Salvia miltiorrhiza prevents deep vein thrombosis via antioxidative effects in endothelial cells

Introduction

Deep vein thrombosis (DVT) is a common clinical problem that affects >250,000 individuals in the USA and 25,000 individuals in Canada annually (1). DVT in the lower extremities is characterized by initial swelling and lower limb pain symptoms, which may be severe. DVT is also a well-recognized cause of pulmonary embolism (PE), which is a potentially life-threatening complication (2,3), for which treatment is necessary. Traditionally, the initial treatment for DVT involves anticoagulation using low-molecular-weight heparin (4,5), followed by long term therapy with vitamin K antagonists, such as warfarin (6). Since the outcome of these treatments in some cases is disappointing (7,8), more effective approaches aimed at reducing DVT have garnered interest in recent years.

Salvia miltiorrhiza is one of the most versatile Chinese herbal drugs, which has been used for hundreds of years to treat numerous ailments (9). S. miltiorrhiza is considered to be highly effective in activating circulation, and dispersing stasis or sludging of blood (10). In recent years, S. miltiorrhiza has been widely used to treat cardiovascular diseases (11). The present study investigated whether S. miltiorrhiza could prevent DVT, using a rabbit ligation model. Furthermore, the underlying mechanism of the protective effects of S. miltiorrhiza on vascular endothelial cells was examined.

Materials and methods

Materials

S. miltiorrhiza was donated by the Pharmacy of the Hubei University of Medicine (Shiyan, China) (no. Z51021303). These were extractions of roots, containing 1.5 g/ml. Malondialdehyde (MDA) and Superoxide Dismutase (SOD) Detection kits were obtained from Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). A total of 30 male Japanese white rabbits, weighing 1.8–2.2 kg, were obtained from the Animal Care Facility at the Hubei Animal Center (Wuhan, China) (no. SYXK 2004-0021). All the rabbits had ad libitum access to food and water and were under a standard 12 h light/12 h dark cycle. All experiments performed were in accordance with animal ethics standards. Prior to the experiment, the rabbits were maintained for several days, to observe their health. The rabbits were randomly divided into three groups (n=10 per group): The control, model and Salvia groups. All procedures were approved by the Animal Research Ethics Board at Hubei University of Medicine.

Modeling method

All surgical manipulations were conducted under aseptic conditions, and the rabbits were anesthetized with 10% chloral hydrate (Weihai Wego Medical Systems Co., Ltd, Weihai, China), by means of an intramuscular injection (3 ml/kg, body weight). Following anesthetization, the two sides of the femoral vein (4 cm) of the rabbits were exposed and separated from the surrounding tissue, taking care not to damage the femoral artery and nerve. Each end of the femoral vein was then completely ligated using 2-0 silk thread (Weihai Wego Medical Systems Co., Ltd; Fig. 1).

Figure 1 Following anesthetization, the femoral vein, artery and nerve of the rabbits were exposed from the surrounding tissue. (A) From left to right are the femoral nerve, artery and vein. (B) Each end of the femoral vein was completely ligated using 2-0 silk thread.

Delivery method

A total of 30 rabbits were randomly divided into three groups, each containing 10 rabbits: Control, model and Salvia. The Salvia group were treated with a daily intravenous injection of 2 g/kg S. miltiorrhiza nto the ear vein prior to generation of the ligation model. The model and control groups received equal amounts of sodium chloride. Following one week of injections the rabbit ligation models were generated, according to the protocol described above. Each group of rabbits received the same treatment as mentioned until the end of the experiment. The response to stimuli (sensitivity to pain stimuli), activity levels and appetite in the rabbits were recorded. The rabbits were sacrificed by aeroembolism seven days after generation of the ligation model.

Specimen collection

Venous blood was collected in the normal and heparin tubes (Weihai Wego Medical Systems Co., Ltd), in order to measure SOD activities, MDA content, coagulation function (including prothrombin, activated partial prothrombin, fibrinogen and thrombin times) and blood rheological parameters (including whole blood viscosity, plasma viscosity and erythrocyte aggregation). Coagulation function was measured by CoaguChek® (Roche Diagnostics, Xinqin, China) and blood rheological parameters were measured using a Blood Rheology Analyzer (SA-9000; Lemon, Zhejing, China). These measurements were made prior to generation of the model, and three and seven days afterwards. At the end of the seven days the ligated femoral veins were placed in 10% formaldehyde solution, fixed for 48 h and paraffin-embedded. Serial sections (4 μm) were collected for hematoxylin and eosin staining. Images of the venous wall and thrombosis were captured under a microscope (BX41; Olympus Corp., Tokyo, Japan). Low magnification (x10) was used to evaluate the vascular wall, while endothelial cells were observed at a higher magnification (x40).

Statistical analysis

The data were analyzed by Student’s t-test and one-way analysis of variance to compare the differences between the groups, using SPSS version 13.0 software (SPSS Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Results

Toxicity and quality of life

The response to stimuli, activity levels and appetite of the model and Salvia groups were similar to that of the control group. Furthermore, no fatal PE occurred in any of the rabbits.

Detection of coagulation function prior to generation of the ligation model

Following one week of S. miltiorrhiza treatment, the coagulation function of the Salvia group was not affected, except for prothrombin time (PT), which was significantly increased, as compared with the control group (P<0.05; Fig. 2).

Figure 2 Effects after one week of intravenous infusion with Salvia miltiorrhiza. (A) Prothrombin time of the Salvia group was delayed compared with the control group (*P<0.05). (B–D) No statistically significant differences were identified in the other measurements of coagulation function, including activated partial prothrombin, fibrinogen and thrombin times (P>0.05).

Measurements of blood rheological parameters and coagulation function, three days following generation of the ligation model

Following generation of the model, blood rheological parameters were measured, and whole blood viscosity (1/sec, 5/sec, 30/sec) and erythrocyte aggregation were shown to be significantly increased in the model group, as compared with the control group (P<0.05). These results suggest that ligation of the femoral vein may result in a hypercoagulable state. There was also a statistically significant increase in whole blood viscosity (1/sec, 5/sec, 30/sec) in the Salvia group, as compared with the model group (P<0.05; Fig. 3). No significant differences were identified in the measurements of coagulation function, except for PT, which was increased in the Salvia group, as compared with the model group (P<0.05; Fig. 4).

Figure 3 Measurements of blood rheological parameters were conducted three days after the generation of the ligation model. (A–C) There was a statistically significant difference in whole blood viscosity (1/sec, 5/sec and 30/sec) and (F) erythrocyte aggregation index between the model and control groups (*P<0.05). This suggested that ligation of the femoral vein results in a hypercoagulable state. (A–C) There was also a delay in the 1/sec, 5/sec and 30/sec whole blood viscosity in the Salvia group compared with the model group (#P<0.05). (D–F) No significant differences were identified in the other blood rheological parameters between the groups, including 200/sec whole blood viscosity, plasma specific viscosity and erythrocyte aggregation index (P>0.05).

Figure 4 Effects three days after the generation of the ligation model. (A) Prothrombin time of the Salvia group was delayed compared with the model group (*P<0.05). (B–D) No significant differences were identified in the other measurements of coagulation function between the groups, including activated partial prothrombin, fibrinogen and thrombin times (P>0.05).

Measurements of blood rheological parameters and coagulation function, seven days following generation of the ligation model

Seven days after generation of the model, blood rheological parameters were measured, and whole blood viscosity (1/sec, 5/sec, 30/sec and 200/sec) was shown to be significantly decreased in the Salvia group, as compared with the model group (P<0.05; Fig. 5). There were no significant differences in the coagulation function between the Salvia and model groups (P>0.05; Fig. 6).

Figure 5 Effects seven days after the generation of the ligation model. (A–D) There was a significant increase in whole blood viscosity (1/s, 5/s, 30/s and 200/s) in the Salvia group compared with the model group (*P<0.05). (E and F) No significant differences were identified in plasma-specific viscosity and erythrocyte aggregation index between the groups (P>0.05).

Figure 6 Seven days after the generation of the ligation model, no significant differences were identified in any of the measurements of coagulation function between the groups (P>0.05).

Antioxidative function

The present study evaluated the antioxidative function of S. miltiorrhiza, the results of which are presented in Fig. 7. The MDA content was significantly decreased (P<0.05), and the activities of SOD were notably increased (P<0.05) in the Salvia group compared with the model group three days after generation of the model. The MDA content was higher on the seventh day (8.00±1.96) in the model group compared with the third day (5.63±1.77). However, in response to treatment with S. miltiorrhiza, MDA content was decreased (P<0.05), and SOD activities were increased (P<0.05) compared with the model group seven days after generation of the ligation model.

Figure 7 In the Salvia group, (A) activities of superoxide dismutase (SOD) were notably increased and the (B) malondialdehyde (MDA) content was significantly decreased compared with the model group three days after generation of the ligation model (*P<0.05). (C) MDA content was higher on the seventh day (8.00±1.96) compared with the third day (5.63±1.77) in the model group. (C and D) In response to treatment with S. miltiorrhiza, SOD activities increased, and MDA content decreased compared with the model group seven days after generation of the model (*P<0.05).

Histopathological observation

Following the sacrifice of the rabbits, the ligated femoral veins were harvested and images of the veins were captured under a microscope. The vascular wall was continuous in the control group, endothelial integrity was maintained and no shedding of endothelial cells was observed. The vascular wall of the model group exhibited discontinuous change, intimal irregularities, endothelial cell shedding, and thrombosis (mixed thrombus and red thrombus), which was attached to the vascular wall. The vascular wall of the Salvia group exhibited complete continuity, regular intima, some endothelial cell shedding and a small amount of intraluminal thrombosis, which was partly attached to the vascular wall (Fig. 8).

Figure 8 Images of the venous wall were captured under a microscope. (A and B) In the control group the vascular wall was continuous, endothelial integrity was maintained and no shedding of endothelial cells was observed. (C and D) In the model group the vascular wall showed discontinuous change, intimal irregularities, endothelial cell shedding and thrombosis (mixed and red thrombi), which was attached to the vein vascular wall. (E and F) In the Salvia group the vascular wall exhibited complete continuity, regular intima, some endothelial cell shedding and a small amount of intraluminal thrombosis, which was partly attached to the vein vascular wall. (A, C and E) Hematoxylin and eosin (H&E); magnification, ×10. (B, D and F) H&E; magnification, ×40.

Discussion

DVT is a well-known major public health problem, which represents a significant clinical and economic disease burden. Up to 21% of cases of DVT may lead to PE, which is a potentially life-threatening complication (12). The conventional treatment for acute DVT is immediate anticoagulation using low molecular-weight heparin, followed by a period (3–6 months) of treatment with oral anticoagulants (13,14). This treatment aims to prevent thrombus propagation, and to reduce the risks of PE and DVT recurrence. However, anticoagulation therapy does not possess significant fibrinolytic activity, and patients with severe, extensive and proximal DVTs remain at high risk (15). In DVT treated with anticoagulants alone, it has been shown that early spontaneous clot lysis frequently results in preservation of valvular function, which may help to reduce post-thrombotic morbidity (16). The present study chose to administer an intravenous injection of S. miltiorrhiza one week prior to the generation of a ligation model in rabbits.

S. miltiorrhiza is a common traditional Chinese medicine used for improving body function, which is capable of promoting circulation and improving blood flow. In addition, it has been used for the treatment of cardiovascular diseases, including coronary heart disease, hyperlipidemia and cerebrovascular disease (17,18). S. miltiorrhiza has also been widely used in the United States (10). When platelets are challenged with outside stimuli to vascular endothelial cells they increase the expression of the adhesion molecule CD31, which may adhere to vascular endothelial cells, resulting in the formation of a soft thrombus (19). Furthermore, the formation of hard thrombi is initiated by the enhanced adhesion of platelets, and the conversion of fibrinogen to fibrin (19). Numerous studies have been conducted regarding the effects of S. miltiorrhiza on platelet aggregation. The inhibitory effects of S. miltiorrhiza have been suggested to be associated with numerous events, including the inhibition of Ca2+ influx in platelets, an increase in the number of fibroblasts in the G0/G1 phase and the attenuation of collagen secretion (20). The present study demonstrated that treatment with S. miltiorrhiza one week after generation of a ligation model, did not affect the majority of measurements of coagulation function, except for PT, which can be used to evaluate the action of five different clotting factors (I, II, V, VII, and X). Blood that takes a long time to clot in a PT test has previously been shown to be an indicator of treatment using warfarin (21). In the present study the PT was significantly increased in the Salvia group on the third day, but not on the seventh day, after generation of the model.

Endothelium secretes factors that control vascular relaxation and contraction, thrombogenesis and fibrinolysis, and platelet activation and inhibition (22). Therefore, maintenance of the functional integrity of endothelium is critical for preservation of blood flow, and the prevention of thrombosis (23). A balance between growth and death of endothelial cells is important for the integrity of the vascular endothelium. An appropriate growth response of endothelial cells helps maintain the integrity of the endothelium, and prevent the development of atherosclerosis (24). The results of the present study indicated that S. miltiorrhiza may protect vascular endothelial cells in vivo. Previous studies have also demonstrated the protective functions of S. miltiorrhiza on human vascular endothelial cells in vitro (25–27).

Oxidative stress is the imbalance between the levels of antioxidants and the production of oxygen-derived species (28). In the present study S. miltiorrhiza exhibited antioxidative functions in the vein ligation model. Chan et al (29) investigated the effects of S. miltiorrhiza on the pharmacodynamics and pharmacokinetics of warfarin in rats. S. miltiorrhiza was shown to potentiate the anticoagulant action of warfarin, by increasing the absorption rate constant, the area under the plasma concentration time curve, and the maximum concentration and half-life of warfarin. In addition, S. miltiorrhiza decreased the clearance and the apparent volume of distribution of warfarin. The present study did not investigate the effects of warfarin, or the effects of a combined treatment of S. miltiorrhiza with warfarin.

In conclusion, previous studies indicated that S. miltiorrhiza promoted circulation and improved blood flow in the treatment of cardiovascular diseases, while the present study revealed that S. miltiorrhiza exhibited antioxidative and protective effects on vascular endothelial cells. The results of the present in vivo study demonstrated that S. miltiorrhiza can decrease blood rheological parameters. S. miltiorrhiza was also shown to exhibit antioxidative and protective effects on vascular endothelial cells. These results suggest that S. miltiorrhiza may have potential applications for the treatment of DVT.

Acknowledgements

The present study was supported by the Hubei Provincial Department of Education (grant no. Q20132108).

References