CN117653730A - Application of dimethylbutyryl carnitine inhibitor in preparation of antithrombotic drugs - Google Patents
Application of dimethylbutyryl carnitine inhibitor in preparation of antithrombotic drugs Download PDFInfo
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- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
The invention belongs to the field of biological medicine, and discloses application of a dimethylbutyryl carnitine inhibitor in preparation of antithrombotic medicines. The invention discovers that dimethylbutyryl carnitine has positive regulation and control effect on thrombus formation, reduces the content of dimethylbutyryl carnitine or inhibits the cell receptor activity of dimethylbutyryl carnitine, and can effectively reduce the thrombus formation. The invention widens the treatment path of thrombus.
Description
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to an application of a dimethylbutyryl carnitine inhibitor in preparing antithrombotic medicines.
Background
Adverse events such as ischemic heart disease and stroke caused by thrombus are the leading cause of death of adults worldwide. In recent years, along with the aging of population, the change of eating habits, life style and the like, the occurrence rate of thrombotic events in China is gradually increased, the life health of people in China is seriously influenced by high disability rate and mortality rate, and a heavy burden is brought to society and economy, so that the thrombotic events become serious public health problems.
In the past, studies on the pathological mechanism of thrombus have focused on the "downstream" pathway of platelet activation, and antithrombotic drugs such as aspirin and clopidogrel developed by targeting this pathway have been able to reduce the risk of occurrence of thrombotic events to some extent, but they have also been accompanied by an increase in the risk of adverse events such as bleeding due to their therapeutic effects while interfering with the normal physiological coagulation function of platelets. Therefore, it is important and difficult to have an in-depth understanding of the "upstream" pathological mechanism that induces thrombosis to search for more physiologically and safe and effective antithrombotic therapeutic agents.
Disclosure of Invention
The invention aims to provide an application of dimethylbutyryl carnitine inhibitor in preparing antithrombotic medicines.
The aim of the invention is achieved by the following technical scheme: the application of the dimethylbutyryl carnitine inhibitor in preparing antithrombotic medicaments is based on the discovery that dimethylbutyryl carnitine has positive regulation and control effects on thrombus formation, reduces the content of dimethylbutyryl carnitine or inhibits the cell receptor activity of dimethylbutyryl carnitine, and can effectively reduce thrombus formation.
The English name of the dimethylbutyryl carnitine is 2-methyl butyryl carnitine, and the molecular formula is C 12 H 23 NO 4 The molecular structural formula is as follows:
the dimethylbutyryl carnitine inhibitor refers to a substance which reduces the dimethylbutyryl carnitine content or inhibits the cell receptor activity of dimethylbutyryl carnitine; preferably an integrin (integrin) α2β1-specific inhibitor or antibiotic.
The integrin alpha 2 beta 1 specific inhibitor is BTT 3033.
The antibiotics are preferably at least one of neomycin, streptomycin and bacitracin; more preferably neomycin, streptomycin and bacitracin in a mass ratio of 1:1:1, and compounding to obtain the product.
The cell receptor is platelet membrane receptor integrin alpha 2 beta 1.
Compared with the prior art, the invention has the following advantages and effects:
(1) According to the invention, through a thrombus model and an in-vitro platelet experiment, the fact that dimethylbutyryl carnitine has a positive regulation effect on thrombus formation is discovered for the first time, namely, dimethylbutyryl carnitine can increase platelet reactivity, promote platelet aggregation, diffusivity in a collagen matrix and a platelet contraction function, accelerate in-vivo thrombus formation and increase occurrence risk of thrombus events.
(2) The invention proves that the platelet membrane receptor integrin alpha 2 beta 1 is a functional receptor for mediating the increase of platelet reactivity of dimethylbutyryl carnitine and promoting thrombosis, and the platelet reactivity increase caused by dimethylbutyryl carnitine can be effectively blocked by adopting the integrin alpha 2 beta 1 specific inhibitor BTT 3033, so that the thrombosis in vivo can be effectively lightened.
(3) The invention proves that dimethylbutyryl carnitine is a metabolic product derived from intestinal flora, and a precursor dimethylbutyric acid can be converted into dimethylbutyryl carnitine through the intestinal flora to promote thrombosis in vivo. And the conversion of dimethylbutyric acid to dimethylbutyryl carnitine can be inhibited by clearing intestinal flora with antibiotic cocktails, thereby inhibiting thrombosis.
Drawings
FIG. 1 shows the reaction of iron trichloride (FeCl) in example 1 3 ) Experimental results of induced carotid artery thrombosis model of mice; wherein A is shown in FeCl 3 The thrombotic formation of carotid artery in different time periods after stimulation, the white part in the figure is platelet thrombotic tissue; b is a statistical analysis result of carotid artery blood flow blocking time of the mice in the experimental group and the control group; * P < 0.0001.
FIG. 2 is a graph of experimental results of a photochemically damaged carotid thrombosis model; wherein, A shows the formation of carotid artery thrombus at different time periods after laser irradiation, and the white part in the figure is platelet thrombus tissue; b is a statistical analysis result of carotid artery blood flow blocking time of the mice in the experimental group and the control group; * P value is less than 0.05.
FIG. 3 is a graph of survival of pulmonary embolism model mice.
FIG. 4 is a graph of the effect of dimethylbutyryl carnitine on platelet function; wherein, A is platelet aggregation experiment, the left graph in A is platelet aggregation curve under ADP stimulation, the right graph in A is statistical analysis result of platelet aggregation rate, and P is less than 0.01; b is a platelet spreading experiment, the left panel in B is a platelet spreading pattern on a collagen matrix coated slide, the right panel in B is a statistical analysis of platelet spreading, P is less than 0.01; c is a blood clot retraction experiment, the left graph in C shows the blood clot retraction situation at different time points under thrombin activation, the right graph in C shows the statistical analysis result of blood clot retraction rate at different time points, wherein P < 0.05 and P < 0.01.
FIG. 5 is a graph showing the results of direct binding experiments of dimethylbutyryl carnitine to integrin α2β1 purified protein.
FIG. 6 is a sample of iron trichloride (FeCl) of example 7 3 ) Experimental results of induced carotid artery thrombosis model of mice; wherein A is shown in FeCl 3 The thrombotic formation of carotid artery of each group of mice in different time periods after stimulation, the white part in the figure is the thrombotic tissue of blood platelets; b is the result of statistical analysis of carotid artery blood flow blocking time of each group of mice, n.s. is no statistical difference, P < 0.05, P < 0.01.
FIG. 7 is a graph showing the results of experiments on the inhibition of the platelet reactivity of BTT 3033 by dimethylbutyryl carnitine (2 MBC); wherein A is the statistical analysis result of the platelet aggregation rate of each group; b is the result of statistical analysis of the platelet diffusivity of each group, n.s. is no statistical difference, P < 0.05, P < 0.01, P < 0.001, P < 0.0001.
FIG. 8 is a graph showing the results of an in vitro fermentation experiment of intestinal bacteria of mice; * P < 0.05.
FIG. 9 is a graph of the results of an experiment for inhibiting the production of dimethylbutyryl carnitine by an antibiotic in mice; * P is less than 0.05.
FIG. 10 is a graph showing experimental results of antibiotic inhibition of dimethylbutyryl carnitine conversion in mice to improve thrombosis; * P is less than 0.01, P is less than 0.001, and P is less than 0.0001.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
EXAMPLE 1 dimethylbutyryl carnitine (2 MBC) versus FeCl 3 Effects of induced carotid thrombosis
16 male C57BL/6J mice (purchased from Zhuhai Baitong Biotechnology Co., ltd.) of 6-8 weeks old were selected and randomly divided into experimental groups (8) and control groups (8). Mice in the experimental group were intraperitoneally injected with 100mg/kg dimethylbutyryl carnitine (2 MBC), while the control group was injected with the same volume of physiological saline, and thrombus molding was performed 2 hours after the injectionAnd (5) molding. The mice were anesthetized with 1% (w/v) sodium pentobarbital, and after satisfactory anesthetic effect, the skin was incised through the middle of the neck, 100 μl rhodamine 6G (0.5 mg/mL) labeled platelets were injected into the right jugular vein, and left carotid artery was completely free exposed. By 7.5% FeCl 3 The infiltrated filter paper (about 2mm by 1.5 mm) was placed on the carotid artery surface for 1 min incubation, followed by real-time observation of thrombus formation in the carotid artery under a fluorescence microscope (Leica M205 FA) and acquisition of pictures, recording the time for complete cessation of carotid blood flow. Results were statistically analyzed using GraphPad9.0 software.
The experiment was performed in 3 replicates, and fig. 1 is an average of 3 replicates, and fig. 1 shows that the average blood flow blocking time of the control group was 791.3 seconds, and the average blood flow blocking time of the experimental group was 367.5 seconds, suggesting that 2MBC significantly accelerates the formation of carotid artery thrombosis in mice.
EXAMPLE 2 Effect of dimethylbutyryl carnitine on photochemical injury-induced carotid thrombosis
10 male C57BL/6J mice (purchased from Zhuhai Baitong Biotechnology Co., ltd.) of 6-8 weeks old were selected and randomly divided into an experimental group (5) and a control group (5). Mice in the experimental group were intraperitoneally injected with 100mg/kg dimethylbutyryl carnitine (2 MBC), while the control group was injected with an equal volume of physiological saline, and thrombus model molding was performed 2 hours after the injection. Mice were anesthetized with 1% (w/v) sodium pentobarbital and, after satisfactory anesthetic effect, tiger red B (100 mg/kg) was injected via the tail vein. Skin was cut centrally along the neck and 100 μl rhodamine 6G (0.5 mg/mL) was injected via the right jugular vein to label platelets, completely free revealing the left carotid artery. The carotid artery was exposed to green laser light, and thrombus formation in the carotid artery was observed in real time and imaged by a MICRON IV retinal imaging microscope, and the time at which carotid blood flow was completely stopped was recorded. Results were statistically analyzed using GraphPad9.0 software.
The experiment was performed in 2 replicates, fig. 2 is an average of 2 replicates, fig. 2 shows that the average blood flow blocking time for the control group was 42 minutes, and the average blood flow blocking time for the experimental group was 25.8 minutes, suggesting that 2MBC significantly accelerates the formation of carotid artery thrombosis in mice.
EXAMPLE 3 Effect of dimethylbutyryl carnitine on thrombin-induced pulmonary embolism
12 female C57BL/6J mice (purchased from the affiliated animal experiment center of the first hospital of the university of Zhongshan) were selected at 8 weeks of age and randomly divided into an experimental group (6) and a control group (6). Mice in the experimental group were intraperitoneally injected with 100mg/kg dimethylbutyryl carnitine (2 MBC), while the control group was injected with an equal volume of physiological saline, and thrombus model molding was performed 2 hours after the injection. Mice were anesthetized with 1% (w/v) sodium pentobarbital, after satisfactory anesthetic effect, the skin was cut open along the middle of the neck, pulmonary embolism was induced by right jugular intravenous thrombin injection (200U/kg), survival time of mice was observed in real time, and final survival rate was defined as the number of mice surviving over 30 minutes. Results were statistically analyzed using GraphPad9.0 software.
The experiment was performed in 2 replicates, and fig. 3 is an average of 2 replicates, and fig. 3 shows that the survival rate of mice in the control group was 66.7%, while the survival rate of mice in the experimental group was reduced to 16.7%, which suggests that 2MBC significantly reduced the survival rate of mice with pulmonary embolism.
Example 4 extraction and preparation of platelet suspensions
(1) Extraction and preparation of mouse platelet suspension:
16 male C57BL/6J mice (from Zhuhai Baitong Biotechnology Co., ltd.) of 6-8 weeks old are selected, 1% (w/v) sodium pentobarbital is used for anesthesia, after the anesthesia effect is satisfied, 600. Mu.L of whole blood is collected by cardiac puncture with a syringe containing 0.38% sodium citrate 100. Mu.L, 500. Mu.L of modified desktop liquid without calcium is added for dilution, and the mixture is left to stand at room temperature for 15 minutes. Centrifugation at 100g for 10 min at 22℃and aspiration of the supernatant, which is Platelet Rich Plasma (PRP). The remaining blood was centrifuged at 1500g for 15 min at 22℃and the supernatant was aspirated, which was Platelet Poor Plasma (PPP). In some experiments, further separation of washed platelets was performed, 100nM PGE1 (prostaglandin E1) was added to the above-extracted PRP, centrifuged at 500g at 22℃for 10 minutes to pellet platelets, and after removal of the supernatant, the platelets were resuspended in sterile PBS, 100nM PGE1 was added and centrifuged at 500g at 22℃for 10 minutes to wash out residual plasma components. To contain 0.35% (w/v) BSAThe washed platelet suspension was prepared without calcium modified tabletop fluid resuspension of platelets. Platelet concentration was measured using a merry animal blood cell tester and adjusted to about 2 x 10 8 /mL, to be used in subsequent experiments.
(2) Extraction and preparation of rat platelet suspension:
male SD rats (purchased from Zhuhai Baitong Biotechnology Co., ltd.) were selected, weighing about 200-250g, were anesthetized with 1% sodium pentobarbital, and after satisfactory anesthesia, were heart-pricked with 100. Mu.L syringe containing 0.38% sodium citrate to collect whole blood (ratio of whole blood to sodium citrate: about 6:1), diluted with an equal volume of modified desktop liquid free of calcium, and allowed to stand at room temperature for 15 minutes. Centrifugation at 200g for 20 min at 22℃and aspiration of the supernatant, which is Platelet Rich Plasma (PRP). The remaining blood was centrifuged at 1500g for 15 min at 22℃and the supernatant was aspirated, which was Platelet Poor Plasma (PPP). In some experiments, further separation of washed platelets was performed, 100nM PGE1 was added to the above-extracted PRP, and centrifugation was performed at 500g for 20 minutes at 22℃to precipitate platelets, after which the supernatant was removed, sterile PBS was added to resuspend platelets, 100nM PGE1 was added, and centrifugation was performed at 500g for 20 minutes at 22℃to wash out residual plasma components. Washed platelet suspensions were prepared by resuspending platelets in a calcium-free modified tabletop fluid containing 0.35% BSA. Platelet concentration was measured using a merry animal blood cell tester and adjusted to about 2 x 10 8 /mL, to be used in subsequent experiments.
Example 5 Effect of dimethylbutyryl carnitine on platelet function
(1) Platelet aggregation assay:
the platelet suspension prepared in example 4 was divided into experimental and control groups. The experimental group was incubated with 0.5. Mu.M dimethylbutyryl carnitine for 30 minutes, while the control group was incubated with an equivalent volume of physiological saline. 400 mu L of platelet suspension is added into a reaction tube, and CaCl with equal concentration is respectively added 2 The reaction tube was placed in a Chrono-log Type700 platelet aggregation apparatus, the inducer was added to stimulate platelet activation, the platelet aggregation curve was observed in real time at 37℃for 1000 revolutions per minute, and the reaction was stopped when the maximum aggregation level was reached. Blood at this pointThe platelet inducer is preferably ADP, thrombin, type I collagen, arachidonic acid. Results were statistically analyzed using GraphPad9.0 software.
(2) Platelet diffusion experiments:
the slide was coated overnight with 20. Mu.g/mL type I collagen in a refrigerator at 4℃and the collagen was discarded and washed 2 times with PBS. The platelet suspension prepared in example 4 was divided into experimental and control groups. The experimental group was incubated at room temperature with 0.5. Mu.M dimethylbutyryl carnitine, while the control group was incubated with an equivalent volume of physiological saline. After 30 minutes incubation, 200 μl of platelet suspension was pipetted onto the slide, and after 60 minutes incubation at 37 ℃ the slide was washed with PBS. Random field acquisition pictures were then selected under a fluorescence microscope (Leica DMI 8), 5-10 random fields were acquired per slide, and platelet diffusivity was quantitatively analyzed using Image J analysis software. Results were statistically analyzed using GraphPad9.0 software.
(3) Clot retraction experiment:
the platelet suspension prepared in example 4 was divided into experimental and control groups. The experimental group was incubated at room temperature with 0.5. Mu.M dimethylbutyryl carnitine, while the control group was incubated with an equivalent volume of physiological saline. After incubation for 30 minutes, 370. Mu.L of platelet suspension was pipetted into a clear reaction tube and 2mM CaCl was added sequentially 2 0.5mg/mL fibrinogen and 1U/mL thrombin. Photographs were taken at 0, 15, 30, 60 minutes, respectively, and clot shrinkage was quantified by Image J analysis software. Results were statistically analyzed using graphpad9.0 software.
The results are shown in FIG. 4: a in FIG. 4 is the platelet aggregation experimental result, which shows that the platelet aggregation rate of the experimental group is obviously increased compared with that of the control group, and the difference has statistical significance; b in FIG. 4 is a platelet diffusion experiment result, showing that the platelet diffusion rate of the experiment group is obviously increased compared with that of the control group, and the difference has statistical significance; c in FIG. 4 is the blood clot retraction test result, showing that the blood clot retraction rate is obviously increased in the test group at 30 minutes and 60 minutes compared with the control group, and the difference is statistically significant. The above results suggest that 2MBC significantly increases platelet reactivity.
EXAMPLE 6 direct binding of dimethylbutyryl carnitine to integrin alpha 2 beta 1
Commercial human integrin alpha 2 beta 1 purified protein (IT 1-H52W 6) is purchased, the purified protein is coupled to a Senor Chip CM7 Chip by using a Biacore T100 system, and different concentrations (0.3125, 0.625, 1.25, 5, 10, 20 and 40 mu M) of dimethylbutyryl carnitine (2 MBC) are respectively added for reaction, and the reaction signal index of the 2MBC and the integrin alpha 2 beta 1 is detected and analyzed. Data analysis was performed using BIA evaluation (Version 4.1) to calculate the binding constant Kd value between the two.
As shown in FIG. 5, it can be seen that the response values of direct binding of 2MBC and integrin alpha 2 beta 1 purified proteins at different concentrations increase with increasing 2MBC concentration, and certain saturation effect is exhibited, and the calculated binding constant Kd value between the two is 10.6 mu M. The above results suggest that 2MBC can bind directly to integrin α2β1.
Example 7BTT 3033 inhibits dimethylbutyryl carnitine mediated thrombotic action
24 male C57BL/6J mice (purchased from Zhuhai Baitong Biotechnology Co., ltd.) of 6-8 weeks old were selected and randomly divided into 4 groups of 6, each of which was Vehicle group (normal control group), 2MBC group, BTT 3033 group and 2MBC+BTT 3033 group. 100mg/kg dimethylbutyryl carnitine (e.g. 2MBC and 2MBC+BTT groups treated with 2 MBC) or equivalent volumes of physiological saline (e.g. Vehicle and BTT 3033) were administered intraperitoneally, respectively, followed by the administration of integrin α2β1-specific inhibitor BTT 3033 (1 mg/kg) (e.g. BTT 3033, 2MBC+BTT 3033) or equivalent volumes of solution without BTT 3033 (e.g. Vehicle and 2MBC groups), respectively, and thrombus modeling was performed 2 hours after the injection. The mice were anesthetized with 1% (w/v) sodium pentobarbital, and after satisfactory anesthetic effect, the skin was incised through the middle of the neck, 100 μl rhodamine 6G (0.5 mg/mL) labeled platelets were injected into the right jugular vein, and left carotid artery was completely free exposed. By 7.5% FeCl 3 The infiltrated filter paper (about 2mm by 1.5 mm) was placed on the carotid artery surface for 1 min incubation, followed by real-time observation of thrombus formation in the carotid artery under a fluorescence microscope (Leica M205 FA) and acquisition of pictures, recording the time for complete cessation of carotid blood flow. Results are takenStatistical analysis was performed by GraphPad9.0 software.
The results are shown in FIG. 6, which shows that the thrombus formation rate of the mice (2 MBC group) in the 2MBC treatment group is significantly faster than that of the mice (Vehicle group) in the normal control group; after treatment with BTT 3033 on the basis of 2MBC (2MBC+BTT 3033 group), carotid blood flow blocking time is almost recovered to the level of a Vehicle mouse, suggesting that BTT 3033 significantly blocks the thrombogenic effect of 2 MBC; while treatment with BTT 3033 alone (BTT 3033 group) had no significant effect on carotid blood flow occlusion time.
Example 8BTT 3033 inhibits dimethylbutyryl carnitine mediated enhancement of platelet reactivity
(1) Platelet aggregation assay:
the platelet suspensions prepared in example 4 were divided into 4 groups. Physiological saline (Vehicle group), 0.5. Mu.M dimethylbutyryl carnitine (2 MBC group), 1. Mu.M BTT 3033 (BTT 3033 group), 0.5. Mu.M dimethylbutyryl carnitine, and 1. Mu.M BTT 3033 (2MBC+BTT 3033 group) were added, respectively, and incubated for 30 minutes. 400 mu L of platelet suspension is added into a reaction tube, and CaCl with equal concentration is respectively added 2 The reaction tube was placed in a Chrono-log Type700 platelet aggregation apparatus, the inducer was added to stimulate platelet activation, the platelet aggregation curve was observed in real time at 37℃at 1000 revolutions per minute, and the reaction was stopped when the maximum aggregation level was reached. The platelet inducer herein is preferably ADP, thrombin, type I collagen, arachidonic acid. Results were statistically analyzed using GraphPad9.0 software.
(2) Platelet diffusion experiments:
the slide was coated overnight with 20. Mu.g/mL type I collagen in a refrigerator at 4℃and the collagen was discarded and washed 2 times with PBS. The platelet suspensions prepared in example 4 were divided into 4 groups. Physiological saline (Vehicle group), 0.5. Mu.M dimethylbutyryl carnitine (2 MBC group), 1. Mu.M BTT 3033 (BTT 3033 group), 0.5. Mu.M dimethylbutyryl carnitine, and 1. Mu.M BTT 3033 (2MBC+BTT3033 group) were added, respectively, and incubated for 30 minutes. 200. Mu.L of the platelet suspension was pipetted onto a slide, incubated at 37℃for 60 minutes and the slide was washed with PBS. Random field acquisition pictures were then selected under a fluorescence microscope (Leica DMI 8), 5-10 random fields were acquired per slide, and platelet diffusivity was quantitatively analyzed using Image J analysis software. Results were statistically analyzed using GraphPad9.0 software.
The results are shown in FIG. 7: FIG. 7A shows that the platelet aggregation rate of the 2MBC group is significantly increased compared with the Vehicle group, and that BTT 3033 can significantly block the platelet aggregation promoting effect of 2MBC, while BTT 3033 alone has no significant effect on the platelet aggregation rate; FIG. 7B shows that the platelet diffusivity of the 2MBC group is significantly increased compared to the Vehicle group, and that BTT 3033 significantly blocks the platelet diffusion promoting effect of 2MBC, whereas BTT 3033 alone has no significant effect.
Example 9 conversion of dimethylbutyric acid by murine enteric bacteria to dimethylbutyryl carnitine
8 male C57BL/6J mice of 6 weeks old were selected, and after 1% sodium pentobarbital (w/v) was anesthetized, the abdominal cavity was cut through the abdomen, the cecal content was taken out under aseptic conditions, and sterile PBS was added to prepare an enteric bacterial suspension. 500g was centrifuged at room temperature for 10 minutes, the supernatant was aspirated and added to thioglycolate medium, and the mixture was placed in an anaerobic incubator and incubated overnight at 37 ℃. The bacterial liquid is measured by an enzyme-labeled instrument at 600nm absorbance to determine the bacterial quantity, and thioglycolate medium is added to adjust the absorbance to 2.0. Subsequently 1mL of the bacterial suspension was removed, centrifuged at 1500g for 10 minutes at room temperature, the supernatant was removed, and the bacterial pellet was resuspended in sterile PBS. 200. Mu.M dimethylbutyric acid or an equal volume of sterile PBS was added, respectively, and incubated in an anaerobic incubator for 1 hour. The concentration of dimethylbutyryl carnitine in the culture supernatant was determined by a high resolution mass spectrometer. Results were statistically analyzed using GraphPad9.0 software.
The results are shown in fig. 8, which shows that the concentration of dimethylbutyryl carnitine in the experimental group is significantly increased as compared to the control group, suggesting that dimethylbutyryl acid can be converted into dimethylbutyryl carnitine by the intestinal bacteria.
EXAMPLE 10 antibiotic cocktail eliminates intestinal bacteria and inhibits the production of dimethylbutyryl carnitine by dimethylbutyric acid in vivo
6 male C57BL/6J mice of 8 weeks old were selected, and an antibiotic cocktail (neomycin 1mg/mL, streptomycin 1mg/mL, bacitracin 1 mg/mL) was added to drinking water to perform in vivo intervention (experimental group) on the mice to clear intestinal bacteria. In addition, 6 male C57BL/6J mice which are 8 weeks old and produced in the same cage are selected as experimental controls (control group), and the antibiotic cocktail is not added into drinking water. After 5 days, mice were perfused with 50 μg/kg dimethylbutyric acid, respectively, and 0, 60, 120 and 180 minute whole blood samples were collected, and plasma was extracted by centrifugation at 3000g at 4 ℃ and the concentration of dimethylbutyryl carnitine in the plasma was detected by high resolution mass spectrometry. In the data analysis, the concentration of dimethylbutyryl carnitine measured at each time point was subtracted from the concentration of dimethylbutyryl carnitine measured at each time point for 0 minutes, and the result was a change in the concentration of dimethylbutyryl carnitine. Results were statistically analyzed using GraphPad9.0 software.
The results are shown in fig. 9, which shows that the increased concentration of dimethylbutyryl carnitine in the plasma of mice in the control group is higher than that in the experimental group at various time points after dimethylbutyrate administration, suggesting that the conversion of dimethylbutyric acid to dimethylbutyryl carnitine can be significantly inhibited by the removal of intestinal bacteria from mice via antibiotic cocktails.
EXAMPLE 11 in vivo antibiotic inhibition of dimethylbutyryl carnitine conversion and improvement of thrombosis
15 male C57BL/6J mice of 6 weeks of age were selected and the mice were divided into 3 groups (5 per group) for pretreatment: abx+2MBA group was given drinking water (neomycin 1mg/mL, streptomycin 1mg/mL, bacitracin 1 mg/mL) intervention to clear intestinal bacteria, whereas Ctrl group (control group) and 2MBA group were not given antibiotic cocktails. After 5 days, mice in groups 2MBA and ABX+2MBA were given 50. Mu.g/kg dimethylbutyric acid lavage, respectively, while Ctrl group was given saline lavage, as a normal control. Then build up of ferric trichloride (FeCl) 3 ) An induced mouse carotid thrombosis model to observe the effect on carotid thrombosis.
The results are shown in fig. 10, which shows that supplementation with dimethylbutyrate shortens the carotid artery blood flow blocking time of mice compared to Ctrl group, suggesting that it promotes thrombosis; and when the antibiotic cocktail is adopted for pretreatment to remove intestinal bacteria and inhibit the conversion of dimethylbutyric acid to dimethylbutyryl carnitine, the carotid artery blood flow blocking time of the mice can be restored to the level of normal control mice. The above results suggest that by targeting intestinal bacteria to reduce the production of dimethylbutyryl carnitine in vivo, the effect of inhibiting thrombosis can be exerted.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (5)
1. The application of dimethylbutyryl carnitine inhibitor in preparing antithrombotic medicine is provided.
2. Use of dimethylbutyryl carnitine inhibitor according to claim 1 for the preparation of antithrombotic medicaments, characterized in that: the dimethylbutyryl carnitine inhibitor is an integrin alpha 2 beta 1 specific inhibitor or an antibiotic.
3. Use of dimethylbutyryl carnitine inhibitor according to claim 2 for the preparation of antithrombotic medicaments, characterized in that: the integrin alpha 2 beta 1 specific inhibitor is BTT 3033.
4. Use of dimethylbutyryl carnitine inhibitor according to claim 2 for the preparation of antithrombotic medicaments, characterized in that: the antibiotic is at least one of neomycin, streptomycin and bacitracin.
5. The use of dimethylbutyryl carnitine inhibitor according to claim 4 for the preparation of antithrombotic drugs, characterized in that: the antibiotics are neomycin, streptomycin and bacitracin according to the mass ratio of 1:1:1, and compounding to obtain the product.
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