CGA (purity ≥98%) was purchased from Solarbio (Wuhan, China). Primary antibodies were purchased as follows: TNF-a (ab66579; Abcam, Cambridge, United Kingdom), iNOS (ab178945; Abcam, Cambridge, United Kingdom), β-Actin (#3700; Cell Signaling Technology, MA, United States), Histone H3 (#4499; Cell Signaling Technology, MA, United States), IkBa (#9242; Cell Signaling Technology, MA, United States), P65 (#8242; Cell Signaling Technology, MA, United States), Nrf2 (#12721; Cell Signaling Technology, MA, United States), HO-1 (#43966; Cell Signaling Technology, MA, United States), IL-1β (#12703; Cell Signaling Technology, MA, United States), Sirt1 (DF6033, affinity, United States), SOD2 (AF5198, affinity, United States). The secondary antibodies of Goat Anti-Rabbit IgG and Alexa Fluor®594 labeled were purchased from Bioworld (OH, United States). Dimethylsulfoxide (DMSO) was purchased from Sigma Chemical Co. (St. Louis, MO, United States). Fetal bovine serum, B27, neurobasal medium, L-glutamine (0.5 mM), and Dulbecco’s modified Eagle medium were purchased from Gibco (Grand Island, NY, United States). Malondialdehyde (MDA) content test kit was purchased from Solarbio (Beijing, China). A catalase (CAT) activity detection kit was purchased from Solarbio (Beijing, China). Cell-Counting Kit-8 (CCK-8) was purchased from Dojindo (Kumano, Japan). The nuclear stain 4′,6-diamidino-2- phenylindole (DAPI) was purchased from Beyotime (Shanghai, China). A nuclear and cytoplasmic protein extraction kit was purchased from Beyotime (Shanghai, China). Bovine serum albumin (BSA) was procured from Beyotime Biotechnology (Shanghai, China).
Neonatal Hypoxic-Ischemic Brain Injury Model and Drug Administration
Sprague Dawley (SD) rats (200-250g) were purchased from the Shanghai Zoological Center of the Chinese Academy of Sciences. Animal breeding and experiments were conducted as per the requirements of the Animal Breeding and Use Committee of Wenzhou Medical University. Adult SD rats mate freely, and male pups were used in the experiment on the 7th day after birth (P 7). A modified Rice-Fannuzzi model was used as described previously , and isoflurane was applied to completely anesthetize and maintain P 7 pups. Subsequently, the left common carotid artery of P 7 young mice was separated within 5 min, ligated, and cut. The pups recovered with the mother 2 hours after the operation. The pups rested enough and were placed in a humidified mixed gas chamber composed of 92% N2 and 8% O2 and ventilated at a flow rate of 3 L/min for 2 hours. The above hypoxia equipment was placed in a constant temperature water bath at 37.5°C. We did not ligate the common carotid artery of the pups in the sham operation group since they did not have hypoxia, but the common carotid artery of the pups of the other groups were ligated and the pups were treated separately. After the end of hypoxia, all the pups were caged to continue feeding, awaiting follow-up experiments. For the next few days, the rats were given drugs daily as follows: The CGA pretreatment group was intraperitoneally injected with different concentrations of CGA (150, 300, or 600 mg/kg) immediately after hypoxia, with an interval of 24 hours between each administration, and the young mice were euthanized to establish the most effective concentration. EX-527 (0.5mg/kg) 5ul each (2 mm rostral, 1.5 mm lateral to bregma, and 2.5 mm below the skull surface) was intracerebroventricularly injected, 30 minutes before hypoxia . The needle was held for another 10 minutes after the injection is completed, and then withdrawn at a speed of 1 mm/minute.
2,3,5-triphenyltetrazolium chloride (TTC) staining
2,3,5-triphenyl tetrazolium chloride (TTC) staining method was employed to measure the volume of cerebral infarction based on the previous experimental procedure for assessing the area of cerebral infarction . Brain tissues were collected from P7 young mice, 24 hours after HI injury, stored at -20°C for 15 minutes, and cut into approximately 2mm thick coronal sections. Then, the coronal brain slices were placed in 1% TTC (Sigma, USA) solution and incubated in a 37°C oven in the dark for 30 minutes. Subsequently, the 11% TTC solution was discarded and the tissues were fixed overnight with 4% paraformaldehyde. Image-Pro Plus software was applied to measure the volume of cerebral infarction.
Brain tissue specimens were collected 7 days after HI injury, whereby rats were anesthetized with isoflurane, and the heart was perfused with 25 ml of sterile saline and further perfused with an equal volume of 4% paraformaldehyde. The brain specimen was immersed in 4% paraformaldehyde, stored at 4°C for 24 hours, and embedded in paraffin. Sections were cut at the thickness of 5 μm from the paraffin block to visually show the hemispherical integrity of functional neurons between the cerebral cortex and the hippocampus coronal slices. The brain sections were then deparaffinized, hydrated, and stained with HE or Nissl solution (Sohrab, Beijing, China). Finally, an optical microscope was employed to assess and record the results of histological staining. ImageJ software was used to analyze the results.
Nuclear protein and cytoplasmic protein extraction
The nuclear protein was extracted for western blot detection of nuclear protein changes . Appropriate amounts of 1mM PMSF cytoplasmic protein extraction reagents A and B were mixed at a 20:1 ratio to prepare a tissue homogenate, which was put in a homogenizer for tissue homogenization. Subsequently, the homogenized liquid was transferred to a 1.5mlep centrifuge tube, left to stand on ice for 15 minutes, and then centrifuged (1,500 g for 5 minutes at 4°C). The supernatant (the cytoplasmic protein) was collected, and the pellet was stored at -80℃ waiting for further processing. The precipitate was added to the cytoplasmic protein extraction reagent A containing 1 mM PMSF in a ratio of 1:10, shaken vigorously for 5 seconds, and left to stand on ice for 15 minutes. Following the addition of cytoplasmic protein extraction reagent B at a ratio of 20:1, mixture was shaken vigorously for 5 seconds, left to stand on the ice for 1 minute, and centrifuged (12,000g at 4°C for 5 minutes). The supernatant (cytoplasmic protein) was collected to save the pellet and merged into the previous cytoplasmic protein. After complete aspiration of the supernatant, 50μl of nuclear protein extraction reagent containing 1mM PMSF was added for precipitation. The mixture was vortexed for 30 seconds, then put in the ice bath, and vortexed again for 15-30 seconds every 2 minutes for 30 minutes. The mixture was centrifuged at 12,000 g at 4°C for 10 minutes, and the supernatant was collected as the nuclear protein.
The extracted cerebral cortex tissue or primary neurons were lysed in RIPA lysis buffer containing 1 mM PMSF. A tweezer was used to tear up the cerebral cortex tissue. The tissue was homogenized using a tissue homogenizer, fully sonicated on ice for 10 minutes, and then centrifuged (4°C at 12,000 rpm at a speed of 20 minutes) to obtain the supernatant. Measurement of protein concentration was taken using the BCA kit (Beyotime) and the protein was prepared. Sodium dodecyl sulfate (SDS)-polyacrylamide separation gel electrophoresis was used to separate the protein, and then the bands were transferred to PVDF membrane (Millipore). After blocking with a 5% skimmed milk solution or a 5% BSA solution diluted with TBST for 4 hours, the membrane was incubated with primary antibodies overnight in a refrigerator at 4°C: iNOS (1:1,000), Sirt1 (1:1,000), TNF-a (1:1,000), p65 (1:1,000), β-actin (1:5,000), IkBα (1:1,000), IL-1β (1:1,000), Histone H3 (1:1,000), Nrf2 (1:1,000), and HO-1 (1:1,000). Subsequently, TBST was washed thrice for 5 minutes each time and incubated with the corresponding secondary antibody (1:10,000) for 90 minutes. Afte washing with TBST for 3 times, the blots were visualized using an ECL Plus chemiluminescence reagent kit (RPN3243; Amersham Bioscience, Bensenville, IL, USA) and quantified by the Imaging System (Bio-Rad).
We washed rat brain tissue with PBS, added PBS at a ratio of 1:9 and homogenized the samples on ice. We centrifuged the homogenate obtained above at 12,000 rpm for 10 minutes at 4°C, and took the supernatant (100 μl) for analysis. The enzyme-linked immunosorbent assay (ELISA) (E-EL-R0012c, Elabscience) was used to detect the content of IL-1β in the brain tissue of rats in each group. We set up blank wells, standard wells and sample wells on the ELISA plate. We filled the blank well with 100 μl of standard/sample diluent. The standard wells contained 100 μl of standard samples diluted in multiples, and the sample wells contained 100 μl of experimental samples. Then the plate was incubated at 37°C for 90 minutes. After being added the biotinylated antibody working solution (100 μl/well), the plate was incubated at 37°C for 60 minutes, followed by being washed with washing solution 3 times. Then the plate was incubated at for 30 min with 100 μl horseradish peroxidase conjugate solution. Next we washed it with washing solution for 5 times, then added 90μl of substrate solution to each well, and incubated it for 15 minutes at 37°C. Finally, we added 50μl of stop solution, and immediately measured the optical density of IL-1β at 450 nm wavelength.
Malondialdehyde (MDA) content test
Lipid peroxidation level was detected based on the level of MDA . Rats were euthanized after deep anesthesia with isoflurane three days after the HI injury, and the cerebral cortex tissues of the young rats were isolated and stored at -80°C for subsequent analysis. We weighed about 0.1g of tissue of each group, added 1mL of extracting solution to homogenize in ice bath, centrifuged it at 8000g at 4℃ for 10min, and took the supernatant for next testing. Subsequently, the MDA was extracted and placed in a microplate reader to detect the absorbance at wavelengths of 450nm, 532nm, and 600nm. Finally, according to the MDA content (nmol/g mass) =5× (12.9× (ΔA532 -ΔA600)-2.58×ΔA450) ÷ W. W: sample mass, g; ΔA450=Δ450 measurement-Δ450 blank; ΔA532=Δ532 measurement-Δ532 blank; ΔA600=Δ600 measurement-Δ600 blank, we measured the concentration of MDA.
Catalase (CAT) activity detection
Catalase (CAT) is the most important hydrogen peroxide scavenging enzyme in the body [31, 32]. Using the catalase (CAT) activity detection kit as per the manufacturer’s instructions and a microplate reader, we detected the CAT content according to the change in absorbance over time at a wavelength of 240nm.
Coronal brain tissue slices were stained with GFAP. Briefly, the slices were dried in an oven at 65°C for 3 hours and immersed in xylene to deparaffinize for 20 minutes. The slices were hydrated in gradient alcohol and washed thrice with PBS (5 minutes each time). Following antigen retrieval with sodium citrate, slices were washed thrice with PBS (5 minutes each time), diluted with 0.3% Triton X-100 (PBS), and reacted for 15 minutes at room temperature. We washed slices 3 times with PBS for 5 minutes each time, blocked them with 10% goat serum (PBS), and then incubated slices overnight at 4°C with GFAP (1:200) primary antibody. On the second day, after 3 times wash with PBS, slices were exposed to a secondary antibody (1:200) labeled with Alexa Fluor® 594for 1.5 hours. After washing thrice with PBS (5 minutes each time), they were incubated with DAPI for 10 minutes, washed with PBS, and mounted with an anti-quenching agent. The samples were taken and preserved in an Olympus fluorescence microscope (Tokyo, Japan). ImageJ software was employed to determine the fluorescence intensity.
Morris Water Maze test (MWM)
The Morris Water Maze Test (MWM) is a behavioral experiment for determining the cognitive function of animals . We applied the MWM test, 21 days after the HI injury, to evaluate the learning and memory abilities of experimental animals, whereby they were put to swim to find a platform hidden underwater. A black circular pool with a diameter of 140 cm and a height of 50 cm was prepared in a room protected from noise and light. The water depth of the pool was 1 cm higher than the movable platform. Water was dyed black with non-toxic black ink and the pool was divided into four equal quadrants. The rats were trained for 6 days, after which the platform was removed. The swimming route, incubation time, and the number of times on stage were recorded. The experiment was performed by the SLY-WMS Morris water maze experiment system.
Primary cortical neuron extraction and culture
Primary cortical neurons were extracted from the cerebral cortex of SD neonatal rats (P 0). Briefly, the newborn rat was immersed in 75% alcohol for 20 minutes, the brain was isolated to separate the cerebral cortex and washed thrice with PBS. Then we used 0.25% papain-EDTA solution to digest cerebral cortex tissue at 37°C for 15 minutes. After centrifugation of the final product above, the pellet was introduced to a 6-well cell culture plate coated with poly-D-lysine according to 1x105, incubated (5% CO2 at 37°C for 6 hours). The culture medium was replaced with 2% B27, 0.5 Neural basal medium of mM L-glutamine, penicillin/streptomycin antibiotics.
The cytotoxicity of CGA to primary cortical neurons was detected by the CCK-8 kit. Briefly, primary cortical neurons were extracted and inoculated in 96-well plates (8,000 cells/well). Then, the cells were treated with CGA in a concentration gradient of 0,100,200,300, and 400nM for 24 hours. Finally, 10 μl of CCK-8 solution was added to each well of the 96-well plate and incubated at 37°C for 1.5 hours. A microplate reader (Leica Microsystem, Germany) was used to detect the absorbance of each well at a wavelength of 450 nm.