CN109125355B - Antiplatelet and antithrombotic pharmacological application of Xinmailong - Google Patents

Abstract

The application of Xinmailong in medicine for resisting platelet and thrombus. The invention relates to an application of Xinmailong in antiplatelet and antithrombotic pharmacology, belonging to the fields of pharmaceutical chemistry and pharmacology. The invention discloses pharmacological actions of cardiovasculum-glonoine in resisting platelet and thrombus, which is researched from double-layer surfaces of molecular and integral animal models, the action of the cardiovasculum-glonoine on platelet aggregation activity is measured through an in-vitro human platelet aggregation experiment, and the action of the cardiovasculum-glonoine on in-vivo thrombotic diseases is researched through a mouse acute pulmonary embolism model. Experiments prove that the Xinmailong has obvious inhibition effect on platelet function and thrombotic diseases, and provides experimental basis for the research on the aspects of platelet resistance and thrombus resistance in the future.

Description

Antiplatelet and antithrombotic pharmacological application of Xinmailong

technical field

The invention relates to the application of Xinmailong in antiplatelet and antithrombotic pharmacology, and belongs to the fields of medicinal chemistry and pharmacology.

technical background

Cardiovascular and cerebrovascular diseases mainly include ischemic heart disease, acute myocardial infarction, pulmonary infarction, cerebral infarction, cerebral infarction and other common diseases, which greatly threaten human health and life, and are the first cause of death for Chinese residents ^[1] . The occurrence and development of cardiovascular and cerebrovascular diseases are mainly a series of cascade reactions of platelet activation, adhesion, aggregation and thrombosis ^{[2, 3]} . Therefore, inhibition of platelet function and thrombosis is crucial for the prevention and treatment of cardiovascular and cerebrovascular diseases.

Xinmailong is a clinical medicine containing biologically active substances made of insect extracts as the main component. Xinmailong is an extract made from cockroach, American cockroach and other extracts and purified. Sticky Candy ^[4] . Xinmailong has a wide range of therapeutic effects on cardiovascular diseases, and has passed acute toxicity tests and long-term toxicity tests ^[4] . Studies have found that Xinmailong mainly has pharmacological effects such as enhancing myocardial contractility, increasing blood pressure, improving microcirculation, anti-atherosclerosis, increasing coronary blood flow, stimulating breathing, increasing renal blood flow and diuresis ^{[5, 6]} . At present, it is widely used in clinical treatment of acute and chronic heart failure, shock, and myocardial ischemic diseases. It can significantly improve microcirculation and achieve good clinical efficacy. However, there is no relevant report on the effect on platelet function and thrombotic diseases.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide an application of Xinmailong Chinese medicine for preparing antiplatelet and antithrombotic preparations.

The Xinmailong Chinese medicine can be used to prepare antiplatelet and antithrombotic preparations.

Said Xinmailong has anti-platelet and anti-thrombotic pharmacological effects, its effect on platelet aggregation activity was determined by in vitro human blood platelet aggregation experiment, and its effect on mouse thrombotic diseases was studied by mouse acute pulmonary embolism model. effect. Experiments have shown that Xinmailong has obvious inhibitory effect on platelet function and thrombotic diseases in mice. The pharmacological action of the novel Xinmailong involved in the present invention has not been reported so far.

The Xinmailong described in the present invention is obtained by dissolving the extract in physiological saline, and the concentration is expressed in m/v.

In the above-mentioned in vitro human blood platelet aggregation experiments, the following various concentrations of 0mg/ml, 0.1mg/ml, 1 mg/ml, 10 mg/ml Xinmailong's effects on human blood platelet aggregation were determined.

In the above mouse acute pulmonary embolism model experiment, the administration concentrations were 0mg/kg, 10mg/kg, 20mg/kg, and the clinical routine antiplatelet drug aspirin 50mg/kg, H&E staining was performed on the left lung of each group of mice , Observe the morphology and distribution of microvascular thrombus in the lung tissue under the light microscope, and the morphological changes of the pulmonary interstitium and parenchyma. At the same time, the survival time of each group of mice was determined to evaluate the effect of Xinmailong on thrombosis.

The present invention conducts research from the molecular and overall animal models, and finds that Xinmailong can inhibit collagen (Collagen), thrombin (Thrombin), arachidonic acid (Arachidonic acid, AA), adenosine diphosphate (ADP), etc. Agonist-induced platelet aggregation can inhibit the formation of acute pulmonary thrombosis in mice and prolong the survival time of mice.

Another biological property of the cardiomyopathy provided by the present invention envisions its many applications.

The present invention can be used for thrombotic diseases: evaluating platelet aggregation function, evaluating the effectiveness of antiplatelet therapy (such as aspirin, clopidogrel, ticagrelor, etc.) now or in the future; at the same time, because of its inhibitory effect on platelet aggregation, Can be used for antithrombotic therapy.

The invention can be used as a scarring agent or as a dermatological emulsion: the in situ migration, adhesion, aggregation and activation of platelets is required for the healing of certain wound scarring or skin diseases.

Description of drawings

Figure 1 shows the platelet aggregation induced by collagen, ADP, arachidonic acid and thrombin detected by different concentrations of Xinmailong (0mg/ml, 0.1mg/ml, 1 mg/ml, 10 mg/ml) by optical turbidimetry Influenced platelet aggregation curve. After the washed platelets were incubated with different concentrations of Xinmailong at 37°C for 10 minutes, inducers such as collagen, thrombin, arachidonic acid, and ADP were added, and the platelet aggregation curve was recorded (mean, n≥3).

Figure 2 shows the platelet aggregation induced by collagen, ADP, arachidonic acid and thrombin in different concentrations of Xinmailong (0mg/ml, 0.1mg/ml, 1 mg/ml, 10 mg/ml) detected by optical turbidimetry Bar graph of impact. [Mean±SE, n≥3; *(P<0.05), **(P<0.01)].

Figure 3 shows the effect of different concentrations of Xinmailong (0mg/kg, 10mg/kg, 20mg/kg) and the clinical routine antiplatelet drug aspirin (50mg/kg) on the survival time of mice after collagen-epinephrine-induced acute pulmonary thrombosis influences. (The abscissa is the grouping, and the ordinate is the survival time: s)

Figure 4 shows the effects of different concentrations of Xinmailong (10mg/kg, 20mg/kg) and clinical routine antiplatelet drug aspirin (50mg/kg) on the left lung tissue of mice after collagen-epinephrine-induced acute pulmonary thrombosis in mice H&E staining, magnification: 10X4.

Detailed ways

1. Preparation of Washed Platelets and Xinmailong Solution

Preparation of washed platelets: Take the cubital venous blood of healthy volunteers in a 3.8% sodium citrate (1:9) anticoagulation tube, dilute an equal volume (1:1) of Tyrode Buffer, and add PGE1 (final concentration: 50ng/ml) at the same time. ); centrifuge at 200×g for 15min, absorb the platelet-rich liquid in the upper layer, add PGE1 (final concentration is 50ng/ml), centrifuge at 1000×g for 10min, discard the supernatant to obtain concentrated platelet clumps, add an appropriate amount of Tyrode Buffer, Add PGE1 (final concentration 50ng/ml) and 1mM EDTA respectively, gently pipette with a Pasteur pipette to resuspend the platelets; centrifuge at 1000 × g for 10 min, discard the supernatant to obtain platelet clumps, add an appropriate amount of Tyrode Buffer, Resuspend the platelets by gentle pipetting; measure the number of platelets with an automatic blood cell analyzer, adjust the number of platelets to 3×10 ⁸ /ml with Tyrode Buffer, and store at room temperature for later use.

Configuration of Xinmailong solution: Take Xinmailong extract and dissolve it in physiological saline at room temperature to prepare Xinmailong liquid of 100 mg/ml, and store it at room temperature for future use.

2. Determination of platelet aggregation in human blood

(1) Platelet aggregation was measured using the principle of turbidimetry, which was performed on a dual-channel platelet aggregator from Chrono-Log. First, the platelet aggregator was preheated to 37°C, and the paper speed of the recorder was set to 1 cm/min. Add 400ul of platelet suspension and stirrer to the turbidimetric tube, add Xinmailong with different concentrations (0.1mg/kg, 1mg/kg, 10mg/kg) and incubate for 10 minutes, take Tyrode Buffer as reference, 37 ℃ constant temperature and constant speed The baseline was traced under magnetic stirring (1200 rpm). After the baseline was stable, different agonists (thrombin, arachidonic acid, collagen, adenosine diphosphate, etc.) were added to observe and record the changes of the aggregation curve. Repeat at least three times and take the average. The platelet aggregation curve was recorded according to the platelet aggregation rate (Fig. 1) and the corresponding bar graph was drawn (Fig. 2). Meanwhile, the platelet aggregation inhibition rate was calculated by the following formula: [(X - Y)/X]*100%. X represents the average aggregation rate of platelets induced by each agonist; Y represents the average aggregation rate of platelets induced by different concentrations of Xinmailong + each agonist.

(2) The experimental data were processed with SPSS 21.0 statistical software. Results are expressed as mean ± standard error. Homogeneity of variance test was performed first for comparison between multiple groups. One-way ANOVA was used for homogeneity of variance, and LSD (least-significant difference) test was used for pairwise comparison; Then the Tamhane's T2 test was performed, and the t test was used for pairwise comparisons. p<0.05 considered the difference to be statistically significant.

It can be seen from the experimental results that within the above-mentioned effective dose range, Xinmailong of the present invention has the effect on human blood induced by thrombin, arachidonic acid (AA), collagen (Collagen) and adenosine diphosphate (ADP). Platelet aggregation was significantly inhibited in a dose-dependent manner (Table 1A and Table 1B, Figure 1, Figure 2).

Table 1A

Table 1B

It can be seen from Table 1A and Table 1B that the Xinmailong described in the present invention is within the above effective dose range, and is effective for human induced by arachidonic acid (AA), thrombin, collagen (Collagen) and adenosine diphosphate (ADP). Blood platelet aggregation has a significant inhibitory effect in a dose-dependent manner.

3. The effect of Xinmailong on collagen-adrenaline-induced acute pulmonary thrombosis in mice

The experimental animals were 50 Kunming mice, weighing 20-30g, provided by Hunan Slike Jingda Laboratory Animal Co., Ltd., license number: SYXK (Dian) 2013-0004.

1) Mice were weighed, recorded and grouped. This experiment was divided into 5 groups: blank group, model group, Xinmailong 10mg/kg group, Xinmailong 20mg/kg group and aspirin 50mg/kg group, 10 cases in each group ;

2) Collagen (1.0mg/kg) and epinephrine (100ug/kg) mixed injection was prepared as model inducer;

3) Mice were administered by tail vein injection by group, the blank control group and model group were all given normal saline, the positive control group was given 50 mg/kg aspirin, and the drug group was given Xinmailong 10 mg/kg and 20 mg/kg respectively;

4) After 30 minutes of administration, the collagen-epinephrine mixture was injected into the tail vein for modeling;

5) Record the behavioral changes of mice and the survival time of mice in each group within 5 minutes of modeling;

6) The left lungs of mice in each group were taken out and fixed with 4% paraformaldehyde solution;

7) Sections were fixed and stained with H&E. The morphology, distribution, and morphological changes of lung interstitium and parenchyma in lung tissue were observed under light microscope.

The experimental data were processed with SPSS 21.0 statistical software. Results are expressed as mean ± standard error. Homogeneity of variance test was performed first for comparison between multiple groups. One-way ANOVA was used for homogeneity of variance, and LSD (least-significant difference) test was used for pairwise comparison; Then the Tamhane's T2 test was performed, and the t test was used for pairwise comparisons. p<0.05 considered the difference to be statistically significant.

The experimental results showed that compared with the model group (152.40±2.31s), Xinmailong 10mg/kg group (306.10±17.51s, p<0.05) and Xinmailong 20mg/kg group (352.70±10.53s, p<0.05) ) and the 50mg/kg aspirin group (367.10±14.39s, p<0.05) could prolong the death time of mice, and there was a significant difference (Figure 3); among them, the 50mg/kg aspirin group and the Xinmailong 20mg/kg group Both can reduce the mortality of mice, and there was no significant statistical difference (p>0.05) (Figure 3), indicating that Xinmailong 20mg/kg group and 50mg/kg aspirin group had comparable effects on inhibiting thrombosis in vivo (Table 2). ).

Table 2

As can be seen from Table 2, Xinmailong provided by the present invention can delay the death time of mice after acute pulmonary embolism induced by collagen-adrenaline mixture, and Xinmailong 20mg/kg group and clinical routine antiplatelet drug 50mg The antithrombotic effect of the /kg aspirin group was comparable.

The results of immunohistochemistry showed that there was no thrombosis in the blank group (Figure 4A), and the model group had obvious thrombosis (as shown by the arrow in Figure 4B), while the aspirin group and the high-dose Xinmailong group (20 mg/kg) significantly reduced thrombosis ( Figure 4E, 4D arrows), small thrombus formation was seen in the Xinmailong low-dose group (10 mg/kg), but less thrombosis than the model group (Figure 4C arrows). Therefore, it is concluded that Xinmailong has an inhibitory effect on collagen-adrenaline-induced thrombus formation in vivo.

references

[1] Chen Weiwei, Gao Runlin, Liu Lisheng, Zhu Manlu, Wang Wen, Wang Yongjun, et al. Summary of "China Cardiovascular Disease Report 2017" [J]. Chinese Journal of Circulation. 2018, (01): 1-8.

[2] George JN. Platelets [J]. Lancet (London, England). 2000, 355(9214): 1531-9.

[3] Jurk K, Kehrel BE.[Pathophysiology and biochemistry of platelets][J].Der Internist.2010,51(9):1086, 8-92, 94.

[4] Li Shunan, Hu Zhong. Xinmailong extract and Xinmailong medicinal preparation [P]. Yunnan: CN1124141, 1996-06-12.

[5] Hu Changjun. Review of the research progress of Xinmailong injection [J]. Modern Chinese Medicine Research and Practice. 2013, (04): 84-5.

[6] Tang Xiaohong. Pharmacological effects of Xinmailong injection and clinical research progress in the treatment of heart failure [J]. China Journal of New Drugs. 2008, (06): 461-4.

Claims (3)

1. The application of Xinmailong Chinese medicine in the preparation of antiplatelet aggregation and antithrombotic preparations. 2. the application of Xinmailong Chinese medicine according to claim 1 in the preparation for preparing antiplatelet aggregation and antithrombosis, it is characterized in that: described application is to measure Xinmailong by in vitro human blood platelet aggregation experiment The effect of traditional Chinese medicine on platelet aggregation activity; including the determination of the effect of Xinmailong Chinese medicine at 0 mg/ml, 0.1 mg/ml, 1 mg/ml and 10 mg/ml on human blood platelet aggregation; the Xinmailong Chinese medicine is based on The extract form is dissolved in physiological saline. 3. the application of Xinmailong Chinese medicine according to claim 1 in the preparation for preparing antiplatelet aggregation and antithrombosis, it is characterized in that: described application is to study Xinmailong by mouse acute pulmonary embolism model The effect of traditional Chinese medicine on thrombotic diseases in mice; In the acute pulmonary embolism model in mice, the administration concentrations of Xinmailong Chinese medicine were 0mg/kg, 10mg/kg and 20mg/kg, and the clinical routine antiplatelet drug aspirin 50mg/kg was used as a positive control. H&E staining was performed to observe the morphology and distribution of microvascular thrombus in the lung tissue, and the morphological changes of the pulmonary interstitium and parenchyma in the lung tissue. .

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