CN117731676A - Application of Sha Ruihuan element in preparation of anti-platelet aggregation medicine - Google Patents
Application of Sha Ruihuan element in preparation of anti-platelet aggregation medicine Download PDFInfo
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- CN117731676A CN117731676A CN202211519752.XA CN202211519752A CN117731676A CN 117731676 A CN117731676 A CN 117731676A CN 202211519752 A CN202211519752 A CN 202211519752A CN 117731676 A CN117731676 A CN 117731676A
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Abstract
The invention provides application of pharmaceutically acceptable salts of saricycline and/or Sha Ruihuan in preparation of medicines for inhibiting platelet collagen receptor glycoprotein VI signal paths. Compared with the prior art, according to the results of non-clinical cell tests and animal drug effect tests, sha Ruihuan element inhibits collagen-mediated platelet aggregation, so that the anti-thrombus effect is exerted, the risk of pathological bleeding is not increased, and the safe and effective anti-platelet aggregation drug is developed.
Description
Technical Field
The invention belongs to the field of pharmacy, relates to a new application of Sha Ruihuan, and in particular relates to an application of Sha Ruihuan in preparation of an anti-platelet aggregation medicine.
Background
Inhibition of platelet aggregation to prevent and treat thrombosis is an important means for the prevention and treatment of thrombotic diseases. During physiological hemostasis and arterial thrombosis, platelets adhere to the exposed subendothelial matrix, so that platelet activation is the initial stage of thrombosis, and platelet aggregation undergoes a release reaction, ultimately leading to thrombosis. Signal transduction and platelet activation following platelet adhesion rely on the collagen-platelet glycoprotein receptor VI (GPVI) axis, and inhibition of this pathway can inhibit platelet activation at an earlier stage, thereby inhibiting thrombosis. In vitro and in vivo experiments find that the inhibitor or antibody developed by taking GPVI as a target spot can inhibit thrombus and inflammatory response thereof, does not interfere normal hemostatic response, is safe and effective, and is beneficial to solving the defect of the current clinical medication, namely, the improvement of the platelet aggregation resisting activity of the medicine is accompanied with the increase of bleeding risk. This has prompted the development of a variety of formulations that regulate or inhibit GPVI-mediated platelet activation and thrombosis. Currently, these drugs are mainly divided into four categories: competitive inhibitors of GPVI, such as GPVI-Fc; anti-GPVI antibodies, such as a class of antibodies that induce endocytosis or cleavage of GPVI (depleting antibodies) include JAQ1, mF1201, mF1232 and cF1232; GPVI blockers, i.e., antibodies that target GPVI to inhibit its function, range from monoclonal antibodies (mAbs) to antigen binding fragments (Fab) thereof, 9O12.3, 204-11, OM2, OM4, m-Fab-F and 1G5; the last category mainly takes GPVI signal paths as targets, such as curcumin, losartan and the like. Among them, the soluble GPVI-Fc dimer protein (revcept) as a competitive inhibitor has been currently subjected to phase 1 clinical trials, and the results indicate that it can safely and effectively inhibit collagen-induced platelet aggregation without extending bleeding time.
Sha Ruihuan (sarcine) is a novel narrow-spectrum oral tetracycline, originally developed by Paratek and Allergen, whose tablets were approved in the United states in month 10 of 2018, and were mainly used to treat non-nodular moderate-severe acne vulgaris in patients 9 years and older, the exact mechanism of treatment being unclear. Sha Ruihuan is a tetracycline ribosomal protein inhibitor that inhibits protein synthesis by interacting with the 70S bacterial ribosome, whereas unlike other tetracyclines, the unique C7 of Sha Ruihuan extends into the messenger RNA (mRNA) channel, forming a direct interaction with the a-codon, thereby interfering with mRNA movement through the channel and/or disrupting a-codon/anticodon interactions. The pharmacological action of the composition is effective on propionibacterium acnes and other gram positive bacteria in vitro, and the composition has an in vitro anti-inflammatory effect. In clinical experiments, two three-period double-blind random control experiments evaluate the curative effect of Sha Ruihuan elements on moderate-severe acne vulgaris, and the results show that the medicament is safe and effective and has good tolerance.
The Sha Ruihuan element has the following structural formula:
the inventor discovers through experimental study for the first time that Sha Ruihuan element can play a role in resisting platelet aggregation as a CPVI inhibitor to treat related diseases.
Disclosure of Invention
Sha Ruihuan (sarcine) is used as a novel narrow-spectrum oral tetracycline for treating non-nodular moderate-severe acne vulgaris in patients 9 years old and older, and the exact mechanism of treatment is not clear.
We have surprisingly found during the course of the study that this drug has the effect of inhibiting the collagen-platelet glycoprotein receptor VI (GPVI) shaft pathway, thereby exerting an antithrombotic effect and cerebrovascular disease.
The invention provides application of pharmaceutically acceptable salts of saricycline and/or Sha Ruihuan in preparation of medicines for inhibiting platelet collagen receptor glycoprotein VI signal paths.
Among them, the pharmaceutically acceptable salt of Sha Ruihuan may be any pharmaceutically acceptable salt known to those skilled in the art, and may be a salt formed by Sha Ruihuan elements and inorganic acid or a salt formed by Sha Ruihuan elements and organic acid, and is not particularly limited, but is preferably an acid addition salt formed by Sha Ruihuan elements and one or more of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, acetic acid, citric acid, maleic acid, oxalic acid, formic acid, benzoic acid, fumaric acid, succinic acid, tartaric acid, lactic acid, pyruvic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and trifluoroacetic acid.
The invention also provides application of the Sha Ruihuan element and/or Sha Ruihuan element pharmaceutically acceptable salt in preparation of anti-platelet aggregation medicines.
Preferably, the Sha Ruihuan element and/or Sha Ruihuan element pharmaceutically acceptable salt is effective to inhibit platelet aggregation by inhibiting platelet collagen receptor glycoprotein VI signaling pathway.
Namely, the medicine takes GPVI as a target spot to inhibit platelet activation and plays a role in resisting platelet aggregation.
Specifically, the platelet collagen receptor is a collagen COL receptor.
Preferably, the concentration of Sha Ruihuan element and/or a pharmaceutically acceptable salt thereof is 1 to 100. Mu.M, more preferably 3 to 100. Mu.M, still more preferably 10 to 100. Mu.M, when anti-platelet aggregation is performed.
The Sha Ruihuan element and/or Sha Ruihuan element pharmaceutically acceptable salt provided by the invention can be applied to preparation of medicines for treating thrombotic diseases and cerebrovascular diseases caused by platelet aggregation.
The invention also provides application of the Sha Ruihuan element and/or Sha Ruihuan element pharmaceutically acceptable salt in preparing a medicament for treating and/or preventing thrombotic diseases.
Preferably, the thrombotic disorder is one or more of arterial thrombotic disorder, venous thromboembolic disorder and thrombotic microangiopathy.
In the present invention, feCl is specifically purified by pharmaceutically acceptable salts of Sha Ruihuan and/or Sha Ruihuan elements 3 The inhibition of induced arterial thrombosis of rats indicates that the pharmaceutically acceptable salts of Sha Ruihuan and/or Sha Ruihuan can be used for preparing medicaments for treating and/or preventing arterial thrombotic diseases.
The invention also provides application of the Sha Ruihuan element and/or Sha Ruihuan element pharmaceutically acceptable salt in preparing a medicament for treating and/or preventing cerebrovascular diseases.
The invention also provides application of the Sha Ruihuan element and/or Sha Ruihuan element pharmaceutically acceptable salt in preparation of medicaments for treating and/or preventing cerebral apoplexy.
The invention also provides application of the Sha Ruihuan element and/or Sha Ruihuan element pharmaceutically acceptable salt in preparing a medicament for treating and/or preventing cerebral small vessel diseases.
The invention also provides application of the Sha Ruihuan element and/or Sha Ruihuan element pharmaceutically acceptable salt in preparing a medicament for treating and/or preventing cerebral thrombosis.
The invention also provides application of the Sha Ruihuan element and/or Sha Ruihuan element pharmaceutically acceptable salt in preparation of cerebral ischemia reperfusion injury drugs.
In the invention, sha Ruihuan and/or Sha Ruihuan pharmaceutically acceptable salts can improve the symptom of neurological deficit and reduce the range of cerebral infarction on a rat focal cerebral ischemia reperfusion model.
The invention also provides application of the Sha Ruihuan element and/or Sha Ruihuan element pharmaceutically acceptable salt in preparation of arterial stenosis drugs; preferably, the arterial stenosis is a carotid stenosis, more preferably a bilateral carotid stenosis.
In particular, the invention provides Sha Ruihuan and/or Sha Ruihuan pharmaceutically acceptable salts which can improve cerebral blood flow, improve learning and memory ability, improve brain protein injury and reduce microglial hyperplasia of cortex and hippocampal region of mice for bilateral common carotid artery stenosis.
The invention also provides a pharmaceutical composition comprising Sha Ruihuan element and/or Sha Ruihuan element pharmaceutically acceptable salts.
Preferably, the composition further comprises pharmaceutically acceptable auxiliary materials.
The pharmaceutical composition can be used for treating thrombotic diseases and cerebrovascular diseases caused by platelet aggregation.
Preferably, the pharmaceutical composition is used for treating thrombotic diseases and cerebrovascular diseases caused by platelet aggregation, and the dosage of the pharmaceutically acceptable salts of the active ingredients of the sarirecycline and/or Sha Ruihuan element is 2-100 mg/kg, more preferably 3-80 mg/kg, still more preferably 3-30 mg/kg.
Preferably, the pharmaceutical composition is used for treating arterial stenosis, the pharmaceutically acceptable salts of active ingredient sariresinol and/or Sha Ruihuan are used in an amount of 2 to 100mg/kg, more preferably 20 to 80mg/kg, still more preferably 40 to 80mg/kg.
The invention provides application of Sha Ruihuan element and/or Sha Ruihuan element pharmaceutically acceptable salt in preparation of medicines for inhibiting platelet collagen receptor glycoprotein VI signal pathway. Compared with the prior art, according to the results of non-clinical cell tests and animal drug effect tests, sha Ruihuan element inhibits collagen-mediated platelet aggregation, so that the anti-thrombus effect is exerted, the risk of pathological bleeding is not increased, and the safe and effective anti-platelet aggregation drug is developed.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
To further illustrate the present invention, the following examples are provided to illustrate the use of Sericycline in the manufacture of an anti-platelet aggregation agent.
The reagents used in the examples below are all commercially available.
EXAMPLE 1 investigation of the in vitro anti-platelet aggregation Activity of 1 Sha Ruihuan
1. Materials and methods
1.1 Animals
Sprague-Dawley (SD) rats, shanghai Laek laboratory animal Co., ltd
1.2 Reagent and consumable
1.3 Formulation and grouping of drug delivery formulations
Sha Ruihuan mg of the powder 4.875mg was weighed precisely one day before the experiment, added with 100. Mu.L of DMF, blown and vortexed, and allowed to dissolve well until clear, giving 100mM of a yellow-brown stock solution. The solution was diluted with DMF to gradient 40mM, 12mM, 4mM, 1.2mM and 0.4 mM. 12. Mu.L of cilostazol 100mM mother liquor was added to 88. Mu.L of DMF and stirred and mixed to obtain a 12mM dilution. The experiment was set up in 6 groups, which were: the vehicle was added to the blank, sha Ruihuan (1. Mu.M), sha Ruihuan (3. Mu.M), sha Ruihuan (10. Mu.M), sha Ruihuan (30. Mu.M), sha Ruihuan (100. Mu.M) and the same volume of vehicle. The number of experiments in each group is more than or equal to 6. In ADP as inducer experiment, cilostazol 30 μm final concentration group is added as positive control.
1.4 preparation of platelet rich plasma
Injecting 7% chloral hydrate (6 mL/kg) into the abdominal cavity of a rat, separating abdominal aorta at anesthesia depth which is suitable for severely stimulating slight reaction of the animal, wiping off residual liquid on the surface of the abdominal aorta, taking blood by using a venous blood taking needle and a sodium citrate anticoagulation tube (1:9), carefully and gently shaking the blood, uniformly mixing the blood, centrifuging the blood at 180g at room temperature for 15min, carefully taking the middle part of upper liquid into an EP tube by a liquid dispenser, and avoiding sucking red blood cells and interface white floccules to obtain upper turbid liquid, namely platelet-rich plasma (PRP); saline was used to zero and dilute PRP.
1.5 measurement of platelet aggregation Rate
The semiautomatic platelet aggregation instrument is powered on, preheated for about 30min, and tested after the temperature is displayed at about 37 ℃ and stabilized. The physiological saline and PRP solution were diluted 1:1 (total volume 290. Mu.L), a stirrer was added, and the mixture was placed in a thermostatic well with a test cup containing physiological saline and preheated for 5min. The TEST drug group is added with the compound to be tested 2min in advance, the instrument is modulated into a TEST state, a physiological saline TEST cup is placed in a TEST channel, an ENT key or a corresponding channel key is pressed, the instrument automatically detects a zero point, and a window displays a certain value, such as P40. After the value is stable, pressing the ENT key or the corresponding channel key. The physiological saline test cup is removed and placed in the PRP test cup, and the window displays another value, such as R30, and the window displays the inducer after pressing the ENT key or the corresponding channel key. And (3) sucking 10 mu L of COL or ADP by using a pipette, adding the mixture into the bottom of the cup, pressing an ENT key or a corresponding channel key, enabling the instrument to enter a platelet aggregation state to be tested, displaying and timing by a window, displaying the current platelet aggregation rate by the window after pressing the channel key, ending the test after 5min, and displaying the maximum platelet aggregation rate by the window.
COL collagen final concentration was 3. Mu.g/mL, ADP final concentration was 5. Mu. Mol/L.
1.6 platelet aggregation inhibition Rate calculation
From the platelet aggregation rate (maximum aggregation rate, MAR) of the measured samples, the platelet aggregation inhibition rate at each concentration of Sha Ruihuan element compared to the control group was calculated. The calculation formula is as follows:
platelet aggregation inhibition (%) = (MAR) Blank control group -MAR Test drug group )/MAR Blank control group ×100％。
1.7 data statistics
Experimental data are expressed as Mean ± standard deviation (Mean ± SD). One-way ANOVA One-way analysis of variance Dunnett's method examined group-to-group differences. P <0.05 is indicated as a significant difference.
2 experimental results
2.1 Effect of Sha Ruihuan on collagen COL-induced platelet aggregation
As shown in table 1, sha Ruihuan was able to inhibit platelet aggregation in a concentration range of 1 to 100 μm in comparison to the blank group. The results of one-way analysis of variance show that Sha Ruihuan element 1 mu M has obvious platelet aggregation resisting effect, and the inhibition rates of the platelet aggregation at the concentrations of 1,3, 10, 30 and 100 mu M are 9.62%, 22.18%, 33.40%, 65.82% and 95.19%, respectively. The IC50 value of the half inhibition concentration of Sha Ruihuan element for inhibiting collagen-induced platelet aggregation is 19.95 mu M.
TABLE 1 influence of Sha Ruihuan on COL-induced platelet aggregation
Data are expressed as mean±sd. P <0.05, < p <0.01, < p <0.001, compared to the model group.
2.2 Effect of Sha Ruihuan on ADP-induced platelet aggregation
As shown in table 2, there was no significant difference in the platelet aggregation rate at each concentration of Sha Ruihuan element compared to the blank group, i.e., no effect on ADP-induced platelet aggregation. Cilostazol 30 μm as a positive control significantly inhibited platelet aggregation (inhibition rate 25-30%).
TABLE 2 influence of Sha Ruihuan on ADP-induced platelet aggregation
Data are expressed as mean±sd. P <0.05, < p <0.01, < p <0.001, compared to the model group.
Example 2 Effect of Sha Ruihuan on collagen binding to platelets
1. Materials and methods
1.1 Experimental animal
Sprague-Dawley (SD) rats, shanghai Laek laboratory animal Co., ltd.
1.2 Reagent and consumable
1.3 Platelet extraction and processing
Rats were intraperitoneally injected with 7% chloral hydrate (6 mL/kg), the depth of anesthesia was such that the animals were gently stimulated, abdominal aorta was isolated, and the surface residual fluid was wiped dry, blood was collected using a venous lancet and sodium citrate anticoagulant tube (1:9), gently shaken carefully to mix them well, centrifuged at 180g at room temperature for 15min, the middle portion of the upper fluid was carefully gently removed by a pipette into the EP tube to avoid aspiration of red blood cells and interface white floc, and the resulting upper turbid fluid, platelet-rich plasma (PRP), was used as zeroing and PRP dilution.
1.4 flow cytometer detection
Platelets (2X 10) 8 .mL -1 ) Incubation with 30. Mu.M Sha Ruihuan or 5. Mu.g/mL Fab 9O12 for 10min at 37℃followed by addition of 10. Mu.g/mL -1 FITC-type I collagen was activated for 20 minutes at Room Temperature (RT) and then multimerized with 2%Formaldehyde solution PFA fixation. Samples were analyzed by flow cytometry using a FACS101 cytometer from Becton Dickinson.
1.5 data statistics
As in example 1.
2 experimental results
As shown in Table 3, the effect of Sha Ruihuan on the binding of FITC-collagen to platelets was further analyzed using FITC-conjugated type I collagen-induced platelet aggregation, and the results showed that 10. Mu.g/mL of FITC-collagen induced platelet aggregation after washing and Sha Ruihuan on the binding of FITC-collagen to platelets was significantly inhibited. The GPVI antibody Fab 9O12 (50. Mu.g/mL) was used as a positive control.
TABLE 3 influence of Sha Ruihuan element on collagen and platelet binding
Data are expressed as mean±sd. P <0.05, < p <0.001 compared to model group.
EXAMPLE 3 inhibition study of the platelet aggregation in rats by Sha Ruihuan element (in vivo validation)
1. Materials and methods
1.1 Experimental animal
Sprague-Dawley (SD) rats, male, SPF grade, weight 250-280g.
1.2 test drug
Sha Ruihuan and He Xiluo tazode are the same as in example 1.
1.3 Experimental methods
1.3.1 grouping and administration of animals
The experimental animals were divided into Sha Ruihuan (1 mg/kg), sha Ruihuan (2 mg/kg), sha Ruihuan (4 mg/kg), sha Ruihuan (8 mg/kg), cilostazol (8 mg/kg) and normal control groups, which were 6 groups in total. Sha Ruihuan and He Xiluo, and the sari Han Suzu and cilostazol group experimental animals were each given 1 time by gastric administration in a 0.5% CMC-Na solution 3h before blood collection, and the normal control group animals were given an equal volume of vehicle, and the blood collection was abdominal aortic blood collection.
1.3.2 blood collection and PRP preparation
Experimental animals were weighed and randomly and equally single-blinded into groups. Performing gastric lavage administration of Sha Ruihuan and cilostazol according to body weight, injecting 7% chloral hydrate (6 mL/kg) into abdominal cavity of a rat after 3h for anesthesia, separating abdominal aorta, wiping off residual liquid and mucous membrane on the surface of the abdominal aorta, taking blood by using an intravenous blood taking needle and a sodium citrate anticoagulation tube (1:9), taking blood volume to be 6mL, carefully and gently shaking the blood, uniformly mixing the blood and the blood, centrifuging the blood and the mucous membrane for 15min at 180g at room temperature, carefully taking the middle part of upper liquid into an EP tube by a liquid-transferring device, avoiding sucking red blood cells, and obtaining an upper turbid liquid, namely platelet-rich plasma (PRP), and marking groups; saline was used to zero and dilute PRP.
1.3.3 measurement of platelet aggregation Rate
The semiautomatic platelet aggregation instrument is powered on, preheated for about 30min, and tested after the temperature is displayed at about 37 ℃ and stabilized. Diluting physiological saline and PRP solution 1:1 (the total volume of the physiological saline and the PRP solution is 290 mu L), adding a stirrer, and placing the stirrer and a test cup of the physiological saline in a constant temperature hole respectively for preheating for 5min. The TEST drug group is added with the compound to be tested 2min in advance, the instrument is modulated into a TEST state, a physiological saline TEST cup is placed in a TEST channel, an ENT key or a corresponding channel key is pressed, the instrument automatically detects a zero point, and a window displays a certain value, such as P40. After the value is stable, pressing the ENT key or the corresponding channel key. The physiological saline test cup is removed and placed in the PRP test cup, and the window displays another value, such as R30, and the window displays the inducer after pressing the ENT key or the corresponding channel key. And (3) sucking 10 mu L of COL by using a pipette, adding the COL into the bottom of the cup, pressing an ENT key or a corresponding channel key, enabling the instrument to enter a platelet aggregation state to be tested, displaying and timing by a window, displaying the current platelet aggregation rate by the window after pressing the channel key, and displaying the maximum platelet aggregation rate by the window after 5min of testing.
COL collagen final concentration was 3. Mu.g/mL.
1.4 data statistics
As in example 1.
2 experimental results
The inhibition rate of each group of animals on collagen-induced platelet aggregation is shown in table 4, and compared with a normal control group, sha Ruihuan element 2mg/kg can obviously inhibit collagen-induced platelet aggregation of rats, and the inhibition effect has dose dependency. Compared with cilostazol group (8 mg/kg), sha Ruihuan showed no significant difference in platelet aggregation inhibition rate of 4 mg/kg. The Sha Ruihuan element has better platelet aggregation resisting effect than cilostazol.
TABLE 4 action of Sha Ruihuan element on platelet aggregation in rats (in vivo validation)
Data are expressed as mean±sd. P <0.05, < p <0.01, < p <0.001, compared to the model group.
EXAMPLE 4 Sha Ruihuan element pair FeCl 3 Inhibition study of induced arterial thrombosis in rats
1. Materials and methods
1.1 Experimental animal
Sprague-Dawley (SD) rats, male, SPF grade, weight 250-280g.
1.2 test drug
Sha Ruihuan the same as in example 1.
1.3 Experimental methods
1.3.1 grouping and administration of animals
Animals in thrombus experiments were randomly and equally divided into 5 groups according to body weight, a sham operation group, a model group, a Sha Ruihuan element group (2 mg/kg), a Sha Ruihuan element group (4 mg/kg) and a Sha Ruihuan element group (8 mg/kg). The Sha Ruihuan element group was suspended in a 0.5% CMC-Na solution for intragastric administration, and the sham-operated group and model group were administered with 0.5% CMC-Na solution once daily for 7 consecutive days in the morning and afternoon, respectively. After the last administration for 30min, experiments were performed.
1.3.2FeCl 3 Induction of arterial thrombosis in rats
After 7% chloral hydrate (6 mL/kg) is injected into the abdominal cavity of a rat for anesthesia, the rat is sheared along the middle line of the neck, the common carotid artery on the right side is passively separated for 1cm long, a sealing rubber strip with the width of 0.6cm is put in, and then 20% FeCl is soaked in the solution 3 A strip of filter paper (1.0 cm. Times.0.5 cm) of the solution was wrapped around the isolated carotid segment and sealed with a sealing strip. After 15min, the strip was removed. After 40min, ligating blood vessels at two ends of the filter paper strip, precisely cutting off blood vessel sections wrapped by the filter paper strip, sucking residual blood in the blood vessel by using clean filter paper, precisely weighing blood vessel wet weight containing thrombus, and weighing the blood vessel after taking out the thrombus, wherein the mass of the thrombus in the blood vessel sections with the length of 0.5cm is obtained by subtracting the two blood vessel wet weight. Saline instead of FeCl for sham group 3 A soaked filter paper strip.
1.4 data statistics
As in example 1.
2 experimental results
Compared with the sham operation group, the model group has the advantages that the carotid artery modeling part of the rat is filled with thrombus embolus, which shows FeCl 3 Can obviously induce carotid thrombosis. The Sha Ruihuan element group (4 mg/kg and 8 mg/kg) significantly reduced thrombus weight (p < 0.05) compared to the model group.
TABLE 5 Sha Ruihuan element pair FeCl 3 Inhibition study of induced arterial thrombosis in rats
Data are expressed as mean±sd. P <0.05, < p <0.01 compared to model group.
Example 5 test of efficacy study of Sha Ruihuan on rat focal cerebral ischemia reperfusion model
1 materials and methods
1.1 laboratory animals
Sprague-Dawley (SD) rats, males, SPF grade, weight 280-300g.
1.2 test drug
Sha Ruihuan the same as in example 1.
1.3 Experimental methods
1.3.1 preparation of focal cerebral ischemia reperfusion model
Rats were placed in the induction box of the anesthesia machine (isoflurane concentration 4%) and the depth of anesthesia was such that the animals were severely stimulated to respond slightly. The rat in an anesthetic state is tightly bound with rubber bands on limbs (the hind limb is fixed above the knee joint and the front limb is fixed above the wrist joint) and the head, the animal is fixed on an operating table in a supine position, the rat head is covered with an anesthetic cover, and the concentration of isoflurane is kept to be 2.5 percent so as to prevent the animal from waking up in the experimental process. The animal shaver is used for shaving hair from the head end to the chest, and the skin is sterilized by alcohol. Median incision of neck and blunt separation of subcutaneous tissue. Separating the thin fascia on the triangular surface of the front of the neck, plucking the lower edge of the low-side collarbone hyoid muscle, seeing the longitudinal pulsating artery parallel to the muscle, opening the arterial shell, exposing the bifurcation of the right carotid artery, separating the right common carotid artery, the external carotid artery and the internal carotid artery, gently peeling the vagus nerve, ligating and cutting off the external carotid artery. The proximal end of the common carotid artery was clamped, an incision was made from the distal end of the ligature of the external carotid artery, a tether was inserted, bifurcated through the common carotid artery into the internal carotid artery, and then gently inserted until there was slight resistance (about 20mm from the bifurcation), blocking all blood supply to the middle cerebral artery. The thread is used for fixing the bolt thread slightly below the external carotid incision, the proximal end of the common carotid artery is loosened to clamp the thread, and the animal is simply sutured and placed in the feeding box. After 90min of right cerebral ischemia, the internal thrombus line of the ischemic animal is gently pulled out, blood supply is restored for reperfusion, the external carotid artery is ligated by the thread of the fixed thrombus line, the skin is sutured, and the skin is disinfected. Placing the rats in clean feed, and observing the general condition and respiration until anesthesia is recovered; adding water for feeding, and conventionally feeding.
1.3.2 grouping and administration of animals
The experimental animals were divided into 5 groups of model group, sha Ruihuan group (3 mg/kg), sha Ruihuan group (10 mg/kg), sha Ruihuan group (30 mg/kg) and edaravone group (6 mg/kg). After preparation of the cerebral ischemia model, animals were assigned to each group with equal probability for single blindness. Animals were dosed 1 time immediately after reperfusion, sha Ruihuan elements were dosed by gavage, edaravone was intravenously injected, and model animals were dosed by gavage with an equal volume of 0.5% CMC-Na. Neurological deficit symptoms were assessed 24 hours after cerebral ischemia, then animals were sacrificed, brains were taken, stained, photographed and cerebral infarct size was determined.
1.3.3 determination of neurological deficit symptom score and cerebral infarction area
The neurological deficit symptoms were evaluated using the modified Bederson 5 assay. And (3) evaluating the neurological deficit symptoms of the rat after cerebral ischemia by adopting a single blind method, namely marking animals by groups by a test designer, wherein a tester for scoring the neurological deficit symptoms does not know the grouping situation of the animals, and after the scoring is finished, the scoring results of various marks are presented to the designer by the scoring designer, and the designer uncovers the blind to obtain the score of each animal in each test group.
The attached table: method for scoring Bederson 5 for neurological deficit symptoms
And (5) measuring the cerebral infarction degree by adopting a TTC staining method. After the animal neurological deficit symptoms are evaluated, CO is used for 2 Killing, taking brains, removing olfactory bulb, cerebellum and low brain stem, flushing cerebral surface blood trace with physiological saline, sucking residual water trace on surface, standing at-20deg.C for 20min, taking out, immediately making a coronal section vertically downward on the intersecting plane of sight line, cutting a slice every 2mm backward, placing the brain slice in 1% TTC dye solution, incubating (37 deg.C for 30 min), staining normal brain tissue into dark red, flushing ischemic brain tissue with physiological saline, rapidly arranging the brain slices in a row from front to back, sucking residual water trace on surface, and photographing.
Calculation of cerebral infarction area: the photo is processed by Image J software, and the corresponding area of the left brain and the non-infarct area of the right brain are calculated according to a formula, so that the percentage of the infarct range is calculated.
Infarct volume calculation method:
V＝t(A1+A2+A3+.........+An)
t is the slice thickness, A is the infarct size.
％I＝100％×(V C -V L )/V C
% I is infarct volume percent, V C For control side (left hemisphere) brain volume, V L Is the infarct side (right brain hemisphere) non-infarct area volume.
1.4 data statistics
Experimental data are expressed as Mean ± standard deviation (Mean ± SD). The individual efficacy indicators were analyzed for one-way variance using the software GraphPad Prism (7.04), dunnett's method to examine group-to-group differences. P <0.05 is indicated as significantly different.
2. Experimental results
2.1 Effects on neurological deficit symptoms
The degrees of the animal neurological deficit symptoms of each group are shown in table 6, statistical differences exist among the groups through single-factor analysis of variance, compared with a model group, the Sha Ruihuan element 3mg/kg, the 10mg/kg and the 30mg/kg can obviously improve the rat neurological deficit symptoms, and the action effect has dose dependency (p= 0.036,0.011,0.000).
TABLE 6 influence of Sha Ruihuan elements on the symptoms of neurological deficit
Data are expressed as mean±sd. P <0.05, < p <0.01, < p <0.001, compared to the model group.
2.2 Effect on cerebral infarction area
The effect on cerebral infarction area is shown in Table 7, and statistical differences exist among the groups through single-factor analysis of variance, and compared with the model group, the Sha Ruihuan element group can obviously reduce the cerebral infarction range of the model rat by 3mg/kg,10mg/kg and 30mg/kg. (p= 0.047,0.028,0.000).
TABLE 7 influence of Sha Ruihuan on cerebral infarction area by the administration of the extract
Data are expressed as mean±sd. P <0.01, p <0.001 compared to model group
Example 6 test of efficacy study of Sha Ruihuan on a model of bilateral common carotid artery stenosis in mice
1. Materials and methods
1.1 Experimental animal
C57BL/6 mice, male, SPF grade, weight 27-30g.
1.2 test drug
Sha Ruihuan the same as in example 1.
1.3 Experimental methods
1.3.1 preparation of mouse bilateral common carotid artery stenosis (BCAS) model
The neck hair of the mice was shaved two to three days prior to surgery. After the test animals were weighed and recorded, the mice were placed in the induction box of the anesthesia machine (isoflurane concentration 3.5%), and the anesthesia depth was such that the animals were severely stimulated with a slight response. The four limbs and the head of the mouse in an anesthetic state are fixed by using an adhesive tape, the supine position is fixed in an operation area of a stereoscopic microscope, the head of the mouse is covered with an anesthetic cover, and the concentration of isoflurane is kept to be 1.5%, so that the animal is prevented from waking up in the experimental process.
After the skin is sterilized by alcohol, the neck is centrally cut, and subcutaneous tissue is blunt-separated. The thin fascia on the anterior triangle surface of the neck was separated, the inferior border of the low-side-collarbone hyoid muscle was lifted, the longitudinal pulsating artery parallel to this muscle was seen, the arterial shell was opened, the common carotid arteries on both sides were exposed and carefully separated, and a wire was placed over and under each artery for lifting the operation, gently dissecting the vagus nerve, taking special care that the operation did not cause vagal nerve reactions. The light wire rod carefully winds the spring ring on the common carotid artery, and hair, fat or tissue adhesion on the common carotid artery is avoided in the winding process. Loosening the common carotid artery to clamp the silk thread, suturing the animal on a heat insulation blanket, observing the general condition and breathing until anesthesia is revived, putting the animal into a feeding box, adding feeding water, and feeding conventionally. The sham operation group performs the same operation procedure as the model group except for the spring coil. During the operation, animals with abnormal conditions due to anesthesia, operation and the like are removed and recorded.
1.3.2 grouping and administration of animals
After the BCAS model was prepared sequentially for experimental animals, animals were randomly and equally single-blinded into groups. The experimental animals were divided into a sham operation group, a model group, a Sha Ruihuan element group (40 mg/kg), a Sha Ruihuan element group (80 mg/kg), and cilostazol (100 mg/kg) for a total of 5 groups. Administration was started once daily on day 3 after molding. All surviving animals were weighed twice a week for the first two weeks after molding, and then once a week. Sha Ruihuan and cilostazol were administered by gavage in the form of a 0.5% CMC-Na suspension, and animals in model group were given equal volumes of 0.5% CMC-Na by gavage.
1.3.3 measurement of cerebral blood flow, behavioural detection, LFB myelin staining pathology score and IBA-1 immunohistochemical assay
1.3.3.1 detection of Cerebral Blood Flow (CBF)
Regional Cerebral Blood Flow (CBF) was measured using a high resolution laser speckle blood flow imager (LSCI). The detection time points are 1,3, 7 and 14 days before and after BCAS operation. After the mice were anesthetized (isoflurane induced at 3.5% concentration and maintained at 1.5% concentration) they were placed in the laboratory bench operating area, head hair was shaved, prone position was fixed, scalp was cut along the midline of the brain, skull was exposed, the fascia on the surface of the skull was cleaned, the photographing height and angle of the blood flow imager were adjusted, the photographing position was fixed in real-time display mode, and the blood flow of the entire brain region was continuously recorded for 5 seconds in photographing mode. Off-line analysis mode the mean blood flow of the region of interest (ROI) stability zone was derived, the mouse incision was sutured and iodophor was applied to prevent infection. Data analysis is expressed as a percentage of baseline values.
1.3.3.2Morris water maze
The Morris water maze experiment is adopted to detect the spatial learning and memory capacity of the experimental mice. Morris water maze experiment was performed 4 weeks after the operation, the diameter of the Morris water maze pool was 120cm, the height was 50cm, and the water depth was 29cm. The platform has a diameter of 6cm and a height of 28cm, and is fixed 1cm below the water surface of the SE quadrant (target quadrant). The camera lens is arranged above the center of the pool and at a distance of 2m from the bottom of the pool, and is used for synchronously recording the movement track of the mice. The water temperature was maintained at 22.+ -. 1 ℃. Experiments total 6 days, including hidden platform experiments and space exploration experiments. The first 5 days are hidden platform experiments, and the last 1 day is space exploration experiments.
Hidden platform test (hidden platform trial)
The experiment is carried out for 5 days, the position of a platform is fixed in a target quadrant (SE quadrant) during the experiment, water is respectively introduced from water inlet points of four quadrants, an animal is lightly put into water towards a pool wall mark during training, the time for the animal to find the platform is recorded, and then the animal stays on the platform for 10s. If no platform is found within 60s, the latency is noted as 60s and the animal is guided to rest on the platform for 10s. After training, the mice were returned to the rearing cage and warmed up. The statistical analysis was performed at 4 water entry points each 1 time per day with the average of four latencies as the performance of the day. The water inlet sequence of each animal is kept consistent every day, but the water inlet sequence of different animals in the same cage is different, so that animal information communication in the group is excluded.
Space exploration test (probe real)
And removing the platform after the test of the hidden platform is finished for 24 hours. Then water is added from the opposite water inlet point of the platform, and the residence time in the target quadrant and the crossing times of the original platform in the mouse 60s are recorded. Observing the space positioning capability of animals and the change rule in the space exploration process.
1.3.3.3Y maze test
The free alternate experiment of the Y maze detects the learning and memory ability of experimental animals. The detection is carried out 6 weeks after the operation, the mice are brought into a behavioural laboratory from an animal house in advance to adapt to experimental environments, the detection environments in the laboratory are ensured to be quiet, and meanwhile, the light source is regulated to avoid direct irradiation of strong light. After the experiment is started, the mice are uniformly oriented and placed in the center of a Y maze, so that the mice freely shuttle through 3 arms, the sequence (or manual record) of the mice entering and exiting each arm within 5-8min is recorded and analyzed by TopscanLite animal behavioural analysis software, 3 different arms are continuously entered as correct selection, the correct alternation times N and the total arm entering times N of each mouse are recorded, and the correct alternation rate of each mouse is calculated. After the test of each mouse is finished, excrement is cleaned, the interior of the Y maze is wiped by 75% medical alcohol until the alcohol volatilizes completely, and then the test of the next mouse is continued, so that the odor is prevented from affecting the shuttle behavior of the subsequent mice.
Correct alternation rate = correct alternation number N/(total arm advance number N-2)
1.3.3.4LFB myelin staining experiment
8 weeks after operation, animals were perfused with normal saline +4% paraformaldehyde to obtain brains, stored in 4% paraformaldehyde at normal temperature, paraffin-embedded with samples and sectioned to obtain coronal sections (4 μm), the sections were selected at the beginning of the hippocampus, and the corpus callosum, the inner sac, the caudal putamen and the optic bundle were in the same visual field plane.
Dewaxing and rehydrating the slices, and placing the slices in a dyeing vat containing LFB dye liquor (Solarbio, beijing, china) for water bath at 56 ℃ for 2h. Excess staining solution was washed with 95% ethanol and then with distilled water. Separating color with firm blue differentiation liquid for 10s, separating color with 70% ethanol for 20s, washing with water, and observing with microscope until gray matter and white matter are well defined. And (5) counterstaining with tar violet staining solution for 30-40s, and washing with water. Air-dried in a fume hood, finally transparent in xylene and sealed with neutral resin.
Come biological microscope was used to shoot at 200 and 400 magnifications. Blue in the field of view of the sections is myelin sheath, pink-purple is neuron, and the severity of white matter damage WML is assessed by the density of stained axonal fibers.
WMLs were evaluated in five brain regions: visual bundle (OPT), inner sac (IC), caudate nucleus (CPU), medial and lateral to the corpus callosum. WMLs severity is classified as normal (grade 0), nerve fiber alignment disorder (grade 1), overt vacuole formation (grade 2), and medullary fiber disappearance (grade 3).
1.3.3.5IBA-1 immunohistochemical detection
IBA-1 proteins are generally considered microglial markers in the central nervous system, reflecting neuroinflammation. IBA-1 was chosen as an index for evaluation of neuroinflammation.
Paraffin sections dewaxed to water: sequentially placing the slices into an environment-friendly dewaxing liquid I10 min-environment-friendly dewaxing liquid II 10 min-environment-friendly dewaxing liquid III 10 min-absolute ethyl alcohol I5 min-absolute ethyl alcohol II 5 min-absolute ethyl alcohol III 5 min-distilled water for washing. Antigen retrieval: repair is shown in the above table, and buffer solution should be prevented from evaporating excessively during this process, and the sheet should not be dried. After natural cooling, the slide was washed 3 times with shaking in PBS (pH 7.4) on a decolorizing shaker for 5min each. (repair fluid and repair conditions are determined by the tissue). Blocking endogenous peroxidases: the sections were incubated in 3% hydrogen peroxide solution at room temperature for 25min in the absence of light, and the slides were washed 3 times with shaking in PBS (pH 7.4) on a decolorizing shaker for 5min each. Serum blocking, namely 3% BSA is dripped into a histochemical ring to uniformly cover tissues, and the tissue is blocked for 30min at room temperature. (primary antibody was goat-derived blocked with rabbit serum, and other sources blocked with BSA). Adding an antibody: the blocking solution is gently thrown away, PBS is dripped on the slice, the slice is horizontally placed in a wet box for incubation at 4 ℃ for overnight. Adding a secondary antibody: the slide was washed with shaking 3 times, 5min each time, in PBS (pH 7.4) on a decolorizing shaker. And (3) dripping secondary antibodies (HRP marks) of corresponding species with the primary antibodies into the circles to cover tissues after the sections are slightly dried, and incubating for 50 minutes at room temperature. DAB color development: the slide was washed with shaking 3 times, 5min each time, in PBS (pH 7.4) on a decolorizing shaker. And (3) dripping freshly prepared DAB color development liquid into the ring after the slice is slightly dried, controlling the color development time under a microscope, and washing the slice with tap water to terminate the color development, wherein the positive color is brown. Counterstaining the nuclei: the hematoxylin counterstain is carried out for about 3min, the water washing is carried out, the hematoxylin differentiation liquid is differentiated for a plurality of seconds, the tap water washing is carried out, the hematoxylin blue returning liquid returns blue, and the running water washing is carried out. And (3) removing the water sealing piece: sequentially placing the slices into 75% alcohol for 5 min-85% alcohol for 5 min-absolute alcohol for II for 5 min-n-butanol for 5 min-xylene for 5min for dehydration and transparency, taking out the slices from the xylene, airing slightly, and sealing the slices by sealing glue. And (5) microscopic examination: and (5) performing result interpretation under a white light microscope. Hematoxylin-stained nuclei were blue and DAB showed positive expression as brown yellow.
The cortex and hippocampus CA1 regions were photographed at 10X 20 and 10X 40 magnification using a Leica biological microscope, and the cortex and hippocampus of each slide were each selected from 5 high power fields (×200) to manually count IBA-1 positive cells, and the average of the counts was taken as the positive cell number of the sample.
1.4 data statistics
Experimental data are expressed as Mean ± standard deviation (Mean ± SD). Each efficacy index was analyzed by single or double factor variance analysis using software GraphPad Prism (9.00) and the LSD method was used to examine the differences between groups. P <0.05 is indicated as significantly different.
2 experimental results
2.1 Effect on BCAS mouse cerebral blood flow
The brain blood flow CBF (n=6-8/group) was measured on days 0, 1,3, 7 and 14 after BCAS surgery and the rate of change of the brain blood flow with time for each group of animals is shown in table 8. Statistical differences were found between the groups as a whole by two-factor anova, and the results showed that the brain blood flow was significantly reduced (p < 0.0001) on days 1,3, 7 and 14 after BCAS in the model group experimental animals compared to the sham group. Compared with the model group, the Sha Ruihuan element He Xiluo tazoie can improve the reduction of the cerebral blood flow of mice, and the Sha Ruihuan element 80mg/kg (D7, D10, p= 0.002,0.017) and cilostazol 100mg/kg (D3, D10, p= 0.032,0.047) have remarkable action effects.
TABLE 8 influence of Sha Ruihuan on cerebral blood flow after cerebral ischemia
Data are expressed as mean±sd. P <0.05, < p <0.01, < p <0.001, compared to the model group.
2.2 Effect on the learning memory Capacity of BCAS mice
The effects on learning and memory are shown in Table 9, table 10 and Table 11. The escape latency was statistically different between groups of water maze experiments by two-factor analysis of variance. The model group and the sham group were significantly different from the sham group in escape latency from day 3 of the bench test. The cilostazol group animals had a significantly shortened escape latency (p=0.016) on day 3 of the cryptotazoie test and 80mg/kg animals had a significantly shortened escape latency (p=0.008) on day 5 of the cryptotazoie test compared to the model group.
The water maze space exploration test has the advantages that through single-factor analysis of variance, statistical differences exist among groups, and compared with a sham operation group, the residence time of an animal target quadrant of a model group is obviously shortened (P=0.031); the Sha Ruihuan-prime group had a significant increase in 80mg/kg target quadrant residence time (p=0.040) compared to the model group. There was no statistical difference in the number of target quadrant platform passes between groups.
The Y maze test has statistical difference between groups through single-factor analysis of variance, and compared with a sham operation group, the correct alternation rate of the action track of animals in the Y maze is obviously reduced (P=0.0008); the correct alternation rate of the course of action in the Y maze was significantly increased in the Sha Ruihuan-prime group 80mg/kg and the cilostazol group 100mg/kg group compared to the model group (p=0.034, p=0.044).
TABLE 9 influence of Sha Ruihuan element on escape latency of mice in the water maze
Data are expressed as mean±sd. P <0.05, < p <0.01, < p <0.001, compared to the model group.
TABLE 10 influence of Sha Ruihuan element on the spatial exploration of the mouse water maze
Data are expressed as mean±sd. P <0.05, < p <0.01 compared to model group.
TABLE 11 influence of Sha Ruihuan element on correct alternation rate of mice in Y maze
Data are expressed as mean±sd. P <0.05, p <0.001 compared to model group
2.3 Effect on white matter lesions
The myelinated nerve axons were stained by Luxol firm blue brain sections, and white matter lesions of BCAS modeling corpus callosum, caudal putamen, endobursa and optic tract areas were seen, and the scores of the animal brain areas WMLs for each group are shown in table 12. The lesions of the corpus callosum, endobursa and optic tract areas observed in mice from the experimental endpoint BCAS model group were significantly higher than those observed in the sham-operated group, as analyzed by T-test. The observed endocapsule and optic tract region WMLs scores were significantly lower in the Sha Ruihuan group of 80mg/kg mice than in the model group of mice (p=0.042, p=0.029), suggesting improved neuropathological changes. The observed beam area WMLs scores were significantly lower in the cilostazol group 100mg/kg group mice than in the model group mice (p=0.021).
TABLE 12 influence of Sha Ruihuan element on white matter damage
Data are expressed as mean±sd. P <0.05, < p <0.01 compared to model group.
2.2 effects on neuroinflammation
Immunohistochemistry for microglial proliferation in the cerebral cortex and hippocampal areas of mice was performed, and statistical differences in the numbers of IBA-1 positive cells were found in each group by single-factor analysis of variance, as shown in Table 13. The BCAS group mice had significantly increased microglial proliferation in the cortex and hippocampal region (p= 0.017,0.003) compared to sham group mice. 80mg/kg of the Sha Ruihuan-element group significantly reduced microglial proliferation in the hippocampal region of the model mice compared to the model group (p=0.028). Cilostazol 100mg/kg significantly reduced microglial proliferation in the cortex and hippocampal areas of model mice (p= 0.35,0.018).
TABLE 13 influence of Sha Ruihuan elements on neuroinflammation
Data are expressed as mean±sd. P <0.05, < p <0.01 compared to model group.
Claims (9)
1. Use of a pharmaceutically acceptable salt of Sha Ruihuan and/or Sha Ruihuan in the manufacture of a medicament for inhibiting platelet collagen receptor glycoprotein VI signaling pathway.
2. The application of Sha Ruihuan and/or Sha Ruihuan pharmaceutically acceptable salts in preparing anti-platelet aggregation medicines.
3. The use according to claim 2, wherein the pharmaceutically acceptable salt of Sha Ruihuan and/or Sha Ruihuan is effective against platelet aggregation by inhibiting platelet collagen receptor glycoprotein VI signaling pathway.
4. Use of pharmaceutically acceptable salts of Sha Ruihuan and/or Sha Ruihuan in the manufacture of a medicament for the treatment and/or prophylaxis of thrombotic disorders.
5. The use according to claim 4, wherein the thrombotic disorder is one or more of arterial thrombotic disorder, venous thromboembolic disorder and thrombotic microangiopathy.
6. Use of pharmaceutically acceptable salts of Sha Ruihuan and/or Sha Ruihuan in the manufacture of a medicament for the treatment and/or prophylaxis of cerebrovascular disease.
7. Application of Sha Ruihuan and/or Sha Ruihuan pharmaceutically acceptable salts in preparation of medicines for treating and/or preventing cerebral apoplexy is provided.
8. The application of Sha Ruihuan element and/or Sha Ruihuan element pharmaceutically acceptable salt in preparing medicaments for treating and/or preventing cerebral small vessel diseases.
9. Application of Sha Ruihuan and/or Sha Ruihuan pharmaceutically acceptable salts in preparing medicines for treating and/or preventing cerebral thrombosis.
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