The Promoting Blood Circulation And Removing Blood Stasis Effects Of The Holotrichia Diompha Lialarvae And It’s Preliminary Mechanism Of Action

Research backgroundCardiovascular disease is a frequently-occurring disease on medicine at present, a serious threat to the patient’s physical and mental health. In recent years, with the improvement of life level, but also because of the deterioration of the environment, the cardiovascular disease incidence shows ascendant trend, and has become the leading death cause of China population at present. Thrombosis is a kind of common disease in cardiovascular system, in recent years, serious harmful to human health, is also one of the reasons causing the highest mortality, such as myocardial infarction, cerebral thrombosis, deep vein thrombosis, cerebral embolism, pulmonary embolism etc. These diseases have become the major diseases that endanger human life and health. According to incomplete statistics, in the population over 40 years old, the incidence rate of myocardial infarction was 39.7/10 million to 64.0/10 million per year, the incidence rate of stroke was 109/10 million per year. Blood coagulation and thrombosis is the important pathological process involved in the pathogenesis of this kind of disease. Therefore, anticoagulation, prevention of thrombosis and dissolving the formed thrombus has become the current clinically important methods of prevention and treatment of thrombotic diseases. At present, the antithrombotic drugs included anticoagulant, fibrinolysis drugs and anti platelet drugs in three categories. Antithrombotic therapy drugs currently used in clinical application mainly was urokinase or tissue plasminogen activator, but because of its expensive, it’s difficult to meet the need of treatment and prevention of thrombosis disease. At the same time, because of the high incidence of thrombosis disease rate and it’s high morbidity and high mortality, there is urgently need prevention and treatment of drug with high curative effect, low toxic and side effect at clinical, however, the drug existing are of different shortcomings. For instance, to the thrombolytic therapy for vascular thromboembolic diseases with Urokinase, Alteplase or Reteplase, the short "time window" become a major problem and the problem has not been resolved in the internation. In view of the above situation, many medical fellow especially in traditional Chinese medicine or the drug industry has made arduous efforts in this respect, although "traditional Chinese medicine thrombolysis effect has not yet been recognized by the medical profession, but it provides us many positive enlightenment. Such as by adjustting the prescription drug selection, dosage and Compatibility skills of Chinese medicine, we find it prolongs the "time window" compared with Western thrombolytic medicine after years of exploration and clinical verification, and see the dawn of the traditional Chinese medicine thrombolysis in practice.Drug resources from insects is abundant, the active protein and polypeptide from insect has become a new research hotspot at present. Holotrichia diomphalia larvae is beetle larvae, it’s main effect was removing blood stasis, scatter tubercle, analgesic, detoxification, it has a variety of applications in traditional Chinese medicine, it can cure tetanus, eye screen, infantile erysipelas etc.. In addition, there are also records that Holotrichia diomphalia larvae was used for promoting blood circulation and removing blood stasis. "Records" in Changsha medicine:Holotrichia diomphalia larvae, can remove the blood stasis, eliminating block. "Compendium of Materia Medica":"Benshi Fang", Holotrichia diomphalia larvae, cure bars hurry, nourishing Dihuang pill, taken as treatment of blood stasis arthralgia also. Thus, Holotrichia diomphalia larvae is medicine for promoting blood circulation and removing blood stasis. But because the research on grass grub active component is extremely poor, the mechanism of promoting blood circulation and removing blood stasis is not clear, which greatly limits the use of white grubs in promoting blood circulation and removing blood stasis. We get the crude extract from the Holotrichia diomphalia larvae luminal contents, which is called " CEHDL " in the next text, to attempts to confirm it’s effect of" promoting blood circulation and removing blood stasis " from the aspect of Western pharmacology of anticoagulation and thrombolysis, and to explore the mechanism of anticoagulation and thrombolysis.Objective1. Explain the effect of promoting blood circulation and removing blood stasis of Holotrichia diomphalia larvae from the perspective of anticoagulation and thrombolysis in western medicine pharmacology2. Investigation the mechanism of anticoagulation and thrombolysis of the Holotrichia diomphalia larvae3. Preliminary identification of active substances of Holotrichia diomphalia larvaeMethods1. Determination of the plasma recalcification time of rats to detect anticoagulant activity in vitro of CEHDLBlood was collected from the abdominal aorta of SD rat’s and plasma was obtained after centrifugation,then plasma was mixed with the negative control (saline), four groups of grubs extract (12.5μg/ml,25 μg/ml,50 μg/ml,100 μg/ml) in 37℃ water bath incubation for 5 minutes, respectively,then the calcium chloride solution (25 mM) was added into the plasma, the plasma recalcification time was the period from adding calcium chloride to the liquid surface solidification2. Determination of bleeding time of mice tail vein to detect anticoagulant activity in vivo of CEHDL30 kunming mice were randomly divided into 5 groups. Each group of mice was injected of saline, three concentrations of CEHDL (10 μg/g,20μg/g,50μg/g), and urokinase. After 2 hours, cut off mice tai of 5 mm, each group of mice tail vein bleeding time was determinated3. Dissolution of blood clot in vitro to test fibrinolytic activity of CEHDLBlood was collected from heart after New Zealand rabbits was anesthesiaed and let it solidified into a blood clot, then blood clot was cut into roughly equal small blood clots, divided into 5 groups, each group of blood clots was incubated with the negative control (saline), three concentration group of CEHDL (200 μg/ml,400 μg/ml, 800 μg/ml), urokinase (100 u/ml).Observe the number of red blood cells released to the supernatant solution because of thrombus dissolving with inverted microscope, and take photos. After 12 h, calculate the dissolving rate of thrombolysis of each group according to the difference between the initial weight and end weight of the blood clot4. Determination of the effect of anti mouse tail vein thrombosis of CEHDL by thrombosis model induced by carrageenan32 kunming mice were randomly divided into 4 groups, each group of mice was tail vein injection of saline, the concentration of two grubs extract (20 μg/g and 50 μg/g), and urokinase, respectively. After 2 h, each group of mice was injection of 0.1% carageen to induce tail vein thrombosis. After 24 hours, statistic thrombosis incidence in each group of mice, at the same time, measure the length of tail vein thrombus using caliper in each group of mice.5. Determination of the degradation of CEHDL on fibrinogen by gel electrophoresisThe degradation of different concentrations of CEHDL on fibrinogen and the degradation order of CEHDL on fibrinogen was all determinated by gel electrophoresis6. Determination of inhibition effect of CEHDL on platelet aggregation induced by ADPBlood was taken from the heart of rats to obtain platelet poor plasma and platelet rich plasma after centrifugation, the aggregation curve of platelet rich plasma with different concentrations of CEHDL during 5 min was determinated using platelet aggregometer, and calculate the inhibition rate of maximum aggregation rate in 5 mm.7. Determination of the degradation of CEHDL on fibrin and its degradation modeWith agarose as carrier, under the action of thrombin, fibrinogen was activated into fibrin to form fibrin plate. Then it was baked in the oven at 85 for 30 min to make into the inactivated plate. Then samples were added onto the fibrin plate, the effect of CEHDL on fibrin and its degradation mode was determinated by observing the formation of the dissolving ring and comparing the size of the dissolving ring.8. Preliminary separation and purification of the CEHDL and identification of properties of fibrinolytic substanceGel electrophoresis experiment was conducted by incubating with fibrinogen to detect the fibrinolytic peak after separation and purification of CEHDL with S-100HR; determination of active substances for anionic or cationic type by observing the combination between CEHDL with materials of DEAE sephadex-A50 or Sephadex-C50; identification of glucide in the CEHDL by Molish reaction.9. Determination of the influencing factors of CEHDL’s fibrinolytic activitySDS-PAGE gel electrophoresis was conducted to determinate the influence of temperature、pH、metal ion and protease inhibitors on fibrinolytic activity of CEHDL.Results1. When the concentration of CEHDL in plasma is above 12.5 μg/mL, it can obviously prolong the plasma calcification time, and the effect increases with the increase of the extract concentration, this suggests that it has good anticoagulant activity in vitro.2. Mouse tail vein bleeding time test show that injection of CEHDL for the mice advancely can obviously prolong the mice tail vein bleeding time compared with negative control, and present of a dose of relevance, this suggest that it has good anticoagulant activity in vivo.3. In the experiment of dissolution of CEHDL to blood clots in vitro, with the increase of concentration of CEHDL, the number of red blood cells released by dissolving blood clot show significantly increasing trend, and after 12 h, the dissolving rate of thrombosis in group of CEHDL is significantly higher than the negative control group, suggestting that CEHDL can dissolve the formed thrombus, that also show that it has good in vitro fibrinolytic activity in vitro.4. CEHDL can significantly reduced the thrombosis rate of mouse tail vein induced by carrageenan, it can also shorten the length of tail thrombosis in mice, this suggest it has good anti thrombus activity in vivo.5. Gel electrophoresis experiments show that CEHDL can not only degrade fibrinogen,but also can degrade all three subunits of fibrinogen, and degradated sequence for the three subunit is α→β→γ.6. CEHDL can slightly inhibited rat plasma aggregation induced by ADP7. The inactived fibrin plate experiment results show that dissolving circle was formed around the hole of CEHDL, suggesting that CEHDL can degradate fibrin by itself, and the degradation increases with the increase of the concentration, but CEHDL can not be activated plasminogen, therefore the CEHDL play the role of thrombolysisis by degradation of fibrin on itself.8. CEHDL fibrinolytic substances is in peak II after separation and purification with S-100HR, fibrinolytic substance is anion, and it does not contain sugar.9. The fibrinolytic activity of CEHDL will be inactivated above 75℃, the fibrinolytic activity can be inhibited partially by serine protease inhibitor, TPSI, aprotinin, leueplin, PMSF, but EDTA has no effect on the fibrinolytic activity, which showed that the active components of fibrinolytic activity contain serine protease but not metal protease, it can keep fibrinolytic activity in (pH3.0～pH10.0) range, and all kinds of metal ions (Mg2+, Ca2+, Zn2+, Fe2+,Fe3+, Cu2+) have hardly affect on the fibrinolysis activity.Conclusion1. This paper studies the activity of the grub from the aspect of western medicine pharmacology, CEHDL can prolong the plasma recalcification time and bleeding time of mouse tail vein, at the same time it can dissolve blood clots in vitro, indicating that has good anticoagulant and fibrinolytic activity, so as to achieve the effect of promoting blood circulation and removing blood stasis.2. The CEHDL play the role of anticoagulation and thrombolysis mainly through the degradation of fibrinogen and fibrin degradation directly. The three chains of fibrinogen were all degraded, and the degradation sequence was α→β→γ, but it can not activated plasminogen, it play a fibrinolytic activity by directly degradation of fibrin.3. Fibrinolytic substance of CEHDL contain serine proteases but does not contain metal protease, metal ion and pH (pH3.0～pH10.0) has little effect on it’s fibrinolytic activity.