Timed pregnant female Sprague–Dawley (SD) rats were purchased from Animal Centre of Kunming Medical University and housed in individual cages under a 12-hour (h) light/dark cycle, with food and water available throughout the study. Seven-day-old postnatal pups were fed for the following experiments. The animal study was legally approved by the Animal Care & Welfare Committee of Kunming Medical University with the approval number: 2015-1A. All experiments conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. Rats were randomly arranged to the four groups for behavioral tests (n = 5/group): HI group (rats were subjected to HI insult), Sham group (rats were subjected to exposure of the right carotid artery only), HI + SCU + GAP43+/+ group (HI rats received SCU administration), HI + SCU + GAP43−/− group (GAP43-knockout rats were subjected to HI insult and received Scu administration), and the two groups for TTC staining (n = 5/group): HI-GAP43+/+ group (rats were subjected to HI insult) and HI-GAP43−/− group (GAP43-knockout rats were subjected to HI insult). Twenty 1-day-old newborn rats were used for the collection and cultures of primary cortical neurons.
Primary cortical neurons cultures
The culture of primary cortical neurons was carried out as previously described . Briefly, the cerebral cortexes of 1-day-old neonatal rats were harvested, homogenated and culture. The complete culture medium composed of DMEM/HIGH GLUCOSE, 10% fetal calf serum and 1% penicillin–streptomycin solution as used for cell culture. Neurons were plated in 6-well plates (Corning, United States) coated with poly-d-lysine and laminin (Sigma-Aldrich, St. Louis, MO, United States) at a density of 2–5 × 105/ml, and incubated at 37 °C, 5% CO2. Moreover, the complete medium was replaced by neuron specific medium (neuron basal + 2% B27, no serum) (Invitrogen, Carlsbad, CA). The culture medium was then changed the next day. Neurons were divided into 4 groups (n = 3 well/group): normal group (neurons without any treatment); OGD group (Neurons were subjected to OGD); Control group (OGD neurons received 1/3000 DMSO); OGD + Scu 3 μM group (OGD neurons received 3 μM Scu administration).
Oxygen–glucose deprivation model
Cultured primary cortical neurons for 5 days were firstly washed with PBS for three times and then placed into the glucose-free medium (Gibco, USA) at 37 °C. Subsequently, the cells were transferred into a hypoxia chamber equipped with a compact oxygen controller to maintain the inner concentration of 95% N2, and 5% CO2 at 37 °C for 2 h. Afterwards, these obtained cells were put back into normal DMEM medium with 95% air and 5% CO2, and incubated for 24 h for reoxygenation.
Scu administration in vitro
Firstly, 150 mg Scu (Batch no.20200602, Longjing Tech, Kunming, China), after being exposed under ultraviolet irradiation for half an hour, were dissolved by 1 ml DMSO into 150 mg/ml and then implanted into the previously cultured normal primary cortical neurons and neurons post OGD. As for vehicle treatment, the equivalent 1/3000 DMSO was added into the normal group and OGD group. SiRNA of GAP43 was performed to verify the role of GAP43 expression on HI injury. In each group, cells were observed at 24 h, 48 h and 72 h respectively after drug administration to monitor the survival of cells.
Stimulation ratio measurement
Cell counting kit-8 (CCK-8) was used to detect the 50% effective concentration (EC50) of primary cortical neurons under Scu administration at concentrations of 0.1 μM, 0.2 μM, 0.5 μM, 1 μM, 3 μM, 10 μM, 15 μM, 50 μM, 100 μM. Following Primary cortical neurons were cultured for 7 days, OGD was performed and then neurons (1 × 105 per well) were seeded into 96-well plate in a total volume of 100 μl medium containing 10% FBS (Hyclone, USA) and 1% PBS (Hyclone, USA). Twenty-four h after culturing, Scu was added into cells for co-incubation. CCK8 solution was subsequently added into a tri-gas incubator for 4 h. Twenty-four h later, the spectrometric absorbance at 490 nm was measured by a microplate reader (Model 680; Bio-199 Rad, Hercules, CA, USA). All the procedures were performed in triplicate and repeated at least three times.
Immunofluorescence staining was performed to observe the status of damaged neurons with Scu administration. Briefly, at 24 h post OGD, the cells in 96-well plate were washed three times with 0.01 M PBS and fixed using 4% paraformaldehyde for 10 minutes (min), followed by subsequent washes with 0.01 M PBS. Then, the cells were blocked with 0.1% sodium citrate and 5% horse-goat serum plus 0.3% TritonX-100 prior to incubation overnight at 4 °C with primary antibodies (Tuj1, mouse, 1:200, Abbkine). After washing three times with 0.1 M PBS (pH 7.4) containing 0.1% Tween 20 for 5 min each time, sections were incubated for 1 h at 37 °C with secondary antibody labeled with fluorescent dyes (anti-mouse, Abbkine, 1:100). Finally, images were taken under a fluorescence microscope and the number of positive cells of Tuj1 was observed.
Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling (TUNEL) staining
TUNEL staining was employed to detect neuronal apoptosis. Sections were fixed with 4% paraformaldehyde for 10 min after rinsed three times with PBS for 5 min each time. Subsequently, being rinsed with PBS again, sections were sealed with PBS containing 0.1% Triton in the ice bath for 2 min. The TUNEL reaction mixture was prepared in the dark: enzyme solution and label solution in a ratio of 1:9 on ice. The specimens were then put into a dark box to incubate at 37 °C for 1 h, and DAPI containing anti-fluorescence quencher were added to stain the cells which were incubated for 3 min at room temperature. A high-content cell imaging system was finally used for picture collection. Positive cells were counted by two researchers blinded to the experiment.
Real-time quantitative polymerase chains reaction (RT-qPCR)
The total RNA fraction was extracted using Trizol reagent (Takara Bio Inc., Otsu, Japan) and reverse transcribed using RevertAid™ First Strand cDNA Synthesis System (Invitrogen). Quantitative PCR reactions were carried out with Power SYBR (DBI Bioscience) according to the manufacturer’s instructions. The expression level of each gene was normalized to that of GAPDH using the 2−ΔΔct method. Primers used for the reactions are as follows:
GAPDH: forward: TGACTTCAACAGCGACAC CCA,
GAPDH: reverse: CACCCTGTTGCTGTAGC CAAA;
GAP43: forward: TGTT GCCGATGGGGTGGAGA,
GAP43: reverse: CCGTTGGAGGCTGGGCTGTT;
PTN: forward: CAGTGGAGTGTGTGTGTGCC;
PTN: reverse: GATTCTGCTTGAGGTT TGGG;
STAT3: forward: GACAAAGACTCTGGGGACG,
STAT3: reverse: ATTG GGGGCTTGGTAAAAA;
JAK2: forward: CGGCTGGGCAGTGGAGAGT,
JAK2: reverse: CGGTGATGGTGCGATTTGG.
PC12 cells cultures
PC12 cell line, purchased from ATCC (ATCC CRL-1721), were maintained in Dulbecco’s modified Eagle’s medium composed of 0.11 g/l sodium-pyruvate, pyridoxine, 10% horse serum, 5% tetracycline-free fetal calf serum, 10 mM l-glutamine, penicillin/streptomycin (100 units/ml and 100 μg/ml, respectively) and 100 μg/ml geneticin. Cells were seeded out at a density of 1.5 × 104 cells/cm2 and incubated at 37 °C with 5% CO2 at 90% humidity. The culture medium was changed every 3 days.
Screening of effective siRNA sequences
Two siRNA sequences and a random control sequence, purchased from Guangzhou Ruibo Company (Guangzhou, China), were designed by referring to the gene database of GAP 43 from NCBI. In short, as PC12 cells grew to 40% confluence, fresh medium containing siRNA fragments was added to cells. Afterward, PC12 cells were randomly divided into the normal group, NC group, Reagent group, F1 group, and F2 group. Moreover, qRT-PCR was employed to verify the interference efficiency 48 h later, and the most efficient siRNA fragment was opted out for the following experiment.
Target interference transfection
After being cultured for 5 days, primary cortical neurons post OGD were randomly divided into 5 groups as described above. Then half of the culture medium was removed before 60 μl 1X buffer, 5 μl siRNA and 5 μl siRNA reagent were added to each well of 6-well plates. Afterward, the neurons were incubated at room temperature for 15–30 min with the addition of 1 ml medium per well 24 h later. After 48 h, the morphology and number of neurons were observed from Leica AF6000 cell station by choosing 5 randomly selected high power fields (200X). The interference efficiency was determined using RT-qPCR following transfection for 4 days.
HI model establishment
A hypoxic-ischemic model of HIE was generated as previously described . Briefly, the 7-day newborn SD rats (weighing 12–15 g, both sexes) were anesthetized with isoflurane (4% for induction, 2% for sustained inhalation anesthesia) and were used for HI model establishment, which were subjected to ligation of right common carotid artery and 2-h hypoxia in an airtight chamber containing 8% O2 and 92% N2. Rats in the sham group were only treated with exposure of the right carotid artery.
Triphenyl tetrazolium chloride (TTC) staining and evaluation of infarction volume
At 24 h after HI, rats were euthanatized under deep anesthesia with 4% isoflurane inhalation for 2 min. After the consciousness of the rats was evaluated by claws clamping, brains were rapidly harvested and sectioned into 2 mm coronal sections in a rat brain matrix (Seino Co., Ltd. Beijing, China) before being immersed in 2% solution of 2,3,5 triphenyl tetrazolium chloride (Sigma Co., St Louis, MO, USA). Afterward, all slices were placed into the incubation chamber at 37 °C for 30 min. Following being fixed by paraformaldehyde, the infarct site was tracked and analyzed by Image J Software (Version 1.43u; National Institutes of Health, Bethesda, MD, USA). Infarct percentage = (infarct area/the whole brain area) × 100%. Brain swelling = the ipsilateral hemisphere area − the contralateral hemisphere area.
Scu administration in vivo
Firstly, 1 g Scu (Batch no.20200602, Longjing Tech, Kunming, China) was dissolved by DMSO into 120 mg/ml after being exposed under ultraviolet irradiation for half an hour, and then diluted into 4 mg/ml by 0.9% normal saline. Subsequently, Scu of 20 mg/kg was administered intraperitoneally at 24 h after HI. The control groups were intraperitoneally injected with the same dose of 1/3000 DMSO. The injection lasted for two weeks. Neurobehavioral scores were performed at 8 weeks after the operation.
Network pharmacological analysis
Traditional Chinese Medicine System Pharmacology Database (TCMSP, http://tcmspnw.com/) was applied for the drug targets of Scu. GO enrichment analysis provides all GO terms that are significantly enriched in targets compared to the genome background and filters the targets that correspond to biological functions. All the targets were mapped into GO terms in the Gene Ontology database (http://www.geneontology.org/), gene numbers were calculated for every term. Pathway enrichment analysis identified significantly signal transduction pathways in targets in the KEGG pathway database (http://www.genome.jp/kegg/). In this study, R software version 3.6.1 (http://www.r-project.org) with several R packages, such as clusterProfiler, org.Hs.eg.db, enrichplot, and ggplot2, was applied to draw the barplot, bubble diagram, and signalling pathway map during GO and KEGG enrichment analysis. The statistics were collected by the ClueGO and CluePedia plugins with the False Discovery Rate (FDR) Correction set as ≤ 0.05 The R packages are available on Bioconductor (https://www.bioconductor.org/).
Construction of GAP43 knockout rat
To confirm the function of GAP43 in HIE, CRISPER/CAS9 technology was applied to constructed GAP43 KO rats which were produced and provided by Cyagen Company (Guangzhou, China). Two targets were designed for GAP43, and two pairs of oligonucleotide chains (TGGGAGAGGTATATCCGGAA and GCTGCCTGG AGACACATCGA) were synthesized to prepare single gRNA. Then, more KO rats were reproduced by mating and detected by genotype identification.
For neonatal rats aging 3–5 days, the toes and tail tips were collected and numbered. Then, rats’ genomic DNA was extracted using Transgen's genomic DNA extraction kit (ee101-12), and PCR detection was performed with the amplification primer: Rat GAP43 forward: TGTTGCCGATGGGGTGGAGA, Rat GAP43 reverse: CCGTTGG AGGCTGGGCTGTT. In addition, the PCR amplification reaction system was as follows: a mixture with 10 µl PCR master mix, 0.6 µl upstream primers, 0.6 µl downstream, 3 µl DNA template and 5.8 µl water. The thermal cycling conditions were performed as: initial denaturation at 94 °C for 5 min, and 35 cycles of denaturation at 94 °C for 30 s, annealing at 59 °C for 30 s, with elongation at 72 °C for 30 s, followed by elongation at 72 °C for 5 min and the storage at 12 °C. Subsequently, agarose gel electrophoresis system was applied to visualize the final genotype detection under U.V after 55 min electrophoresis at 150 V.
Zea-longa score test was performed daily after HI for 2 weeks to verify the successful establishment of the HI model . In terms of long-term neurological function, Morris water maze, Y-maze test, open field test and Rotarod test were performed at 8 weeks post HI as described in our previous study .
GeneMANIA (http://genemania.org), known as a flexible user-friendly web site for single gene queries, multiple gene queries and network search, has been extensively used to generate hypotheses about gene function, analyze gene lists and prioritize genes for functional assays . To further determine the potential network related to GAP43, GeneMania (http://genemania.org/) was applied in this study. The detailed connections among GAP43 and related genes will be returned after query.
Experimental data were analyzed by SPSS 17.0 software. Graphs were generated using Graphpad Prism 6. For comparing multiple groups, a one-way analysis of variance with post hoc Tukey’s test was used. The data between two groups were analyzed by two-tailed Student's t-test. The water maze data were analyzed by two-way analysis of variance with Dunnett’s multiple comparisons test. All the experimental animals and cells were randomly grouped, and the samples were normally distributed. Tukey post-Hoc test was used when assuming homogeneity of variance, otherwise Dunnett’s T3 test was used instead. Differences were considered statistically significant when p < 0.05, and the significance level is shown by *p < 0.05; **p < 0.01; ***p < 0.001.