Materials
PTS (Lot No.:20190318) was provided by Chengdu Huasun Technology Group (Chengdu, China). Aspirin Enteric-coated Tablets (Lot No.:BJ45216) was purchased from Bayer Health Care (Milano, Italy). Xuesaitong Soft Capsule (Lot No.:20190322) with 75% content of PNS (saponins which were extracted from notoginseng, including Ginsenoside Rg1, Ginsenoside Rb1, Ginsenoside Re, Notoginsenoside R1, and Ginsenoside Rd) was purchased from Kunming Shenghuo Pharmaceutical (Kunming, China). Reference Standards of Ginsenoside Rg1, Ginsenoside Re and Notoginsenoside R1 were purchased from 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 analytic grade and used as received. The kits for determining cAMP, TXA2, TXB2, PGI2, 6-keto-PGF1α was 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). CM5 Chip, Peptide Coupling Kit, GST Capture Kit and other buffers used in SPR test were purchased from GE Healthcare Life Sciences.
All animals in our research were provided by the Experimental Animal Center of Chengdu University of Traditional Chinese Medicine, and raised in SPF-class housing of laboratory with a controlled condition (20-22℃, relative humidity of 50-60%, and 12 h light-dark cycles). They were fed with water and commercial rat pellet diet. All animal experiments were permitted by the Sichuan Provincial Committee for Experimental Animal Management. After acclimation, the adult male SD rats (300±20 g) were given the preventive drug at 10 mL/kg by gavage, once a day for 6 consecutive days. And then the animals were divided into 7 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 combined treatment (PTS at 50 mg/kg and aspirin at 21 mg/kg) groups. Specifically, the MCAO model of rats was established at 30 min after gavage on Day 6, and the animals were irradiated using incandescent light to maintain their anal temperature at (37±1)℃ until recovery of activity. The rats underwent fasting for a period of 12 hours prior to the experimental procedures with water available ad libitum, and were anesthetized. The intraoperative and postoperative room temperature was maintained at about 25℃, reperfusion was performed after 2 h of completely blocking the blood supply from the middle cerebral artery. Determination of a successful model: (1) positive Horner’s syndrome on the right side; (2) hemiplegia on the left side mainly manifesting as forelimb movement abnormality. In the sham surgery group, all the procedures were performed except no insertion of a fishing line. The animals were dosed twice in process of model establishment, at 2 hours after ischemia before reperfusion and 6 hours after reperfusion, respectively, and then the bloods and 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 about 2 mm were taken from brain tissues, and each side of the ischemic brain tissue was cut into 5 pieces. Then, the pieces of brain were immediately placed in 2% TTC solutionand incubated for 30 min at 37 °C in the dark. After staining, the normal brain tissue was rosy, while the cerebral infarct area was white. The total area and infarcted area of ischemic brain tissue in each slice were calculated by Nodus DanioScope Version 1.0.109. The percentage of the brain in infarct area to the total brain was taken as the infarct size (%). The stained brain tissue was weighed before and after dried in an oven at 105 °C for 48 h to a constant weight, respectively, and the water content in brain tissue was calculated according to the following formula:
water content in brain tissue (%) = 100%.
Histomorphological observation
Sections were taken from the same part of brain tissue for rats in each group. The tissue samples were fixed with 4% paraformaldehyde, embed with paraffin, and then cut into pieces (4 µm) using a Leica RM2235 microtome. Subsequently, H&E and Nissl's staining were implemented for observing histopathological changes using the microscopic imaging system, and the Nissl body numbers were quantitatively analyzed by Image Pro Plus 6.0 software.
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 for investigating the following indexes: cAMP, Ca2+, NO, NOS, TXA2, TXB2, PGI2 and 6-keto-PGF1α. In which, cAMP, Ca2+, NO and NOS was determined using corresponding test kit, and the content of TXA2, TXB2, PGI2 and 6-keto-PGF1α was determined using ELISA. Besides, platelets were collected with platelet isolation kits from the blood. Platelets were also collected with platelet isolation kits from the rats in each group. Besides, the total proteins were collected with a kit for the rest, and the expression level of GP1BA and PAF was determined by Western Blot.
Methods of SPR Test
After coupling captured molecule and pre-concentration of ligand, these results indicated that GP1BA could be optimally captured at pH 5.0. The relevant parameters were 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). GP1BA and samples (Rg1, Re, R1) with different concentrations were prepared and injected for test, the contact time and the flow rate were set according to system instructions. The obtained data were analyzed and fitted by PLEXERA SPR Date Analysis Module (DAM). Kinetic data such as fitting constant, dissociation constant and equilibrium dissociation constant was calculated based on the fitting curve 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 by using density functional method [20, 21] at the B3LYP level of theory [22, 23] with the 6-31G(d) basis set in Gaussian 16 package. The 3D crystal structure of human platelet receptor GP1BA was obtained from RCSB Protein Data Bank (PDB ID: 1P9A) [24]. All docking simulations were performed with the Lamarckian genetic algorithm in Autodock 4.2 software [25]. The docking images were generated by PyMOL.
Statistical analysis
The experimental data are subject to one-way ANOVA after being processed with SPSS 17.0 statistical package. Data presented as mean±standards deviation (n = 8). If P<0.05 or P<0.01, it indicates that there is significant difference.