Materials
L-Borneolum (95% purity) is a crystal made by extraction and processing of fresh branches and leaves of Blumea balsamifera (L.) DC, was purchased from Luodian (Guizhou, China). Nimodipine was obtained from Yabao Pharmaceutical Group Co., Ltd (Shanxi, China). Ethyl carbamate (chemically pure) was obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Tween 80 was purchased from Tianjin Chemical Reagent Co., Ltd. (Tianjin, China). Benzylpenicillin sodium for injection was obtained from Harbin Pharmaceutical Group Holding Co., Ltd. (Heilongjiang, China).
Drug preparation
The dose of l-borneolum in Chinese Pharmacopoeia (2020 edition) was 0.3 g/d (adult in 60 kg). The 10, 20 and 40 times of the clinical dose of l-borneolum were used as the low, medium and high dose group of l-borneolum and it was administrated as 0.05 g/kg, 0.1 g/kg, and 0.2 g/kg as our previous study[14]. Both nimodipine and l-borneolum were dissolved in 5% tween 80 solvent.
Molecular docking
The corresponding crystal structure of the protein is obtained from the protein data bank (https://www.rcsb.org/). The protein is pretreated by the Protein Preparation Wizard module in Schrödinger 2020 (Schrödinger, LLC, New York, NY, 2020), including add hydrogen and missing side chains, remove all water molecules, assign protonation states and partial charges through OPLS2005 force field. After l-borneolum were preprepared using the LigPrep (LigPrep, Schrödinger, LLC, New York, NY, 2020), the original small molecule in the protein is used as the ligand space to dock with the small molecule of l-borneolum. Finally, l-borneolum were docked into the binding site of proteins and evaluated by using the standard precision (SP) scoring function. The interaction relationship between receptor-ligand in molecular docking was visualized with Pymol 1.7.
Ethics statement
All animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory. This study was performed in the Chinese Medicine Pharmacology Research Laboratory of Chengdu University of Traditional Chinese Medicine certified by the State Administration of Traditional Chinese Medicine (Approval NO: TCM-09-315).
Animal handing and experiment design
Male Sprague-Dawley rats, body weight 250-270 g, were obtained from the Chengdu Dashuo Biological Technology Co., Ltd. (Chengdu, China) (Permission No. SYXK (Chuan) 2014-124). In this study, an automatic temperature and humidity monitoring system was used to strictly control the breeding environment of rats. The temperature of the housing room was 25 ± 0.5 ℃. The humidity was 55 ± 5%. The light duration was 12 h: 12 h light-dark cycle. All animals were acclimatized for five days, and were free access to food and water before the experiment.
Rats were randomly divided into seven groups (twelve rats in each group), including control group, sham group, model group, vehicle group, nimodipine group (1.08 × 10-2 g/kg), l-borneolum low dose group (0.05 g/kg), l-borneolum medium dose group (0.1 g/kg), and l-borneolum high dose group (0.2 g/kg). Both the sham group and model group were given an equal volume of normal saline by gavage. The vehicle group was intragastrically administered with the same volume of 5% tween 80 solution. Rats in nimodipine and l-borneolum group were intragastrically administered with corresponding drugs for three consecutive days before modeling.
In this study, the pMCAO model was established after 30 minutes on the third day of prophylactic administration (Figure 2). Rats were anesthetized intraperitoneally with 20% ethyl carbamate solution and then fixed in the supine position. The left common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) of the rats were divided sequentially. Next, the fishing line was inserted 20 mm into the ICA through the beginning of the middle cerebral artery (MCA) to the proximal end of the anterior cerebral artery (ACA), and finally the CCA was ligated. At this moment, the time when cerebral ischemia occurred was recorded. Rats were injected with benzylpenicillin sodium for injection (100,000 units) intramuscularly. No treatment was performed for the control group. For the sham operation group, the neck skin of the rat was incised and the CCA, ECA and ICA were stripped, but the fishing line was not inserted, and the rest of the operation was the same as in other groups.
After the operation, the limbs of rats were hemiplegic to the right, and the right forelimb was bent to the inside when the tail was lifted. Rats were placed on the ground and turned to the right, or even to the right hemiplegia (as shown in Figure 1B below). The above-mentioned neurological deficits in rats indicated that the model was successful.
Rats with successfully prepared CIS model were given the corresponding drugs for three consecutive days. After the final administration, rats were sacrificed under 20% ethyl carbamate solution. Also, blood samples and brain tissue were collected. Brain samples were fixed in 10% neutral buffered formalin or stored at -80 ℃ for the detection of mRNA and protein expressions.
Recovery time detection
The righting reflex disappeared about 3 minutes after the rats were anaesthetized with ethyl carbamate, and the time point was recorded. After the CIS model was prepared, the righting reflex of the rats recovered, and the time point was recorded. The difference between the before and after time points is the time to wake up the rats after anesthesia.
Rectal temperature detection
The rectal temperature of rats in each group was measured 0.5 h before the first preventive administration, and then 0.5 h after each administration (a total of 8 measurements). The rectal temperature of rats in each group was measured every day. The rectal temperature difference before and after administration (Δt), before and after modeling (Δt), and before and after drug withdrawal (Δt) were calculated, respectively.
Evaluation of neurological function score
The mNss and Longa scoring methods were used to assess the neurobehavioral scores of rats. At 96 h after the pMCAO surgical ischemia, the rats in each group were neurologically scored according to the mNss neurological score table. In addition, 96 h after pMCAO surgery ischemia and awakening, the rats in each group were again scored for neurological deficits according to the Zea Longa five-level 4-point method. The blinding method was adopted. Three people scored each rat in parallel, and the average value was taken for statistical analysis.
Cerebral infarction rate detection
Rats were anesthetized by intraperitoneal injection with 20% ethyl carbamate and then sacrificed. The cerebral tissue was quickly taken out, and the olfactory bulb, brain stem, and cerebellum were removed. The remaining tissue was cut into 2 mm serial sections and incubated at 37 ℃ for 30 min in 2% TTC solution (dissolved in physiological saline). These tissues were finally fixed in 4% paraformaldehyde solution for 24 h. The infarct area is white, and the normal area is red. The infarcted white area of each rat was carefully peeled off and weighed. Simultaneously, the total weight of the brain slice of each rat was weighed. The calculation formula of cerebral infarction rate is as follows: cerebral infarction rate = weight of infarct area/total weight of slice × 100%.
Morphological examination
HE staining was used to detect the morphological changes of rat cerebral tissue. The brainstem, cortex and hippocampal CA3 area of cerebral tissue were quickly collected and then fixed in neutral formalin. The paraffin-embedded brainstem, cortex and hippocampal CA3 area were cut into sections with an average of 5 μm. Finally, the brainstem, cortex and hippocampal CA3 area sections were deparaffinized, dehydrated and stained with hematoxylin eosin (H&E) for pathological evaluation. The pathological changes of brainstem, cortex and hippocampus CA3 area were observed under a 200 folds optical microscope.
Observation of the ultrastructure of rat brain cortex
Rats were anesthetized by intraperitoneal injection with 20% ethyl carbamate after the last administration for 24 h. The heart and great blood vessels of the rats were exposed. The syringe needle infused with physiological saline was inserted into the ascending aorta from the apex of the left heart of the rat. The left atrial appendage of the rat was incised as perfusion fluid. The blood in the liver of the rats was washed until the color becomes lighter or even white. Rats were perfused and fixed with 4% paraformaldehyde until the whole body became stiff, which was maintained for about 60 min. After the fixation was sufficient, the brain was removed by craniotomy, and the corresponding cerebral cortex on the ischemic side was removed. The surface water was absorbed by filter paper and fixed in 2.5% glutaraldehyde solution, and stored at 4 ℃ for later use. After the rat brain cortex was fixed, dehydrated, embedded, sliced and stained, the microvessels and blood-brain barrier of the sample are photographed using Hitachi H-600 IV transmission electron microscope (8000 ×).
Protein extraction and Western blotting analysis
Total protein of cerebral tissue was extracted by RIPA buffer (Lot. No. 20180515, Solarbio, Beijing, China) supplemented with 1/100 phenylmethylsulfonyl fluoride (PMSF) (Lot. No. 20180313, Solarbio, Beijing, China). The lysate was centrifuged at 15, 000 g for 10 min at 4°C and the supernatant was collected. Protein concentration was determined with a BCA protein assay kit (Lot. No. 20180218, Solarbio, Beijing, China) in line with the manufacturer's recommendations. Protein samples were separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels, and then transferred to polyvinylidene difluoride (PVDF) membranes (0.45 μm, Millipore, MA). The membranes were washed twice with TBS containing 0.1% Tween 20 (TBST) and incubated with blocking solution for 2 h at room temperature. The membranes were incubated overnight at 4 °C with the following primary antibodies: anti-Notch1 rabbit polyclonal antibody (3608s, Cell signaling technology, dilution: 1:1,000), anti-DLL-4 rabbit polyclonal antibody (NB600-892, Novous, dilution: 1:1,000), anti-VEGF-A mouse monoclonal antibody (ab1316, Abcam, dilution: 1:200), anti-Hes1 rabbit polyclonal antibody (D6P2U, Cell signaling technology, dilution: 1:1,000), anti-Hes5 rabbit polyclonal antibody (NBP2-27174, Novous, dilution: 1:100), anti-Hey1 rabbit polyclonal antibody (NBP1-82377, Novous, dilution: 1:1,000), anti-p65 rabbit polyclonal antibody (8242s, Cell signaling technology, dilution: 1:1,000), anti-β-actin polyclonal antibody (dilution: 1:5,000). The second day, blots were washed five times with TBST for 20 min and incubated with goat anti-rabbit IgG (H+L)/HRP secondary antibody (dilution:1:1,000) for 1 h at room temperature. Blots were again washed five times with TBST and then visualized using ECL Plus detection kit (Amersham, UK). Quantification of bands was carried out by densitometric analysis using Bio-Rad Quantity One. β-actin was used for an internal control to normalize the data.
Immunohistochemical staining
The expression of Caspase-3 levels in cerebral tissue was measured by immunohistochemical staining. Immunohistochemistry staining was performed as follows. The cardiac tissue was fixed with 4% paraformaldehyde and embedded in paraffin. anti-Caspase-3 rabbit polyclonal antibody (9662s, Cell signaling technology) was used. The images were photographed on Nikon Eclipse Ni-U microscope plus Imaging Software NIS-Elements 4.0 (Nikon, Japan) at 100 magnification.
Statistical analysis
SPSS statistical analysis software (version 17.0, SPSS Inc., Chicago, IL, USA) was used for one-way ANOVA analysis. Data were expressed as mean ± standard deviation (`X ± SD). GraphPad Prism software (version 8.2.0) was used to visualize the results. Differences were statistically significant at P < 0.05 and highly significant at P < 0.01. Other matters related to materials and methods are showed in the figure legend.