Materials
20(S)-panaxadiol (PD, purity ≥98% by HPLC) was obtained from Shanghai yuanye Bio-Technology Co., Ltd (Shanghai, China). Hemocoagulase was obtained from Jinzhou Ahon Pharmaceutical Co, Ltd. (Liaoning, China). Prothrombin (PT), activated partial thromboplastin time (APTT), thrombin time (TT), and fibrinogen (FIB) kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Luciferin/luciferase reagent and thrombin were purchased from Chrono-Log Corporation (Pennsylvania, USA). Vorapaxar, ticagrelor, and seratrodast were obtained from MedChemExpress (New Jersey, USA). Cyclic adenosine monophosphate (cAMP) ELISA kit was obtained Sino Best Biological Technology Co., Ltd. (Shanghai, China). FITC-conjugated anti-human CD62P (P-selectin) and FITC-conjugated anti-human PAC-1 were obtained BioLegend (California, USA). Fluo-3 AM Calcium indicators were purchased from Beyotime Biotechnology (Shanghai, China). Akt, p-Akt, PI3K, p-PI3K, GSK3β, p-GSK3β, and β-actin were purchased from Abcam (Cambridge, Britain).
Animals
Male Kunming mice (weighting 20.0±2.0 g) and male Wistar rats (weighting 190.0±10.0 g) were purchased from Liaoning Changsheng Biotechnology Co., Ltd (Animal license No. SCXK (Ji)-2016-0003). The animals were housed under controlled temperature (25±1℃), relative humidity (60±5%), and a 12 h light/dark cycle with ad libitum access to food and water. This experiment was approved by the Bioethics Committee of the Changchun University of Chinese Medicine and the Institutional Animal Care (Approval NO. 20190133), which was conducted based on the guideline for the use of laboratory animals.
Bleeding time measurement
The measurements of bleeding time in mouse tail amputation and liver scratch models were established, according to previous methods [32] with some modifications. Briefly, 40 male Kunming mice (20.0±2.0 g) were randomly divided into 5 groups, NS group (1 % sodium carboxymethyl cellulose-normal saline), HC group (1 KU/mL hemocoagulase), and 2, 4, 8 mg/kg PD groups. After the drugs with subcutaneously injected for 4 h, mice were anesthetized with 4 % pentobarbital sodium via intraperitoneal injection (IP). In the tail amputation model, the tails of mice were transected with a sterile razor blade at the site that 10 mm apart from the tip, and then immersed in 37 ℃ normal saline. The bleed time was defined as the time from the start of transection to bleeding cessation. Stop time of tail bleeding over 30 s was considered as bleeding time. In the liver scratch model, the liver injury was established by scratching the left lateral lobe with a 2-mL syringe about 1 cm, to cause the liver to bleed. Then the incision was dipped with filer paper at 10 s intervals until hemostasis. All of the mice were euthanized via cervical dislocation under anesthesia at the end of each experiment.
Routine blood test
1 % CMCNa-normal saline (NS group), 1 KU/mL hemocoagulase (HC group), and 2, 4, 8 mg/kg PD were subcutaneously injected into 5 groups of Wister male rats (n=8), respectively. After 4 h treatment, the rats were anesthetized with 4% pentobarbital sodium via IP to withdrawn blood samples from the aorta abdominal, and then placed in plastic tubes with EDTA. The XT-2000i automated hematology analyzer (Sysmex Corporation, Japan) was used to detect routine blood test.
Plasma coagulation assay
Rat blood samples were withdrawn from the aorta abdominal and then placed in a 3.8 % sodium citrate vacuum tube with a blood/coagulant ratio of 9:1, and then centrifuged at 3000 rpm for 15 min to obtain plasma. Plasma mixtures with 1.45 mL plasma and 0.05 mL different concentration of PD were incubated at 37 ℃ for 10 min, which were used to detect PT, APTT, TT, and FIB concentration, according to the manufacturer's protocols using the H1201 automatic coagulation analyzer (Jiangsu Horner Medical Instrument Co., Ltd., China).
Platelets analysis
Human and rat washed platelets preparation
As described above, blood from healthy consented volunteers and male Wistar rats were collected into an anticoagulant tube of 3.8 % sodium citrate, respectively. Platelet-rich plasma (PRP) was isolated as the supernatant from centrifugation at 800 rpm for 5 min. Human/rat washed platelets were prepared as before [33]. PRP was centrifuged at 3,000 rpm for 5 min and washed twice with Tyrode's buffer (137 mM NaCl, 2 mM KCl, 12 mM NaHCO3, 5 mM HEPES, 0.35 % BSA, PH 7.4) to obtain human/rat washed platelets.
Platelet aggregation assay and ATP release assay
Human/rat washed platelets were adjusted to 3 × 108 /mL with Tyrode's buffer including 1mM CaCl2. After the incubation at 37 ℃ for 5 min, the platelets were stimulated by various concentrations of PD or thrombin, respectively. Platelet aggregation was performed by using a platelet aggregometer (Chrono-Log 700, Chrono-Log Co., USA) by measuring the changes in light transmission. ATP release was measured using luciferin/luciferase reagent (Chrono-lume). Additionally, vorapaxar (VP, a PAR-1 antagonist of thrombin, 10 μM), ticagrelor (TG, a P2Y12 receptor antagonist of ADP, 10 μM) and seratrodast (ST, a potent and selective thromboxane A2 receptor antagonist, 10 μM) were used to further analyze the possible mechanism of PD on platelet activity by platelet aggregation assay.
P-selectin secretion and glycoprotein (GP) IIb/IIIa activation on the surface of platelets by flow cytometric analysis
Human washed platelets were incubated with different concentrations of PD at 37 ℃ for 5 min, and then incubated with FITC-conjugated CD62P (P-selectin marker) or FITC-conjugated PAC-1 (activated GP IIb/IIIa receptor marker) antibodies in the dark for 20 min. After stopping by adding 200 μL of phosphate-buffered saline (PBS), the samples were immediately analyzed with a BD FACSAria II flow cytometer (BD Biosciences, USA). A total of 10,000 events in triplicated from different groups were analyzed the platelet P-selectin secretion and glycoprotein IIb/IIIa activation, which was repeated at least three times to ensure reliability.
Determination of the intracellular calcium concentration [Ca2+]i
As previously reported [34], human washed platelets were incubated with Fluo-3 AM (5 μM) at 37 ℃ for 60 min in the dark condition, and washed two times and suspended in Tyrode's buffer. Platelets at the final concentration of approximately 3×108/mL were added to the 96-well microplates (Nunc F96, ThermoFisher Scientific, Waltham, USA) and incubated with PD. After adding PD, Fluo-3 fluorescence was determined at 17 seconds intervals for 20 min on Cell Imaging Multi-Mode Reader (Cytation 5, BioTek, Vermont, USA) with an excitation wavelength of 488 nm and an emission wavelength of 525 nm to draw calcium kinetic curve. The [Ca2+]i is calculated by the previous method [35] as follows: [Ca2+]i in cytosol = 525 nM×(F-Fmin)/(Fmax-F), where 525 nM is the dissociation constant of the Fluo-3, F represents the fluorescence value of the sample. Fmin and Fmax are minimum and maximum fluorescence values, are measured after the treatment with 10 mM EGTA and 0.1 % Triton X-100, respectively.
Measurement of cAMP
Human washed platelets were incubated with methanol or PD at 37 ℃ for 10 min, and then added the 10 mM EDTA to terminate the reaction. After freezing at -80℃ and thawing at 37 ℃ for 5 times, the solution was centrifuged at 3,000 rpm for 10 min at 4 ℃, and the supernatant for detecting the concentrations of cAMP using the ELISA kit according to the manufacturer's protocol. To evaluate whether the cAMP production was involved in VP inhibited the platelet aggregation. Human washed platelets were pretreated with VP (10 μM) for 5 min, then treated with PD to detect the cAMP concentration.
Western blot
To assess the effect of PD on the downstream signaling pathway of PAR1, western blot experiments were performed. The washed human platelets were pretreated with methanol or PD at 37 ℃ for 15 min, lysis buffer (PRO-PREP; iNtRON Biotechnology, Seoul, Korea) with 100 μL RIPA buffer and protease/phosphatase inhibitor cocktail (Beyotime Biotechnology, Shanghai, China) was added to the mixture. Thirty micrograms of the cellular proteins were resolved by electrophoresis in 10 % SDS-polyacrylamide gel, and subsequently transferred to polyvinylidene difluoride (PVDF) membrane. Following 1 h incubation in a fresh TBS buffer containing 0.1 % Tween-20 and 5 % BSA, the blots were probed with specific antibodies including p-Akt, Akt, p-PI3K, PI3K, p-GSK3β, GSK3β and β-actin overnight at 4℃. After incubation with appropriate secondary antibodies for 1 h at room temperature, protein bands were visualized and analyzed using a chemiluminescent imaging system (FluorChem, ProteinSimple, San Jose, CA, United States)
Statistical analysis
Data from all experiments are presented as the mean ± standard deviations (SD). Ordinary one-way ANOVA of variance was used to analyze the differences among the groups by GraphPad Prism 8.0 software. p<0.05 was considered as statistical significance.