CN103919763A - Application of quercetin in preparing medicine for treating focal cerebral ischemia reperfusion injury - Google Patents
Application of quercetin in preparing medicine for treating focal cerebral ischemia reperfusion injury Download PDFInfo
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Abstract
The invention relates to a new application of quercetin, and in particular relates to an application of the quercetin in preparing a medicine for treating focal cerebral ischemia reperfusion injury. Furthermore, the invention provides an optimized quercetin pharmaceutical preparation, i.e., a DMSO preparation of quercetin, for preparing a medicine for treating focal cerebral ischemia reperfusion injury. The invention provides a new way for effective treatment of focal cerebral ischemia reperfusion injury and for improving cognition and motor functions of patients.
Description
Technical field
The new purposes, particularly Quercetin that the present invention relates to Quercetin relate to the purposes for the preparation of the medicine for the treatment of focal brain ischemia-reperfusion injury.
Background technology
Along with the change of social environmental factor and dietary structure, the sickness rate of mankind's ischemic cerebrovascular increases day by day; Focal cerebral ischemia often causes the delayed ischemic neurological deficits such as patient's sensory disturbance, transience are dizzy, aphasia, and severe patient can cause hemiplegia, disturbance of consciousness or the outbreak etc. of dampinging off; In general, thromboembolism treatment occurs in latter 4.5 hours in cerebral ischemia is effectively, but only has a few patients to obtain medical treatment in the meantime, and traditional thromboembolism treatment can increase the risk of intracranial hemorrhage, and bottleneck has been arrived in the treatment of cerebral ischemia at present.Free radicals a large amount of after acute cerebral ischemia generate, and cause the reinforcement of brain inner lipid peroxidation, cause cell membrane, mitochondrial membrane and blood-brain barrier damage, cause and increase the weight of ischemia and cerebral tissue edema.Cerebral tissue is very responsive to the damage of free radical, and in ischemic brain injury, ischemia process no doubt can produce a large amount of free radicals, but quantity research shows between flush phase, to generate more again greatly.In ischemic brain injury, radical reaction was started in ischemic stage, but obviously peroxidization occurs in flush phase again, therefore larger in the damage of flush phase cerebral tissue again.Inflammatory reaction and the oxidative stress damage found after effective method prevention cerebral tissue ischemia are significant.
Quercetin is a kind of in flavone compound, and chemistry Quercetin by name, is polyphenol compound, chemical formula C
15h
10o
7, molecular weight 302.24, is extensively present in fruit, vegetable and some Chinese medicine.Quercetin dissolves in dimethyl sulfoxide (DMSO), methanol, ethanol, glacial acetic acid, acetone etc., but water insoluble.The pharmacological action of current certified Quercetin has: eliminating phlegm and relieving asthma, treatment chronic bronchitis, the propagation that suppresses cell or cell death inducing (as tumor), antiinflammatory, antioxidation etc.A large amount of experiment in vitro shows that Quercetin can, by approach performance antioxidations such as direct Scavenger of ROS free radical, inhibition low-density lipoprotein Pseudobulbus Bletillae (Rhizoma Bletillae) DNA oxidative damages, have inhibitory action to inflammatory reaction and oxidative stress due to various factors.
Summary of the invention
The object of the present invention is to provide Quercetin for the preparation of the purposes of the medicine for the treatment of focal brain ischemia-reperfusion injury, for effectively treatment focal brain ischemia-reperfusion injury, the cognition that improves patient and motor function provide new approach.
" focal cerebral ischemia " rat model prepared by the present invention is simulating human Imaging in Patients with Cerebral Ischemia Disease.In embodiments of the present invention, focal cerebral ischemia in rats can cause the early stage edema of ishemic part, inflammation and later stage atrophy, and after recovery blood reperfusion, inflammation further increases the weight of, and ischemia injury is more obvious, causes the defect of rat cognition and motor function; Quercetin has significant antiinflammatory action, can alleviate ischemia edema and the inflammatory reaction of acute stage, suppresses the release of inflammatory factor in body, reduces the volume at cerebral ischemia infarction position, thereby alleviates the atrophy degree of ischemia later stage cerebral tissue; Cerebral ischemia can stimulate generation and the propagation of Adult Mammals central nervous system neural stem cell, but these neural stem cell major parts are dead at about 2 weeks, cannot normally play a role, if use Quercetin obviously to extend the time-to-live of neural stem cell to Level In Rats With Focal Cerebral Ischemia, ischemia in the time of 3～4 weeks these cells still survive, and neurad glial cell or the conversion of neuron direction, bring into play certain function.
The present invention further provides the preferred Quercetin pharmaceutical preparation of medicine for the preparation for the treatment of focal brain ischemia-reperfusion injury, it is the DMSO preparation of Quercetin, make full use of the effects such as anti-inflammatory analgetic, blood circulation promoting and diuresis that DMSO itself has, and DMSO has strong penetration, can increase drug absorption, promote that medicine plays a role in vivo fast.
Because Quercetin is fat-soluble medicine, water insoluble, dissolve in DMSO, but volume is used DMSO to have certain toxicity to human body, therefore, the present invention is through groping, when guaranteeing curative effect, reduce as far as possible the working concentration of DMSO, determined following preferred preparation scheme:
(1) take Quercetin appropriate, be dissolved in aseptic DMSO, making its final concentration is 0.66mol/L, is placed in 4 ℃ of refrigerators and keeps in Dark Place, standby as storage liquid;
(2) with warm in advance physiological saline solution, storing solution is diluted to 100 times before use, working concentration is 10mg/kg.
The present invention adopts the method for first preparing storing solution, diluting with normal saline more effectively to reduce the use amount of DMSO, therefore, DMSO-Quercetin medicament forms prepared by the present invention both can guarantee that Quercetin brought into play drug effect to greatest extent, can effectively reduce the toxicity of DMSO to organism again.
Mechanism of action about Quercetin of the present invention for the preparation of the medicine for the treatment of focal brain ischemia-reperfusion injury, inventor thinks has substantial connection with antiinflammatory, the antioxidation of Quercetin.And inventor's experiment in vitro shows, Quercetin can suppress the GFAP expression of impaired astrocyte, obviously suppresses the formation of colloid cicatrix, promotes the recovery of damaging cells function, reduces the death of injured nerve cell.In the industry, glial cell is one of main cell in brain, accounts for the more than 90% of brain inner cell total amount, aspect nutrition, support and neuron regeneration, has important function; But after cerebral ischemia, damage location easily forms colloid cicatrix, affects the performance of normal neurons function.Therefore, Quercetin is prepared into the medicine for the treatment of focal brain ischemia-reperfusion injury, contributes to suppress the formation of the colloid cicatrix of damage location, recover normal neurons function.
Accompanying drawing explanation
Fig. 1 is ischemic brain tissue T TC dyeing picture, shows postoperative the 2nd day of Focal Ischemia-Reperfusion in Rats, brain tissue slice TTC coloration result; In figure, arrow indication is rat cerebral ischemia position.
Fig. 2 is rat brain nuclear magnetic resonance, NMR (MRI) image.
Fig. 2 A is postoperative 7 days brain MRI images of DMSO matched group, and Fig. 2 B is postoperative 28 days brain MRI images of DMSO matched group, and Fig. 2 C is postoperative 7 days brain MRI images of Quercetin treatment group, and Fig. 2 D is postoperative 28 days brain MRI images of Quercetin treatment group.In Fig. 2 A to Fig. 2 D, arrow indication is rat cerebral ischemia position.
Fig. 3 is rat brain PET-CT image.
Fig. 3 A is postoperative the 7th day brain PET-CT image of DMSO matched group, and Fig. 3 B is postoperative the 28th day brain PET-CT image of DMSO matched group, and Fig. 3 C is postoperative the 7th day brain PET-CT image of Quercetin treatment group, and Fig. 3 D is postoperative the 28th day brain PET-CT image of Quercetin treatment group.In Fig. 3 A to Fig. 3 D, arrow indication is rat cerebral ischemia position.
Fig. 4 is rat water maze laboratory result figure.
Fig. 5 is the tired experimental result picture of rat transfer rod.
Fig. 6 A is the expression figure of IL-4 in rat blood serum.
Fig. 6 B is the expression figure of IL-6 in rat blood serum.
Fig. 6 C is the expression figure of IL-13 in rat blood serum.
Fig. 6 D is the expression figure of TNF-alpha factor in rat blood serum.
Fig. 7 is rat ischemia brain tissue slice immunofluorescence dyeing picture.
Fig. 7 A is 7 days GFAP immunofluorescence dyeing pictures of DMSO matched group, Fig. 7 B is 28 days GFAP immunofluorescence dyeing pictures of DMSO matched group, Fig. 7 C is 7 days GFAP immunofluorescence dyeing pictures of Quercetin treatment group, and Fig. 7 D is 28 days GFAP immunofluorescence dyeing pictures of Quercetin treatment group.In Fig. 7 A to Fig. 7 D, green is GFAP positive staining, and blueness is DAPI transfect cell core, and microscope amplification is 200 times.
Fig. 7 E is 7 days β-tubulin immunofluorescence dyeing pictures of DMSO matched group, Fig. 7 F is 28 days β-tubulin immunofluorescence dyeing pictures of DMSO matched group, Fig. 7 G is 7 days β-tubulin immunofluorescence dyeing pictures of Quercetin treatment group, and Fig. 7 H is 28 days β-tubulin immunofluorescence dyeing pictures of Quercetin treatment group.In Fig. 7 E to Fig. 7 H, green is β-tubulin positive staining, and blueness is DAPI transfect cell core, and microscope amplification is 200 times.
The specific embodiment
In order to make the technical problem to be solved in the present invention, technical scheme and beneficial effect clearer, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not intended to limit the present invention.
Embodiment 1: focal brain ischemia-reperfusion injury in rats model experiment
Focal brain ischemia-reperfusion injury in rats model adopts line bolt method to make.Ready line bolt before modelling, with nylon wire self-control, the about 4cm of length, head end viscose glue slightly expands, and can block better middle cerebral artery initial part, and line bolt soaks with physiological saline solution before using.During modelling, choose SPF level SD male rat, weight 220-240g, 10% chloral hydrate intraperitoneal injection of anesthesia (3.5ml/kg body weight), rat is fixed on to surgical console, routine disinfection, get neck median incision, cut skin subcutaneous tissue, expose and cut off digastric belly of muscle, separated right common carotid artery, ligation common carotid artery proximal part, upwards minute prong of internal carotid artery and external carotid artery is found in separation, from 45 ° of angular cuts of external carotid artery, insert line bolt, through a minute prong, enter internal carotid artery, from internal carotid artery, line bolt is delivered to intracranial, meet slight resistance and stop, now line bolt head end has reached middle cerebral artery initial part, line bolt is stopped to this position, blocking-up blood flow of middle cerebral artery, make medium-sized artery blood supply cerebral tissue ischemia, simply sew up the incision, after 2 hours, pull out Outlet bolt, realize ischemic tissue of brain and pour into again, carefully sew up wound, postoperatively give suitable nursing, enough diet are fed, and guarantee temperature, humidity and the illumination of feeding environment.
Modelling is complete after animal is clear-headed, observes the behavior performance of animal, more blunt, the torpescence of the successful animal performance of molding; Damage branch hole ball is more outstanding; During action, around damage side, turn-take, can not straight line moving, there is hemiplegia symptom; Carry and when tail is hung by the feet, damage side forelimb and can not firmly stretch forward, illustrate that damage side extremity motor function is impaired.
After modelling, because animal injury is larger, affect its feed, rat body weight alleviates gradually, until within 2～3 weeks, could recover its original body weight.
The compound method of Quercetin comprises the steps:
(1) electronic balance takes Quercetin powder 200mg, is dissolved in the aseptic DMSO solution of 1ml, mixes, and making its final concentration is 0.66mol/L, is placed in 4 ℃ of refrigerators and keeps in Dark Place, standby as storage liquid;
(2) to drawing 100ul storing solution with pipettor before animal injection, with 10ml warm physiological saline solution dilution in advance; Because DMSO has certain toxicity, with this, reduce DMSO for the concentration in body.
All laboratory animals are divided into three groups, and wherein one group is sham operated rats, only equally with animal pattern do identical surgical incision, but do not insert line bolt, and block blood flow, does not treat yet; Another two groups is Focal Cerebral Ischemia Reperfusion animal pattern, wherein treat (hereinafter referred to as " Quercetin treatment group ") for one group, ischemia-reperfusion is brought into use the above-mentioned Quercetin preparing after 1 hour, with the dosage of 10mg/kg (for example, weight is that the rat of 200g gives Quercetin diluent 1ml) lumbar injection, every day 1 time, be used in conjunction 7 days, note drug injection not being arrived to animal intestinal wall, otherwise easily cause intestinal adhesion; Another group model animal does not give Quercetin (hereinafter referred to as " DMSO matched group "), only gives DMSO diluent (only with 10ml physiological saline solution dilution 100ul DMSO, not containing Quercetin) lumbar injection, every day 1 time, is used in conjunction 7 days, in contrast.
During treatment, from the performance substantially of animal, the rat state of Quercetin treatment group is significantly better than DMSO control rats, and behavior is more active, and hemiplegia symptom early disappears; The appetite of Quercetin treatment group animal is significantly better than DMSO control animals, and the degree losing weight and mortality rate are all lower, and concrete numerical value is in Table 1.The body weight change of laboratory animal during table 1 treatment
In order to verify the reliability of Quercetin treatment focal brain ischemia-reperfusion injury in rats, further carried out following experiment.
Embodiment 2:TTC dyeing
1. experimental principle
TTC(2,3,5-triphenyltetrazolium chloride) be the proton acceptor of pyridine-nucleoside structure enzyme system in respiratory chain, take on a red color with the dehydrogenase reaction in normal structure, and in ischemic tissue, dehydrogenase activity declines, and can not react, therefore tissue is pale asphyxia.TTC dyeing can be distinguished normal structure and ischemic tissue.
2. experimental technique
(1) Focal Cerebral Ischemia Reperfusion postoperative 24 hours sham operated rats, Quercetin treatment group, DMSO matched groups are respectively got a rat, and broken end is got brain, because cerebral tissue is not fixed with paraformaldehyde, thus softer, the careful integrity that keeps brain while getting brain; After taking-up, brain is placed in to-20 ℃ of refrigerators freezing 0.5～1 hour, is convenient to section.
(2) during freezing brain, prepare 2%TTC dye liquor, take TTC powder 0.9g, be dissolved in phosphate buffer (PBS) 45ml of pH value 7.4, mix; By in 45ml dye liquor mean allocation to three small beaker, for dying cerebral tissue.
(3) brain tissue slice, is cut into 6 by cerebral tissue, every 2mm, cuts a slice; Section is placed in to TTC dye liquor, and masking foil covers beaker mouth, puts into 37 ℃ of water-baths 20～30 minutes, during often stir brain sheet, allow brain sheet even contact to dye liquor.
The rat that more than different groups are got in experiment is repeated 3 times.
3. experimental result
Sham-operation rat cerebral tissue is dyed to normal redness, and animal pattern is infarction under right side cerebral cortex and cortex, and Quercetin treatment rat cerebral tissue infarct size is significantly less than DMSO control rats, the results are shown in accompanying drawing 1.Illustrate that Quercetin can obviously reduce the infarcted region of cerebral tissue.
Embodiment 3: rat brain NMR (Nuclear Magnetic Resonance)-imaging (MRI)
NMR (Nuclear Magnetic Resonance)-imaging is used rf wave and high-intensity magnetic field can obtain gem-pure rat cerebral tissue picture, shows position and the area of ischemic infarction.The nuclear magnetic resonance analyser model that the present invention uses is " PHILIPS MR Achieva/Intera ".
Quercetin treatment group, DMSO matched group are respectively got 3 rats, within postoperative 7 days, 14 days and 28 days, carry out brain magnetic resonance imaging imaging respectively in Focal Cerebral Ischemia Reperfusion.Image shows the variation along with the time, and the brain infarction area of Quercetin group rat diminishes gradually, and DMSO group rat cerebral infarction area change is little or infarct size increases a little.Result is referring to Fig. 2 A to Fig. 2 D.
Embodiment 4: rat brain Positron Emission Computed Tomography (PET-CT)
PET-CT can merge functional image and structural images, and function and form is combined, and can be used for observing the ability of cells in vivo picked-up energy.By to rat injection 18F-FDG, observe the function that Nerve Cells of Rat Brain absorbs FDG; The various metabolic activities of normal organism all need the participation of glucose energy, and FDG and glucose seemingly, can be absorbed by organism.FDG is metabolism 30 minutes in vivo, and image shows that normal rat cerebral tissue picked-up FDG position is highlighted, and ischemic tissue of brain picked-up FDG functional defect, picked-up position does not work.The PET-CT instrument model that the present invention uses is " SIEMENS Inveon ".
Quercetin treatment group, DMSO matched group are respectively got 3 rats, within postoperative 7 days, 14 days and 28 days, carry out PET-CT scanning imagery respectively in Focal Cerebral Ischemia Reperfusion.Image shows the variation along with the time, and the brain tissue cell ingestion of glucose function of Quercetin group rat is organized rat significantly better than DMSO.The function that Quercetin can recovered part brain tissue cell is described.Result is referring to Fig. 3 A to Fig. 3 D.
Embodiment 5: water maze laboratory
1. experiment purpose
Investigate the Learning and Memory function of experimental rat to locus.
2. experimental technique
(1) experimental provision
In the round pool that is 180cm at diameter, pack water into, depth of water 30cm, adds dark dye, makes water opaque; In pond, placing the small circular platform that a diameter is 12cm, is highly 28cm, and because water is opaque, rat cannot be seen platform under water, and in experimentation, position of platform immobilizes; Pond surrounding is pulled on the door curtain made of cloth, can on the door curtain made of cloth, paste the pattern of different colours and shape, so that rat explanation direction.
Mouse is innately able to swim, but do not like staying in water, and do not stop swimming extremely consume one's strength, therefore they can instinct find the recreating facility in water, in searching process, relate to a complicated Memory Process, comprise and collect the visual information relevant to space orientation, then arrange and remember, object is successfully to find as early as possible the platform in water, to depart from the environment in water.Adopt video camera tracking technique, computer software can record the swimming track of mouse in water automatically, and records the time that mouse is found platform.
(2) experimentation
Laboratory animal was carried out training for three days on end before ischemia model operation, put into water from 4 place of entry by rat respectively towards pool wall every day, put into position and get at random one of four original positions in east, south, west, north, allow rat find platform under water, record the time that rat is found platform; In front training several times, if rat can not find platform under water, rat is directed on platform, and is allowed to condition on platform and stopped for 10 seconds.After ischemia model operation, sham operated rats, Quercetin treatment group, DMSO matched group are respectively got 5 rats, respectively after surgery within the 1st, 4,7,14,21,28 days, carry out water maze laboratory, get and respectively organize the meansigma methods that rat finds the underwater platform time and compare.
3. experimental result
Through training, rats in sham-operated group can search out platform under water very fast, and the animal of two model group obviously will spend the more time and find, and the Quercetin treatment group rat used time will obviously be less than DMSO control rats.Illustrate that Quercetin can strengthen space learning and the memory function of focal brain ischemia-reperfusion injury rat.The results are shown in accompanying drawing 4.
Embodiment 6: transfer rod fatigue experiment
1. experiment purpose
Investigate the extremity coordination exercise ability of experimental rat.
2. experimental technique
The rotating speed of transfer rod is set to: 0～10rpm is speedup gradually, totally 120 seconds time; 10～20rpm is speedup gradually, totally 120 seconds time; 20～0rpm slows down gradually, totally 60 seconds time, totally 300 seconds observing time.Observe the time that rat moves on transfer rod, once fall down from transfer rod, computer records the time that rat stops on transfer rod automatically.
Laboratory animal was carried out training for three days on end before ischemia model operation, after operation, sham operated rats, Quercetin treatment group, DMSO matched group are respectively got 5 rats, respectively after surgery within the 1st, 4,7,14,21,28 days, carry out transfer rod fatigue experiment, get and respectively organize rat on transfer rod the meansigma methods of the time of staying compares.
3. experimental result
Through training, the time that rats in sham-operated group stops on transfer rod is obviously longer than the animal of two model group, and the movement time of Quercetin group rat is obviously longer than DMSO control rats.Illustrate that Quercetin can strengthen the extremity coordination exercise ability of focal brain ischemia-reperfusion injury rat.The results are shown in accompanying drawing 5.
Embodiment 7: the expression of inflammatory factor level in rat blood serum
Focal Ischemia-Reperfusion in Rats is postoperative, and body produces inflammatory reaction, in blood, can discharge various inflammatory factors, and the present invention has detected the expression of IL-4, IL-6, IL-13, these several inflammatory factors of TNF-α.TNF-α occurs the earliest in inflammatory reaction, is also most important inflammatory mediator, can activate neutrophilic granulocyte and lymphocyte, regulates other tissue metabolisms' activity and impels the synthetic of other factors and discharge; IL-6 can induce B cell differentiation and produce antibody, and inducing T cell activation and proliferation and differentiation, participates in the immunne response of body, is the agent that inspires of inflammatory reaction.
1. experimental technique:
(1) specimen taken
Sham operated rats, Quercetin treatment group, DMSO matched group are respectively got 3 rats, and difference the 1st, 4,7 days etherizations after surgery, get eye socket posterior vein blood 0.5～1ml; Separation of serum, is sub-packed in the EP pipe of 0.5ml, is placed in-80 ℃ of Refrigerator stores, avoids multigelation; Unified detection after specimen collection is neat.
(2) detect
The Luminex Luminex of employing double-antibody sandwich detects the expression of the various factors.Luminex liquid-phase chip technology is based on the multiple polystyrene microsphere that is marked with different fluorescent dyes, and microsphere band is magnetic, and is coated with IL-4, IL-6, IL-13, TNF-Alpha antibodies, and once experiment can detect the expression of these several factors simultaneously.2. experimental result
Serum il-4 of model group rat, IL-6, IL-13, TNF-alpha levels are all higher than sham operated rats, and Quercetin group rat is starkly lower than DMSO control rats, and the release that Quercetin can the inflammation-inhibiting factor is described, have good antiinflammatory action.Result is referring to Fig. 6 A to Fig. 6 D.
Embodiment 8: rat cerebral tissue's frozen section immunofluorescence dyeing
1. experimental technique
(1) perfusion is fixed and is drawn materials
Sham operated rats, Quercetin treatment group, DMSO matched group is respectively got 3 rats, the 28th day after surgery, by the excessive deep anaesthesia (5.0ml/kg of rat, 10% chloral hydrate intraperitoneal anesthesia) after, fixing rat, make breast median abdominal incision, carry and press from both sides out ensiform process of sternum, to back upper place, both sides, cut off every flesh and thoracic wall, open pericardium, expose heart, from the apex of the heart, insert perfusion syringe needle to left ventricle, cut off right auricle simultaneously, be close to spinal column folder and close ventral aorta, quick filling normal saline 150～200ml, rinse liquid in body, treat that trickle becomes limpid, after observation conjunctive bulbi and oral mucosa are pale, with 4% paraformaldehyde solution 400ml, pour into first quick and back slow more fixing, while just starting to pour into, rat forelimb performance is acutely twitched, then forelimb and cervical region become stiff.After having poured into, cut off rat head and neck skin and expose skull and cervical vertebra, from cervical vertebra, cut off neck marrow, rear musculi colli is removed in separation; With curved forceps, carefully reject skull, avoid cerebral dura mater to scratch cerebral tissue, from oblongata, start slowly separated basis cranii tissue, take out Mus brain, be placed in 4% paraformaldehyde solution, after 4 ℃, fix 6 hours, then proceed to 4 ℃ of placement 12-24 hour in 150ml30% sucrose solution, after cerebral tissue sinks to the bottom, with OTC embedding cerebral tissue, carry out frozen section.(2) frozen section
Freezing microtome operating temperature is adjusted to-26 ℃, the section of ischemic injuries region, the thick 8um of sheet, gets one, use anticreep microscope slide bonding die every four.
(3) section immunofluorescence dyeing
1) section is placed in to ice acetone and fixes 15 minutes;
2) take out section, PBS rinsing 3 times, each 5 minutes;
3) 30 minutes ruptures of membranes of 0.3%Triton-X100 room temperature; PBS rinsing 3 times, each 5 minutes;
4) 3%H2O2 room temperature is 10 minutes, removes intrinsic oversxidase active; PBS rinsing 3 times, each 5 minutes;
5) normal goats serum sealing, room temperature 30 minutes;
6) primary antibodie is hatched (the anti-glial fibrillary acidic protein GFAP of 1:100 mice, the anti-'beta '-tubulin β-tubulin of 1:100 mice), is placed in 4 ℃, wet box and spends the night;
7) take out section, PBS rinsing 3 times, each 5 minutes next day;
8) two anti-(1:100Alexa fluor488 goat anti-mouse IgG), lucifuge, the room temperatures 1 hour of hatching; PBS rinsing 3 times, each 5 minutes;
9) DAPI redyes core, lucifuge, room temperature 10 minutes; PBS rinsing 3 times, each 5 minutes;
10) anti-fluorescent quenching mountant mounting, fluorescence microscopy Microscopic observation, takes pictures.
2. experimental result
DMSO group rat section GFAP positive cell quantity is obviously more than Quercetin group, and β-tubulin positive cell quantity is significantly less than Quercetin group.Illustrate that Quercetin can obviously suppress the formation of ishemic part colloid cicatrix after cerebrum ischemia reperfusion injury, increase neuronic quantity, impel the recovery of function after rat brain damage.Result is referring to Fig. 7 A to Fig. 7 H.
Claims (3)
1. Quercetin is for the preparation of the purposes of the medicine for the treatment of focal brain ischemia-reperfusion injury.
2. purposes according to claim 1, is characterized in that, the DMSO preparation that described medicine is Quercetin.
3. purposes according to claim 2, is characterized in that, the DMSO preparation of described skin element is prepared by the following method:
(1) take Quercetin appropriate, be dissolved in aseptic DMSO, making its final concentration is 0.66mol/L, is placed in 4 ℃ of refrigerators and keeps in Dark Place, standby as storage liquid;
(2) with warm in advance physiological saline solution, storing solution is diluted to 100 times before use, working concentration is 10mg/kg.
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| CN107243037A (en) * | 2017-08-12 | 2017-10-13 | 河南中医药大学 | Application of the field pennycress general flavone in treatment focal cerebral ischemia medicine is prepared |
| CN107441135A (en) * | 2017-08-12 | 2017-12-08 | 河南中医药大学 | Field thistle general flavone is preparing the application in treating cerebral ischemia and focal brain ischemia medicament repeatedly |
| CN108295082A (en) * | 2018-01-25 | 2018-07-20 | 遵义医学院 | Application of the trifloroside in preventing cerebral ischemia re-pouring injured drug |
| CN113230260A (en) * | 2021-05-19 | 2021-08-10 | 复旦大学附属华山医院 | Method for constructing animal disease model of persistent gouty arthritis |
| CN114767666A (en) * | 2022-03-31 | 2022-07-22 | 东北大学 | Application of tamarisk flavine in preparation of medicine for preventing or treating ischemic encephalopathy |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107243037A (en) * | 2017-08-12 | 2017-10-13 | 河南中医药大学 | Application of the field pennycress general flavone in treatment focal cerebral ischemia medicine is prepared |
| CN107441135A (en) * | 2017-08-12 | 2017-12-08 | 河南中医药大学 | Field thistle general flavone is preparing the application in treating cerebral ischemia and focal brain ischemia medicament repeatedly |
| CN108295082A (en) * | 2018-01-25 | 2018-07-20 | 遵义医学院 | Application of the trifloroside in preventing cerebral ischemia re-pouring injured drug |
| CN113230260A (en) * | 2021-05-19 | 2021-08-10 | 复旦大学附属华山医院 | Method for constructing animal disease model of persistent gouty arthritis |
| CN114767666A (en) * | 2022-03-31 | 2022-07-22 | 东北大学 | Application of tamarisk flavine in preparation of medicine for preventing or treating ischemic encephalopathy |
| CN114767666B (en) * | 2022-03-31 | 2024-01-23 | 东北大学 | Application of tamarigenin in preparing medicament for preventing or treating ischemic encephalopathy |
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