Materials and Reagents
Cyclovirobuxine D (CVBD) (>98% purity) was purchased from Sichuan Weikeqi Biological Technology Co., Ltd. (Chengdu, China). Soybean lecithin (injection grade, >98% purity), cholesterol, polysorbate 80 and DL-α-Tocopherol were purchased from Shanghai Aladd in Biochem Technology Co., Ltd. (Shanghai, China). Angiopep-2 (TFFYGGSRGKRNNFKTEEY) was synthesized by GL Biochem Co., Ltd. (Shanghai, China). DSPE-PEG-2000 was purchased from Xi'an ruixi Biological Technology Co., Ltd. (Xian, China). Nimodipine (NMDP) was purchased from Bayer Healthcare Co., Ltd. (Beijing, China). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and 0.25% (w/v) trypsin solution were purchased from Gibco (Grand Island, New York, USA). All other chemicals were of analytical or chromatographic grade.
Eighty male Sprague-Dawley rats of SPF grade (250-270 g, approval No. SYXK 2018-0001) were provided by the Experimental Animal Center of Guangzhou University of Chinese Medicine. All rats had free access to a standard diet and drank water, and they were housed in a room at 24.0 ± 0.5°C with a 12 h/12 h cyclic lighting schedule. This study was approved by the Animal Ethics Committee of Guangzhou University of Chinese Medicine (Approved No. S2018034).
Cerebral Artery Occlusion (MCAO)-Induced Focal CIRI Model
MCAO-induced CIRI was established as follows . Rats were anesthetized via an intraperitoneal injection of pentobarbital sodium. Then, the left common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA) were carefully exposed. The ECA needed to be ligated at a proximal location. The CCA and ICA were temporarily nipped. A suture was inserted from an incision on the CCA into the ICA until the head of the suture arrived at the bifurcation of the ICA and middle cerebral artery (MCA). The length of the nylon suture was approximately 18 mm from the bifurcation of the CCA/ECA. After 1.5 h, the suture was removed to restore blood supply to the MCA area via reperfusion. After the surgery, neurological deficits were evaluated and scored. The rats in the Sham group underwent the same surgical procedure without a suture occlusion.
All animals were randomly and averagely divided into four groups (n = 20) as follows: Sham (distilled water, p.o.), Model (distilled water, p.o.), NMDP (NMDP at a dosage of 0.03 g/kg, p.o.), and The T80-An2-CVBD-Lps group (CVBD) (CVBD at the dosage of 0.2mL/per rat, Nasal drip). Distilled water, NMDP or CVBD were administered once daily for a week after surgery.
Neurological Behavior Assessment
Neurologic deficit scoring was performed on days 1, 3 and 7 after surgery. The criteria were as follows: (1) lifting rats by the tail and observing the flexion-extension of their front legs (0-4 points); (2) placing rats on the ground and examining the resistance force of their shoulders (0-3 points); (3) placing rats on a metal net and examining the muscular tension of their front legs (0-3 points); and (4) circling to the left or right (0-3 points). Higher scores indicated more severe neurological deficits.
Cerebral Infarction Size
Rats were sacrificed using anesthesia, and then whole heads were removed via decapitation for sample collection. Cerebrums were completely peeled off, and other tissues were carefully removed, including cerebella, olfactory bulbs, and low brain stems. The sample-collecting process was performed on ice. Samples were quickly frozen at -20°C for 30 min and later removed to be made into coronal sections. These sections were further preprocessed as follows: stained in 2% TTC (2,3,5-triphenyl-2H-tetrazolium chloride, Amresco, center valley, PA, USA), incubated in the dark for 20 min, and then fixed in 4% paraformaldehyde (Servicebio, Wuhan, Hubei, China) for 1 h. The TTC staining results were digitally scanned and analyzed using Image-Pro Plus 6.0.
Six rats from each group were used for pathological analysis. The cerebral tissue around the optic chiasma were sliced into coronal sections, fixed in 4% paraformaldehyde, dehydrated through a graded alcohol series, mounted in paraffin, cut into cross-sections at 4 μm, and stained with hematoxylin & eosin (HE staining) or 0.1% cresyl violet-luxol (Nissl staining). HE staining was used to evaluate the pathological changes of cerebral tissue, and Nissl staining was used to evaluate the morphology, amount, and distribution changes of Nissl bodies.
The MRI study of the characteristics of the ischemic injury of rat brains and blood vessels was carried out using small animal 7T PharmaScan70/16 US (Bruker BioSpin, USA). One animal from each group was randomly selected for the MRI scan on the 7th day after the MCAO operation. The rats were anesthetized with 5% isoflurane-nitrogen/oxygen (70% / 30%) mixed gas and maintained with 1% isoflurane. The specific parameters of T2 WI imaging are as follows: TE=30.5ms, TR=3000 ms, layer thickness=0.8 mm. The parameters of 3D-TOF MRA imaging are set as follows: TE=2.7 ms, TR=15 ms, layer thickness=0.3 mm. After scanning, the raw data obtained by ParaVision 6.0.1, and analysed by RadiAnt DICOM Viewer.
The impact of CVBD on oxidative stress in acute ischemic stroke models was assessed by lipid peroxidation (LPO), superoxide dismutase (SOD), malondialdehyde (MDA) and catalase (CAT). The measurements and calculations were strictly detected with commercial kits (Jian-cheng, Nanjing, Jiangsu, China).
Western blot was applied to detect protein expressions of LC-3, Nrf2, HO-1, NQO1, GCLM, p62 and Beclin1 (Abcam, MA, USA). Briefly, tissues were lysed in RIPA buffer (Cwbio, Beijing, China) and centrifuged at 12,000×g, 4°C for 10 min. After dilution, degeneration and centrifugation, the supernatant was added to a prepared sodium dodecyl sulfate-polyacrylamide gel to separate proteins via electrophoresis. Proteins were then transferred to a polyvinylidene difluoride membrane. After blocking with 5% bovine serum albumin for 1 h, the membrane was probed overnight at 4°C with anti-NQO1, anti-LC3B, anti-GCLM, anti-active p62, anti-Beclin1, anti-Nrf2, anti-heme oxygenase 1 (Abcam, MA, USA), anti-GAPDH or anti-β-actin antibodies (Cwbio, Beijing, China). The membrane was incubated with secondary antibody (IRDye 800CW Goat anti-Rabbit or IRDye 800CW Goat anti-Mouse were purchased from LI-COR, Inc (Lindon, Nebraska, USA)). Bands were quantified using Image-Pro Plus 6.0.
HT22 cells were kindly gifted by Prof. Wang Lisheng from Guangzhou University of Chinese Medicine. Cells were cultured in DMEM supplemented with 10% fetal bovine serum (v/v) and antibiotics (100 units/l penicillin, 100 mg/l streptomycin) at 37°C in an incubator with 5% CO2.
Establishment of OGD/R Cell Model and Cell Treatment
After the culture medium was replaced with glucose-free DMEM at 37°C in an incubator with 5% O2 for 9 h, the cells were placed with complete medium and then incubated with 20% O2 for 1 h to finish the process of OGD/R. Cells were divided into the following groups: Normal group (NC) (cells were cultured in normal environment without any stimuli); OGD/R group; OGD/R + 9.38 μM CVBD liposomes (CVBD-L); OGD/R + 18.75 μM CVBD liposomes (CVBD-M); OGD/R + 37.5 μM CVBD liposomes (CVBD-H); OGD/R + chloroquine (CQ); OGD/R + CQ + 37.5 μM CVBD liposomes. CQ (S4157, Selleck, TX, USA) was the specific inhibitor of autophagy and the applied concentration in the study was 25 μM. Cell viability was measured by CCK-8 kits. ROS was detected by commercial kits (E004, Jian-cheng, Nanjing, China) and analyzed by Real-Time Live-Cell Imaging System (IncuCyte ZOOM, Essen Bioscience, USA).
Mrfp-GFP-LC3 Adenovirus Transfection
After transfected, cells were treated with/without OGD/R according to the mentioned above. mRFP-GFP-LC3 adenoviral vectors were purchased from HanBio Technology (Shanghai, China). Adenoviral infection was performed according to the manufacturer’s instructions. LC3 puncta were examined with Zeiss LSM710 confocal microscope (Carl Zeiss) fitted with a 40× oil immersion objective  .
Data were analyzed using SPSS 22.0 statistical software and presented as the mean ± SD. A t-test was used to compare two independent samples, whereas one-way analysis of variance was applied to analyze between-group differences. In addition, a least significant difference test was used for two-sample comparisons between groups. P<0.05 was considered statistically significant. Graphs were created using GraphPad Prism 6.0.