.
Transmission electron microscopy (TEM)
Tissues were immersed in 2% glutaraldehyde and 1% osmium tetroxide (Sigma–Aldrich; Merck KGaA) for 2 h at 4 °C, and then dehydrated via a graded ethanol series. Following the displacement of ethanol with propylene oxide, the tissues were embedded in Epon (both from Sigma–Aldrich) and sectioned along the coronal plane with a diamond knife (FernAnclez-hIorln 1953; Ivan Sorvall, Inc., New York, NY, USA) at a thickness of 60 nm. The sections were stained with lead citrate and observed using a CM-120 electron microscope (Philips, Eindhoven, Netherlands). In order to quantify the alteration of the number of the autolysosomes, the area of the cell cytoplasm was measured by using Image-Pro Plus 6.0.
Western blot analysis
Proteins (50 µg) were separated by 12% SDS–PAGE and transferred onto a nitrocellulose membrane. The membrane was blocked with 5% nonfat milk at room temperature for 2 h and then incubated overnight at 4 °C with primary antibodies against microtubule-associated protein light chain 3 (LC3) (1:500, PM036, MBL, Nagoya, Japan), p62 (1:1,000, ab101266, Abcam, Cambridge, MA, USA), beclin 1 (1:1,000, ab137161, Abcam), Keap1 (1:1,000, ab118285, Abcam), Nrf2 (1:1,000, ab62352, Abcam), beta-actin (1:1,500, A1978, Sigma, USA), and PCNA (1:500, ab92552, Abcam). The gray values of the protein bands were measured using ImageJ software and normalized to that of β-actin that was used as an internal control.
Frozen cross-sections (15 µm) were prepared and examined. The sections were incubated with primary antibodies against Keap1 (1:200, ab139729), p62 (1:200, ab56416), and Nrf2 (1:1,000, ab62352) (all from Abcam) overnight at 4 °C, and then with a mixture of FITC- and TRITC-conjugated secondary antibodies for 2 h at room temperature. Images were captured under a ﬂuorescence microscope (Olympus, Japan).
Real-time PCR analysis
Total RNA was extracted using Trizol reagent (Invitrogen, USA) following the manufacturer's instructions. Quantitative PCR was performed on a Bio-Rad Cx96 Detection System (Bio-Rad, USA) using a SYBR green PCR kit (Applied Biosystems, USA) and gene-specific primers (Additional file 2).
Detection of oxidative parameters
The generation of reactive oxygen species (ROS) was determined by fluorescence-labeled dihydroethidium (DHE). Frozen cross-sections (15 µm) were incubated in DHE for 30 min at 37 °C in a dark humidified chamber. The sections were rinsed three times in PBS and observed using an inverted fluorescence microscope (Olympus, Japan). Malondialdehyde (MDA) levels were measured using the thiobarbituric acid reactive substances (TBARS) assay. The activities of superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) were determined using SOD, CAT, and GSH assay kits, respectively (Nanjing Jiancheng Bioengineering Institute, China).
Primary cortical neuron culture
The skull, blood and meninges were carefully removed from fetal mouse brains. After the cortical tissue was digested in 0.25% trypsin (BI, Israel) for 5 min at 37 °C, the suspensions, containing fetal bovine serum (BI, Israel), were passed through filters with a 0.22-μm pore size (Millipore, USA) and then centrifuged at 1,500 rpm for 5 min. The cells were distributed in poly-D-lysine-coated plates. The medium was replaced with neurobasal medium supplemented with streptomycin, penicillin, HEPES, glutamate, and B27 (BI, Israel). The cells were exposed to different doses of calcitriol (1nM, 10nM, 100nM, 500nM) for 24 h. The cells were pre-treated for 6h with 25 μM CQ and then treated with the indicated doses of calcitriol.
Cell viability analysis
MTT was added to each well of a 24-well plate followed by incubation at 37 °C for 1 h. The purple formazan crystals formed through the reduction of MTT were then dissolved in 500 µL DMSO, and the absorbance of the wells was recorded at 590 nm. Cell viability was calculated by the absorbance ratio of the treated group to that of the control.
Autophagic flux analysis
Autophagic flux was detected by using the RFP-GFP-LC3 adenovirus (Hanbio, China). After plating the cells in a 24-well plate at a density of 1 × 104 cells/ dish and incubating with mRFP-GFP-LC3 adenovirus for 24 h. Autophagic flux was observed under an inverted fluorescent microscope (Olympus, Japan). The yellow puncta indicated autophagosomes, and the red puncta indicated autolysosomes.
The results were expressed as means ± SD. All the analyses were performed using SPSS 17.0 software. Statistical significance was determined using one-way analysis of variance (ANOVA), and the Student–Newman–Keuls post hoc test was used to determine differences among different groups. A P-value < 0.05 was considered statistically significant.