Anticoagulant effect of Huisheng Oral Solution in a rat model of thrombosis.

OBJECTIVE: To investigate whether Huisheng Oral Solution has an anticoagulant effect in a rat model of thrombosis.
MATERIALS AND METHODS: A total of 40 male SD rats were equally and randomly divided into four groups: blank group, model group, and two treatment groups (A and B). Rats were subcutaneously injected with carrageenan to induce thrombosis. Rats in the treatment group A were intragastrically administered with Huisheng Oral Solution at a dose of 2 ml/100 g body weight (once per 8 hours), 72 hours after carrageenan injection, while those in the treatment group B were administered with Huisheng Oral Solution both 72 hours before and after induction of thrombosis. Blood samples were collected 24, 48, and 72 hours after carrageenan injection for measurements of prothrombin time (PT), activated partial thromboplastin time (APTT), international normalized ratio (INR), fibrinogen (FIB), prothrombin activity (PTA), platelets (PLT), fibrin degradation products (FDPs), and D-dimer. Lung, liver, and mesentery samples were taken 72 hours after carrageenan injection for histopathological analysis. The numbers of microthrombi in sections of different tissue samples were counted under a microscope. Blood parameters among each group were compared using the Welch test, the Kruskal-Wallis test, or the SNK test after testing for normality, while the number of microthrombi was compared using the Bonferroni test.
RESULTS: Compared to those in the model group, PT, APTT, and INR were significantly prolonged or increased while FIB was significantly reduced at the majority of time points in the two treatment groups (P < 0.05 for all). The levels of FDPs and D-dimer and PLT counts at the majority of time points were significantly lower (P < 0.05 for all), and the numbers of microthrombi in lung, liver, and mesentery samples were significantly decreased (P < 0.05 for all) in the two treatment groups. The above parameters at the majority of time points showed no significant differences between the two treatment groups.
CONCLUSIONS: Huisheng Oral Solution can significantly improve coagulation parameters, fibrinolysis parameters, and PLT count, and reduce blood hypercoagulability and microthrombosis, suggesting that Huisheng Oral Solution has an anticoagulant effect in a rat model of thrombosis.

Introduction

Environmental pollution and population aging in modern society have greatly increased the incidence of malignant tumors and other diseases. Many patients with malignant tumors are in a hypercoagulable state. Venous thromboembolism, pulmonary embolism, and mild disseminated intravascular coagulation (DIC) are common manifestations of a hypercoagulable state.[12] Studies have shown that the incidence of venous thromboembolism and pulmonary embolism in patients with malignant tumors is approximately 1.5%.[3] About 25% of patients with thromboembolic diseases have malignant tumors.[4]

Huisheng Oral Solution was developed based on a classical recipe named Huazheng Huisheng Dan that was recorded in the Detailed Analysis of Epidemic Warm Diseases, a medical book written in Qing Dynasty. Its main ingredients include ginseng, rhizoma cyperi, angelica, motherwort, rhizoma sparganii, trogopterus dung, turtle shell, frankincense, saffron, Ligusticum wallichii, peach kernel, rhubarb, leech, clove, and ferula asafoetida.[5] To promote flow of Qi and blood circulation, eliminate phlegm and dampness, warm Yang and dredge meridians, and replenish Qi and blood, Huisheng Oral Solution is effective and safe in the treatment of tumors.

Treatment with Huisheng Oral Solution combined with radiotherapy or chemotherapy can improve the quality of life and prolong survival in patients with advanced cancers. Coagulation disorders are common complications of malignant tumors, mainly manifesting as thrombosis caused by hypercoagulability.[6] Huisheng Oral Solution contains ingredients that are able to promote flow of Qi and blood circulation and can therefore be used to treat coagulation disorders in clinical practice. In this study, we investigated the effect of Huisheng Oral Solution on coagulation function in a rat model of venous thrombosis.

Materials and Methods

Animals

Written ethics committee approval was provided by our institution. Forty 7- to 9-week-old male Sprague-Dawley (SD) rats, weighing 180 to 220 g, were provided by the Laboratory Animal Center of Chinese Academy of Medical Sciences. The animals were raised in a temperature-controlled room (24 ± 1°C; humidity ≥40%) on a 12-hour light–dark cycle, with free access to food and water.

Reagents and equipment

Huisheng Oral Solution (batch No. 110203) was provided by the Chengdu Diao Group Tianfu Pharmaceutical Co., Ltd. Carrageenan was purchased from Sigma-Aldrich. The main equipments used in this study included electronic balance (BS 423S, Sartorius), microscope, automatic blood analyzer (LH750, Beckman, USA), and automated coagulation analyzer (Acl-Top, IL, USA).

Animal groups

A total of 40 SD rats were equally and randomly divided into four groups: blank group, model group, and two treatment groups (A and B). Rats in the treatment group A were given Huisheng Oral Solution 72 hours after induction of thrombosis, while those in the group B were given Huisheng Oral Solution both 72 hours before and after induction of thrombosis.

Model of thrombosis

Rats were subcutaneously injected in the sole of the rear foot with 2% carrageenan (20 mg/ml, in normal saline) at a dose of 20 mg/kg of body weight. The rats were placed at room temperature after injection. A dark red thrombus formation area appeared in the tail tip of the majority of animals and gradually expanded toward the tail root. Approximately 48 to 72 hours later, the sites of thrombus formation became black, and the tail was shed. Pathological evaluation revealed the presence of great amounts of fiber and few white blood cells and platelets (PLT) in the small tail veins and capillaries, nuclear condensation in vascular endothelial cells (cube-shaped), and adherence of neutrophils to the inner walls of the blood vessels 24 hours after carrageenan injection. At 72 hours, inflammatory changes along with mixed thrombus formation were visible in bigger blood vessels.

Treatments, measurements, and histopathology

All rats were observed for 72 hours. Rats in the treatment group A were intragastrically administered with Huisheng Oral Solution at a dose of 2 ml/100 g body weight (once per 8 hours), 72 hours after carrageenan injection, while those in the treatment group B were initially given Huisheng Oral Solution at the same dose 72 hours before carrageenan injection, followed by injection 72 hours after carrageenan injection. Blood samples were collected 24, 48, and 72 hours after carrageenan injection for routine blood tests, coagulation tests (PT, APTT, INR, FIB, and PTA), and measurements of D-dimer and fibrin degradation products (FDPs). Lung, liver, and mesentery samples were taken 72 hours after carrageenan injection, fixed in 10% formalin, dehydrated, and embedded in paraffin. Sections (4 μm) were then cut using tissue samples from the upper lobe of the left lung, right liver lobe, and middle part of the mesentery, stained with hematoxylin and eosin, and observed under a microscope. After choosing the field that contained the maximum number of microthrombi under low magnification (×10), the number of microthrombi was counted under high magnification (×40). Due to specimen problems or technical reasons, some specimens could not meet the requirements of testing and some data were missing. In this case, new data were obtained by performing repeated experiments in a random manner.

Statistical Analysis

Numerical data were expressed as mean ± standard deviation (SD). Statistical analysis was performed using SAS 9.1 (for analysis of blood parameters) and SPSS 9.1 (for analysis of the number of microthrombi). Blood parameters among each group were compared using the Welch test, Kruskal-Wallis test, or SNK test after testing for normality. The number of microthrombi was compared using the Bonferroni test. P values <0.05 were considered statistically significant.

Results

Coagulation Parameters

At all the three time points, prothrombin time (PT) and activated partial thromboplastin time (APTT) were significantly shorter and prothrombin activity (PTA) was significantly longer in the model group than in the blank group [P < 0.05 for all, Table 1]. There was no significant difference in international normalized ratio (INR) between the blank group and model group. At 48 and 72 hours, fibrinogen (FIB) was significantly higher in the model group than in the blank group [P < 0.05 for both, Table 1]. These data suggest that rats in the model group were in a hypercoagulable state.

Table 1

Coagulation parameters in rats of each group (mean ± SD)

Compared to the blank group, PT showed no significant differences at 24 and 48 hours but was significantly shorter at 72 hours in the two treatment groups. However, PT was significantly longer at all the three time points in the two treatment groups than in the model group [P < 0.05 for both, Table 1]. There was no significant difference in PT between the two treatment groups. These data suggest that hypercoagulable states were significantly improved in the two treatment groups compared to the model group.

Compared to the blank group, APTT did not differ significantly at 72 hours in the treatment group B [P > 0.05, Table 1] but was significantly different in the two treatment groups at all the other time points. APTT at 48 and 72 hours were significantly longer in the two treatment groups than in the model group [P < 0.05 for both, Table 1]. In addition, APTT at 72 hours were significantly longer in the treatment group A than in the treatment group B.

Compared to the model group, INR did not differ significantly at 24 hours in the treatment group A [P > 0.05, Table 1] but was significantly higher in the two treatment groups at all other time points [P < 0.05 for all, Table 1]. Compared to the blank group, INR was significantly higher in the two treatment groups at all the other time points except at 24 hours in the treatment group A. There was no significant difference in INR between the two treatment groups.

Compared to the model group, FIB did not differ significantly at 72 hours in the treatment group A and at 24 hours in the treatment group B, but was significantly higher in the two treatment groups at all the other time points [P < 0.05 for all, Table 1]. There was no significant difference in INR between the blank group and the two treatment groups. INR was significantly lower at 72 hours in the treatment group B than in the treatment group A, but no significant difference was shown at the other time points between the two treatment groups.

Compared to the model group, FIB was significantly higher at 48 hours in the treatment group A, but showed no significant differences at other time points in the two treatment groups [P > 0.05 for all, Table 1]. FIB was significantly higher at all the time points in the two treatment groups than in the blank group. PTA was significantly lower at 48 hours in the treatment group A than in the treatment group B but showed no significant difference at other time points between the two treatment groups.

The above results suggest that the majority of blood coagulation parameters were significantly improved in the two treatment groups compared to the model group.

Fibrinolysis Parameters

At all the three time points, the levels of FDPs and D-dimer were significantly higher in the model group than in the blank group [P < 0.05 for all, Table 2], indicating that the fibrinolytic activity was high in rats in the model group. This result indirectly suggests that rats of the model group were in a hypercoagulable state.

Table 2

Fibrinolysis parameters in rats of each group (mean ± SD)

At all the three time points, the levels of FDPs were significantly lower in the two treatment groups than in the model group [P < 0.05 for all, Table 2]. Compared to the blank group, the levels of FDPs were significantly higher at 24 and 48 hours in the treatment group A [P < 0.05 for both, Table 2], but showed no significant difference at other time points in the two treatment groups. The levels of FDPs were significantly lower at 24 and 48 hours in the treatment group B than in the treatment group A [P < 0.05 for both, Table 2].

At 48 and 72 hours, the levels of D-dimer were significantly lower in the two treatment groups than in the model group [P < 0.05 for all, Table 2]. Compared to the blank group, the levels of D-dimer were significantly higher at all time points in the two treatment groups [P < 0.05 for all, Table 2]. There was no significant difference in the levels of D-dimer between the two treatment groups.

The above results suggest that the majority of fibrinolysis parameters were significantly improved in the two treatment groups compared to the model group.

Platelets

At all the three time points, platelet counts were significantly higher in the model group than in the blank group and in the two treatment groups [P < 0.05 for all, Figure 1]. Compared to the blank control group, platelet counts were significantly higher at 24 hours, showed no significant difference at 48 hours, and were significantly lower at 72 hours in the treatment group A. At all the three time points, platelet counts were significantly lower in the treatment group B than in the blank control group. Additionally, platelet counts were significantly lower at 24 and 48 hours in the treatment group B than in the treatment group A.

Figure 1

Platelet counts in rats of each group. *P < 0.05 vs Blank group; *P < 0.05 vs Control group; ▴P < 0.05 vs Treatment group A.

Histopathology

Seventy-two hours after carrageenan injection, lung, liver, and mesentery tissue samples were taken, stained with hematoxylin and eosin, and examined by microscopy. Extensive microthrombus formation was visible in the lung, liver, and mesentery of rats in the model group. Additionally, capillary hemorrhage and interstitial neutrophil infiltration in the lung, cloudy swelling, degeneration, and necrosis of hepatocytes in local areas in the liver, as well as local congestion and hemorrhage in the mesentery were seen in rats in the model group [Figure 2].

Figure 2

Histopathological changes in the lung, liver, and mesentery of rats in each group.

We next chose the field that contained the maximum number of microthrombi under low magnification (×10), counted and compared the number of microthrombi under high magnification (×40) among each group. The numbers of microthrombi in the lung, liver and mesentery were significantly lower in the two treatment groups than in the model group. There were no significant differences in the numbers of microthrombus in the lung, liver, and mesentery between the two treatment groups [Figure 3].

Figure 3

Comparison of the numbers of microthrombi in the liver, lung, and mesentery of rats of each group. *P < 0.05 vs Control group

Tissue samples were taken 72 hours after carrageenan injection, fixed, dehydrated, embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined by microscopy. Thrombus formation in the small blood vessels was indicated (arrows). A, lung of a normal rat; B, lung of a rat in the model group; C, liver of a normal rat; D, liver of a rat in the model group; E, mesentery of a normal rat; and F, mesentery of a rat in the model group. (magnification, ×40).

Discussion

Hypercoagulable states in patients with malignant tumors are a focus of novel research in medicine. Many factors may contribute to hypercoagulable states, including increased levels of physiological coagulation factors, increased activity of pathological coagulation factors, elevated fibrin monomer levels in blood, coagulation factor transfusion, decreased ability to inhibit and eliminate activated coagulation factors, diminished fibrinolytic activity, platelet factors, and other genetic factors.[7] Hypercoagulable states can be congenital, acquired, or both.[8] For cancer patients, central venous catheterization, surgery, or chemotherapy during cancer treatment may cause vascular endothelial cell injury, increased levels of adhesion molecules, adhesion of blood cells to each other, and release of procoagulants, thereby resulting in the formation of hypercoagulable states.[9] Chemotherapy-induced reactive thrombocytosis is also a cause of hypercoagulable states in cancer patients.[10] A previous study has shown that increased blood concentrations of tissue factor-positive microparticles are associated with the pathogenesis of hypercoagulable states in cancer patients.[11]

More than 90% of patients with malignant tumors have subclinical coagulation disorders, and many of them are in a hypercoagulable state, which manifests as elevated levels of FIB, PLT, coagulation factors V, VIII, IX, and X, FDP, and plasminogen activator inhibitor-1, and decreased levels of coagulation factor inhibitors (antithrombin, protein C, and protein S).[121314] In the present study, we chose PT, APTT, INR, FIB, and PTA to comprehensively assess coagulation function changes in subject rats. PT is a sensitive screening test for the extrinsic coagulation pathway. APTT reflects the integrity of the endogenous pathways of the procoagulant cascade (VIII, IX, and XI). The INR is calculated by normalizing the PT ratio to the power of the international sensitivity index (ISI). It standardizes the PT across different reagents of varying sensitivity and can better reflect the true nature of coagulation disorders. FIB positively correlates with whole blood viscosity, plasma viscosity, erythrocyte sedimentation rate, and platelet aggregation. Elevated blood levels of FIB can increase blood viscosity, promote red blood cell aggregation and platelet aggregation, and thereby result in the formation of hypercoagulable states and thrombosis.

In the five coagulation parameters detected in this study, approximately 66.7% (20/30) of data values in the two treatment groups were significantly different from those in the model group. In contrast, only about 20% (3/15) of data values were significantly different between the two treatment groups.

Platelet count is also closely associated with the coagulation system and has been used as an important parameter to assess coagulation function in many studies.[15161718] In this study, we found that platelet counts were significantly reduced in the treatment groups compared to the model group. In addition, a significant difference in platelet count between the two treatment groups was observed only at 72 hours.

A hypercoagulable state is often associated with hyperfibrinolysis, which manifests as increased levels of FDPs and D-dimer. In the present study, we found that approximately 83.3% (10/12) of data values in the two treatment groups were significantly different from those in the model group. In contrast, only 1/3 (2/6) of data values were significantly different between the two treatment groups.

Histopathological analysis revealed obvious vascular microthrombosis in both the model group and the two treatment groups. However, the numbers of microthrombi in the lung, liver, and mesentery were significantly lower in the two treatment groups than in the model group, but showed no significant difference between the two treatment groups.

Huisheng Oral Solution has been traditionally used as an anticancer drug in clinical practice. There has been no evidence so far about whether it can improve coagulation or not. In this study, we found that the majority of coagulation parameters (66.7%), fibrinolysis parameters (83.3%), and platelet counts (100%) in the two treatment groups were significantly improved compared to the model group. In contrast, there were no significant differences in these parameters between the two treatment groups. These findings were also confirmed by histopathological analysis. The Solution is derived from a variety of Chinese herbs that have anti-tumor effects. A previous study showed that it has significant anti-cancer effects in mice bearing transplanted Lewis lung carcinoma.[19] Ma et al.[20] found that Huisheng Oral Solution is beneficial in improving cellular immune function in elderly cancer patients. We surmise that the anticoagulant effect of Huisheng Oral Solution observed in this study may be due to the fact that it contains many blood-invigorating ingredients. Yin et al.[21] found that angelica and its active ingredient ferulic acid could significantly inhibit thrombin-induced platelet aggregation. Zhang et al.[22] investigated the effect of motherwort on mouse platelet cAMP and cGMP and PGI-like substance in rat carotid artery wall and found that it could significantly inhibit platelet aggregation. In addition, rhizoma sparganii, saffron, rhubarb, and leech can suppress platelet aggregation, prolong thrombosis time, reduce blood viscosity, and improve microcirculation. Hirudin, the main active ingredient of leech, is by far the strongest natural thrombin inhibitor in the world and has strong anticoagulant effect.[23242526]

In this study, we found that Huisheng Oral Solution could significantly reduce blood hypercoagulability, microthrombosis, and secondary fibrinolysis in a rat model of thrombosis. This may be due to the fact that it contains a variety of ingredients that have anti-clotting effects. However, we did not find that Huisheng Oral Solution had preventive effects against the pathogenesis of hypercoagulable states.