Materials
PTS (Lot No.: 20190318) was provided by Chengdu Huasun Technology Group (Chengdu, China). Aspirin enteric-coated tablets (Lot No.: BJ45216) were purchased from Bayer Health Care (Milano, Italy). Xuesaitong soft capsules (Lot No.: 20190322) with a 75% content of PNS (saponins extracted from notoginseng, including Ginsenoside Rg1, Ginsenoside Rb1, Ginsenoside Re, Notoginsenoside R1, and Ginsenoside Rd) were purchased from Kunming Shenghuo Pharmaceutical (Kunming, China). Reference Standards of Ginsenoside Rg1, Ginsenoside Re and Notoginsenoside R1 were purchased from the National Institutes for Food and Drug Control (Beijing, China). Toluidine Blue O was purchased from Scientan (Beijing, China). TTC, and other chemical reagents were purchased from Chengdu Chron Chemicals (Chengdu, China). All chemicals were of analytical grade and used as received. The kits for determining cAMP, TXA2, TXB2, PGI2, and 6-keto-PGF1α were purchased from Elabscience Biotechnology (Wuhan, China). The platelet protein extraction kit was obtained from Bestbio Biotechnology (Shanghai, Chinese). The kits for determining NO and NOS were purchased from Nanjing KeyGen Biotech (Nanjing, China). The peripheral blood platelet separation kit was obtained from Haoyang Biological Manufacture (Tianjin, China). PAF was purchased from Abcam (Cambridge, USA). GP1BA was purchased from Proteintech (Rosemont, USA). The CM5 Chip, Peptide Coupling Kit, GST Capture Kit and other buffers used in the SPR test were purchased from GE Healthcare Life Sciences.
Division of groups and the middle cerebral artery occlusion model
All research animals were provided by the Experimental Animal Center of Chengdu University of Traditional Chinese Medicine and raised in SPF-class housing in a laboratory with controlled conditions (20-22℃, relative humidity of 50-60%, and 12 h light-dark cycles). The animals were fed with water and a commercial rat pellet diet. All animal experiments were approved by the Sichuan Provincial Committee for Experimental Animal Management. After acclimation, testing drugs were orally administrated orally to the adult male SD rats (weighing 300±20 g) at 10 mL/kg by gavage, once a day for six consecutive days. Then the animals were divided into seven groups with different forms of administration: sham surgery and model control (distilled water), positive control (PNS at 28 mg/kg), high-, medium- and low-dose PTS (100, 50 and 25 mg/kg, respectively) as well as the combined treatment (PTS at 50 mg/kg and aspirin at 21 mg/kg). Specifically, the MCAO model of rats was established at 30 min after gavage on Day 6, the operation of modeling was improved according to Longa’s method [23] as described below: the rats underwent fasting for a period of 12 hours prior to the experimental procedures with water available ad libitum, and were anesthetized intraperitoneally using 10% chloral hydrate at 35 mg/100 g body weight. After the righting reflex disappeared, the animals were fixed in a supine position for neck fur removal and disinfection, and then incised at the midline of the neck. After the left common carotid artery (CCA) was located, the external carotid artery (ECA) and internal carotid artery (ICA) were separated while injury to vagus was avoided. Then the proximal CCA, and ECA and all its branches were ligated to separate the trunk. A fishing line with a diameter of 0.20 mm and a polished tip end was advanced into the ICA under direct vision through an incision which was made 4 mm from the bifurcation proximally. The insertion went to about (18.5±0.5) mm till the proximal anterior cerebral artery, thus completely blocking the blood supply from the middle cerebral artery. Reperfusion was performed after 2 h of ischemia. At reperfusion, the nylon fishing line was gently withdrawn by a length of 10mm. The intraoperative and postoperative room temperature was maintained at about 25℃, and the animals were irradiated using incandescent light to maintain their anal temperature at 37±1°C until recovery of activity. A model was determined to be successful based on the following criteria: (1) positive Horner’s syndrome on the right side and (2) hemiplegia on the left side mainly manifesting as forelimb movement abnormality. In the sham surgery group, all procedures were performed except there was no insertion of a fishing line. The animals were dosed twice in the process of establishing the model: two hours after ischemia before reperfusion and six hours after reperfusion, respectively. Then, the blood was taken from the femoral artery of the leg and the brains were collected after reperfusion for 22 h.
Determination of cerebral infarct size and water content in brain tissue
After reperfusion for 22 h, the rats were decapitated and their rhinencephalon, lower brain stem, and cerebellum were removed. After weighing, coronal sections with a thickness of approximately 2 mm were obtained from the brain tissues and each side of the ischemic brain tissue was cut into five pieces. Then, the pieces of brain were immediately placed in a 2% TTC solution and incubated for 30 min in the dark at 37 °C. After staining, the normal brain tissue was rosy, whereas the cerebral infarct area was white. The total area and the infarcted area of the ischemic brain tissue in each slice were calculated by Nodus DanioScope Version 1.0.109. The percentage of the brain in the infarct area to the total brain was taken as the infarct size (%). The stained brain tissue was weighed before and after being dried in an oven at 105 °C for 48 h and the water content in the brain tissue was calculated according to the following formula:
See formula 1 in the supplementary files.
Histomorphological observations
Sections were obtained from the same area of the brain tissue for rats in each group. The tissue samples were fixed with 4% paraformaldehyde, embedded with paraffin, and then cut into pieces (4 µm) using a Leica RM2235 microtome. Subsequently, H&E and Nissl's staining were implemented to observe histopathological changes using the microscopic imaging system. The number of Nissl bodies were quantitatively analyzed via Image Pro Plus 6.0 software and the evaluation was performed by a blinded investigator.
Determination of related factors and platelet receptor expression
Blood samples were collected and centrifuged at 3500 rpm for 10 min to obtain serum and plasma to investigate the following: cAMP, Ca2+, NO, NOS, TXA2, TXB2, PGI2 and 6-keto-PGF1α. cAMP, Ca2+, NO and NOS were determined using corresponding test kits and TXA2, TXB2, PGI2 and 6-keto-PGF1α were determined using ELISA. Also, platelets were collected from the serum of the rats in each group using platelet isolation kits. Total protein was collected with a kit for the rest. GP1BA and PAF protein expression was determined by Western Blot.
Methods for the SPR test
After coupling captured molecule and pre-concentration of ligand, we determined that the results indicated that GP1BA could be optimally captured at pH 5.0. The relevant parameters were set as follows: Rmax = analyte Mw/ligand Mw×RL× Sm; anti-GST antibody: 26kDa; GP1BA: 56.7 kDa; Rmax: typically, 100 RU; Sm: stoichiometric ratio, typically 1 (actual captured > RL); GP1BA: pI=5.63, pH of sodium acetate buffer: 3.5< pH< pI. The GP1BA and different concentrations of samples (Rg1, Re, R1) were prepared and injected for the test. The contact time and flow rate were set according to the system instructions. The data collected were analyzed and fitted by PLEXERA SPR Date Analysis Module (DAM). Kinetic data such as fitting constant, dissociation constant, and equilibrium dissociation constant were calculated based on curve fitting to obtain the specificity of molecular binding and the binding process.
Method of molecular docking
The geometric structures of Ginsenoside Rg1, Re, and Notoginsenoside R1 were optimized using the density functional method [24, 25] at the B3LYP level of theory [26, 27] with the 6-31G(d) basis set in Gaussian 16 package. The 3D crystal structure of the human platelet receptor GP1BA was obtained from the RCSB Protein Data Bank (PDB ID: 1P9A) [28]. All docking simulations were performed with the Lamarckian genetic algorithm in Autodock 4.2 software [29]. The docking images were generated by PyMOL.
Statistical analysis
The experimental data were subject to one-way ANOVA after being processed with the SPSS 17.0 statistical package. Data are presented as mean ± standard deviations (n = 8). If P<0.05 or P<0.01, it indicated a significant difference. The homogeneity test of variance was compared in each group. The least significant difference (LSD) test was conducted when the variance was homogeneous, and Tamhane’s T2 test was conducted when the variance was heterogeneous.