Antithrombotic effect and action mechanism of Salvia miltiorrhiza and Panax notoginseng herbal pair on the zebrafish

According to the WHO World Health Statistics 2018, an estimated 17.9 million deaths accounting for 44% of noncommunicable diseases (NCDs) occurred due to the cardiovascular diseases [1]. Thrombosis, which plays an important role in cardiovascular disease, seriously threatens human health and life [2, 3]. Currently, the most commonly used anti-thrombotic drugs are aspirin, warfarin and heparin, but they are accompanied by bleeding and drug resistance [4, 5]. Complex pathological conditions such as thrombosis require combinational therapies that can act on multiple biological targets to efficiently manage the underlying mechanistic pathways [6]. Considering that traditional Chinese medicines (TCMs) have the advantages of limited adverse effects, significant efficiency and having active-target diversity, therefore, TCMs become an important research direction to search for anti-thrombotic drugs [7].

Zebrafish, as an animal model has many advantages including rapid propagating and high homology with human genes, has been successfully applied in developmental biology, genetics and human diseases studies [8‐10]. In fact, zebrafish are especially suitable for the study of thrombus due to ease observation of circulation system without invasive methods and its good response to anti-coagulant drugs commonly used in clinical treatment [11, 12]. From previous reports, the arachidonic acid (AA)/FeCl₃-induced zebrafish thrombosis model had been used for evaluation of antithrombotic activity of rosmarinic acid and p-coumaric acid in Danhong injection [13] and natural terpenoid glycosides from the leaves of Crataegus pinnatifida [14].

Salvia miltiorrhiza (Danshen, DS) and Panax notoginseng (Sanqi, SQ), as the traditional medicinal materials that can be used in healthy food, have been focused an increasing number of attentions [15, 16]. The Danshen–Sanqi herbal pair (DS–SQ) has been popular used as the Chinese herbal medicine for prevention and treatment of cardiovascular diseases, including stroke, myocardial infarction and angina pectoris [17]. Recently, DS–SQ was reported having a variety of biological and physiological effects such as protective effects on cell survival in the human cardiovascular endothelial [18], anti-inflammatory activity [19] and inhibition of retina cell apoptosis [20]. A study by Yue et al. revealed that DS–SQ showed cardio-protective effects against ischemia–reperfusion injury [21]. Liu et al. confirmed that DS–SQ with a combination ratio of 10:3 could markedly inhibit platelet aggregation and adhesion in normal rabbit [22]. Although DS–SQ showed the anti-thrombotic activity, there is still a lack of sufficient scientific evidence to illustrate the optimum combination ratio of these two herbs as well as its action mechanisms. Thus, in the present study, taking advantage of transparent zebrafish larvae, a phenylhydrazine (PHZ)-induced zebrafish thrombosis model was developed to evaluate the anti-thrombotic activity of DS–SQ and their nine compounds, and the underlying action mechanism was also explored.

Cardiovascular endothelial injury and the changes of hemodynamics and blood coagulation function are the most important causes of thrombosis [33]. DS–SQ is known as a commonly used TCM for the treatment of cardiovascular diseases such as thrombosis for a long time. Pharmacological research revealed that DS–SQ has the effect of promoting blood circulation and antagonizing myocardial ischemia and reperfusion injury, can be used for the treatment of coronary heart disease and angina pectoris [34, 35]. Herein, the optimum compatibility proportion and the underlying action mechanism of DS–SQ on anti-thrombotic effect were further investigated in this study.

There were extensive studies on phytochemical of DS and SQ [29]. DS contains two types of major bioactive components, including water-soluble phenolic acids and fat-soluble diterpenoid quinones. As the major bioactive components, protopanaxadiol and protopanaxatriol saponins are abundant in SQ [28]. Many analytical methods have been developed for the component identification of the crude drug of DS and SQ. For example, Hong et al. developed an analytical method for the identification and quantification of six phenolic acids in S. miltiorrhiza [36]. Yuan et al. identified new trace triterpenoid saponins from the roots of P. notogiseng by LC–ESI–MS/MS [31]. In the present study, thirty-three chemical compounds, including twelve phenolic acids, seven diterpenoid quinones and fourteen saponins were identified by LC–ESI–MS/MS from the extract of DS–SQ. It was reported that protocatechuic aldehyde and salvianolic acid A, as the water-soluble phenolic acids of DS, and 15, 16-dihydrotanshinone I, cryptotanshinone and tanshinone IIA, as the lipophilic diterpenoid quinones of DS, showed the direct thrombin inhibition activities [37‐39]. While, GRg1, as a high content saponin in SQ, could inhibit platelet activation via the inhibition of PKC and ERK pathway and attenuate arterial thrombus formation in vivo [40].

PHZ can promote vascular endothelial injury and platelets aggregation, which eventually result in the formation of thrombosis [41]. In the present study, a PHZ-induced thrombosis model in zebrafish was established. On the other hand, ASP can inhibit arachidonic acid release in platelets and reduce the production of thromboxane A2 by inhibiting the effects of epoxidase, peroxidase and thromboxane synthase, thereby inhibiting the adhesion and aggregation of platelets and preventing blood coagulation and thrombosis [42]. Therefore, ASP was selected as a positive control drug in the present study. Results indicated that the thrombus could be significantly decreased after the treatment of ASP. Furthermore, DS–SQ at the ratios of 10:1 decreased the PHZ-induced thrombosis in a dose-dependent manner, which indicated that there is a significant synergistic effect in anti-thrombotic activity of DS–SQ herbal pair. However, these two herbs are mixed together before decoction in practical application, which is different from the present study. Therefore, further study can be focused on the impact of preparation methods on the chemical components in DS–SQ and its anti-thrombotic activity. Meanwhile, MA, LA and SAB from DS also showed an anti-thrombotic effect. It is speculated that DS may play a major role in inhibiting thrombosis [43]. However, SQ showed a dual efficacy in hemostasis [44] and anti-thrombosis [45].

Subsequently, the possible anti-thrombotic molecular mechanism of DS–SQ was further studied. The protein kinase C family of serine/threonine kinases plays a well-established and critical role in platelet function and thrombosis [46]. Both of PKCα and PKCβ are two conventional PKC isoforms expressed in platelets. It was reported that PKCα is an important positive regulator of platelet function and a crucial regulator of thrombosis in vitro and in vivo. Likewise, PKCβ showed the functions of regulation the outside-in signaling through the α_IIbβ₃ integrin and positive regulation of thrombosis in vivo [47, 48]. The coagulation cascade is a series of sequential reactions of limited proteolysis of protein factors resulting in generation of thrombin, while fibrinogen, which is encoded by fga, fgb and fgg genes, is clearly one of the key proteins in coagulation cascade [49]. On the one hand, fibrinogen acts as the precursor of the fibrin net that gives structure to blood clots, on the other hand, it acts as the promoter of platelet aggregation and fibrinolysis [50]. The vWF, which is an adhesive multimeric glycoprotein that is crucial for the hemostasis, is synthesized in both endothelial cells and megakaryocytes. Previous report showed that vWF aggravates thrombotic disease such as stroke via a GPIb-dependent mechanism [51]. The result of RT-qPCR indicted that PHZ could upregulate the mRNA expression of PKCα, PKCβ, fga, fgb, fgg and vWF, while the expression quantity were significantly restored after the treatment of DS–SQ extract. These results indicated that the underlying anti-thrombotic mechanism of DS–SQ is related to the regulation of mRNA expression of these genes.

In summary, thirty-three components were identified in DS–SQ by HPLC–ESI–MS/MS analysis. On the PHZ-induced zebrafish thrombosis model, DS–SQ at the combination ratio of 10:1 showed a significant synergistic effect in anti-thrombotic activity with a dose-dependent manner. Furthermore, MA, LA and SAB showed good thrombosis inhibitory activity, indicating that they may be the primary anti-thrombotic active compounds in DS, which further confirmed that DS played a major anti-thrombotic role in the DS–SQ herbal pair. In addition, the anti-thrombotic effect of DS–SQ may through the downregulation of PKCα, PKCβ, fga, fgb, fgg and vWF expressions.