Preventive and therapeutic effect of brozopine on stroke in Dahl Salt-sensitive hypertensive rats

Accepted Manuscript Research report Preventive and therapeutic effect of brozopine on stroke in Dahl Salt-sensitive hypertensive rats Yuan Gao, Yan Wang, Miao Li, Yali Liu, Junbiao Chang, Hailing Qiao PII: DOI: Reference:

S0006-8993(17)30318-9 http://dx.doi.org/10.1016/j.brainres.2017.07.019 BRES 45433

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Brain Research

Received Date: Revised Date: Accepted Date:

31 May 2017 11 July 2017 24 July 2017

Please cite this article as: Y. Gao, Y. Wang, M. Li, Y. Liu, J. Chang, H. Qiao, Preventive and therapeutic effect of brozopine on stroke in Dahl Salt-sensitive hypertensive rats, Brain Research (2017), doi: http://dx.doi.org/10.1016/ j.brainres.2017.07.019

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Preventive and therapeutic effect of brozopine on stroke in Dahl Salt-sensitive hypertensive rats Yuan Gao , Yan Wang , Miao Li , Yali Liu , Junbiao Changb*, Hailing Qiaoa* a

*

a

a

a

a

Institute of clinical pharmacology, Zhengzhou University, Henan, China;

b

College of Chemistry and Molecular Engineering, Zhengzhou University, Henan, China.

Co-corresponding author: the first corresponding author is Hailing Qiao, [email protected] and

the second corresponding author is Junbiao Chang, [email protected]. Abstract Our aim was to explore the preventive and therapeutic effects of sodium (±)-5-bromo-2-(α-hydroxypentyl) benzoate (brand name: brozopine, BZP) on stroke in Dahl Salt-sensitive (Dahl-SS) hypertensive rats. Dahl-SS rats were fed a high-salt diet to observe the effect of BZP on blood pressure, and brain, heart, and kidney tissues. Additionally, the incidence of stroke was recorded according to the neurological score. The relative mechanisms investigated included anti-oxidative effects and anti-platelet aggregation. BZP reduced the incidence of stroke, neuronal necrosis in the brain, and cell swelling and inflammatory infiltration in the kidney. Its mechanisms were related to the increased activities of gluthatione peroxidase and catalase and the decreased level of plasma nitric oxide. BZP inhibited arachidonic acid (AA) - induced platelet aggregation (IC50: 12 µM) rather than that of adenosine diphosphate (ADP) - and/or thrombin-induced platelet aggregation in vitro. Interestingly, BZP inhibited ADP-, thrombin-, or AA-induced platelet aggregation and elevated the level of AMP-activated protein kinase, cyclic guanosine monophosphate, and vasodilator-stimulated-phosphoprotein, and attenuated ATP contents and mitogen-activated protein kinase levels in platelet and inhibited thrombus formation in a carotid artery thrombosis model, dose-dependently, in Dahl-SS hypertensive-induced stroke rats. In conclusion, BZP can have therapeutic and preventive effects on stroke in Dahl-SS hypertensive rats, the mechanisms of which may be related to anti-oxidant, anti-platelet aggregation and anti-thrombus formation. Key words: brozopine; Dahl Salt-sensitive rat; oxidative; platelet aggregation 1. Introduction Hypertension has become one of the most common cardiovascular diseases and has been an independent risk factor of stroke in developing and developed countries. Stroke has a high incidence in China and exhibits a rising trend annually. Salt has been considered one of the most important environmental factors of hypertension, and high salt intake is closely related to blood pressure. Platelet activation was thought to play a vital role in the initiation and maintenance of atherosclerosis (Garcia Carrascal et al., 2017) and was implicated in the pathogenesis of vascular diseases. Moreover, these changes were exaggerated during sodium loading, especially in salt-sensitive hypertension (Somova and Mufunda, 1993). Patients with hypertension revealed that platelets were more sensitive to platelet aggregation inducer (Mugellini et al., 2005) and showed an elevation in their intracellular free calcium (Rendu et al., 1993). This later potentiated platelet activity and increased the risk of thromboembolic diseases such as myocardial infarction 1

and peripheral artery disease (Dogne et al., 2002). Platelet activation was abnormal in cerebral ischemia, but usually returned to normal with anti-platelet therapy. This activation of platelets might contribute to the clinical manifestations of occlusive vascular diseases. Platelet activation, induced by platelet agonists such as adenosine diphosphate (ADP), thrombin, and arachidonic acid (AA) at the site of the injury, was a vital process for platelet adhesion and aggregation (Kazianka et al., 2017). Currently, anti-platelet therapy is considered one of the valid approached to ameliorate cerebral ischemia injury (Furie and Furie, 2005; Huo and Ley, 2004; Li et al., 2007). To date, several anti-thrombotic drugs have been developed and widely used in clinical practice. Generally, the acetylsalicylic acid (ASA),which is a cyclooxygenase inhibitor, preventing thromboxane A2 synthesis, has been used as an anti-platelet agent in the prevention of ischemic stroke. However, its use became greatly limited due to severe adverse effects and drug resistance in many patients (Fitzgerald and Pirmohamed, 2011; Fong et al., 2011). Sodium (±)-5-bromo-2-(α-hydroxypentyl) benzoate (BZP) is derived from 1-3-n-butylphthalide (NBP) and its chemical structure is similar to that of ASA. Based on our previous findings, BZP has the ability to protect from focal cerebral ischemia-reperfusion injury in rats, through mechanisms including anti-apoptosis, anti-inflammation, and promoting synaptic plasticity (unpublished data). Currently, BZP is used in phase I clinical trials with encouraging efficacy results. The preventive and therapeutic effect of BZP on stroke in Dahl-SS hypertensive rats have not been studied yet. Accordingly, we assessed the modulatory effects of BZP on Dahl-SS hypertensive rats at 28 days after stroke. 2. Results 2.1 No difference was observed in BP in Dahl-SS hypertensive-induced stroke rats treated with BZP The mean arterial pressure was significantly higher in the Dahl-SS group as compared with the SS-13BN group (135.66 ± 6.18 mmHg vs 99.33 ± 3.96 mmHg, respectively) after 2 weeks of high salt diet. The systolic (SBP) and dystolic blood pressure (DBP) were significantly increased in the Dahl-SS group compared with the controls (SBP: 155.18 ± 4.58 mmHg vs 115.19 ± 3.39 mmHg, respectively; DBP: 125.88 ± 7.94 mmHg vs 91.40 ± 5.23 mmHg, respectively). After establishing the hypertensive model, the drug was administrated by caudal vein once daily for the next 28 days. The results indicated no statistical difference between the BZP (0.75, 3, 12 mg/kg) and Model group (P > 0.05), indicating that BZP is not an anti-hypertensive drug. (Data was shown in supplementary Fig.1) 2.2 Behavioral testing The prehensile traction, beam-walking test, and neurological deficit (Bederson) score were examined before administration. Subsequently, all rats were tested subjected to behavioral tests at least every 7 days. Compared with the Control group that exhibited an intact performance, the Dahl-SS hypertensive-induced stroke rats displayed behavioral deficits following cerebral lesions on the beam-walk test as evidenced by a reduced time on the beam. In contrast, rats in the BZP groups remained longer time on the beam compared with the Model group. Moreover, the Model 2

group demonstrated a decrease in the time spent on the rope in the prehensile traction test, whereas an increase was observed in rats treated with different doses of BZP. There were significant differences in the time spent on the rope between the BZP and Model groups. Additionally, the BZP (12 mg/kg) group exhibited an improvement in their motor function that was more prevalent than in the NBP-K (9.6 mg/kg) and Br-NBP (10.5 mg/kg) groups. A significant group effect in the grip strength was observed at days 7, 14, and 28, whereby rats treated with BZP (0.75, 3, and 12 mg/kg) achieved significantly lower scores than the other experimental groups (Fig. 1). 2.3 BZP reduced the incidence of stroke and increased the survival rate in Dahl-SS hypertensive rats Stroke was defined by comprehensive scores equal or greater than three points according to the motor changes. Following the intake of high salt diet, the incidence of stroke increased gradually in the Model group, which tended to be higher than in the other groups (Fig. 2A). The incidence of stroke was 83.3% in the Model group with apathetic, lethargy, and motor dysfunction (such as circling behavior) after 4 weeks of high salt diet. In contrast, the incidence of stroke was obviously lower in the BZP-treated rats (0.75, 3, and 12 mg/kg). This observation was dose -dependent manner and rats displayed a good physiological state and no circling behavior. The highest stroke incidence was merely 8.3% and was decreased with prolonged medication. As shown in Fig. 2B, there was a decreased survival rate (58.3%) in the Model group, while BZP (0.75, 3, and 12 mg/kg) remarkably increased the survival rate (83.3%, 91.7%, and 100%, respectively) in a dose-dependent manner. These findings indicated that BZP could reduce the incidence of stroke and increase the survival rate in Dahl-SS hypertensive rats. 2.4 Increased GSH-px and CAT activities and decreased NO level in the plasma of Dahl-SS hypertensive rats treated with BZP The blood samples were obtained from the abdominal aorta to detect the oxidative stress using biochemical assays. As shown in Fig. 3, the activities of GSH-px and CAT were obviously decreased (P < 0.01), while the level of NO was significantly increased (P < 0.01) in the Model group as compared with controls; Following treatment with BZP, the activities of GSH-px and CAT increased in the BZP (12 mg/kg) group (P < 0.05 and P < 0.01 respectively), and the level of NO was significantly decreased in the BZP (3 and 12mg/kg) groups (P < 0.01) as compare with the Model group. 2.5 Histological alterations Hematoxylin-Eosin staining was performed on brain, kidney, and heart tissues at day 28 after drug administration. As shown in Fig. 4, rats treated with BZP (0.75, 3, and 12 mg/kg) had a significantly reduced swelling and necrosis of the brain than the Model group. Additionally, the glomerulus displayed an increased the hemorrhagic spot and capillary basement membrane with inflammatory cells infiltration in the Model group. Treatment with BZP significantly reduced the edema and inflammation. The results also indicated that the cardiomyocytes and cell nuclei were 3

arranged irregularly in the Model group, while the BZP groups (0.75, 3, and 12 mg/kg) displayed significantly reduced necrosis. 2.6 BZP inhibited platelet aggregation following stimulation of platelets with various agonists in vitro Our data indicated no changes in ADP- or/and thrombin-induced platelet aggregation (Fig. 5A, B). The maximal rate of aggregation was reached in the AA-induced platelet aggregation (73.13 ± 3.42%). BZP potently inhibited the platelet aggregation induced by 3.6 mM AA in vitro in a concentration-dependent manner (Fig. 5C). The IC50 was computed to be 12 µM by PROBIT analysis (data was shown in supplementary Fig. 3). Under the same situation, the aggregation of NBP-K (12 µM) was about 51.90%. 2.7 BZP inhibited platelet aggregation following platelets stimulation with various agonists ex vivo Intravenous injection of agents in advance for 30 min had been utilized to identify whether BZP is effective also in vivo. Platelet aggregations induced by ADP, thrombin, and AA were observed in rat platelet-rich plasma (PRP). As shown in Fig. 6, BZP at different doses could significantly inhibit ADP-, thrombin-, and AA- induced platelet aggregation. 2.8 Significant correlation between AA-induced platelet aggregation and comprehensive score in Dahl-SS hypertensive-induced stroke rats We performed a correlation analysis between AA-induced platelet aggregation and comprehensive scores in Controls and BZP (0.75, 3 and 12 mg/kg) groups. As shown in Fig. 7, AA-induced platelet aggregation and comprehensive scores were directly related, indicating that the higher the rate of platelet aggregation, the more severe the neurological deficit was. 2.9 Effect of BZP on the AMPK, ATP, cGMP, VASP, MAPK, and VASP levels in AA-induced platelets As shown in Fig.8, the level of AMPK in the Model group was lower than the Control group, and BZP (3 and 12 mg/kg) could elevate AMPK level distinctly (P < 0.05). There was no significant difference between the BZP 0.75 mg/kg and Model groups (P > 0.05). The level of ATP in the Model group was higher than the Control group, and BZP (3 and 12 mg/kg) could attenuate the ATP level significantly (P < 0.01). The level of cGMP in the Model group was lower than the Control group, and BZP (12 mg/kg) could significantly elevate the cGMP level (P < 0.05). Furthermore, BZP (3 and 12 mg/kg) could increase the expression of VASP stimulated by AA. However, BZP (0.75 mg/kg) had no effect on VASP expression. To investigate the inhibitory mechanisms of BZP in platelet activation stimulated by AA, we further detected the MAPK signaling including MAPK levels. The ELISA analysis revealed that BZP (3 and 12 mg/kg) attenuated the MAPK level stimulated by AA. However, BZP (0.75 mg/kg) had no effect on MAPK expression. 2.10 Effect of BZP on the cerebral thrombosis in Dahl-SS hypertensive-induced stroke rats In this study, we used the absorbance value (A) / brain weight to determine the degree of cerebral thrombosis. The ratio in the Model group was significantly increased compared with 4

controls (P < 0.05) as show in Fig. 9. However, when compared with the Model group, the ratio was markedly decreased (P < 0.05) in the BZP groups (0.75, 3, and 12 mg/kg). Additionally, there was no difference between ASA (7 mg/kg) and BZP (12 mg/kg). These findings indicated that BZP can reduce the cerebral thrombosis in Dahl-SS hypertensive-induced stroke rats. 3. Discussion Although a diet with high level sodium caused a significant elevation in the BP in Dahl-SS rats, our findings demonstrated that BZP had no significant effect on reducing BP. The neurobehavioral performance was significantly improved compared with untreated ischemic rats, as evidenced by the results on the prehensile traction test, beam walking test, and Bederson score.BZP could also reduce necrosis in the brain, heart, and renal tissue and alleviate inflammatory infiltration. However, little is known about the mechanism of BZP in stroke prevention/treatment in Dahl-SS hypertensive rats. Therefore, we further observed the effects of BZP on anti-oxidation and platelet aggregation. Based on our findings, BZP inhibited oxidative stress and AA-induced platelet aggregation, but not platelet aggregation induced by ADP and thrombin in vitro. This may be due to the fact that the chemical structure of BZP was similar to ASA. Astonishingly, BZP could inhibit ADP-, thrombin-, or AA-induced platelet aggregation and thrombosis formation in Dahl-SS hypertensive rats in a dose dependent manner. These results were consistent with a previous report (Ma et al., 2012);because BZP was converted into Br-NBP in plasma (Tian et al., 2016). This mechanism was related to increased intracellular levels of AMPK, cGMP, and VASP and decreased ATP contents and MAPK levels caused by AA. The antioxidant activity of plasma might be an important factor providing protection from neurological damage induced by stroke associated with oxidative stress (El Kossi and Zakhary, 2000; Leinonen et al., 2000). Various free radicals and reactive oxygen species (ROS), such as peroxides, superoxide, and hydroxyl radicals, caused lipid peroxidation, thus resulting in the loss of cell membrane integrity and mitochondrial dysfunction, and further inducing biological cell damage. The production of excessive NO was an important mediator in hypoxic or cerebral ischemia injury. The expression of inducible nitric oxide synthase (iNOS) was elevated when stroke occurred, accompanied by the production of a large quantity of NO. Moreover, NO has been considered as a primary contributor to the post-ischemic inflammatory response (Brown et al., 2008). Antioxidant enzymes like GSH-px and CAT maintained the equilibrium between oxidation and anti-oxidation. GSH-px was known to be involved in the detoxification of H2O2 by utilizing GSH as a substrate (Hoehn et al., 2003). Additionally, CAT substantially detoxified the H2O2 and protected the brain from more toxic OH− when SOD was insufficient to remove O−2. Previous studies have demonstrated that the brain was protected against oxidative stress damage via endogenous antioxidant enzymes like GSH-px and CAT. Furthermore, reduced GSH-px and CAT levels were associated with an increased stroke risk (Weisbrot-Lefkowitz et al., 1998). Our results showed that NO levels were significantly increased in the Model group, whereas BZP groups (3 and 12 mg/kg) reduced the NO levels compared with the Model group. Furthermore, our findings indicated a significant decrease in GSH-px and CAT levels in the Model group. BZP 5

(12 mg/kg) increased the levels of GSH-px and CAT, mainly due to the enhanced body antioxidant property of BZP. The pathogenesis of anti-oxidative is very complicated (Cervantes Gracia et al., 2017; Li et al., 2014; Love, 1999). Hence, its mechanism and the target of BZP will be explored in the process of further research. Platelets are important in the formation of thrombosis and are key factors in the development and progression of cardiovascular diseases (Jackson, 2007). ROS played a pivotal role in controlling various cell functions and was related to the formation and progression of thrombotic disorders, which included atherosclerosis, transient ischemic attack and myocardial infarction (Iuliano et al., 1997). O2−, a kind of ROS, caused platelet aggregation and is considered to affect platelet function by reacting with nitric oxide, a natural vasodilating substance released by endothelium or platelets (Higashi et al., 2009). There is plausibility that antioxidants could attenuate platelet activation. Platelet activation involved a complex signal transduction cascade reaction. AMPK exists widely in eukaryotic cells. It is a heterotrimeric serine/threonine protein kinase consisting of a catalytic subunit (α) and two regulatory subunits (β and γ), which exists as multiple isoforms and splice variants, resulting in the generation of 12 possible heterotrimeric combinations. AMPK activates endothelial nitric oxide synthase (eNOS) via phosphorylation at the activating site. The eNOS-nitric oxide (NO)/soluble guanylate cyclase (sGC)-cGMP/cGMP-dependent protein kinase (PKG) signaling axis is a major anti-aggregation mechanism residing in platelets. It is generally referred to as a “metabolite-sensing kinase”, because it is activated by a low energy status such as a decrease in the cellular ATP: AMP ratio, which triggers a switch from an ATP-consuming anabolic condition to an ATP producing catabolic condition (Hardie, 2007). The activation of AMPK leads to eNOS stimulation followed by the generation of NO, which ultimately results in aggregation inhibition. Some important mediators such as cAMP and cGMP might regulate platelet activation. cGMP and cAMP are produced by soluble guanylate cyclase (sGC) and soluble adenylate cyclase, which are activated by nitric oxide (NO) and prostacyclin, respectively. Subsequent phosphorylation of VASP, a PKG substrate, promotes platelet activation. The findings in this study demonstrated that BZP was able to promote the activation of the AMPK/eNOS-nitric oxide/sGC-cGMP/PKG-VASP signaling pathway. In conclusion, BZP could have an effect on the treatment and prevention of cerebral ischemic stroke. We speculated that the relevant mechanisms may inhibit platelet aggregation and thrombosis. Further studies of this unifying hypothesis are still warranted to validate our conclusions. 4. Materials and methods 4.1 Reagents BZP and 3-butyl-6-bromo-1(3H)-isobenzofuranone (Br-NBP) were synthesized by the Department of Chemistry, Zhengzhou University. Purities were 99.4% and 99.8%, respectively. Potassium 2-(1-hydroxypentyl)-benzoate (NBP-K) and ASA were purchased from the National

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Institutes for Food and Drug Control, China. ADP, thrombin, and arachidonic acid (AA) were obtained from Sigma Chemical Co., USA. The catalase (CAT) assay kit, nitric oxide (NO) assay kit, and glutathione peroxidase (GSH-px) assay kit were purchased from Nanjing Jiancheng Co., China. The enzyme-linked immunoassay (ELISA) Kits for cyclic guanosine monophosphate (cGMP), vasodilator-stimulated phosphoprotein (VASP), mitogen-activated protein kinase (MAPK), AMP-activated protein kinase (AMPK) and ATP were all purchased from R&D Co., USA. 4.2 Animals A total of 72 male Dahl salt-sensitive (Dahl-SS) rats (7 weeks old) were purchased from Beijing Vital River and kept in individually ventilated cages (IVC) laboratory animal facility at 22-24 °C with free access to food (8% salt AIN-76a diet) and water. As normotensive sham controls,12 male SS-13BN rats (7 weeks old), were purchased from Beijing Vital River and were also kept in the IVC laboratory animal facility at 22-24 °C with free access to food (0.3% salt 5L79 diet) and water. The animal experiments were approved by the institutional guidelines of the Experimental Animal Center of the Chinese Academy of Medical Science (Certification number: SCXK (Jing) 2012-0001). 4.3 Experimental setup The Dahl-SS rats adapted to the new environment for 1 week and were subsequently given the high-salt diet (8% salt AIN-76a diet) for 2 weeks. Monitoring of blood pressure (BP) and neurological scores were recorded. The BP was measured in all animals in quiet conditions using the tail-cuff method. The apparatus platform was warmed up to 37 °C for 10 min. Subsequently, the rats were placed in restrainers and a BP tail-cuff was attached to the base of the tail. After several training sessions to habituate to the procedure (two animals at a time), each rat was subjected to successive measurements. The occurrence of cerebral apoplexy symptoms in rats indicated that the model was successfully established; the incidence of stroke was then recorded. After the establishment of the hypertensive model, all solutions were applied through the caudal vein in the Dahl-SS hypertensive rats once a day for the next 28 days. The Dahl-SS hypertensive rats were randomly divided into six experimental groups consisting of 12 rats each: Model group, non-treated Dahl-SS hypertensive rats; BZP-treated groups, rats treated with 0.75, 3, or 12mg/kg BZP; Br-NBP group (10.5mg/kg); and NBP-K group (9.6mg/kg).The Control group consisted of 12 SS-13BN rats. In the ex vivo platelet aggregation assay, similar groups as described above were assigned. Each group consisted of six samples. Blood samples were obtained via the abdominal aorta from the Dahl-SS hypertensive rats 30 min after the last injection. 7

4.4 Behavioral assessment All animals were subjected to behavioral trainings at least every seven days to adapt them to behavioral tests. The behavioral assessment was performed between 8:30 and 12:00 in the morning under standard laboratory conditions (temperature 22-24 °C and relative humidity 45-50%) by a person blinded to the rats’ experimental groups. 4.4.1 Beam-walking test Rats were trained to walk on a wooden beam (2.5 × 2.5 × 80cm), elevated 60 cm above the floor, to reach their home cage. Their performance was evaluated using a modified scale: score 0, the rat traversed the beam with no foot slip; score 1, the rat traversed with grasping of the lateral side of the beam; score 2, the rat showed disability of walking on the beam but could traverse; score 3, the rat took a considerable amount of time to traverse the beam because of difficulty walking; score 4, the rat was unable to traverse the beam; score 5, the rat displayed difficulty to move the body or any limb on the beam; score 6, the rat was unable to stay on the beam for 10 s. (Urakawa et al., 2007) 4.4.2 Prehensile traction test Rats were trained to grab a wire (diameter, 0.15 cm and length, 100 cm). The wire was elevated 70 cm above the floor and placed over a 5-cm- thick foam pad in case the rats fall. After placing the front paws on the wire, we recorded the time latency until the rat lost hold of the wire. The performance was evaluated on a four-grade score: score 0, hanging on the wire over 5 s and the hind legs placed on the ropes; score 1, hanging on the wire over 5 s; score 2, hanging on the wire for 3-4 s; score 3, hanging on the wire for 0-2 s. (Hattori et al., 2000) 4.4.3 Neurological deficit score According to the method described by Bederson et al (Bederson et al., 1986) deficits were scored on a five-point scale: 0, normal with no neurological deficits; 1, failure to extend fully the right forepaw; 2, circling to the right; 3, falling to the right; 4, unable to walk spontaneously and decreased levels of consciousness. 4.5 Preparation of the platelet-rich plasma (PRP) The Dahl-SS hypertensive rats were anesthetized with 10% chloral hydrate and blood was collected from the abdominal aorta. Whole blood was anticoagulated with citrate (3.8%; 1:9, v/v) and centrifuged at 500 rpm for 3 min at room temperature. Platelet-rich plasma (PRP) was obtained from the resulting supernatant. The residue was centrifuged at 4,000 rpm for another 10 min to attain platelet-poor plasma (PPP). 4.6 In vitro study 4.6.1 In vitro platelet aggregation assay Platelets were pelleted by centrifugation at 2,500 rpm for 10 min at room temperature and washed twice with a modified Tyrode HEPES buffer (129 mM NaCl, 2.8 mM KCl, 8.9 mM NaHCO3, 0.8 mM MgCl2, 0.8 mM KH2PO4, 2 mM EGTA, 5.6 mM glucose, 10 mM HEPES, 0.35% BSA, pH 7.4; Fan et al., 2010). Samples were centrifuged again and gently resuspended in Tyrode-HEPES buffer containing 1 mM CaCl2 to make a concentration of 4 × 108 platelets/mL, 8

which were stored at room temperature. A total of 0.3 ml of the washed platelets were placed in a micro-cuvette and stirred with a rotor at 37 °C for 5 min. Subsequently, the washed platelets were incubated with vehicle, BZP (5-20 µM), NBP-K (12 µM), ASA (12 µM), or Br-NBP (12 µM) for 5 min, respectively. Aggregation was stimulated by ADP, thrombin, and AA at a final concentration of 15 µM, 1.05 U/L, and 3.6 mM measured turbidimetrically at 37 °C with a platelet aggregometer (LBY-NJ4, Pulisheng Instrument Co. Ltd., China) (Born, 1962). The aggregometer was calibrated by using PPP to determine the 100% light transmittance. In order to exclude the artificial effects, the concentration of the vehicle was kept within 0.5% (v/v). The inhibition of the aggregation was calculated as follows: Inhibition of aggregation = (rate of aggregation in control group - rate of aggregation in treated group) / rate of platelet aggregation in control group × 100% 4.7 Ex vivo study 4.7.1 Ex vivo platelet aggregation assay The Dahl-SS hypertensive rats were assigned randomly to seven groups consisting of six rats each: Control; BZP-treated (0.75, 3, and 12mg/kg); ASA (7 mg/kg); NBP-K (9.6 mg/kg), and Br-NBP (10.5 mg/kg). Sample groups were injected intravenously with the corresponding agents; at the same time, the Control group received an intravenous injection of 0.9% NaCl. Thirty minutes after the last injection, blood samples were obtained from the rats’ abdominal aorta. PRP and PPP were obtained as described above. The PRP was adjusted with PPP in order to obtain platelet counts of 4 × 108 platelets/mL. PRP was placed in a cuvette and stirred with a rotor at 37 °C for 5 min. The ex vivo aggregation was tested after irritating the platelets by 15 µM ADP, 1.05 U/L thrombin, and 3.6 mM AA for 5 min with a platelet aggregometer. The inhibition of the aggregation was calculated as follows: Inhibition of aggregation = (rate of aggregation in control group - rate of aggregation in treated group) / rate of platelet aggregation in control group × 100%. 4.7.2 Determination of the cyclic guanosine monophosphate (cGMP) level Animals were grouped and treated as in the ex vivo aggregation study. PRP was divided into seven groups: Control, BZP-treated (0.75, 3, and 12mg/kg), ASA-treated (7 mg/kg), NBP-K-treated (9.6 mg/kg), and Br-NBP-treated (10.5 mg/kg). After stimulation by 3.6 mM AA for 5 min, the action was terminated by adding 10 mM EDTA and boiling for 3 min followed by cooling to 4 °C. After centrifugation of the washed platelets at 4,000 rpm for 10 min, the supernatant was used to determine the cGMP level using a cGMP ELISA kit following the procedure recommended by the manufacturer. 4.7.3 Determination of the platelet vasodilator-stimulated phosphoprotein and mitogen-activated protein kinase concentrations The Control group was assayed directly without the addition of agents or AA stimulation. The groups were injected intravenously with the agents and were induced by 3.6 mM AA for 5 min before determining the VASP and MAPK contents in the rat PRP (4 × 108 platelets/mL). Briefly, the precipitated protein was sedimented by centrifugation (3,000 rpm for 10 min) in an 9

Eppendorf microcentrifuge and the supernatant was collected to be immediately assayed. The kinase activity was measured in the supernatants using an MAPK kit (R&D System Co.) at 37 °C, according to the recommendations of the supplier. VASP was measured by using a VASP phosphorylation assay kit according to the manufacturer’s instructions. 4.7.4 AMPK activity assay PRP were assigned to seven groups and treated as described above. The kinase activity was measured using an AMPK kit (R&D System Co.) at 37 °C according to the manufacturer’s instructions. The system was a solid-phase sandwich ELISA designed to detect and quantify the level of kinase proteins. 4.7.5 Determination of the ATP concentration PRP were assigned to seven groups and treated as described above. The PRP samples were diluted with PPP to obtain platelet counts of 4 × 108 platelets/mL. All groups, except the Control group, were induced by 3.6 mM AA for 5 min before the measurements. ATP concentration was measured using the ATP assay kit (R&D System Co.) according to the manufacturer’s recommendations. Briefly, the platelets were incubated for 3 min at 37°C and subsequently stimulated with AA for 5 min. The reaction was halted, platelets were centrifuged, and supernatants were used in the assay. 4.8 Experimental cerebral thrombosis Model One hour after the treatment, the rats were anesthetized with 10% chloral hydrate. Dahl-SS hypertensive rats were inserted the three-limb tubes on the right common carotid artery to avoid touching the heart. The hypertensive rats were injected with mixed inducers. In contrast, the control rats were injected with normal saline at the same volume. The severity of the thrombus was estimated by calculating the ratio of absorbance and brain weight (Cahn and Borzeix, 1982; Harada et al., 1971). 4.9 Statistical analysis Data were processed and analyzed using SPSS Statistics 17.0 and expressed as mean ± standard deviation of the independent experiments carried out. Significant differences between groups with different treatments were evaluated using one-way analysis of variance (ANOVA). Statistical significance was considered at a P value less than 0.05. IC50 values were determined by PROBIT analysis. Acknowledgements We acknowledge the National Significant new drug creation “the twelfth-five–plan” new drug candidates (No. 2012ZX09103101-011) and the National Natural Science Foundation of China (No. 81330075) for the financial support. References Bederson, J.B., Pitts, L.H., Tsuji, M., Nishimura, M.C., Davis, R.L., Bartkowski, H., 1986. Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination. Stroke. 17, 472-6. Born, G.V., 1962. Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature. 10

194, 927-9. Brown, C.M., Dela Cruz, C.D., Yang, E., Wise, P.M., 2008. Inducible nitric oxide synthase and estradiol exhibit complementary neuroprotective roles after ischemic brain injury. Exp Neurol. 210, 782-7. Cahn, J., Borzeix, M.G., 1982. The sodium arachidonate-induced cerebral infarct in the rat: a model for the study of drugs. J Cereb Blood Flow Metab. 2 Suppl 1, S74-7. Cervantes Gracia, K., Llanas-Cornejo, D., Husi, H., 2017. CVD and Oxidative Stress. J Clin Med. 6. Dogne, J.M., de Leval, X., Benoit, P., Delarge, J., Masereel, B., David, J.L., 2002. Recent advances in antiplatelet agents. Curr Med Chem. 9, 577-89. El Kossi, M.M., Zakhary, M.M., 2000. Oxidative stress in the context of acute cerebrovascular stroke. Stroke. 31, 1889-92. Fan, H.Y., Fu, F.H., Yang, M.Y., Xu, H., Zhang, A.H., Liu, K., 2010. Antiplatelet and antithrombotic activities of salvianolic acid A. Thromb Res. 126, e17-22. Fitzgerald, R., Pirmohamed, M., 2011. Aspirin resistance: effect of clinical, biochemical and genetic factors. Pharmacol Ther. 130, 213-25. Fong, J., Cheng-Ching, E., Hussain, M.S., Katzan, I., Gupta, R., 2011. Predictors of biochemical aspirin and clopidogrel resistance in patients with ischemic stroke. J Stroke Cerebrovasc Dis. 20, 227-30. Furie, B., Furie, B.C., 2005. Thrombus formation in vivo. J Clin Invest. 115, 3355-62. Garcia Carrascal, P., Garcia Garcia, J., Sierra Pallares, J., Castro Ruiz, F., Manuel Martin, F.J., 2017. Numerical Study of Blood Clots Influence on the Flow Pattern and Platelet Activation on a Stented Bifurcation Model. Ann Biomed Eng. 45, 1279-1291. Harada, M., Takeuchi, M., Fukao, T., Katagiri, K., 1971. A simple method for the quantitative extraction of dye extravasated into the skin. J Pharm Pharmacol. 23, 218-9. Hardie, D.G., 2007. AMP-activated protein kinase as a drug target. Annu Rev Pharmacol Toxicol. 47, 185-210. Hattori, K., Lee, H., Hurn, P.D., Crain, B.J., Traystman, R.J., DeVries, A.C., 2000. Cognitive deficits after focal cerebral ischemia in mice. Stroke. 31, 1939-44. Higashi, Y., Noma, K., Yoshizumi, M., Kihara, Y., 2009. Endothelial function and oxidative stress in cardiovascular diseases. Circ J. 73, 411-8. Hoehn, B., Yenari, M.A., Sapolsky, R.M., Steinberg, G.K., 2003. Glutathione peroxidase overexpression inhibits cytochrome C release and proapoptotic mediators to protect neurons from experimental stroke. Stroke. 34, 2489-94. Huo, Y., Ley, K.F., 2004. Role of platelets in the development of atherosclerosis. Trends Cardiovasc Med. 14, 18-22. Iuliano, L., Colavita, A.R., Leo, R., Pratico, D., Violi, F., 1997. Oxygen free radicals and platelet activation. Free Radic Biol Med. 22, 999-1006. Jackson, S.P., 2007. The growing complexity of platelet aggregation. Blood. 109, 5087-95. Kazianka, L., Drucker, C., Skrabs, C., Thomas, W., Melchardt, T., Struve, S., Bergmann, M., Staber, 11

P.B., Porpaczy, E., Einberger, C., Heinz, M., Hauswirth, A., Raderer, M., Pabinger, I., Thalhammer, R., Egle, A., Wendtner, C.M., Follows, G., Hoermann, G., Quehenberger, P., Jilma, B., Jaeger, U., 2017. Ristocetin-induced platelet aggregation for monitoring of bleeding tendency in CLL treated with ibrutinib. Leukemia. 31, 1117-1122. Leinonen, J.S., Ahonen, J.P., Lonnrot, K., Jehkonen, M., Dastidar, P., Molnar, G., Alho, H., 2000. Low plasma antioxidant activity is associated with high lesion volume and neurological impairment in stroke. Stroke. 31, 33-9. Li, H., Horke, S., Forstermann, U., 2014. Vascular oxidative stress, nitric oxide and atherosclerosis. Atherosclerosis. 237, 208-19. Li, S., Li, X., Li, J., Deng, X., Li, Y., Cong, Y., 2007. Experimental arterial thrombosis regulated by androgen and its receptor via modulation of platelet activation. Thromb Res. 121, 127-34. Love, S., 1999. Oxidative stress in brain ischemia. Brain Pathol. 9, 119-31. Ma,

F.,

Gao,

Y.,

Qiao,

H.,

Hu,

X.,

Chang,

J.,

2012.

Antiplatelet

activity

of

3-butyl-6-bromo-1(3H)-isobenzofuranone on rat platelet aggregation. J Thromb Thrombolysis. 33, 64-73. Mugellini, A., Rinaldi, A., Zoppi, A., Lazzari, P., Fogari, E., Corradi, L., Fogari, R., 2005. Effect of manidipine as compared to atenolol on platelet aggregation in elderly patients with isolated systolic hypertension and type II diabetes mellitus. Journal Of Cardiovascular Pharmacology. 45, 310-313. Rendu, F., Bachelot, C., Molle, D., Caen, J., Guez, D., 1993. Indapamide inhibits human platelet aggregation in vitro: comparison with hydrochlorothiazide. J Cardiovasc Pharmacol. 22 Suppl 6, S57-63. Somova, L., Mufunda, J., 1993. Platelet activity and salt sensitivity in the pathogenesis of systemic (essential) hypertension in black Africans. Clin Exp Hypertens. 15, 781-96. Tian, X., Liu, B., Zhang, Y., Li, H., Wei, J., Wang, G., Chang, J., Qiao, H., 2016. LC-MS/MS Analysis and Pharmacokinetics of Sodium (+/-)-5-Bromo-2-(alpha-hydroxypentyl) Benzoate (BZP), an Innovative Potent Anti-Ischemic Stroke Agent in Rats. Molecules. 21, 501. Urakawa, S., Hida, H., Masuda, T., Misumi, S., Kim, T.S., Nishino, H., 2007. Environmental enrichment brings a beneficial effect on beam walking and enhances the migration of doublecortin-positive cells following striatal lesions in rats. Neuroscience. 144, 920-33. Weisbrot-Lefkowitz, M., Reuhl, K., Perry, B., Chan, P.H., Inouye, M., Mirochnitchenko, O., 1998. Overexpression of human glutathione peroxidase protects transgenic mice against focal cerebral ischemia/reperfusion damage. Brain Res Mol Brain Res. 53, 333-8.

Figure legends: Fig. 1 Stroke administration of BZP improved motor dysfunction and decreased neurological scores in Dahl-SS hypertensive-induced stroke rats. (A) Prehensile traction test. (B) Beam-walking test. (C) Neurological defect (Bederson) scoring. (D) Comprehensive scoring. X -bar represents the day for administration, “0” means the day before administration, and “7” 12

means 7 days after administration. Fig. 2 Incidence of stroke and survival rate after high-salt diet in Dahl-SS hypertensive rats. (A) The incidence of stroke. (B) The survival rate. X -bar represents the day of administration, “0” means the day before administration, and “7” means 7 days after administration. Fig. 3 Effect of BZP on the activities of GSH-px and CAT and the level of NO in blood plasma on Dahl-SS hypertensive-induced stroke rats. (A) Activity of CAT. (B) Activity of GSH-px. (C) Level of NO. Data were presented as mean ± standard deviation. One-way ANOVA test was used to determine the statistical significance between groups. 0.01 vs Model group; &P < 0.05,

&&

##

P <0.01 vs Control; *P < 0.05, **P <

P < 0.01 vs NBP-K group; ▲P <0.05, ▲▲P <0.01 vs Br-NBP

group. Fig. 4 Effect of BZP on histological changes in the brain, kidney and heart in Dahl-SS hypertensive-induced stroke rats. (A) The brain histological changes. (B) The kidney histological changes. (C) The heart histological changes. The different animal treatments are shown in: (a) Control, (b) Model group, (c) NBP-K (9.6 mg/kg), (d) Br-NBP (10.5 mg/kg), and (e-g) BZP (0.75, 3, and 12 mg/kg). Arrows in (A) show that nerve cells sparse, irregular, even disappeared, nucleus shrinkage with neuronal necrosis. Arrows in (B) show that glomerular capillary fibrosis, atrophy and disappearance, accompanied by inflammatory cell infiltration. Arrows in (C) showed that the cardio myocytes were arranged in disordered, the intercellular space was markedly enlarged and with the muscle fibers fracture. BZP treatment markedly attenuated these histological changes as compared with the Model group. Fig. 5 Effect of BZP on ADP-, thrombin-, and AA-induced platelet aggregation in vitro. (A) ADP-induced platelet aggregation. (B) thrombin-induced platelet aggregation. (C) AA-induced platelet aggregation. Rat washed platelets (4 × 108 platelets/mL) were pre-incubated with BZP for 5 min, and then stimulated with 15 μM ADP, 1.05 U/L thrombin, and 3.6 mM AA to stimulate. Data are expressed as mean ± standard deviation (n = 6); One-way ANOVA test was used to determine statistical significance between groups. ##P < 0.01 vs Control group; *P < 0.05, **P < 0.01 vs ASA group; &P < 0.05 vs NBP-K group; ▲P < 0.05, ▲▲P < 0.01 vs Br-NBP group. The line charts show BZP on AA-induced rat platelet aggregation in vitro. X -bar represents the time of aggregation, Y -bar represents the percent of platelet aggregation. Fig. 6 Effect of BZP on ADP-, thrombin-, and AA-induced platelet aggregation in Dahl-SS hypertensive-induced stroke rats. (A) ADP-induced platelet aggregation. (B) thrombin-induced platelet aggregation. (C) AA-induced platelet aggregation. Data is expressed as mean ± standard deviation (n = 6); One-way ANOVA test was used to determine statistical significance between 13

groups. Note: #P < 0.05, ##P < 0.01 vs Control; &P < 0.05, &&P < 0.01 vs NBP-K; △ P < 0.05, △△P < 0.01 vs ASA; ▲P < 0.05, ▲▲P < 0.01 vs Br-NBP. Fig. 7 Correlation between AA-induced platelet aggregation and comprehensive score in Dahl-SS hypertensive-induced stroke rats. (A) Control group. (B) BZP (12 mg/kg) group. (C) BZP (3 mg/kg) group. (D) BZP (0.75 mg/kg) group. r2 means the correlation coefficient of platelet aggregation and comprehensive score. P < 0.05 and P < 0.01 denote statistical significances. Fig. 8 Effect of BZP on AA-induced AMPK, ATP, cGMP, MAPK, and VASP levels in platelets. (A) the level of AMPK. (B) The level of ATP. (C) The level of cGMP. (D) The level of MAPK. (E) the level of VASP. Data are expressed as mean ± standard deviation (n=6); One-way ANOVA test was used to determine statistical significance between groups. Note: #P < 0.05 vs Control; *P < 0.05, **P < 0.01 vs Model; ▲P < 0.05 vs Br-NBP. Fig. 9 Effect on cerebral thrombosis of BZP in Dahl-SS hypertensive-induced stroke rats. Data are expressed as mean ± standard deviation (n=10); One-way ANOVA test was used to determine statistical significance between groups. Note: #P < 0.05 vs Control; *P < 0.05 vs Model group; &P < 0.05 vs NBP-K group; △ P < 0.05, △△ P < 0.01 vs ASA; ▲P < 0.05, ▲▲P < 0.01 vs Br-NBP.

14

Highlights:  BZP reduces the incidence of stroke, neuronal necrosis in the brain, and cell swelling and inflammatory infiltration in the kidney. 

BZP inhibits arachidonic acid (AA) - induced platelet aggregation (IC50: 12 µM) rather than that of adenosine diphosphate (ADP) - and/or thrombin-induced platelet aggregation in vitro.



BZP inhibits ADP- or thrombin- or AA-induced platelet aggregation ex vivo.



BZP inhibits thrombus formation in Dahl-SS hypertensive-induced stroke rats.



BZP could have an effect on the treatment and prevention of cerebral ischemic stroke in Dahl-SS hypertensive rats.

15

2.0

prehensile traction score

Control

B

Model NBP-K 9.6 mg/kg Br-NBP 10.5 mg/kg BZP 0.75 mg/kg

1.6

1.2

BZP 3 mg/kg BZP 12 mg/kg

0.8

0.4

3.0 Control

beam-walk ing test score

A

0.0

2.5

Model NBP-K 9.6 mg/kg

2.0

Br-NBP 10.5 mg/kg BZP 0.75 mg/kg BZP 3 mg/kg

1.5

BZP 12 mg/kg 1.0 0.5 0.0

0

7

14

21

28

0

7

Day

14

21

28

Day

D 7

2.5 Control Model

BZP 3 mg/kg BZP 12 mg/kg

1.0

0.5

Control Model

6

comprehensive score

NBP-K 9.6 mg/kg Br-NBP 10.5 mg/kg BZP 0.75 mg/kg

1.5

NBP-K 9.6 mg/kg 5

Br-NBP 10.5 mg/kg BZP 0.75 mg/kg

4

BZP 3 mg/kg BZP 12 mg/kg

3 2 1

0.0

0 0

7

14

21

28

0

7

Day

14

21

28

Day

Fig.1 Stroke administration of BZP improved motor dysfunction and decreased neurological scores in Dahl-SS hypertensive-induced stroke rats. (A) Prehensile traction test. (B) Beam-walking test. (C) Neurological defect (Bederson) scoring. (D) Comprehensive scoring. X -bar represents the day for administration, “0” means the day before administration, and “7” means 7 days after administration.

A

B

100

Control Model NBP-K 9.6 mg/kg Br-NBP 10.5 mg/kg BZP 0.75 mg/kg BZP 3 mg/kg BZP 12 mg/kg

90 the incide nce of stroke (%)

Bederson score

2.0

80 70 60 50 40 30 20

110 Control Model NBP-K 9.6 mg/kg Br-NBP 10.5 mg/kg BZP 0.75 mg/kg BZP 3 mg/kg BZP 12 mg/kg

100 90 Pe rce nt survival

C

80 70 60 50 50

10 0

0 0

7

14

21

28

0

Day

25 25

30

35

40

45

day on high-salt diet

Fig.2 Incidence of stroke and survival rate after high-salt diet in Dahl-SS hypertensive rats. (A) The incidence of stroke. (B) The survival rate. X -bar represents the day of administration, “0” means the day before administration, and “7” means 7 days after administration.

16

A

B1500

15

**

*

&& ▲▲

GSH-px (μmol/L)

10

## 5

1000

##

500

N

kg P

Z B

B

Z

P

3

12

m

m

g/

g/

kg

g/ kg m

m P

B r-

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.5

m 9. 6 K

N

B

P-

B Z

B

g/ kg

el od

ol on tr

P

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C

m

m

g/

g/

kg

kg

g/ kg 3 Z P

m

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P

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N 4

m

g/ kg

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el

m

ol

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C

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**

&& ▲

3

&& ▲▲

2

1

kg P

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m

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Z

Z

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Fig.3 Effect of BZP on the activities of GSH-px and CAT and the level of NO in blood plasma on Dahl-SS hypertensiveinduced stroke rats. (A) Activity of CAT. (B) Activity of GSHpx. (C) Level of NO. Data were presented as mean standard deviation. One-way ANOVA test was used to determine the statistical significance between groups. ##P < 0.01 vs Control; *P < 0.05, **P < 0.01 vs Model group; &P < 0.05, &&P < 0.01 vs NBP-K group; ▲P < 0.05, ▲▲P < 0.01 vs Br-NBP group.

A a

b

c

d

e

f

g

B

a

b

c

d

e

f

g 50μm

50μm

C a

b

c

d

e

f

g 50μm

Fig.4 Effect of BZP on histological changes in the brain, kidney and heart in Dahl-SS hypertensive-induced stroke rats. (A) The brain histological changes. (B) The kidney histological changes. (C) The heart histological changes. The different animal treatments are shown in: (a) Control. (b) Model group. (c) NBPK (9.6 mg/kg). (d) Br-NBP (10.5 mg/kg). and(e-g) BZP (0.75, 3, and 12 mg/kg). Arrows in (A) show that nerve cells sparse, irregular, even disappeared, nucleus shrinkage with neuronal necrosis. Arrows in (B) show that glomerular capillary fibrosis, atrophy and disappearance, accompanied by inflammatory cell infiltration. Arrows in (C) showed that the cardio myocytes were arranged in disordered, the intercellular space was markedly enlarged and with the muscle fibers fracture. BZP treatment markedly attenuated these histological changes as compared with the Model group.

17

A

B 100

Platelet aggregation (%)

Platelet aggregation (%)

100

80

60

40

20

0

60

40

20

0 149 μM

Control

264 μM

470 μM

Control

C 80

# **

# **

&

&

# **

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# **

# *

#

#

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& ▲▲

# 20

μM

μM BZ P

20

μM BZ P

14

μM

10

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BZ P

Br -N

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5

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μM

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12 BP

12 BP -K N

A

SA

C

12

on tr

μM

ol

0

A

149 ¦ ÌM

264 ¦ ÌM

470 ¦ ÌM

Fig.5 Effect of BZP on ADP-, thrombin- and AA-induced platelet aggregation in vitro. (A) ADP-induced platelet aggregation. (B) thrombin-induced platelet aggregation. (C) AA-induced platelet aggregation. Rat washed platelets (4 108 platelets/mL) were pre-incubated with BZP for 5 min, and then stimulated with 15 μM ADP, 1.05 U/L thrombin, and 3.6 mM AA to stimulate. Data are expressed as mean standard deviation (n = 6); One-way ANOVA test was used to determine statistical significance between groups. ##P < 0.01 vs Control group; *P < 0.05, **P < 0.01 vs ASA group; &P < 0.05 vs NBPK group; ▲P < 0.05, ▲▲P < 0.01 vs Br-NBP group. The line charts show BZP on AA-induced rat platelet aggregation in vitro. X -bar represents the time of aggregation, Y -bar represents the percent of platelet aggregation.

100

Platelet aggregation (%)

80

B 100

60

#

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#

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#

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# #

#

▲

#

#

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m

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B

Z

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P Z B

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m

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g/ kg

m g/ kg

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m

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Z

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P

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3

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m

m

g/ kg

g/ kg

g/ kg m 10 .5 P B

B

N

rN

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7

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m g/ kg

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80

Platelet aggregation(%)

100

C 80

# △ △ ▲▲

60

#

#

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40

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#

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#

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20

kg g/

kg

m

g/

12

3

Z

P

P

B

Z B

0. 75 P B

Z

m

m

m .5 10 P B rN

Fig.6 Effect of BZP on ADP-, thrombin- , and AAinduced platelet aggregation in Dahl-SS hypertensiveinduced stroke rats. (A) ADP-induced platelet aggregation. (B) thrombin-induced platelet aggregation. (C) AA-induced platelet aggregation. Data is expressed as mean standard deviation (n = 6); One-way ANOVA test was used to determine statistical significance between groups. Note: #P < 0.05, ##P < 0.01 vs Control; &P < 0.05, &&P < 0.01 vs NBP-K; △P < 0.05, △△P < 0.01 vs ASA; ▲P < 0.05, ▲▲ P < 0.01 vs Br-NBP.

B

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m

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Platelet aggregation (%)

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18

A

B 30

Platelet aggregation (%)

Platelet aggregation (%)

85

r 2=0.83, P < 0.05

80

75

70

65

r 2=0.958, P < 0.01 25

20

15 4

5

6

7

8

0.4

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comprehensive score

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60

Platelet aggregation (%)

Platelet aggregation (%)

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r 2=0.97, P < 0.01

35

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1.8

55 r 2=0.701, P < 0.05 50 45 40 35 30 1.0

2.0

1.5

2.0

2.5

comprehensive score

comprehensive score

Fig.7 Correlation between AA-induced platelet aggregation and comprehensive score in Dahl-SS hypertensiveinduced stroke rats. (A) Control group. (B) BZP12mg/kg group. (C) BZP 3 mg/kg group. (D) BZP 0.75 mg/kg group. r2 means the correlation coefficient of platelet aggregation and comprehensive score. P < 0.05 and P < 0.01 denote statistical significances.

B

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9. 6

6

7

8

2.0

10

#

*

▲

8

#

MAPK(U/L)

cGMP(nmol/ml)

**

50

el

ATP(nmol/L)

10

C

C

**

60

M od

AMPK(U/L)

#

70

12

M od

A

1.0

*

*

6

*

**

4

0.5

2

*

*

800

600

400

200

kg g/ 12

m

m g/ 3 B

Z

P

P Z B

0. 75 P B

Z

kg

m g/ kg

g/ kg .5 10

P B rN

B

m

g/ kg m 9. 6

PK

SA A

N

g/ kg

g/ kg

m

g/ kg P Z B

Z

P

3

12

m

m 75 0.

P Z B

B

g/ kg m

10 .5 P rN B

PK

Fig.8 Effect of BZP on AA-induced AMPK, ATP, cGMP, MAPK and VASP level in platelets. (A) the level of AMPK. (B) The level of ATP. (C) The level of cGMP. (D) The level of MAPK. (E) the level of VASP. Data are expressed as mean standard deviation (n = 6); One-way ANOVA test was used to determine statistical significance between groups. Note: #P < 0.05 vs Control; *P < 0.05, **P < 0.01 vs Model; ▲ P < 0.05 vs Br-NBP.

B

el

m g/ kg 7

M od

on tr

ol

0

C

9. 6

m 7 N B

B

*

*

#

m g/ kg

g/ kg

od el A SA

12 Z P B

B

1200

1000

VASP(ng/L)

M

g/ kg m

g/ kg m 3

B Z

Z P

P B

B

rN B

N

P

0. 75

.5 10

9. 6 PK

SA A

m g/ kg

m g/ kg

g/ kg m

el

g/ kg 7

m

M od

l on tr o C

E

C on tr ol

0

0.0

19

0.4

A/Brain weight/g

# * △△ ▲

0.3

*

*

△

*

*

*

△

& ▲▲

&

0.2

0.1

kg

g/

m 12

m

m

3

P

P B

Z

Z B

0. 75 P B

Z

g/

kg

g/ kg

g/ kg m

g/ kg

.5

m 9. 6

P N rB

N

B

B

P-

A

K

SA

10

el

g/ kg

od 7

m

M

C

on tr

ol

0.0

Fig.9 Effect on cerebral thrombosis of BZP in Dahl-SS hypertensive-induced stroke rats. Data are expressed as mean standard deviation (n = 10); One-way ANOVA test was used to determine statistical significance between groups. Note: #P < 0.05 vs Control; *P < 0.05 vs Model group; &P < 0.05 vs NBP-K group; △P < 0.05, △△P < 0.01 vs ASA; ▲P < 0.05, ▲▲P < 0.01 vs Br-NBP.

20