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Extract from Rostellularia procumbens (L.) Nees Inhibits Thrombosis and Platelet Aggregation by Regulating Integrin β3 and MAPK Pathways

Aim of study: The main objective of this study was to investigate the antithrombotic and antiplatelet effect of the extract from Rostellularia procumbenss (L.) Nees and understand the mechanisms by which it exerts its antithrombotic and antiplatelet mechanisms. Materials and methods: The antithrombotic effective parts (RPE) were isolated using D101 macroporous adsorption resin and potential active ingredients (JAC) were isolated using the preparative liquid-phase method. The lactate dehydrogenase kit was used to determine the toxicity of RPE and JAC to platelets. The antiadhesion effect of RPE and JAC on platelets was observed by fluorescence microscopy with rhodamine phalloidin. Antithrombotic efficacy of RPE and JAC in vivo was evaluated by establishing a rat tail thrombosis model. Contents of p-selectin, TXB2, and 6-keto-PGF1α in rat serum were measured using an enzyme-linked immunosorbent (ELISA) assay, and the rat black tail rate was measured to prove the protective effect of RPE and JAC on the tail thrombus rat model. Western blot was used for detection of serum-related proteins in the tail thrombus rat model. Results: The results showed that RPE had antithrombotic and antiplatelet effects. RPE and JAC have no toxicity to platelets. In vitro experiments showed that RPE and JAC had antiadhesion effects on platelets. In vivo experiments showed that RPE significantly inhibited the increase of p-selectin and TXB2 and significantly increased the content of 6-keto-PGF1α in the serum of rats. Western blot results demonstrated that RPE and JDB significantly inhibited the phosphorylation of the MAPK protein family in the platelets of rats, and RPE also significantly inhibited the phosphorylation of β3 protein. Conclusions: RPE has antithrombotic and antiplatelet activity in vivo and vitro. Its mechanism may be via preventing integrin αIIbβ3 activation, which in turn leads to the inhibition of the phosphorylation of the MAPK family and further suppresses TXA2, which leads to the antithrombotic and antiplatelet effects.


INTRODUCTION
Thrombotic disease which is the general term for diseases caused by the two pathological processes of thrombosis and thromboembolism is a disease which seriously endangers human health and life. 1,2 The pathogenesis of thrombotic diseases is very complex and is not very well understood. Recent studies have shown that factors such as the vascular endothelial status, platelet number, and blood coagulation play a role in the pathogenesis of thrombotic diseases. 3 Antiplatelet therapy has become one of the important treatments for thrombotic diseases. Aspirin, clopidogrel, and antiplatelet aggregation drugs which are widely used in clinic often have a single mechanism of action and serious side effects. For example, when aspirin is taken for a long time, it can lead to gastrointestinal bleeding and death. 4 Compared with chemical drugs, traditional Chinese medicines have great advantages. Many researchers have found that traditional Chinese medicines such as Salvia miltiorrhiza have strong antithrom-botic activity and the potential to be developed into antiplatelet aggregation drugs compared to drugs such as aspirin. 5 Information about Rostellularia procumbens (L.) Nees, a medicinal plant of the Acanthaceae family, was first published in Sheng Nong's Herbal Classic. It was also included in the Chinese Pharmacopoeia (1977). It is mainly composed of lignans, flavones, and triterpenes 6 which are clinically used to treat colds, fevers, flu, malaria, and hepatitis. Studies have demonstrated that multiple bioactive effects including   antitumor, antichronic glomerulonephritis, and bacteriostatic activities are exerted by active compositions of R. procumbens (L.) Nees. 7 Previous work done by our lab has demonstrated that R. procumbens (L.) Nees has the effect of antiplatelet aggregation at the network pharmacology level. 8 The objective of this study was to verify the antiplatelet aggregation and antithrombotic efficacy of the active extract of Rostellularia procumbent (L.) Nees and understand the mechanism of its action at the cellular and molecular level.

RESULTS
2.1. Main Active Ingredients in RPE. It was found that there were five potential effective chemical components in RPE by HPLC ( Figure 1A). The reference products had a purity of more than 95% found by NMR. By comparing with the reference products, we determined that the five components were 6′-hydroxy justicidin B, justiscidin B (JDB), chinensinaphthol methyl ether (CME), neojusticidin B, and neojusticidin A (chemical structures in Figure 1B).
2.2. RPE and JAC Have No Toxicity to Platelets. Results of the LDH release rate are shown in Figure 2; it was found that compared with the blank control, each concentration of RPE and JAC had no effect on the release rate of platelets, which means that the RPE and JAC have no toxicity to platelets.
2.3. RPE and JAC Resist Platelet Adhesion In Vitro. Next, the effect of RPE and JAC on platelet adhesion in vitro was evaluated. The rhodamine phalloidin fluorescence was observed, and the sections were analyzed using Image-Pro Plus 6.0 ( Figure 3). There was a significant augmentation of fluorescence staining percentage in the collagen group compared with the control group (P < 0.01). In addition, there was a significant decrease in fluorescence staining percentage in JAC, RPE-M, and RPE-H groups compared with the collagen group (P < 0.01).
2.4. In Vivo Protective Effect of RPE and JAC on Tail Thrombosis in Rats. From Figure 4 and Table 1, we could see that at the 12th h after thrombus modeling, no thrombus was in the tail of the control group at 12 h post modeling. However, a blood thrombus was formed in both the model group and the treatment groups. Compared with the model group, the black tail rate of rats in the treatment groups was significantly decreased (p < 0.05).
The liver of animals in the model group and the treatment groups was swollen and the liver coefficient was increased. The liver was normal in the animals in the control group; the liver coefficient of the model group was significantly higher when compared with that of the control group (p < 0.05), while the liver coefficient of the treatment groups was significantly lower when compared with that of the model group (p < 0.05). This indicated that the drug had a protective effect on tail thrombosis.
2.5. Effects of RPE and JAC on P-Selectin Expression. P-selectin is a member of the selectin family of adhesion molecules. It is a glycoprotein on the α-granule membrane of platelets. On platelet activation, P-selectin is expressed on the cell surface and released as particles, leading to adhesion to leukocytes. 9 Compared with the control group, the expression of P-selectin in the model group increased significantly (p < 0.05), which was significantly alleviated in the treatment groups (p < 0.05) (Figure 5a). These results demonstrated that activation of platelets after thrombus modeling led to the release of p-selectin particles. Administration of RPE and JAC might be beneficial in reducing this increase.
2.6. Effects of RPE and JAC on TXB 2 and 6-Keto-PGF 1α Expression. TXA 2 and PGI 2 are a pair of antagonistic endogenous substances, which play a major role in the regulation of platelet function. We analyzed the expression of 4.007 ± 0.181 c a p < 0.01 versus the control group. b p < 0.05 versus model group. c p < 0.05 versus model group.

ACS Omega
http://pubs.acs.org/journal/acsodf Article TXB 2 and 6-keto-PGF 1α in the serum because they are stable hydrolysis products of TXA 2 and PGI 2 (Figure 5b,c). 10 Compared to the model group, treatment with JDB and RPE significantly downregulated the expression of the TXB 2 level and upregulated the expression of the 6-keto-PGF 1α level in the serum (p < 0.05). 2.7. Reduction in β 3 Phosphorylation by RPE in Carrageenan-Induced Rats. Previous studies have demonstrated that integrin β 3 is the main receptor responsible for regulating platelet aggregation and thrombosis. 11 We measured the content of β 3 proteins in rat platelet protein in order to understand the antithrombotic mechanism of RPE and JAC. Compared to the control group, there was a significant upregulation of β 3 in the model group (p < 0.05). Treatment with ASP, RPE-L, and RPE-M significantly decreased the expression of β 3 protein in the induced rats (p < 0.05). The expression of integrin β3 was also significantly downregulated by RPE-H treatment (p < 0.01) (Figure 6b).
2.8. Reduction in MAPK Phosphorylation by RPE in Carrageenan-Induced Rats. MAPK is widely involved in various physiological processes, such as inflammatory response, oxidative stress, and apoptosis. 12 In our study, after platelet activation, α IIb β 3 is activated, which further leads to phosphorylation of JNK, ERK, and p38 in the MAPK family. The expression of the p-MAPK family in platelet proteins in model rats was analyzed by us using western blot. Compared to the control group, the expression of p-ERK, p-JNK, and p-p38 protein was notably increased in the model group (p < 0.05) (Figure 6c). The treatment groups decreased the p-ERK, p-JNK, and p-p38 expression to some extent. Treatment with RPE-H and RPE-L significantly decreased ERK phosphorylation (p < 0.01). Treatment with ASP and CEM also decreased the ERK phosphorylation significantly (p < 0.05), while JDB had no effect on ERK phosphorylation. JNK phosphorylation was significantly downregulated by ASP, JDB, RPE-L, RPE-M, and RPE-H (p < 0.01). There was no effect of CME on JNK phosphorylation. ASP, RPE-M, and RPE-H significantly decreased p38 phosphorylation (p < 0.01), while there was no effect of JDB and CME on p38 phosphorylation.

DISCUSSION
Most cardiovascular and cerebrovascular diseases develop as a result of thrombotic diseases, such as stroke, myocardial infarction, pulmonary embolism, and so on. 13 Thrombotic diseases have a complex etiology and a long course. Substantial progress has been made in the use of TCM for the treatment of thrombotic and platelet aggregation diseases. R. procumbent (L.) Nees is a traditional herbal medicine with the efficacy of detoxicating, activating blood and pain relieving, diuretic swelling, and so on. Previous studies have demonstrated its efficacy in inhibiting platelet aggregation. 14 However, it is not clear if R. procumbens (L.) Nees has any protective effects on thrombotic diseases. Therefore, in this study, we extracted the antithrombotic effective parts (RPE) and potential active parts (JAC) through the proven methods in the preliminary experiment in our lab. JAC is a general term for two compounds, Justicidin B (JDB) and Chinensinaphthol methyl ether (CME). Finally, we found that all these three parts have an antithrombotic effect to a certain degree. Investigation of the potential mechanisms of action revealed that the antithrombotic effects are achieved by RPE through the inhibition of integrin α IIb β 3 activation, which in turn inhibits the phosphorylation of the MAPK family and further suppresses TXA 2 so as to inhibit the activity of platelet aggregation.
Previous laboratory studies have found that lignans are the main antiplatelet aggregation compounds. Free lignans are lipophilic and insoluble in water. It has been reported that lignans can have metabolic reactions such as glucosaldehydation, sulfuration, hydroxylation, hydroxylation, reductive glucosaldehydation, and reductive sulfation in animals, ACS Omega http://pubs.acs.org/journal/acsodf Article changing their pharmacological effects. 15 Neojusticidin A, taiwanin E methyl ether, chinensinaphthol methyl ether, taiwanin E, and justicidin B have been reported to have inhibitory effects on arachidin-induced platelet aggregation in rabbits. 6 However, the effect of secondary metabolites of these compounds on platelet aggregation in animals has not been reported, which deserves further study.
In the process of drug research and development, it is necessary to investigate the toxicity of drugs. In our study, the LDH release test was carried out to determine whether the RPE and JAC have toxic or side effects on platelets and to understand the way in which the RPE and JAC act, whether they directly kill platelets or induce platelet inactivation through some mechanisms. 16 The results showed that RPE, JDB, and CME had no cytotoxicity, and the release rate of LDH was less than 3%. The effect of drugs on platelet adhesion was studied in this study because platelet adhesion is closely related to platelet aggregation. 17 In our study, rhodamine phalloidin was used to stain platelets coated with collagen to observe the adhesion of platelets to collagen. The results showed that the treatment groups had a certain platelet adhesion effect, among which the effect of RPE and JDB groups was stronger (P < 0.01), while that of CME was weak (P < 0.05).
Previous studies done by our group demonstrated that the extract had the activity of antiplatelet aggregation in vitro. 8 The in vivo activity in animals was however not analyzed. Platelets often accumulate abnormally in animals with thrombotic diseases, which leads to the formation of a large number of thrombi and results in blockage of blood vessels and embolus formation. 18 Therefore, our group used a rat tail model of the thrombus through intragastric administration to prove that RPE and JAC prevented platelet aggregation in animals and helped in treatment of thrombotic diseases. P-selectin is a glycoprotein present on the platelet α granule membrane. After platelet activation, P-selectin is rapidly expressed on the cell surface and releases α particles, which leads to adhesion to leukocytes. 19 In the tail thrombus rat experiment, the expression of P-selectin in the serum of the model group was increased when compared with the control group, while the expression of P-selectin in the serum of the treated group was lower when compared with the model group. This indicated that the drug reduced the expression of P-selectin in the serum of rats. 20 TXA 2 is a thromboxane synthesized and released from platelet microsomes. It has a ACS Omega http://pubs.acs.org/journal/acsodf Article strong biological activity to promote vasoconstriction and platelet aggregation. 21 PGI 2 is a derivative of arachidonic acid and an antagonist of thromboxane. It significantly inhibits platelet aggregation. 22 TXB 2 and 6-keto-PGF 1α are stable forms of thromboxane A 2 (TXA 2 ) and prostacyclin (PGI 2 ), respectively. 23 The content of TXB 2 in the serum of the model group increased, while the content of 6-keto-PGF 1α decreased in the tail thrombosis experiment in rats. The content of TXB 2 in the serum decreased and the content of 6keto-PGF 1α increased in the treatment group when compared with the model group, except for the low-dose group of RPE and CME groups. It is suggested that RPE and JDB can reduce the production of TXA 2 and increase the content of PGI 2 so as to inhibit platelet aggregation. However, antiaggregation effects are not exerted by CME in a similar manner. Integrin α IIb β 3 is a platelet membrane protein. When platelets are activated by inducers, α IIb β 3 is activated. This exposes its binding site to fibrin and finally platelets aggregate (Figure 7). 24 Integrin α IIb β 3 consists of two type I transmembrane glycoproteins, α II and β 3 , which are connected by disulfide bonds. Integrin α IIb β 3 is the main receptor regulating platelet aggregation and thrombosis. 25 Previous work done by our group found through the gene chip technology that the extract affected the expression of α IIb β 3 protein on platelets. 14 Thus, it was suggested that the extract had the potential to develop into antiplatelet aggregation drugs. Results from the current study demonstrated via the western blot method that RPE significantly inhibited the expression of integrinβ 3 . After platelet activation, integrin α IIb β 3 activation will further lead to the phosphorylation of JNK, ERK, and p38 in the MAPK family. 26 This will result in the production of TXA 2 and the increase of Ca 2+ concentration. Results from our study demonstrated that RPE significantly inhibited the phosphorylated expression of ERK and JDB and RPE. It also significantly inhibited the phosphorylated expression of JNK, JDB, and p38.

CONCLUSIONS
RPE exerts antithrombotic and antiplatelet activity via inhibition of integrin α IIb β 3 activation, by reducing the expression of P-selectin through inhibition of the phosphorylation of the MAPK family, by suppressing production of TXA 2 . Antiplatelet aggregation and antithrombotic effects are also exerted by JDB and CME. Both of them could suppress production of TXA 2 and downregulate the phosphorylation of JNK and ERK. However, their effect on p38 phosphorylation and integrin β 3 was not significant. Our study elucidated the antithrombotic and antiplatelet mechanism of JDB, CME, and RPE by regulating integrin β3 and MAPK pathways. It may provide a basis for the further study of R. procumbent (L.) Nees.

Plant
Material. Fresh R. procumbens (L.) Nees was collected from different regions in Hubei Province of China in October 2018 and was identified by Professor Hezhen Wu of Hubei University of Chinese Medicine. Fresh herbs are dried in the sun and stored at 25°C, away from light and in a ventilated place. The plants were dried and powdered, and the powder was percolate-extracted with ethanol. It was then decompressed to recover the ethanol. Lignans were then enriched with macroporous adsorption resin and the effective part (RPE) was obtained at 24°C. 27 The HPLC method was used to analyze the potentially effective components in the effective part RPE. JDB and CME were obtained by liquidphase preparation, with purity greater than 98%. We called these two parts as JAC below for convenience. Previous studies in the laboratory have proved that these three parts are nontoxic for cells and animals.
5.2. Animals. Two male New Zealand rabbits (general class, weight 2−2.5 kg) were purchased from the Hubei Provincial Center for Disease Control and Prevention; 64 male SD rats (SPF class, weight 190−230 g) were purchased from the Laboratory Animal Center of China Three Gorges University (Yichang, China). The animals were given sufficient water and feed and housed in plastic cages with the ambient temperature to 20°C, and relative humidity was maintained between 50 and 60%. Animals were allowed a 2 week quarantine and acclimation period prior to start of the study. All experimental procedures were approved by the Animal Care and Use Committee of Institute of Materia Medica, People's Republic of China.
5.4. Toxicity Assay of RPE and JAC on Platelets. Rabbit blood was obtained from rabbit ear veins; the blood was added to a vacuum blood collection tube containing 3.8% sodium citrate to obtain anticoagulant blood. Platelet-rich plasma (PRP) was obtained by centrifugation of anticoagulant blood (180g, 10 min). PRP was incubated with a blank solution and PRE (0.25−4.00 mg/mL), JDB (0.05−0.80 mg/mL), and CME (0.06−0.96 mg/mL) at 37°C for 10 min, and then, the LDH kit was used to measure the content of LDH in each group. The following is the calculation formula of the release rate Figure 7. Flow chart of thrombus formation. The platelets were activated by the inducer, deformed, and released dense granules; α IIb β 3 on the surface of the activated platelet was activated, linking with fibrinogen to form a network structure, which locked blood cells into it and eventually formed a thrombus.
LDH release rate (%) sample LDH content total LDH content 100% = × 5.5. Platelet Adhesion Assay by Rhodamine Phalloidin Staining. The round coverslips were placed in a 24-well plate. A total of 200 μL of collagen solution (5 μg/mL) was added to each well. After overnight aspiration, 200 μL of 1% BSA solution was added to each well and sealed for 60 min, and each well was washed three times with PBS. The PRP prepared in the previous item of 200 and 200 μL of different concentrations of drug solution and blank control solution was added to each well; the concentration of each administration was ASP (2.0 mg/mL), JDB (0.20 mg/mL), CME (0.24 mg/ mL), RPE-L (0.25 mg/mL), RPE-M (0.50 mg/mL), and RPE-H (1.0 mg/mL). After the drug solution and the platelets were incubated for 90 min, the round coverslips were fixed with 4% paraformaldehyde for 20 min, and finally, 250 μL of rhodamine phalloidin was added for staining. The staining state was observed with a fluorescence microscope in the dark room. The percentage of fluorescent staining was calculated using Image-Pro Plus 6.0. 5.6. In Vivo Carrageenan-Induced Tail Thrombosis Model. Similar to the pulmonary embolism model, the tail thrombus model experiment was also divided into the same eight groups. The rats in the respective groups were treated intragastrically with either 0.5% CMC-Na solution, 20 mg/kg ASP, 4.2 mg/kg JDB, 5.1 mg/kg CME, 5 mg/kg, 10 mg/kg, or 20 mg/kg RPE for 7 days. One hour after the last dose of the abovementioned drugs, rats in the model group and the treatment groups were given an intraperitoneal injection of 0.4% carrageenan solution. 28 Rats in the blank control were given an intraperitoneal injection of the same amount of 0.9% normal saline. The tail thrombosis length of rats was observed 12 h after thrombus modeling; the black tail rate and liver coefficient were calculated as follows. L A is the length of the black tail; L B is the body length of mice. W C is the weight of the liver; W D is the body weight of the mice 29 Test of p-Selectin, TXB 2 , and 6-Keto-PGF 1α in the Serum of Rats with Tail Thrombi. We preadministered for 7 days and then injected carrageenan to make a model rat. At 12 h after the modeling, rats were anesthetized to take the blood and internal organs and finally executed. A total of 12 h after the abovementioned model rats were anesthetized with urethane, 8 mL of blood was taken from the abdominal aorta; 6 mL of blood was added with an anticoagulant for anticoagulation centrifugation, and 2 mL of blood was added without an anticoagulant for centrifugation to get the serum. The content of p-selectin, TXB 2 , and 6-keto-PGF 1α from the un-anticoagulated serum was measured using an ELISA assay as per the instruction manual provided by the manufacturer.
5.8. Preparation of Platelet-Rich Plasma (PRP). The anticoagulated blood obtained from the animals as described in the previous section was then centrifuged at 180g for 10 min. Plasma containing platelets in the supernatant was taken and the remaining sample was centrifuged at 1800g for 18 min. Platelet-rich plasma (PPP) was obtained from the upper platelet plasma by adjusting the platelet plasma solubility with PPP, so that the platelet number was 3.5 × 10 −8 /mL, to obtain PRP finally. 30 5.9. Western Blotting. PRP was centrifuged at low speed to obtain platelet precipitation. Total proteins were extracted using RIPA lysis buffer (Beyotime Institute of Biotechnology, China). Samples were separated by SDS PAGE and electrotransferred onto the NC membrane (Millipore, USA). They were then incubated overnight at 4°C with the specific primary antibodies. The membranes were washed three times with TBST for 10 min. After washing, the membranes were incubated with HRP anticonjugated secondary antibodies for 1 h. The membranes were washed in the same manner as described above. The membranes were scanned using the FluorChem FC3 system (Protein Simple, USA).
5.10. Statistical Analysis. Experimental data were presented as mean x ± SD. Statistical significance was measured using one-way ANOVA. A value less than 0.05 is considered to be statistically significant.