Materials
The human wild-type CST, hChgA352–372(SSMKLSFRARGYGFRGPGPQL), was purchased from Phoenix Pharmaceuticals (Belmont, CA, USA). Anti-Cytochrome C (SAB4502234) antibody was obtained from Sigma-Aldrich. Anti- Caspase-9 (9502), anti- cleaved-Caspase-9 (9505), anti-p-PERK (3179), anti-IRE1 (3294), Anti-ATF4 (D4B8)(#11815), Anti-CHOP (L63F7) (#2895), and anti- PERK (5683) were commercially obtained from Cell Signaling Biotechnology. Anti-IRE1 (phospho S724) (ab48187), and Anti-GADD34 antibody (ab126075) were purchased from Abcam, and anti-b-actin(sc-10731) were obtained from Santa Cruz Biotechnology.
Animals, ischemic/reperfusion model, and Cst administration
All animal procedures complied with the Animal Management Rule. The care and use of the laboratory animals were approved by the Laboratory Animal Ethics Committee of Suqian First People's Hospital. Experiments were performed on adult male Sprague–Dawley (SD) rats (180–200 g, 8 weeks), supplied by Jining Medical University, raised at a constant temperature. The scientific project was supervised and approved by Suqian First People's Hospital.
The schedule of Cst treatment and the behavioral test was graphically presented as in Fig 1A. All rats were randomly divided into four groups: control group, I/R group, I/R and Cst-treatment group, I/R, and vehicle-treatment group. Each group contained 6 independent animals.
The surgical procedures were described previously[18]. In brief, after being anesthetized, transient cerebral ischemia was induced by four-vessel occlusion (4-VO) before rats' both vertebral arteries were occluded permanently by electrocautery and common carotid arteries were exposed, subsequently, rats were recovered for 24 h and fasted overnight. To induce cerebral ischemia, aneurysm clips were used to occlude both carotid arteries. After 15 min of the occlusion, the aneurysm clips were removed for reperfusion.
The administration of Cst into the rat was described previously and with slight modulation[40]. In brief, rats were started to be administered with catestatin 2 days after adaptation to the experimental environment by intraperitoneal injection. Considering the half-life of catestatin (12 hours), its administration was done daily for 7 days (0.25 mg/kg per day B.W).
Open field test
The open field apparatus consisted of a square, opaque acrylic container, and a video camera fixed 1 m above the arena for tracking the rats' movement. A computerized tracking system was used to analyze the images and measure the speed and distance of movement. The individual rat was placed in the middle of the chamber for each trial. After adaptation, the behavior of each rat was recorded. During the interval between trials, rats were returned to its home cage in the same room and the open field was wiped clean with a slightly damp cloth. The number of rearing events, grooming sessions, total distance, and speed traveled were recorded. Meanwhile, to assess the anxiety of rats in the open field, the time spent in the central area was recorded. All rats were randomly divided into four groups: control group, I/R group, I/R and Cst-treatment group, I/R, and vehicle-treatment group. Each group contained 6 independent animals.
Morris water maze (MWM) testing
MWM testing was employed to evaluate the memory and learning capacity on the 7th day after reperfusion and ischemia. In brief, the rats were given 3 swimming trials per day and lasted for 4 consecutive days. The rat was given a maximum of 120 s to find the hidden platform and allowed to stay on it for 20 s. The proportion of time and swimming distance that the rat spent in the quadrant where the platform previously located was recorded and used as a measure of memory. Morris maze performance was analyzed for latency and distance using the ANY-maze video tracking system (Stoelting, Wood Dale, IL, USA) with a CCD camera. Each group contained 6 independent animals.
Rats were anesthetized with chloral hydrate and underwent perfusion with PBS, followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB). Brains were removed, post-fixed overnight in paraformaldehyde, processed, and embedded in paraffin. Coronal brain sections were cut and deparaffinized in xylene and rehydrated in a gradient of ethanol and distilled water. The sections were stained using hematoxylin and eosin and examined with a light microscope. The neuronal density of the hippocampal CA1 pyramidal cells represented the number of cells per 1 mm length which was counted under a light microscope. Each group contained 6 independent animals, and 12 slides from per rat were randomly selected for statistic analysis.
Immunofluorescence and TUNEL assay
Tissue sections were deparaffinized in xylene and rehydrated in a gradient of ethanol and distilled water and then washed in PBS, permeabilized in 0.25% PBS-Triton X-100 and blocked with 3% BSA for 30 min. Then, the slices were incubated overnight at 4 °C with the primary antibody. Following PBS washes, secondary antibodies were incubated at room temperature for 1 h. Slices were mounted on Fluroshield with DAPI and coverslipped. TUNEL assay was performed following the manufacturer’s instruction for a kit (#G3250, Promega, Madison, WI, USA). Slides were examined using a confocal laser-scanning microscope (Flouview FV 1000, Olympus, Japan), and the photos were analyzed using ImageJ software. Each group contained 6 independent animals, and 6 sections from per rat were randomly selected for statistic analysis.
Western blot
The concentration of proteins from tissue homogenates or cell lysates were quantified using Easy II Protein Quantitative Kit (based on BCA, Transgen Biotech). Equal protein samples were electrophoresed using SDS-PAGE gel and then were transferred to the PVDF membrane. After blocking using 5% (w/v) BSA in TBST for 60 min at room temperature, primary antibodies were incubated in recommended dilution at 4°C overnight, and the HRP-conjugated secondary antibodies were incubated. Proteins were detected using Tanon™ High-sig ECL Western Blotting Substrate Kit (Tanon, CAT: 180-501) according to recommended instructions. The protein bands were scanned and analyzed using Bio-Rad ChemiDoc MP (Bio-Rad CAT: 170-8280).
RNA Extraction and Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR)
Total RNA was extracted from rats hippocampus neurons by Qiagen RNeasy Mini Kit according to the manufacturer’s instruction. Equal amount RNA was subjected to reverse transcription for cDNAs using Takara PrimeScript RT-PCR Kit (Takara, Dalian, China).
Real-time PCR was carried out using the TB Green Fast qPCR Mix (Takara, Dalian, China). Quantification was done using ΔΔCt values and reference gene GAPDH was used as the internal control. The primers used in the present study were listed as follows: ATF4 forward, 5′-ATG GCG CTC TTC ACG AAA TC-3′; ATF4 reverse: 5′-ACT GGT CGA AGG GGT CAT CAA-3′; CHOP forward: 5′- GTC CTG TCC TCA GAT GAA ATT GG-3′; CHOP reverse: 5′- GCA GGG TCA AGA GTA GTG AAG GTT-3′; GADD34 forward: 5′- TTT CTA GGC CAG ACA CAT GG-3′; GADD34 reverse: 5′- TGT TCC TTT TTC CTC CGT GG-3′; GAPDH forward: 5′-ATC ACT GCC ACC CAG AAG AC-3′; GAPDH reverse: 5′-ATG AGG TCC ACC ACC CTG TT-3′.
Measurement of Reactive Oxygen Species(ROS) Production
The intracellular reactive oxygen species (ROS) was determined using 2,7-dichlorodihydrofluorescein diacetate (H2-DCFDA), an intracellular ROS indicator, as previously reported[26]. Cultured cortical neurons were incubated with 10 nM H2-DCFDA for 1 hat 37_C in a dark place. Then, the neurons were resuspended with PBS before being read by a fluorescence plate reader (excitation wavelength of 480 nm, the emission wavelength of 530 nm).
Measurement of Mitochondrial Membrane Potential (MMP)
To monitor the mitochondrial membrane potential (MMP), the neurons were incubated in 10 mM fluorescent dye rhodamine 123 (RH 123), and subsequently, washed with PBS three times before reading the fluorescence with a fluorescence plate reader (excitation wavelength of 480 nm, the emission wavelength of 530 nm)[26].
Statistical evaluation
For animal experiments, six animals were independently selected as samples in all groups for behavior tests, western blotting, and histology assays. Image J software was used for semi-quantitative analysis of the bands. Values were expressed as the means ± SEM. Student’s t-test was used for the mean comparison between two groups, and two-way ANOVA was used for multiple group comparisons followed by Bonferron correction post hoc test. p-value < 0.05 were considered to be statistically significant.