2.1 Animals
C57/B6 mice (8-weeks-old) weighing 20–24 g, were obtained from the Experimental Animal Center of Nanjing Medical University. The mice were housed at a suitable temperature and humidity on a 12-h / 12-h light / dark cycle. All the mice had free access to food and water. In total, 118 healthy mice of either sex were used in this study. No specific statistical methods were performed for sample calculation and previous studies were used as a reference(Carneiro et al., 2018). Neurological Severity Score (mNSS) tests were performed on 118 C57/B6 mice (8 weeks old) weighing 20-24 g each. One mouse with an abnormal score was excluded from the experiment and replaced with one of the same sex. In the first part of this study, mice were divided into five groups (n = 14 animals/group): the sham group, the TBI 1d group, the TBI 3d group, the TBI 7d group and the TBI 14d group. In the second part of this study, mice were divided into four groups (n = 12 animals/group): the sham group, TBI+vehicle group, the TBI+SK group, the TBI+TEPP46 group. No randomization was performed to allocate subjects in the study. Behavioural experiments were performed between 2 and 5 p.m., and treated animals were assessed prior to control animals. During experiments and analysis, the investigators were blinded to experimental group. Animals were identified by earmarks that assigned numbers to each, which were announced to the investigator only after finishing experiments and analysis. Animals were killed using cervical dislocation at the time of biochemical analysis.
All procedures were conducted in strict accordance with the ARRIVE Guidelines (Animal research: reporting of in vivo experiments) and approved by the Ethics Committee of Nanjing Medical University College and the National Institutes of Health Guide for the Care and Use of Laboratory Animals (#IACUC-2011010). The study was not pre-registered.
2.2 Experimental TBI model
A controlled cortical impact model was adopted to establish TBI groups based on previously published research(Ren et al., 2020). To ensure that there were no adverse effects on the physiological state of mice, 50mg/kg dose of pentobarbital were administered intraperitoneally for anesthesia. Each anesthetized mouse was placed on a stereotactic machine with its skull exposed. A portable drill and a 3.5 mm ring bit were used to perform a craniotomy in the right parietal temporal cortex, preserving the integrity of the dura. Then, a convex tip (diameter of 3 mm) was used to generate a 1.2 mm depression at a speed of 4.5 m/s causing mild damage. The bone flap was discarded and the scalp was sutured and closed. After each TBI or sham procedure, mice were immediately placed on a heating pad (37°C) for at least 15 minutes to maintain the body temperature. An air heater was also settled to keep the animals warm. Then the mice were allowed to settle in cages until they recovered from anesthesia. No analgesics were applied due to their potential to induce mitochondria dysfunction, which might interfere with the effects of the drugs we used in the experiment. Still, to ease the pain caused by the procedure, jelly was given to the mice to supplement energy and water. And after surgery, 0.15 mg/kg buprenorphine hydrochloride (Recktt Benckiser Healthcare Ltd., Kingston-upon-Thames, UK) was injected subcutaneously to each of mice to reduce pain after surgery. In addition, the same procedure, excluding the hit, was performed in the sham group. All mice regained consciousness within 1 h of surgery. Nine mice from TBI group died during recovery before tests and were excluded from the study.
2.3 Drug treatment
Shikonin was purchased from MedChemExpress Biochemical (Cat. No. No. HY-N0822, Shanghai, China,2021). TEPP46 was purchased from MedChemExpress Biochemical (Cat. No. No. HY-18657, Shanghai, China, 2021). Mice were continuously administered intraperitoneal injections of 5 (shikonin) or 50 mg/kg/day (TEPP46) 24 h after brain injury. The doses were selected based on the results of previous studies(Xie et al., 2016; Angiari et al., 2020). The sham and TBI groups received the same dose of drug solvent. Cells were treated in vitro with different concentrations for 30 min.
2.4 Cortical lesion volume
Cortical sections were obtained every 0.01 mm to determine lesion volume. Lesion volume was calculated from the summation of the defect areas on each tissue slice using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
2.5 Behavioral tests
Behavioral tests were usually performed between 1 and 3 pm. Fear conditioning tests (FCTs) were used to evaluate memory and cognitive function in mice(Cibelli et al., 2010; Terrando et al., 2015). The experimental environment was a test box, and the bottom of the box was energized to establish an environment of fear using noise and electric shock. The experimental procedure included three stages: adaptation, training, and testing, which were completed at the same time point for three consecutive days. During the adaptation stage, the mice were placed in a box and allowed to adapt for 5 min without any stimulation. During the training phase, mice were subjected to 30 s (75 dB, 2.8 kHz) sound stimulation and then 2 s electric shock (1 mA) at the 3rd and 5th minutes, respectively. The entire process lasted 7 min. During the testing stage, there was only sound stimulation, and the participants’ fear of the environment was evaluated by the percentage of freezing time (freezing). This test was conducted on the third, seventh, and 14th day after TBI.
Morris water maze (MWM) test was performed to evaluate spatial learning and reference memory of mice. The device of MWM consisted of a circular pool with a diameter of 120 cm and height of 70 cm and a cylindrical platform with a diameter of 8cm. The pool was filled with opaque water maintained at 20 ± 1 °C and equally divided into four quadrants. The platform was placed in the center of one of the quadrants. During the initial 5 days, mice were trained to found the hidden platform when started from random locations around the pool. The escape latency of the mice was recorded by TopScan Realtime Option system and analyzed in 5-day training session. On sixth day, the platform was removed and the probe test was conducted. The mice was allowed to freely explore the pool for 60s and the number of mice crossing the platform was recorded and analysed.
2.6 Immunohistochemistry and hematoxylin-eosin staining (HE)
All animals that underwent behavioral tests were anesthetized with 4% isoflurane and perfused through the heart with 4% paraformaldehyde. The brain tissue was fixed with 4% paraformaldehyde for 24 h and then embedded in paraffin. Subsequently, the specimens were cut into 10 μm-thick sections. The sections were dewaxed in xylene, rehydrated using decreasing concentrations of ethanol, and washed in phosphate-buffered saline (PBS). The sections were stained with hematoxylin and eosin (HE).
2.7 Immunofluorescence and Confocal Microscopy
All animals that underwent behavioral tests were anesthetized with 4% isoflurane and perfused through the heart with 4% paraformaldehyde. Brain tissue was fixed with 4% paraformaldehyde for 24 h, followed by 30% sucrose solution for continuous dehydration at 4 °C for three days. Sucrose solution was replaced every 24 h to ensure that the brain tissue was fully dehydrated. The tissue was then embedded with optimal cutting temperature (OCT) compound and cut into 10-um-thick sections using a frozen sectioning machine (Leica, CM1860). The sections were incubated overnight at 4 °C with NeuN (RRID:AB_2532109, 1:300, Abcam), Iba1 (RRID:AB_2832244, 1:200, Abcam), and GFAP (RRID:AB_561049, 1:300; Cell Signaling Technology). Sections were washed and incubated with the following secondary antibodies: donkey anti-rabbit IgG (RRID: AB_2535792, 1:1000, Thermo) and donkey anti-mouse IgG (RRID: AB_2732856, 1:200, Abcam) for 1 h at room temperature, and then washed four times with PBS. Cell nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI, Southern Biotech, Cat. No. 0100–20, 2020). Finally, each section was imaged using a confocal microscope (Zeiss, LSM800). Images were analyzed using ImageJ software.
2.8 Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) Staining
The level of apoptosis in mouse neurons was detected using the TUNEL Bright Green Apoptosis Detection Kit (Vazyme, Cat. No. A112-01, 2020). The brain tissue sections were fixed in 4% paraformaldehyde (prepared in PBS) for 30 min at room temperature. The sections were washed four 4 times with PBS, incubated with 0.2% Triton-X100 for 10 min, and then incubated with 1% BSA for 1 h at room temperature. Following this, an equilibration solution (100 μL of 1 × equilibration buffer) was added to each sample to equilibrate for 20 min, and then 50 μL working solution (ddH2O, 5 × equilibration buffer, recombinant TdT enzyme) was added for 1 h at 37 °C. Sections were washed four 4 times with PBS, incubated overnight at 4 °C with neuronal antibodies (RRID:AB_2532109, 1:300, Abcam), and then incubated with donkey anti-mouse IgG(RRID:AB_2732856, 1:200, Abcam) secondary antibodies. All sections were imaged using a confocal microscope (Zeiss, LSM800). Images were analyzed using ImageJ software.
2.9 Brdu labeling
Bromodeoxyuridine (BrdU), a nucleoside analog that commonly used to detect proliferating cells, was applied to evaluate neurogenesis after TBI. BrdU (50mg/kg, Cat. No. HY-15910) was intraperitoneally injected to mice twice a day at 6h interval for 14 consecutive days before euthanized. The brain of mice was harvested for immunofluorescence assays.
2.10 Western blot analysis
Mice were sacrificed by cervical dislocation and the edema zone around the injured cortex of the brain was collected. RIPA Lysis buffer (Beyotime, Cat. No. P0013B, 2021) 200 μL containing 1 μM PMSF was added to each sample, and subsequently homogenized at 12000 × g for 20 min at 4 °C. After centrifugation, the supernatant was collected into a new tube. BCA kit (Beyotime, Cat. No. P0012, 2021) was used to normalize the amount of protein at 40 μg per lane. Equal amounts of protein were separated by 8–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene difluoride membrane. Following this, the membranes were blocked for 2 h in 5% non-fat dry milk (NFDM) and incubated with the following primary antibodies: NLRP3 (RRID:AB_2490202, 1:1000, AdipoGen), ASC (1:1000, Abcam, ab283684), Caspase1 (RRID:AB_2889889, 1:1000, Abcam), IL-1β (RRID:AB_2715503, 1:1000, CST), PKM2 (RRID:AB_1904096, 1:1000, CST), p-PKM2 (RRID:AB_1950369, 1:1000, CST), MFN2 (RRID:AB_2266320, 1:1000, Proteintech), β-actin (RRID:AB_2797445, 1:10000, Bioworld), and lamin B1(RRID:AB_443298, 1:1000, Abcam) overnight at 4 °C. The next day, the membranes were washed three times with TBS-T(TBS + 0.1% Tween-20) for 10 min, probed with an HRP-linked secondary antibody for 1 h at room temperature, and washed three times with TBS-T. The membranes were then incubated with enhanced chemiluminescence (ECL) (Sparkjade, Cat. No. ED0015-C, 2020), and visualized using the Tanon-5200Multi system (17T15NBFLI6-953). The raw density of the bands was analyzed using image J software. All values were normalized to those of β-actin and lamin B1.
2.11 Quantitative RT-PCR
Total RNA was extracted using TRIzol reagent (Invitrogen, Cat. No. 15596018, 2020), according to the manufacturer’s protocol. Next, 1 μg of total RNA was used to synthesize cDNA using a cDNA synthesis kit (Vazyme, Cat. No. R223-01, 2020). All PCRs were performed on a Light Cycler 96 thermocycler (Roche). Primer sequences for the target genes are listed in Table 1. Melting curve analysis was performed to confirm the specific PCR products of interest. Relative gene expression was calculated using the ΔΔCt method and normalized to β-actin expression.
2.12 Transmission electron microscopy (TEM)
The animals were anesthetized with 4% isoflurane and perfused through the heart with 4% paraformaldehyde. The edema zone around the injured cortex of the brain was collected, cut into 2–3 mm pieces, and soaked in glutaraldehyde solution for seven days at 4 °C. After fixation, all samples were washed three times for 15 min and then fixed with 0.1% osmium acid at 25 °C for 2 h. All samples were dehydrated with a concentration gradient ethanol solution (50, 70, 80, 90, and 95%) for 15 min and treated with pure acetone for 20 min. Following this, samples were gradient infiltrated with an embedding agent, heated, and polymerized, trimmed, sectioned (50 nm) with an ultrathin microtome, stained with uranyl acetate 50% ethanol saturated solution for 1 h, and rinsed with ddH2O. Representative areas were chosen to generate ultrathin sections, which were viewed using a transmission electron microscopy (TEM) system at an accelerating voltage of 80 kV.
2.13 Cell extraction
Primary microglia were obtained from one- to three-day-old mice pups. In total, fifty mice pups were sterilized in alcohol and killed by cutting both carotid arteries without decapitating the head, then the cerebral cortex were carefully removed, and placed in serum-free F12 medium (Wisent, Cat. No. 319-075-CL, 2020). The brains membranes were peeled off with micro tweezers under a visual microscope. Following this, microscissors were used to cut the cortical tissue into approximately 1 mm × 1 mm sections. Three to four cerebral cortices were mixed 5 mL 0.25% pancreatin and DNase were added to the medium, and the tissue was placed in a 37 °C incubator for 20 min. After digestion, 2 mL of serum was added, and the mixture was transferred to a 50 mL centrifuge tube. The suspension was sieved twice with a 200-mesh cell sieve, transferred to a T75 cell culture flask, and then placed in a 37 °C cell incubator. After 7–10 days, the culture medium was collected to obtain the primary microglia.
2.14 Cell culture
Primary microglial cells were cultured in T75 cell culture flasks in F12/DMEM (Wisent, Cat. No. 319-075-CL, 2020) containing 10% fetal bovine serum (Gibco, Cat. No. 10099141C, 2020), 100 U/mL penicillin, and 0.1 mg/mL streptomycin. Mouse neuro-2a neuroblastoma cells were cultured in T75 cell culture flasks in DMEM (Wisent, Cat. No. 319-005-CL, 2020) containing 10% FBS (Gibco, Cat. No. 10099141C, 2020), penicillin (100 U/mL), and streptomycin (100 μg/mL). After the cell density reached approximately 90%, cells were passaged and plated. The specific steps were as follows: washing twice with PBS, adding 5 mL of 0.25% pancreatin, incubation at 37 °C for 5 min, adding the culture medium, transferring the cell suspension into a 15 mL centrifuge tube, centrifuging at 1000 rpm for 5 min, resuspending the cells in fresh medium, and counting the cells. The 12-well plate was plated at 1 × 105 cells/well, the 6-well plate was plated at a density of 1×106 cells/well, and the cells were passaged at a ratio of 1:3. Mouse Neuro-2a neuroblastoma cells (ATCC® CCL-131TM, RRID: CVCL0470) (the cell line is not listed as a commonly misidentified cell line by the International Cell Line Authentication Committee) were grown in ATCC-formulated Eagle's minimal essential medium (ATCC®, Cat. No. 30–2003, 2019). The maximum number of passages for Neuro-2a was 15.
2.15 Cell Transfection
Primary microglia were seeded into 12-well plates at a density of 6 × 104 cells/well after trypsinization and passage. Cell transfection was conducted after cell growth was stabilized using lipofectamine reagent (Thermo Fisher Scientific, Cat. No. STEM00015, 2021), according to the manufacturer’s instructions. Small interfering RNA (siRNA) was synthesized by the GenePharma Biological Company (Gene Pharma, China).
2.16 Cell processing
Primary microglia were divided into control, LPS (1 μg/mL), LPS+Shikonin (1 μM), LPS+Shikonin (5 μM), and LPS+Shikonin (10 μM) groups. The LPS group was stimulated with LPS for 12 h and the LPS + SK group was incubated with different concentrations of shikonin for 30 min after 12 h of LPS stimulation. After treatment, cells were harvested for subsequent experiments.
2.17 Cell Counting Kit-8 (CCK-8) assay
A CCK-8 cell counting kit (Vazyme, Cat. No. A311-01, 2021) was used to detect the cell viability of primary microglia treated with different concentrations of drugs to determine the degree of drug toxicity. Primary microglia were seeded in 96-well plates. After the cells attached, they were grouped and treated with a concentration gradient of shikonin (1, 5, 10, or 20 μM). After 30 min of treatment, 10 μL of CCK-8 solution was added and the samples were incubated in the dark for 2 h at 37 °C. Absorbance was measured at 450 nm using a microplate reader.
2.18 Annexin V-FITC/PI apoptosis detection
Apoptosis of neuronal N2A cells was detected using an Annexin V-APC/PI apoptosis analysis kit (Absin, Cat. No. abs50009, 2021). First, primary microglia and Neuro-2a neuroblastoma cells were seeded on a 12-well plate, and the microglia were grouped into control, LPS (1 μg/mL), and LPS + shikonin (5 μM) groups. Samples were treated with LPS for 12 h and shikonin for 30 min, the medium was discarded, fresh medium was added, the medium was collected after 12 h, and N2A cells were added to conditionally culture N2A cells for 12 h. After the conditioned culture was complete, the cells were washed twice with PBS, and 1 mL of trypsin was added to each well and incubated for 3 min to detach. Cells were collected in a flow tube and centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the pellet was washed twice with PBS. Then, 100 μL of 1 × binding buffer was added and the individual samples were mixed by pipetting, and 5 μL of Annexin V-FITC and 5 μL PI Staining Solution were added in the dark. The cells were then incubated for 10 min at room temperature. The cells were analyzed using a FACSCalibur flow cytometer, and the final data were processed using FlowJo flow cytometry analysis software.
2.19 Mito-Tracker Red assay
The morphology of cell mitochondria was observed by labeling mitochondria with MitoTracker Red (AAT Bioquest, Cat. No. 22698, 2020). Microglia were seeded in a confocal dish. After the cells were attached, LPS (12 h) and LPS+Shikonin (30 min) stimulations were performed. The samples were washed twice with PBS, 10 μM MitoTracker Red was added, and the cells were incubated for 20 min. Following this, the cells were added to a medium containing 1 μM Hoechst, incubated for 1 min in the dark, and mitochondrial morphology was observed under a laser confocal microscope (ZEISS, LSM800).
2.20 Mitochondrial membrane potential (MTΔΨ)
A mitochondrial membrane potential detection kit (JC-1) (Beyotime, Cat. No. C2006, 2021) was used to detect mitochondrial membrane potential. First, the cells were seeded into a 12-well plate and divided into the control, LPS (1 μg/mL), and LPS+Shikonin (5 μM) groups. The samples were treated with LPS for 12 h and shikonin for 30 min, collected in a flow tube, and centrifuged at 1000 rpm for 5 min. Next, 1 mL of JC-1 staining working solution was added and the cells were incubated for 20 min. After incubation, the cells were washed twice with 1 × JC-1 staining buffer and 500 μL of 1 × JC-1 staining buffer was added. Finally, the mitochondrial membrane potential was determined using a FACSCalibur flow cytometer, and the final data were processed using the FlowJo flow cytometry analysis software.
2.21 PKM2 nuclear translocation by immunofluorescence staining
Microglia were seeded into a 12-well plate and then divided into control, LPS (1 μg/mL), and LPS+Shikonin (5 μM) groups. The cells were fixed with 4% paraformaldehyde for 15 min. After washing with PBS, cell membranes were lysed with 0.5% Triton for 20 min at room temperature. The cells were blocked with PBST containing 1% BSA for 30 min. Then, the cells were incubated with PKM2 antibodies (RRID: AB_1904096, 1:200, CST) at 4 °C overnight. and The next day, after washing, the cells were incubated with the corresponding fluorescent secondary antibody (RRID:AB_2722623, 1:200, Abcam) for 1 h at room temperature. Finally, 4ʹ-6-Diamidino-2-phenylindole (DAPI; Southern Biotech, Cat. No. 0100–20, 2020) was added to each plate, and the protein expression of PKM2 was observed using an inverted fluorescent microscope (OLYMPUS).
2.22 Co-immunoprecipitation (Co-IP)
BV2 cells were seeded into 12-well plates. Cells were lysed using 200 μL of immunoprecipitation lysis buffer and 10 μL of the lysates were collected as input samples. PKM2 antibody (RRID: AB_1904096, 1:100, CST) was added to the remaining lysates and incubated at 4 ℃ overnight. Subsequently, 20 μL Protein A/G magnetic beads (MCE, Cat. No. HY-K0202, 2021) were added at 4 ℃ for 2 h. After washing and boiling, immunoprecipitates were separated by SDS-PAGE and probed with an MFN2 antibody (RRID: AB_2266320, 1:300, Proteintech).
2.23 Statistical Analysis
GraphPad Prism 8 software was used for all statistical analyses. The Shapiro-Wilk test was used to determine that the data were normally distributed. Two-way analysis of variance was used to compare two groups in multiple groups, and the Bonferroni method was used to compare the differences between the two groups. The results were presented as mean ± SD of ≥ 3 independent biological replicates. Significant differences were defined as P < 0.05.