WO2019192339A1 - 一种预防和/或治疗缺血再灌注损伤的中药组合物 - Google Patents
一种预防和/或治疗缺血再灌注损伤的中药组合物 Download PDFInfo
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
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- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
- A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/25—Araliaceae (Ginseng family), e.g. ivy, aralia, schefflera or tetrapanax
- A61K36/258—Panax (ginseng)
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
- A61K36/481—Astragalus (milkvetch)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
- A61K36/537—Salvia (sage)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
- A61K38/482—Serine endopeptidases (3.4.21)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
Definitions
- the invention relates to the field of traditional Chinese medicine, in particular to a traditional Chinese medicine composition for preventing and/or treating ischemia-reperfusion injury.
- ischemic reperfusion injury when the tissue cells were re-supplied after hypoperfusion ischemia, not only did the tissue ischemic damage be alleviated or restored, but the lack of weight Blood damage. It is a common phenomenon in the higher animal body after ischemia and reperfusion. Reperfusion injury may occur after cardiac surgery, coronary artery bypass grafting, organ blood supply for infarction, organ transplantation, and shock organ hypoperfusion correction. The degree of injury is closely related to the time of ischemia, the condition of collateral circulation, the degree of aerobics, and the conditions of reperfusion.
- the primary is to restore blood perfusion, in order to relieve the state of hypoxia and nutrient supply, to prevent the development of or prevent the recovery of ischemic injury.
- the current drugs for treating ischemia-reperfusion injury include free radical scavengers and antioxidants, calcium antagonists and channel inhibitors, anti-inflammatory drugs and the like.
- Danshen polyphenolic acid the clinically used dosage form is salvian polyphenolic acid for injection, and its function is to promote blood circulation, phlegm and blood stasis.
- Clinically used for coronary heart disease stable angina pectoris, grade I, II, angina pectoris symptoms are mild, moderate, TCM syndrome differentiation for heart and blood stasis syndrome, symptoms of chest pain, chest tightness, palpitations.
- Panax notoginseng saponins (Sanqi total saponins), the clinical dosage form is Xuesaitong for injection, its pharmacological effects are: dilating coronary and peripheral blood vessels, reducing peripheral resistance, slowing heart rate, reducing and reducing myocardial oxygen consumption, increasing Myocardial perfusion, increase cerebral blood flow, improve myocardial and cerebral ischemia; significantly inhibit platelet aggregation, reduce blood viscosity, inhibit thrombosis; in addition, this product also has hypolipidemic, anti-fatigue, It is resistant to hypoxia, enhances and enhances macrophage function. Clinically, it is mainly used for cerebral vascular sequelae, central retinal vein occlusion, and anterior chamber hemorrhage.
- Astragalus saponins clinically used for antithrombotic, can enhance immunity, supplement the role of Qi.
- the traditional Chinese medicine composition is composed of salvian polyphenolic acid, panax notoginseng saponins and total astragalus saponins.
- the weight ratio of salvianolic acid, total saponins of Panax notoginseng and total saponins of Astragalus membranaceus is (1-16): (1-16): (1-16).
- the weight ratio of salvianolic acid, panax notoginseng and total astragaloside in the traditional Chinese medicine composition is (4-16): (1-8): (1-16).
- the weight ratio of salvianolic acid, panax notoginseng and total astragaloside in the traditional Chinese medicine composition is (4 to 16): (1 to 4): (1 to 16).
- the weight ratio of salvian polyphenolic acid, panax notoginseng saponins, and total astragalus saponins in the traditional Chinese medicine composition is (4-8): (1 to 4): (1 to 16), or (8 to 16): (1 to 4): (1 to 16).
- the weight ratio of salvian polyphenolic acid, total saponins of total notoginseng, and total saponins of astragalus in the traditional Chinese medicine composition is (4-8): 1: (5-16), or (4-8): (1) ⁇ 2): (1 to 5).
- Salvia polyphenolic acid is an extract containing salvianolic acid B, danshensu sodium, rosmarinic acid, lithospermic acid and salvianolic acid E.
- the weight percentage of each component is 40-95%, 20-95%, 3- 15%, 2-10% and 0.2-2.2%.
- Salvia polyphenolic acid can be prepared by the following method: Danshen decoction pieces are extracted with ethanol at a concentration of 20-90%, and the extract is concentrated to no alcohol. The extract is subjected to polyamide chromatography, and the impurities are washed away with water or ethanol having a concentration of 30% or less. Elevate the eluate with a concentration of 30-95% ethanol or a concentration of 0.05-0.3% sodium bicarbonate or a concentration of 0.01-0.3% sodium carbonate, adjust the pH to 1-5.5, pass weak polarity or Non-polar macroporous resin, washed with water or 20% ethanol to remove impurities, eluted with 30-95% ethanol, collected eluate, concentrated, and dried.
- the total saponin of Panax notoginseng is an extract containing the components: the content of notoginsenoside R 1 is 5-20%, the content of ginsenoside Rb 1 is not less than 20%, the content of ginsenoside Rd is 3-15%, and the content of ginsenoside Rg 1 is not. Less than 30%, the ginsenoside Re content is not less than 2%, and the sum of the five saponins is not less than 80%.
- the total saponins of Panax notoginseng can be prepared by the following method: taking the medicinal materials of Panax notoginseng, pulverizing into coarse granules, adding 3-10 times the concentration of 20-80% ethanol to reflux two times, each time for 2-5 hours, combining the extracts, The extract is concentrated under reduced pressure until alcohol-free, centrifuged, and the supernatant is enriched by macroporous adsorption resin, and the sugar component and part of the pigment are washed away in 3-10 column volumes, followed by 5-10 times column volume 30-70%. The ethanol was eluted, and the alcohol eluate was collected, and concentrated under reduced pressure to dry powder to obtain a total saponin of Panax notoginseng.
- the total saponin of Astragalus membranaceus is an extract, and the total content of total saponins of Astragalus membranaceus is 20-100%, and the content of Astragaloside IV is 20-95%.
- the total saponin of Astragalus membranaceus can be extracted by the following method: taking Huangqi decoction pieces, pulverizing into coarse particles, and extracting twice with 3-10 times the concentration of 20-70% ethanol (containing 0.1-0.5% sodium hydrogencarbonate), each time 2 -5 hours, the extract is combined, the extract is concentrated under reduced pressure to 2-5 times the volume of the medicinal material, centrifuged, and the supernatant is enriched by macroporous adsorption resin, first with a concentration of 2-8 column volumes of 0.1-1 Rinse with % NaOH solution, elute with 2-8 column volumes of 20-60% ethanol, and finally elute with 2-8 column volumes of 70-95% ethanol.
- the concentration is 70- 95% ethanol eluent. Then, it is decolorized by macroporous resin, and the concentration of 2-5 column volumes is 70-95% ethanol.
- the effluent and the eluate are collected, concentrated under reduced pressure to a small volume, and the solid is crystallized, and the solid is collected and dried to obtain total jaundice. Saponin.
- Another object of the present invention is to provide a preparation comprising the traditional Chinese medicine composition, which preparation consists of a traditional Chinese medicine composition and a pharmaceutically acceptable carrier.
- the pharmaceutically acceptable carrier refers to a conventional pharmaceutical carrier in the pharmaceutical field, selected from the group consisting of fillers, binders, disintegrators, lubricants, solubilizers, suspending agents, wetting agents, pigments, solvents, surface active agents.
- a dose or a flavoring agent One or more of a dose or a flavoring agent.
- the filler is selected from the group consisting of starch, pregelatinized starch, dextrin, glucose, sucrose, lactose, lactitol, microcrystalline cellulose, mannitol, sorbitol or xylitol;
- the binder is selected from the group consisting of sodium carboxymethylcellulose, hydroxypropylmethylcellulose, ethylcellulose, povidone, starch syrup, sucrose, powdered sugar, syrup, gelatin or polyethylene glycol;
- the disintegrant is selected from the group consisting of croscarmellose sodium, crospovidone, low substituted hydroxypropyl cellulose or sodium carboxymethyl starch or starch;
- the lubricant is selected from the group consisting of magnesium stearate, talc, micronized silica gel, PEG4000, PEG6000 or sodium lauryl sulfate;
- the solubilizing agent is selected from the group consisting of sodium hydroxide, potassium hydroxide, sodium hydrogencarbonate, meglumine, L-lysine or L-arginine;
- the suspending agent is selected from the group consisting of micronized silica gel, beeswax, cellulose or solid polyethylene glycol;
- the wetting agent is selected from the group consisting of glycerin, Tween-80, ethoxylated hydrogenated castor oil or lecithin;
- the solvent is selected from the group consisting of ethanol, liquid polyethylene glycol, isopropanol, Tween-80, glycerin, propylene glycol or vegetable oil, and the vegetable oil is selected from the group consisting of soybean oil, castor oil, peanut oil, blended oil, and the like;
- the surfactant is selected from sodium dodecylbenzenesulfonate, stearic acid, polyoxyethylene-polyoxypropylene copolymer, fatty acid sorbitan or polysorbate (Tween);
- the flavoring agent is selected from the group consisting of aspartame, sucralose, flavor, stevioside, acesulfame, citric acid or sodium saccharin.
- Another object of the invention is to provide the use of the traditional Chinese medicine composition.
- the ischemia-reperfusion injury of the present invention includes, but is not limited to, cerebral ischemia-reperfusion injury, myocardial ischemia-reperfusion injury, renal ischemia-reperfusion injury, lower limb ischemia-reperfusion injury, spinal cord ischemia-reperfusion injury, Retinal ischemia-reperfusion injury, skin ischemia-reperfusion injury.
- Ischemia-reperfusion injury including but not limited to ischemia-reperfusion injury caused by thrombolysis, ischemia-reperfusion injury caused by arterial bypass surgery, ischemia reperfusion caused by percutaneous transluminal coronary angioplasty Ischemia-reperfusion injury caused by perfusion injury, ischemia-reperfusion injury caused by cardiopulmonary bypass, ischemia-reperfusion injury caused by cardio-pulmonary resuscitation, replantation of limbs and organ transplantation.
- the traditional Chinese medicine composition provided by the present invention can be used for thrombolysis in combination with tPA (tissue plasminogen activator).
- tPA tissue plasminogen activator
- the traditional Chinese medicine composition and tPA are in a weight ratio of 10: (5) -20) Combine.
- the traditional Chinese medicine composition can alleviate the exudation and hemorrhage caused by tPA, increase the survival rate, and reduce the nerve damage.
- the traditional Chinese medicine composition provided by the invention has synergistic effect, can treat and/or prevent ischemia-reperfusion injury, and is used for thrombolysis in combination with tPA, with good effect and few side effects.
- Figure 1A is a TTC staining image of brain tissue after 24 hours of reperfusion in each group of rats;
- Figure 1B is a statistical diagram of infarct size of different groups of TTC
- 2A and 2B show the results of neurological function scores of rats in each group after 3h and 24h reperfusion
- Figure 3 shows the dynamic changes of thrombus in the common carotid artery of each group of mice.
- the initial value is the base value before FeCl 3 stimulation.
- Ten minutes indicates 10 minutes after the start of FeCl 3 stimulation (wherein FeCl 3 filter paper was removed after 3 minutes of wrapping around the common carotid artery).
- 4.5 hours indicates the time after the start of the administration of the FeCl 3 filter paper around the blood vessel, that is, the start of administration.
- 5.5 hours indicates 1 hour after administration.
- 6.5 hours means 2 hours after administration, and 24 hours means 24 hours after administration;
- Figure 4 is an image of blood perfusion on the surface of a mouse brain detected by a laser Doppler blood flow meter.
- the initial value is the base value before FeCl 3 stimulation.
- Ten minutes indicates 10 minutes after the start of FeCl 3 stimulation (wherein FeCl 3 filter paper was removed after 3 minutes of wrapping around the common carotid artery).
- 4.5 hours indicates the time after the start of the administration of the FeCl 3 filter paper around the blood vessel, that is, the start of administration.
- 5.5 hours indicates 1 hour after administration.
- 6.5 hours means 2 hours after administration, and 24 hours means 24 hours after administration;
- Figure 6 shows the vascular permeability of the posterior capillaries of the cerebral surface of the ischemic penumbra; in the figure: * compared with the sham operation group, p ⁇ 0.05; # compared with the tPA thrombolysis group p ⁇ 0.05; ⁇ 6;
- Figure 7 shows the changes of cerebral perivascular edema, microvascular opening number and dry-to-wet weight ratio at 24 hours after administration; in the figure: * compared with sham operation group, p ⁇ 0.05; # compared with tPA thrombolysis group p ⁇ 0.05 ;N ⁇ 6;
- Figure 8 shows changes in connexin in brain microvascular endothelial cells 24 hours after administration, transmission electron microscopy images of gap junctions in cerebral vascular endothelial cells, and arrows indicating vascular endothelial cell junction (TJ).
- TJ vascular endothelial cell junction
- Figure 9 shows changes in connexin after hypoxia-reoxygenation of endothelial cells in vitro.
- Brain microvascular endothelial cells are hypoxic for 4.5 hours, reoxygenation is given to tPA and/or T541, and tight junctions between endothelial cells are Claudin-5, JAM-1 and sticky.
- Protein immunoblotting with VE-cadherin is attached to the map and statistics.
- Figure 10 is a graph showing cerebral hemorrhage 24 hours after administration, and cerebral hemorrhage and infarction after 24 hours of administration.
- the mouse brain tissue was cut into 1 mm thick slices to record the bleeding, and the infarct size was recorded by TTC staining.
- the graph A is the representative of cerebral hemorrhage and infarction after 24 hours of reperfusion.
- Figure B is the hemoglobin spectrophotometric test box. A histogram of hemoglobin content in the right hemisphere of each group was examined.
- Panel C shows the infarct size statistics for each group. *p ⁇ 0.05 compared with the sham group; #p ⁇ 0.05 compared to the background group; p ⁇ 0.05 compared to the tPA thrombolytic group. N ⁇ 6;
- Figure 11 shows changes in the basement membrane and related proteins of the cerebral ischemic cortex 24 hours after administration.
- Figure A shows the changes in the basal membrane of the cerebral cortex by scanning electron microscopy, indicated by white arrows.
- Basement membrane, basement membrane, BM. N 3.
- Panel B Western blotting shows the expression levels of type IV collagen (Collagen IV) and laminin (Laminin) in the right cerebral ischemic penumbra cortex 24 hours after administration.
- Figure 13 is the oxidative stress damage of brain tissue after 24 hours of administration, in the figure: * compared with the sham operation group, p ⁇ 0.05; p ⁇ 0.05 compared to the tPA thrombolytic group. N ⁇ 6;
- Figure 17 shows apoptosis of endothelial cells in vitro after hypoxia/reoxygenation.
- * compared with the normal control group, p ⁇ 0.05; # compared with the model group, p ⁇ 0.05.
- N 6.
- Example 1 A traditional Chinese medicine composition
- the weight ratio of salvianolic acid, panax notoginseng and total astragalus saponin is 1:2:4.
- Example 2 A traditional Chinese medicine composition
- the weight ratio of Salvia polyphenolic acid, Panax notoginseng saponins and Astragalus total saponins is 1:4:16.
- Example 3 A traditional Chinese medicine composition
- the weight ratio of Salvia polyphenolic acid, Panax notoginseng saponins and Astragalus total saponins is 2:16:1.
- Example 4 A traditional Chinese medicine composition
- the weight ratio of Salvia polyphenolic acid, Panax notoginseng saponins and Astragalus total saponins is 8:1:16.
- Example 5 A traditional Chinese medicine composition
- the weight ratio of salvianolic acid, panax notoginseng and total astragalus saponin is 16:4:1.
- Example 6 A traditional Chinese medicine composition
- the weight ratio of Salvia polyphenolic acid, Panax notoginseng saponins and Astragalus total saponins is 16:8:4.
- Example 7 A traditional Chinese medicine composition
- the weight ratio of salvianolic acid, panax notoginseng saponins and total saponins of astragalus is 4:1:5.
- Table 1 Salvia polyphenolic acid, Panax notoginseng saponins, and Astragaloside IV ratio
- Group Salvia polyphenolic acid (a) Panax notoginseng saponins (b) Astragalus total saponin (c) 1 1 2 4 2 1 4 16 3 2 16 1 4 8 1 16 5 16 4 1 6 4 2 1
- the effects of the 7 groups were verified by experiments.
- the total dose for each group was 20 mg/kg.
- the middle cerebral artery occlusion (MCAO) was induced by suture method in rats, and the middle cerebral artery type I/R was established.
- the details are as follows: anesthesia is given 5 minutes before the experiment with anesthesia (intraperitoneal injection), the rats are fixed in the supine position, the neck is defibrated in the neck, 75% alcohol is disinfected, and the neck is about 3 cm in length.
- the midline incision is along the sternocleidomastoid muscle. After separating the muscles and fascia, avoid the thyroid on both sides of the trachea and the superior parathyroid glands, and carefully separate the total, external and internal carotid arteries.
- the 6-0 surgical suture is attached to the proximal end of the external carotid artery, electrocoagulation of the external carotid artery and small branches.
- the carotid artery and the common carotid artery were temporarily clipped with a small arterial clip, and all the mouth was cut by microsurgery on the external carotid artery.
- the prepared tip end was covered with a silicone wire (linear body diameter 0.38 mm) through the neck.
- the arterial incision is slowly inserted into the internal carotid artery to the beginning of the middle cerebral artery, blocking the blood supply to the middle cerebral artery.
- the length of the incoming line is approximately 1.8 cm - 2.2 cm from the bifurcation of the common carotid artery.
- the suture was pulled out and the skin was sutured to establish a rat brain I/R model.
- each experimental group (10 rats in each group) was administered with a different ratio of the traditional Chinese medicine composition in the above table.
- the sham operation group used the same surgical procedure but did not insert a thread plug.
- the anus temperature of the rats was maintained during the whole operation (37.0 ⁇ 0.5°C), and the anus temperature was maintained by the heating blanket after the operation until the activity was resumed. After 24 h, the rats were anesthetized again and sacrificed.
- Rats with cerebral infarction after I/R injury were detected by TTC (2,3,5-triphenyltetrazolium chloride) staining.
- the specific method was as follows: after reperfusion for 24 hours, the brains of each group of rats were taken out, and cut into 5 slices from the front to the back in a brain positioner and placed in 2% TTC and incubated at 37 ° C for 15 minutes for TTC staining. . The non-infarcted area was stained red, and the infarcted area was not colored and remained white.
- the TTC staining images of the brain slices were taken, and the infarct area of the TTC staining accounted for the percentage of the whole brain area was calculated by the image analysis software IamgeJ (Bethesda, MD, USA) to evaluate the degree of cerebral infarction.
- Stable balance posture 0 Catch the balance beam edge 1 Holding the balance beam, one limb falls from the balance beam 2 Hold the balance beam and the two limbs hang from the balance beam or rotate on the balance beam (>60 seconds) 3 Trying to balance on the balance beam but falling (>40 seconds) 4 Trying to balance on the balance beam but fall (>20 seconds) 5 Dropped, not trying to balance on the balance beam ( ⁇ 20 seconds) 6 Loss of reflection and abnormal movement 4 Auricle reflex (shaking head when touching the external auditory canal) 1 Corneal reflex (blinking when tapping the cornea with cotton) 1 Panic reflex (sports reaction to the noise of fast-crank cardboard) 1 Epilepsy, myoclonus, dystonia; 1
- mice 18 points improved mouse neurological score (mNSS), normal mice scored 0 points, the maximum score of 18 points, the higher the score indicates the more serious neurological deficit, the specific results are shown in Table 3.
- mNSS mouse neurological score
- Fig. 1A is a TTC staining image of brain tissue pieces after reperfusion for 24 hours in each group of rats. The white areas in the figure indicate the infarct area.
- Figure 1B is a statistical plot of infarct size in different groups of TTC (see Table 4 for specific results).
- Fig. 2A and Fig. 2B The results of neurological scoring are shown in Fig. 2A and Fig. 2B (representing the results of neurological function scores of rats in each group after 3 h and 24 h reperfusion) and Table 4.
- the neurological scores of the rats were significantly decreased at 3h and 24h after reperfusion, and the 7 compatibility groups did not improve the neurological score of 3h after reperfusion, but at 24h after reperfusion , 8:1:16, 16:4:1, 4:2:1, 4:1:5 four compatibility groups can significantly improve the neurological score of rats.
- the ratio of the three drugs of salvianolic acid polyphenolic acid (a), panax notoginseng saponin (b) and total astragalus saponin (c) is in the range of (4 to 16): (1 to 8): 1 to 16), preferably (4 to 16): (1 to 4): (1 to 16), which has a good effect of improving ischemia-reperfusion injury.
- the following experiments take the ischemia-reperfusion injury caused by thrombolysis as an example, and select different groups within the optimal ratio range determined in the above experiment to investigate the protective effect of the traditional Chinese medicine composition on ischemia-reperfusion injury after thrombolysis. And promote the role of thrombolysis.
- this does not limit the scope of protection of the present application, because ischemia-reperfusion injury caused by any cause will be manifested as excessive free radicals, cellular calcium overload, inflammatory reaction, etc., and the treatment methods are also consistent.
- the experiments of the present invention confirmed that the traditional Chinese medicine composition of the present application can treat ischemia-reperfusion injury caused by thrombolysis, and it can be deduced that the traditional Chinese medicine composition of the present application can also treat ischemia-reperfusion injury caused by other causes.
- mice Male C57BL/6 clean mice weighing 21 ⁇ 2 grams were purchased from the Experimental Animal Center of Peking University Medical School. The animals were kept at a temperature of 23 ⁇ 2 degrees Celsius and a relative humidity of 45 ⁇ 5%. Drinking water was freely eaten, and 12 hours of light/dark alternating. Animals were fasted and free to drink water 12 hours before the experiment.
- the animals were anesthetized with sodium pentobarbital (2%, 45 mg/kg) and the skin of the neck was sterilized.
- a median neck incision was made to expose and separate the common carotid artery.
- a waterproof gasket was used to isolate the common carotid artery and surrounding tissue about 2.5-3 mm long.
- the separated common carotid artery was wrapped about 1 mm wide with a filter paper soaked with 10% FeCl 3 . After 3 minutes, the filter paper was removed, and the outer side of the blood vessel was washed with 0.9% physiological saline. Remove the waterproof gasket. Suture the neck wound.
- the initial stimulation of Fe 3+ was defined as the onset of ischemia.
- the largest diameter of the neck thrombus blocked the blood vessels.
- the blood flow of the distal end of the carotid artery was less than 20% of the baseline value, and the ischemia was 4.5 hours (ie, The thrombus was stable before reperfusion, and the animals with the largest diameter of the thrombus area blocked the carotid artery diameter of 60% or more were enrolled.
- mice femoral vein cannulation left femoral vein pumped tPA (tissue plasminogen activator) or normal saline
- right femoral vein pumped with the traditional Chinese medicine composition or physiological saline the total dose of 10% bolus, the remaining 90% of the vein was continuously pumped for 1 hour at a rate of 0.1 ml/hr.
- the traditional Chinese medicine composition of the present invention selects three matching groups, which are respectively:
- mice C57BL/6 mice were divided into the following 10 groups according to the random number table:
- T541 high-dose background group (T541 20 mg/kg, referred to as background group) on the basis of sham operation group;
- T541 high-dose thrombolytic group (T541 20 mg/kg alone);
- T541 low dose group (administered tPA + T541 5 mg / kg);
- T541 middle dose group (administered tPA + T541 10 mg / kg);
- T541 high dose group (administered tPA + T541 20 mg / kg)
- T141 group (administered tPA + T141 12 mg / kg);
- tPA was given a clinical equivalent dose of 10 mg / kg. See Table 5 for the mode of administration of each group.
- Table 5 Grouping methods and methods of administration of animal experiments
- the left femoral vein was given a clinically equivalent dose of 10 mg/kg of tPA or an equal volume of saline control.
- Several groups of the traditional Chinese medicine compositions of the present invention were administered to the right femoral vein, respectively, T541 low, medium and high dose groups, T141 group, T582 group or an equal volume of normal saline control.
- the T541 low-dose group, the middle-dose group, the high-dose group, and the T141 group and the T582 group are in the form of simultaneous administration with tPA, so that the thrombolytic effect of the traditional Chinese medicine composition of the present invention can be evaluated, and the Chinese medicine of the present invention can also be examined.
- the therapeutic effect of the composition on ischemia-reperfusion injury caused by thrombolysis, the observation indicators in animal experiments and the number of animals used in each group are shown in Table 6.
- the 24-hour survival rate and neurological score were not less than 6 in each group.
- the number of thrombus observation, triphenyltetrazolium chloride (TTC) infarct size staining, cerebral blood flow and bleeding were observed in each group.
- the sham operation group, T541 high-dose background group, tPA thrombolytic group and optimal drug concentration group were selected for further observation.
- the number of Evans blue exudation, microcirculation dynamic visualization observation, model lateral brainstem wet weight ratio and molecular biological index detection was 6; the number of immunofluorescence staining and electron microscope animals was 3.
- the 24-hour survival rate, neurological deficit score, carotid thrombosis area and cerebral blood flow perfusion were recorded after reperfusion in mice.
- the optimal drug concentration was selected for further analysis.
- the dynamic microcirculation observation system of the mice was used to observe the dynamic changes of mouse microcirculation, and the albumin exudation of the middle cerebral artery region, the anterior artery region and the cerebral venules of the cerebral surface was recorded.
- mice After 24 hours of reperfusion, the mice were sacrificed to observe the hemorrhage and infarct size of the brain tissue, and the hemoglobin content in the brain tissue after hemorrhage was measured. The dry-to-wet weight ratio of brain tissue and the degree of exudation of Evans blue were detected to reflect the edema of the tissue.
- Tissue TUNEL staining was used to observe the severity of apoptosis; connexins Occludin, Claudin-5, JAM-1, ZO-1, VE-cadherin and basement membrane layer laminin protein immunoblot analysis and partial connexin and laminin immunofluorescence staining
- connexins Occludin, Claudin-5, JAM-1, ZO-1, VE-cadherin and basement membrane layer laminin protein immunoblot analysis and partial connexin and laminin immunofluorescence staining
- MDA 8-OHdG, ATP/ADP/AMP and mitochondrial complex I in brain tissue by enzyme-linked immunosorbent assay Changes in II and IV activity.
- TPA was used in combination with T541 and T541 in separate components or in combination, which were tPA + total saponin (tPA+HQ), tPA+danshen polyphenolic acid (tPA+DS), tPA+trisodium saponin (tPA+).
- Table 7 Grouping of T541 splits and administration status of each group
- rat brain endothelial cells were purchased from ATCC, and normal cultures were passaged until 5 to 6 passages.
- T541 high dose group hypoxia 4.5 Hours, reoxygenation for 3 hours, tPA 20 ⁇ g/ml and T541 400 ⁇ g/ml at the beginning of reoxygenation
- T541 high dose administration group hypoxia for 4.5 hours, reoxygenation for 3 hours, reoxygenation at the beginning
- T541 medium dose group hypoxia for 4.5 hours, reoxygenation for 3 hours, reoxygenation start with tPA 20 ⁇ g/ml and T541 4 ⁇ g/ml
- T541 low-dose administration group hypoxia for 4.5 hours, reoxygenation for 3 hours, re-oxygenation at the beginning of tPA 20
- the Cellcounting Kit-8 (CCK-8, MedChem Express, China) kit was used to test the difference between cell viability and normal cells under different drug treatments.
- T541 high-dose drug background group normal cells plus T541 400 ⁇ g/ml for 3 hours
- high-dose drug background group normal cells plus T541 40 Microgram/ml treatment for 3 hours
- medium dose drug background group normal cells plus T541 4 ⁇ g/ml for 3 hours
- low dose drug background group normal cells plus T5410.4 ⁇ g/ml treatment 3 hours
- Low-dose drug background group normal cells were treated with T541 0.04 ⁇ g/ml for 3 hours.
- Cell-Counting Kit 8 (CCK8) was used to test cell viability, reflecting the effects of different concentrations of drugs on normal endothelial cells; detection of connexin Claudin-5 (Claudin-5), JAM-1 (Linking adhesion molecule type I) , VE-cadherin (vascular endothelial cell cadherin) content changes; partial connexin and cytoskeleton F-actin immunofluorescence staining; Western blot analysis of apoptosis-related protein Bax (B-cell lymphoma 2 related X protein), Bcl -2 (B-cell lymphoma-2) content changes; Western blot analysis detected changes in matrix metalloproteinase (MMP)-3 and its precursor pro-MMP-3 content.
- MMP matrix metalloproteinase
- the carotid artery thrombosis in mice was observed dynamically using an upright microscope.
- the anesthetized mice were placed supine on the rat plate, and a mid-neck incision was made.
- the common carotid artery was exposed and isolated.
- the acridine red fluorescently labeled platelets were administered to the femoral vein to observe pre-ischemia (ie, baseline value), ischemia for 10 minutes, and ischemia 4.5.
- Changes in carotid artery thrombosis in mice (hours before reperfusion), 1 hour of reperfusion, 2 hours of reperfusion, and 24 hours of reperfusion. There was no thrombus before ischemia (the basal value).
- the maximum diameter of the thrombus was blocked at 10 minutes after ischemia.
- the blood flow of the distal end of the carotid artery was reduced to 20% of the basal value and the ischemia was 4.5 hours (ie, reperfusion).
- the thrombus was stable before and the thrombus area blocked the carotid vascular area by 60% or more as the successful standard of carotid thrombosis model, and the input power reached 90.9%. Except for the sham operation group and each background group, the other groups were randomly grouped after the model was successful.
- the cerebral cortical blood flow in the left and right middle cerebral artery innervation area was measured by a laser Doppler flow perfusion imager connected to a computer. Anesthetize the mouse in a prone position on the mouse plate, cut the skin on the left and right parietal bones to completely expose the parietal bone, and place a low-energy He-Ne laser probe connected to the computer on the parietal bone at 16-18 cm to detect the ischemia. Baseline value), ischemia for 10 minutes, ischemia for 4.5 hours (ie, before reperfusion), reperfusion for 1 hour, reperfusion for 2 hours, and reperfusion for 24 hours, the left and right cerebral arterial blood supply area of the soft meningeal blood flow. Cerebral cortical blood flow in the same size region dominated by the left and right MCA was calculated using cerebral blood flow analysis software LDPIwin 3.1 (PeriScan PIM3 System, PERIMED, Sweden).
- mice were placed in a 25 x 15 cm squirrel cage and their trajectories were recorded within 3 minutes.
- the mice were essentially inactive as 0 points, rarely active and did not touch any wall of the squirrel cage. For 1 point, touch any wall or 2 points for both walls, touch 3 walls and above for 3 points;
- the tail of the mouse was lifted, and the left and right body movement trajectories were observed.
- the left and right asymmetry was 0, the asymmetry was 1 point, the moderate asymmetry was 2 points, and the symmetry was 3 points.
- the left and right sides of the mouse were touched with a small wooden stick. Compared with the right side, the left side non-responder was recorded as 1 point, the left side weak reaction was recorded as 2 points, and the left side reaction was recorded as 3 points. .
- the mouse neurological score was calculated as the sum of the scores of the above five experimental items, 15 was divided into normal mice, and 0 was the death of the mice.
- mice were perfused with PBS buffer pre-cooled at 4 ° C, the brain was craniotomy, and 5 slices were cut from the front to the back in a brain stereotaxic device, and the bleeding was quickly taken by a stereo microscope.
- mice were perfused with PBS buffer pre-cooled at 4 degrees Celsius, the brain was craniotomy, and 5 slices were cut from the front to the back in a brain stereotaxic device, and the bleeding was quickly photographed under low temperature conditions.
- the cells were placed in 2% triphenyltetrazolium chloride (TTC) and incubated at 37 ° C for 15 minutes and stained with TTC. The non-infarcted area was stained red, and the infarcted area was not colored and remained white.
- TTC triphenyltetrazolium chloride
- TTC staining images of brain slices were taken, and image analysis software ImageJ (Bethesda, MD, USA) was used to calculate the infarct area of TTC staining as a percentage of the total brain area, and the degree of cerebral infarction was evaluated.
- Plasma albumin leakage Under a stereo microscope, a mouse femoral vein cannula (PE/08, outer diameter 0.36 mm, inner diameter 0.20 mm). The mouse was fixed in the prone position on the mouse microcirculation observation plate, the mouse cranial skin was cut off, the right parietal bone was exposed, and under the stereo microscope, the entire parietal bone was thinned with a portable cranial drill until the parietal bone remained. A soft layer of cortical bone. FITC-labeled plasma albumin (excitation wavelength 420-490 nm, emission wavelength 520 nm) was slowly bolused through the femoral vein at a dose of 50 mg/kg.
- Evans blue exudation 21 hours after reperfusion, Evans blue dye (2%, 4 ml/kg) dissolved in physiological saline was slowly bolused into the mice via the femoral vein. After 3 hours, the heart was perfused with normal saline 15-20 ml (the right atrial appendage was stopped when the liquid was colorless), then the brain was taken to 4% paraformaldehyde for 3 hours, and the brain was cut into 5 pieces after taking out. And separate the left and right brains and weigh them separately. The left and right brains were separately added to 1 ml of a 50% trichloroacetic acid EP tube, homogenized, and the supernatant was centrifuged. The Evans blue standard was configured and the Evans blue content was detected by a multi-function microplate reader (excitation light 620 nm, emission light 680 nm). The Evans blue content is expressed in Evans blue micrograms per gram of brain tissue.
- Brain tissue dry-to-wet weight ratio Immediately after reperfusion for 24 hours, the mice were anesthetized, the brain was decapitated, and the left and right brains were weighed separately (denoted as wet weight). The brain was then placed in a 60 ° C dry box for 72 hours to dry and weighed (denoted as dry weight). The brain wet weight ratio of the mouse was calculated as (wet weight - dry weight) / wet weight ⁇ 100%.
- mice After 24 hours of reperfusion, the anesthetized mice were perfused with 3% glutaraldehyde for 3 minutes at a rate of 3 ml/min through the left ventricle, and the brain was removed.
- the right cortex of the mice was cut into 1 mm ⁇ 1 mm ⁇ 1 mm. Small pieces, 3% glutaraldehyde were fixed at room temperature for 30 minutes or 4 degrees Celsius overnight.
- the fluid was perfused with saline heart, and the penic penumbra of the right cerebral cortex was taken for Western blot analysis.
- the brain tissue of the mice was taken out, the cerebellum and the prefrontal lobe were cut off, and the brain tissue of the right brain near the left and right hemispheres was cut 1 cm along the sagittal plane. The remaining tissues were cut at an angle of 45 degrees to incision penumbra, and the penumbra was removed.
- the right cerebral cortex was taken, and 1 ⁇ RIPA lysate and Cocktail protease inhibitor (1:100, Cell Signaling Technology, USA) were added and fully lysed and sonicated, and centrifuged at 17,000 g for 30 minutes. The supernatant was taken, and 5 x loading buffer was added in volume, boiled in boiling water for 20 minutes, and stored at -80 ° C. The excess supernatant was quantified by BCA protein quantification solution (Thermo Scientific, USA), and the absorbance value was measured at 560 nm by a microplate reader, and the protein concentration was calculated from the standard curve.
- the secondary antibody diluted with 5% skim milk powder was added to incubate for 1 hour at room temperature, and a luminescent agent (Plylai, China) was added for color development, and development was performed in a dark room.
- the electrophoretic band density values were analyzed using the image analysis software Quantityone. All protein band sizes are expressed as relative values compared to the internal reference ⁇ -actin or the total target protein.
- the cerebral cortex penumbra MDA malondialdehyde
- 8-OHdG (8-hydroxy-2'-deoxyguanosine
- ATP adenosine triphosphate
- ADP adenosine diphosphate
- Each set of data uses at least six samples for independent experiments that are repeated at least three times.
- mice were perfused with PBS buffer pre-cooled at 4 ° C, the brain was craniotomy, fixed for 12 hours, dehydrated with 30% sucrose, embedded in OTC, and frozen slicer (CM1800, Leica, Bensheim) , Germany) cut into 10 micron thick frozen sections, air-dried, placed in 0.1 mol / liter PBS for 5 minutes, 600 watts, 90 degrees Celsius, in the microwave with 0.01 mol / liter of sodium citrate for antigen retrieval After 10 minutes, it was naturally cooled at room temperature for 2 hours.
- frozen slicer CM1800, Leica, Bensheim
- the membrane was immersed for 5 minutes ⁇ 3 times in PBS buffer, pepsin digestion for 15 minutes, and PBS buffer was washed again for 5 minutes ⁇ 3 times.
- the sheep serum was blocked for 30 minutes, rinsed in PBS buffer for 5 minutes ⁇ 3 times, plus primary antibody vWF (1:100, Millipore, Temecula, CA, USA) + JAM-1/Occludin/Laminin (1:50, Invitrogen, Camarillo) , CA, USA), 4 degrees Celsius overnight.
- the warm-up was taken every other day for 1 hour, rinsed with PBS buffer for 5 minutes ⁇ 3 times, and after adding the secondary antibody for 2 hours, the cells were labeled with Hoechst 33342 (1:100, Molecular Probes) and incubated for 10 minutes at room temperature in the dark.
- the slide was mounted with an anti-fluorescence quencher.
- the laser scanning confocal microscope (TCS SP5, Leica, Mannheim, Germany) was used to observe and photograph under a 63-fold objective.
- the results in Table 10 show that the sham group and the background group did not die during the observation period.
- the survival rate of the thrombus group at 18 and 24 hours after administration was 81.82%.
- the 24-hour survival rate of the T541 high-dose thrombolytic group was 90%.
- the 24-hour survival rate after tPA administration was 50%, and the low and medium doses of T541 did not improve the 24-hour survival rate caused by tPA.
- the 24-hour high-dose 24-hour survival rate was 81.82%, and the survival rate was significantly improved compared with the thrombus group (81.82% vs 50%).
- T141 and T582 also did not improve the survival rate reduction caused by tPA.
- the 18-point modified mouse neurological score was used (normal mice scored 0 points, the higher the score indicates the more serious the neurological deficit); 15 points scoring criteria (15 was normal mice, 0 was mouse death) .
- Table 11 24-hour neurological deficit scores for each group of mice after administration
- the results in Table 11 show that the 24-hour neurological scores of the thrombus group were 9.82 (18 points improvement score) and 6.2 (15 points score), respectively, compared with the sham operation group and the background group.
- the T541 high dose group was 4 (18 points improvement score).
- tPA did not improve neurological scores.
- the low, medium, and high dose groups of T541 significantly improved the neurological score, and the effect of high dose was significantly better than the low dose.
- the T541 high-dose group was selected as the optimal drug use concentration by survival rate, neurological defect score, neck thrombus area and cerebral blood flow perfusion.
- Fig. 6 and Table 12 show that, 24 hours after the thrombolytic administration, the vascular permeability of the posterior venule of the cerebral surface of the ischemic penumbra in the ischemic penumbra of the mouse was observed by dynamic visualization. The intravascular albumin leakage was observed within 30 minutes. Plasma albumin in the tPA thrombolytic group was significantly exuded, and the high dose T541 group significantly inhibited plasma albumin exudation.
- Transmission electron microscopy images showed that the sham operation group and the T541 high-dose background group had intact vascular structure, and the vascular endothelium was smooth and continuous, and was closely connected with the surrounding tissues.
- the vascular endothelium was rough, and there were obvious edema voids between the surrounding tissues, and mitochondria edema in the surrounding tissues.
- the vascular endothelium was relatively smooth, the perivascular edema was significantly reduced, the mitochondrial structure was dense, and there was no edema in the perivascular tissue. (See the first and second lines of Figure 7)
- Table 12 Evans blue content, albumin exudation ratio, dry-wet weight ratio
- the histograms of Table 12 and Figure 7 show the statistical results of brain tissue Evans blue, plasma albumin exudation, dry to wet weight ratio 24 hours after administration.
- tPA can cause Evans blue exudation, increased plasma albumin exudation, decreased number of brain microvascular opening, and increased dry-wet weight ratio.
- T541 can suppress the above changes caused by tPA. The results suggest that tPA thrombolysis can cause brain microvascular exudation and cerebral edema, while T541 can inhibit brain microvascular exudation and brain edema caused by tPA.
- T541 can improve the changes of connexin in brain microvascular endothelial cells 24 hours after administration of cerebral cortex in the penumbra. Transmission electron microscopy showed that the tight junction structure of the cerebral cortex in the penumbra of the tPA thrombolysis group was opened, and the use of T541 combined with tPA could close the open endothelial cell junction and restore normal cell junction. (See Figure 8)
- Table 13 Quantitative statistics of connexin in brain tissue 24 hours after administration
- rat brain microvascular endothelial cells were cultured in vitro, treated with hypoxia for 4.5 hours, reoxygenated for 3 hours, simulated in vivo ischemia and after thrombolysis. In the reperfusion state, reoxygenation was given to both tPA and T541.
- the CCK8 kit was used to test the changes of the activity of T541 in normal cells and normal cells. It was found that the T541 high-dose drug background group had an inhibitory effect on endothelial cell activity, while the other T541 group had no endothelial cell activity compared with the normal control. Significant differences.
- T541 In brain endothelial cells cultured in vitro, the administration of tPA after hypoxia/reoxygenation caused a decrease in the content of connexins VE-cadherin, Claudin-5 and JAM-1, and different concentrations of T541 (400, 40, 4, 0.4 0.04 ⁇ g) /ml) can restore the expression of connexin to varying degrees, 40 ⁇ g / ml T541 has a recovery effect on the reduction of VE-cadherin, Claudin-5 and JAM-1 protein.
- the combination of T541 and tPA can reduce the amount of cellular connexin and help maintain the normal cell morphology of endothelial cells.
- T541 can improve the damage of connexin after hypoxia/reoxygenation (H/R) in cultured endothelial cells in vitro
- H/R hypoxia/reoxygenation
- Table 15 Hemoglobin content and infarct size in brain tissue
- the brain tissue of each group was cut into 1 mm thick slices, and hemorrhage and infarction were observed in the lateral cortex and parenchymal areas of the tPA thrombolytic group model. This phenomenon can be alleviated by the combined use of T541 and tPA (see Figure 10).
- the right hemi-brain tissue of each group was tested for hemorrhage, and the hemoglobin content of tPA thrombolytic group was significantly increased, while the high dose of T541 decreased the elevated hemoglobin level and could be restored to the level of sham operation group and background group (see Figure 10 and Table 15).
- Table 17 ATP, ADP, AMP content in brain tissue 24 hours after administration
- T541 Total saponins of Astragalus, Salvia polyphenolic acid, Panax notoginseng saponin single component and two-two combination group and T541.
- the ATP, ADP and AMP contents in the cerebral cortex of the ischemic penumbra were measured by enzyme-linked immunosorbent assay (ELISA). The ratios of ADP/ATP and AMP/ATP were calculated. After 24 hours of administration, the ATP content in the brain tissue of the tPA thrombolytic group was significantly reduced, and the levels of ADP and AMP were increased.
- T541 combined with tPA could restore the reduced ATP and decrease the increased ADP and AMP.
- the ratio of ADP/ATP to AMP/ATP increased in the tPA thrombolytic group.
- the former was reversed by T541 plus tPA, the latter in the tPA + jaundice group, tPA + 37 group, two-dose group and tPA combined with T541 high-dose treatment group. Don't get better.
- T541 improved brain tissue oxidative stress damage 24 hours after ischemic administration. Take the three main components of T541: total saponins of Astragalus, Salvia polyphenolic acid, Panax notoginseng saponin single component and two-two combination group and T541. The changes of MDA and 8-OHdG content in the cerebral cortex of the ischemic penumbra and the activities of mitochondrial complexes I, II and IV were detected by enzyme-linked immunosorbent assay (ELISA).
- ELISA enzyme-linked immunosorbent assay
- Table 18 MDA, 8-OHdG content and mitochondrial complex activity in brain tissue 24 hours after administration
- the T541 high-dose group can significantly inhibit the 8-hydroxydeoxyguanosine monophosphate (8-OHdG) in the cortex of the cerebral ischemic penumbra increased in the tPA thrombolytic group, except for the tPA+xanthine+37 group, which can achieve therapeutic effects.
- tPA plus Astragalus, Salvia, Sanqi single-flavored medicine or Astragalus membranaceus, Salvia miltiorrhiza, Salvia miltiorrhiza + Sanqi two herbs can not achieve significant results.
- tPA+danshen+sanqi group can significantly restore the mitochondrial complex I activity after thrombolysis
- tPA+xanthine+37 can restore mitochondrial complex II activity
- T541 high dose group can restore mitochondrial complex I, II and IV activity at the same time.
- the T541 high-dose group can also restore the mitochondrial complex I, II, IV activity to the level of the sham operation group, which is the effect that the single-flavor drug and the two-two compatibility group can not achieve.
- T541 plays different roles, especially in the improvement of mitochondrial complex activity. More interestingly, even if there is no significant difference between the total saponins of Astragalus, Salvia polyphenols and Panax notoginseng, the combination of T541 can reduce the MDA in brain tissue and restore mitochondrial complex I. The role of IV activity. It shows that the compatibility of multiple drugs is not only a simple addition result, but a synergistic effect.
- Fig. 15 and Table 20 show that there is a large number of TUNEL positive cells in the tPA thrombolysis group after thrombolytic administration.
- the number of TUNEL positive cells decreased significantly after T541 combination, which may mean that T541 can reduce tPA alone.
- the number of apoptotic cells after thrombolysis ( Figure 15). Transmission electron microscopy has similar results. It can be seen that the tPA thrombolysis group has shrinkage and deformation of the cell body.
- the organelle structure in the cytoplasm is abnormal and the density is obviously increased.
- the nuclear membrane is uneven and the heterochromatin appears in the nucleus. After T541, the neurons recovered their normal cell density, and the organelles such as mitochondria recovered and the nuclear membrane was smooth.
- Table 21 Quantification of apoptosis proteins Bax and Bcl-2 in brain tissue
- tPA thrombolytic group 6 1.51 ⁇ 0.14 5.52 ⁇ 0.78* 3.82 ⁇ 0.57* T541 high dose group 6 1.41 ⁇ 0.12 1.43 ⁇ 0.20# 1.14 ⁇ 0.24# tPA+ ⁇ 6 1.47 ⁇ 0.14 3.17 ⁇ 0.35*# 2.32 ⁇ 0.39 tPA+danshen group 6 1.35 ⁇ 0.17 3.29 ⁇ 0.48*# 2.65 ⁇ 0.48 tPA+37 6 1.17 ⁇ 0.20 4.10 ⁇ 0.93* 4.09 ⁇ 1.34* tPA+ ⁇ + ⁇ 6 1.14 ⁇ 0.15 1.78 ⁇ 0.08# 1.79 ⁇ 0.37 tPA+ ⁇ + ⁇ 6 1.12 ⁇ 0.03 2.68 ⁇ 0.24# 2.37 ⁇ 0.16 tPA+danshen+sanqi group 6 1.43 ⁇ 0.10 2.01 ⁇ 0.10# 1.43 ⁇ 0.12
- FIG. 16 and Table 21 show that T541 improves brain tissue apoptosis 24 hours after ischemic administration.
- T541 takes the three main components of T541: total saponins of Astragalus, Salvia polyphenolic acid, Panax notoginseng saponin single component and two-two combination group and T541.
- the content of apoptosis-related proteins Bax and Bcl-2 and their ratio changes in the cerebral cortex of the ischemic penumbra were detected by Western blotting. After 24 hours of tPA thrombolysis, the expression of Bax in the extracted protein of mouse brain tissue was significantly increased.
- Total saponins of Astragalus, Salvia polyphenols, Panax notoginseng saponins, and their combination of two and three, can inhibit the high expression of Bax.
- FIG. 17 and Table 22 show that T541 can improve the degree of apoptosis in endothelial cells after hypoxia/reoxygenation.
- Western blotting was used to detect the expression of Bcl-2 and Bax and their ratio changes in cultured brain microvascular endothelial cells after hypoxia/reoxygenation.
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Abstract
Description
组别 | 丹参多酚酸(a) | 三七总皂苷(b) | 黄芪总皂苷(c) |
1 | 1 | 2 | 4 |
2 | 1 | 4 | 16 |
3 | 2 | 16 | 1 |
4 | 8 | 1 | 16 |
5 | 16 | 4 | 1 |
6 | 4 | 2 | 1 |
7 | 4 | 1 | 5 |
试验评分标准 | 分数 |
提尾试验 | 3 |
前肢屈曲 | 1 |
后肢屈曲 | 1 |
头部在30s内偏离垂直轴>10° | 1 |
将大鼠放置于地板上(正常值=0;最大值=3) | 3 |
正常行走为 | 0 |
不能直线行走 | 1 |
向轻瘫侧转圈为 | 2 |
向轻瘫侧倾倒 | 3 |
感觉试验 | 2 |
放置试验(视觉和触觉测试) | 1 |
本体感觉试验(深感觉,向桌子边缘压鼠爪刺激肢体肌肉 | 2 |
平衡木试验(正常值=0;最大值=6) | 6 |
稳定平衡姿势 | 0 |
紧抓平衡木边缘 | 1 |
紧抱平衡木,一肢体从平衡木垂落 | 2 |
紧抱平衡木,二肢体从平衡木垂落或在平衡木上旋转(>60秒) | 3 |
试图在平衡木上平衡但跌落(>40秒) | 4 |
试图在平衡木上平衡但跌落(>20秒) | 5 |
跌落,未尝试在平衡木上平衡(<20秒) | 6 |
反射丧失和不正常运动 | 4 |
耳廓反射(接触外耳道时摇头) | 1 |
角膜反射(用棉丝轻触角膜时眨眼) | 1 |
惊恐反射(对快弹硬纸板的噪音有运动反应) | 1 |
癫病、肌阵挛、肌张力障碍; | 1 |
0分 | 1-6分 | 7-12分 | 13-18分 |
正常小鼠 | 轻度损伤 | 中等损 | 严重损伤 |
左股静脉给药 | 右股静脉给药 | 黄芪总皂苷:丹参多酚酸:三七总皂苷 | |
假手术组 | 生理盐水 | 生理盐水 | - |
T541高剂量本底组 | 生理盐水 | T541 20毫克/千克 | 5:4:1 |
血栓组 | 生理盐水 | 生理盐水 | - |
T541高剂量溶栓组 | 生理盐水 | T541 20毫克/千克 | 5:4:1 |
tPA溶栓组 | tPA 10毫克/千克 | 生理盐水 | - |
T541低剂量组 | tPA 10毫克/千克 | T541 5毫克/千克 | 5:4:1 |
T541中剂量组 | tPA 10毫克/千克 | T541 10毫克/千克 | 5:4:1 |
T541高剂量组 | tPA 10毫克/千克 | T541 20毫克/千克 | 5:4:1 |
T141组 | tPA 10毫克/千克 | T141 12毫克/千克 | 1:4:1 |
T582组 | tPA 10毫克/千克 | T582 15毫克/千克 | 5:8:2 |
左股静脉给药 | 右股静脉给药 | 数量 | |
假手术组 | 生理盐水 | 生理盐水 | 6 |
T541高剂量本底组 | 生理盐水 | T541 20毫克/千克 | 6 |
tPA溶栓组 | tPA 10毫克/千克 | 生理盐水 | 6 |
tPA+HQ溶栓组 | tPA 10毫克/千克 | 黄芪总皂苷10毫克/千克 | 6 |
tPA+SQ溶栓组 | tPA 10毫克/千克 | 三七总皂苷2毫克/千克 | 6 |
tPA+DS溶栓组 | tPA 10毫克/千克 | 丹参多酚酸8毫克/千克 | 6 |
tPA+HQ+SQ溶栓组 | tPA 10毫克/千克 | 黄芪总皂苷10毫克/千克,三七总皂苷2毫克/千克 | 6 |
tPA+HQ+DS溶栓组 | tPA 10毫克/千克 | 黄芪总皂苷10毫克/千克,丹参多酚酸8毫克/千克 | 6 |
tPA+SQ+DS溶栓组 | tPA 10毫克/千克 | 三七总皂苷2毫克/千克,丹参多酚酸8毫克/千克 | 6 |
T541高剂量组 | tPA 10毫克/千克 | T541 20毫克/千克 | 6 |
合计 | 54 |
组别(平均值±标准误) | 数量 | Bcl-2 | Bax | Bax/Bcl-2 |
假手术组 | 6 | 1.00±0 | 1.00±0 | 1.00±0 |
tPA溶栓组 | 6 | 1.51±0.14 | 5.52±0.78* | 3.82±0.57* |
T541高剂量组 | 6 | 1.41±0.12 | 1.43±0.20# | 1.14±0.24# |
tPA+黄芪组 | 6 | 1.47±0.14 | 3.17±0.35*# | 2.32±0.39 |
tPA+丹参组 | 6 | 1.35±0.17 | 3.29±0.48*# | 2.65±0.48 |
tPA+三七组 | 6 | 1.17±0.20 | 4.10±0.93* | 4.09±1.34* |
tPA+黄芪+丹参组 | 6 | 1.14±0.15 | 1.78±0.08# | 1.79±0.37 |
tPA+黄芪+三七组 | 6 | 1.12±0.03 | 2.68±0.24# | 2.37±0.16 |
tPA+丹参+三七组 | 6 | 1.43±0.10 | 2.01±0.10# | 1.43±0.12 |
组别(平均值±标准误) | 数量 | Bcl-2 | Bax | Bax/Bcl-2 |
正常对照组 | 6 | 1.00±0 | 1.00±0 | 1.00±0 |
模型组 | 6 | 0.69±0.12 | 2.44±0.51 | 4.57±0.75* |
T541高剂量给药组 | 6 | 0.79±0.10 | 1.92±0.27 | 2.60±0.25 |
T541次高剂量给药组 | 6 | 0.88±0.10 | 2.01±0.51 | 1.78±0.18# |
T541中剂量给药组 | 6 | 0.99±0.16 | 2.33±0.47 | 2.31±0.45 |
T541次低剂量给药组 | 6 | 0.83±0.11 | 2.72±0.67 | 3.41±0.83* |
T541低剂量给药组 | 6 | 0.56±0.11 | 2.81±0.48 | 4.55±0.47* |
Claims (10)
- 一种预防和/或治疗缺血再灌注损伤的中药组合物,其特征在于,中药组合物由丹参多酚酸、三七总皂苷、黄芪总皂苷组成,其重量比为(1~16):(1~16):(1~16)。
- 根据权利要求1所述的中药组合物,其特征在于,所述中药组合物中丹参多酚酸、三七总皂苷、黄芪总皂苷的重量比(4~16):(1~8):(1~16)。
- 根据权利要求2所述的中药组合物,其特征在于,所述中药组合物中丹参多酚酸、三七总皂苷、黄芪总皂苷的重量比为(4~16):(1~4):(1~16)。
- 根据权利要求3所述的中药组合物,其特征在于,所述中药组合物中丹参多酚酸、三七总皂苷、黄芪总皂苷的重量比为(4~8):(1~4):(1~16),或为(8~16):(1~4):(1~16)。
- 根据权利要求4所述的中药组合物,其特征在于,所述中药组合物中丹参多酚酸、三七总皂苷、黄芪总皂苷的重量比为(4~8):1:(5~16),或为(4~8):(1~2):(1~5)。
- 根据权利要求5所述的中药组合物,其特征在于,所述中药组合物由丹参多酚酸、三七总皂苷、黄芪总皂苷按照重量比4:1:5组成。
- 含权利要求1-6任一项所述的中药组合物的制剂,其特征在于,该制剂由中药组合物和药学上可接受的载体组成。
- 权利要求1-6任一项所述的中药组合物在制备治疗和/或预防缺血再灌注损伤或促溶栓的药物中的应用。
- 根据权利要求8所述的应用,其特征在于,所述缺血再灌注损伤,包括但不限于脑缺血再灌注损伤、心肌缺血再灌注损伤、肾缺血再灌注损伤、下肢缺血再灌注损伤、脊髓缺血再灌注损伤、视网膜缺血再灌注损伤、皮瓣缺血再灌注损伤,或由以下原因引起的缺血再灌注:溶栓,动脉搭桥术,经皮腔内冠脉血管成形术,心脏外科体外循环,心肺脑复苏,断肢再植和器官移植等。
- 根据权利要求8所述的应用,其特征在于,在促溶栓时,所述药物为权利要求1-6任一项所述的中药组合物和tPA按照10:(5-20)的配比组合。
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CA3093031A CA3093031A1 (en) | 2018-04-04 | 2019-03-25 | Traditional chinese medicinal composition for preventing and/or treating ischemic reperfusion injury |
RU2020134524A RU2020134524A (ru) | 2018-04-04 | 2019-03-25 | Лекарственная композиция традиционной китайской медицины для предупреждения и/или лечения ишемического реперфузионного повреждения |
EP19781599.6A EP3777871A4 (en) | 2018-04-04 | 2019-03-25 | COMPOSITION OF TRADITIONAL CHINESE MEDICINE FOR PREVENTION AND/OR TREATMENT OF ICHEMIC REPERFUSION INJURY |
KR1020207028942A KR20200140277A (ko) | 2018-04-04 | 2019-03-25 | 허혈 재관류 손상 예방 및/또는 치료용 한약 조성물 |
JP2020554106A JP7148629B2 (ja) | 2018-04-04 | 2019-03-25 | 虚血再灌流損傷を予防及び/又は治療する漢方薬組成物 |
US17/041,795 US20210038668A1 (en) | 2018-04-04 | 2019-03-25 | Traditional chinese medicine composition for preventing and/or treating ischemic reperfusion injury |
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CN111888349B (zh) * | 2020-07-29 | 2021-10-22 | 温州医科大学附属第二医院、温州医科大学附属育英儿童医院 | 印楝素在制备促进缺血超长随意皮瓣存活药物的作用 |
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CN1686462A (zh) * | 2005-05-13 | 2005-10-26 | 张海峰 | 一种复方血塞通注射制剂及其制备方法 |
CN1919252B (zh) * | 2005-08-24 | 2011-09-21 | 天津天士力制药股份有限公司 | 一种治疗心脑血管疾病的药物 |
CN1857388A (zh) * | 2006-03-17 | 2006-11-08 | 崔彬 | 三七茎叶总皂苷组合物的制备及应用 |
CN102526423A (zh) * | 2012-01-06 | 2012-07-04 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | 一种治疗缺血性心脏病的药物组合 |
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