CN103405408A - Application of chrysin in preparation of medicines for treating cerebral arterial thrombosis - Google Patents
Application of chrysin in preparation of medicines for treating cerebral arterial thrombosis Download PDFInfo
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- CN103405408A CN103405408A CN2013102635300A CN201310263530A CN103405408A CN 103405408 A CN103405408 A CN 103405408A CN 2013102635300 A CN2013102635300 A CN 2013102635300A CN 201310263530 A CN201310263530 A CN 201310263530A CN 103405408 A CN103405408 A CN 103405408A
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- chrysin
- ischemia
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Abstract
The invention discloses an application of chrysin in the preparation of medicines for treating cerebral arterial thrombosis. Experimental results of the chrysin show that the chrysin has a relatively good nerve protection effect on focal cerebral ischemia-reperfusion injury, can be used for improving the neurological function loss after the cerebral ischemia-reperfusion injury, reducing the water content in brains, the size of cerebral infarction and the content of malonaldehyde (MDA) and improving the activity of superoxide dismutase (SOD); after focal cerebral ischemia-reperfusion, the chrysin can be used for reducing the relative expression quantity of nuclear factor-kappaB (NF-kappaB) of ischemia-reperfusion brain tissues, inducible nitric oxide synthase (iNOS) and a cyclooxygenase-2 (COX-2) gene; and the chrysin can also be used for reducing the expression of GFAP and Iba1 in the ischemia-reperfusion brain tissues, inhibiting the activation of astrocyte and microglial cells and meanwhile reducing the expression of pro-inflammatory cytokines such as IL-1alpha, IL-1beta, IL-6, IL-12, IL-17A, TNF-alpha and IFN-gamma, thus the excessive inflammatory response of cerebral ischemia tissues is alleviated, and the neuroprotective effect after ischemia is realized.
Description
Technical field
The present invention relates to the new purposes of compound chrysin, relate in particular to the application of chrysin in preparation treatment ischemic cerebral apoplexy Chinese medicine.
Background technology
Cerebrovascular is one of three large diseases that cause mankind's death, and its high mortality, high disability rate have been brought white elephant to society and family.As one of the most serious disease of symptom, cerebral infarction is very harmful to patient health in cerebrovascular, often causes irreversible brain injury.Interruption because of cerebral arterial blood flow and oxygen supply, significantly damage the brain physiologically active, thereby the pathophysiological process that has caused series of complex, comprise Energy Metabolism of Brain Tissue disorder, toxicity of excitatory amino acid, radical damage, inflammatory reaction, apoptosis etc.After the cerebral ischemia certain hour recovered blood supply, its function not only failed to recover, and more serious cerebral disturbance occurred on the contrary, i.e. cerebral ischemia reperfusion injury.The enzymatic cascade reaction that ischemical reperfusion injury physiological pathology process is a too many levels, multifactor, multipath damages, relate to brain cell energy metabolism disorder, toxicity of excitatory amino acid, intracellular calcium overload, oxidative stress damage and neuronal apoptosis etc.
After the cerebral tissue ischemia, microglia, astrocyte are activated, and produce a large amount of cytokine and chemokine.Cytokine raises the cerebrovascular endothelial cell expression of adhesion molecule, leukocyte in circulation is under the effect of adhesion molecule and chemotactic factor, stick and migrate to the brain essence of damage, together with glial cell, discharge the cytokines such as a large amount of il-1 (IL-1), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), thereby reinforcement leukocyte infiltration, the inflammatory mediators such as while arachidonic acid metabolite roll up, increase the weight of local damage, finally cause neuronal apoptosis, necrosis.
Cerebral ischemia normally causes brain large artery trunks obstruction to cause by thrombosis or thromboembolism, and the regional cerebral blood flow in this tremulous pulse blood supply district after obstruction (regional cerebral blood flow, rCBF) descends rapidly.The decline of ischemic area blood flow is unbalanced, the blood flow at ischemia center descends the most serious, be called as the ischemia center, the cerebral blood flow threshold value of ischemia center is 10ml/(100gmin), neuron membrane ionic pump and cellular energy metabolism exhaustion, the infringement of cerebral tissue generation irreversibility, and the CBF of peripheral region, ischemia center decline is relatively light, is called as ischemia half blanking bar.The cerebral blood flow of ischemia half blanking bar is between electric failure [be about 20ml/(100gmin)] and energy decline [be about 10ml/(100gmin)], local brain tissue exist the residual blood flow of large artery trunks and (or) collateral circulation, the neuron of large number of viable is still arranged.Therefore, ischemia penumbra is that an organizational structure integrity retains but the low perfusion area of miopragia or disappearance.Ischemia center and ischemia half blanking bar are dynamic pathophysiological processes, and along with the prolongation with Ischemia Time that increases the weight of of degree of ischemia, the both central necrotic district enlarges gradually, and ischemia half blanking bar dwindles gradually, and the cerebral infarction scope increases.But if the blood flow ，Gai district cerebral tissue function that can recover at short notice ischemia half blanking bar is reversible, neurocyte can be survived and restore funcitons.Therefore, recover as early as possible the blood supply of ischemia penumbra, can reduce cerebral infarction volume, improve the prognosis of cerebral apoplexy patient.Save the key of ischemia half blanking bar except timely recovery blood flow, also be to suppress the ischemic cascade reaction.
In prior art, the strategy that cerebral infarction is treated mainly comprises two aspects, the one, and recover brain and fill with again, improve brain blood for (thrombolytic); The 2nd, the blocking-up Cascade of Injury, prevent neuronal damage (brain protection).For the complexity of the pathomechanism of cerebral infarction, except super early stage thrombolytic has definite curative effect, a kind of neuroprotective that sure curative effect is arranged for the evidence-based medicine EBM proof is arranged not yet.The target of neuroprotective is the physiological pathology cascade reaction of intervening ischemia penumbra, saves the still great-hearted cerebral tissue of tool, prevents or postpones cell death.Chrysin, chrisin, claim again chrysin.It is a kind of of flavone compound, is present in seed, the peel of stem of Bignoniaceae plant Semen Oroxyli, the heart wood of pinaceae plant western white pine, the heart wood of bristlecone pine etc.Prior art shows, chrysin has the various biological characteristics such as antitumor, antiinflammatory, but chrysin there is no report for the therapeutical effect to cerebral infarction so far.
Summary of the invention
The objective of the invention is to disclose the application of chrysin aspect preparation treatment ischemic cerebral apoplexy Chinese medicine.The present invention has determined that by lot of experimental data the effective dose that chrysin is used for the treatment of cerebral infarction is the 47-53mg/Kg body weight/day, and then effective dose is preferably to the 50mg/Kg body weight/day.
At present, the screening zoopery of neuroprotection activity is to adopt standby mouse brain Middle cerebral artery occlusion (MCAO) focal cerebral ischemia in rats of internal carotid artery line bolt legal system to estimate the curative effect of anti-cerebral ischemia drugs according to conventional method.The present invention uses experimental animal model to carry out a large amount of experiments, result shows: the preventative chrysin that gives has neuroprotective preferably to focal cerebral ischemia-reperfusion in mice, can improve the neurological deficit after cerebral ischemia reperfusion injury, alleviate brain water content, reduce cerebral infarction volume, increase superoxide dismutase (SOD) vigor, reduce the content of malonaldehyde (MDA); After Focal Cerebral Ischemia Reperfusion, chrysin can be lowered nuclear Factor-Kappa B (NF-κ B), the inducible nitric oxide synthase (iNOS) of reperfusion brain, the relative expression quantity of COX-2 (COX-2) gene; Chrysin also can reduce the expression of GFAP in reperfusion brain, Iba1; suppress astrocyte and microglial activation; reduce simultaneously the expression of the Pro-inflammatory Cytokines such as IL-1 α, IL-1 β, IL-6, IL-12, IL-17A, TNF-α, IFN-γ; thereby alleviate the excessive inflammatory response of ischemic tissue of brain, realize the neuroprotective after ischemia.
The accompanying drawing explanation
Fig. 1 is that chrysin (Chrysin) improves the neurologic impairment figure caused after the middle cerebral artery ischemia; Illustrate that chrysin can improve the neurologic impairment due to cerebral ischemia reperfusion injury, has neuroprotective.
Fig. 2 is that chrysin (Chrysin) alleviates mouse brain tissue water content figure; Illustrate that chrysin has neuroprotective.
Fig. 3 is the Infarction volume figure after chrysin (Chrysin) alleviates focal brain ischemia-reperfusion injury; Illustrate that chrysin has the cerebral tissue protective effect.
Fig. 4 is the pathological change figure that chrysin (Chrysin) improves brain after ischemia reperfusion; Illustrate that the chrysin focal cerebral ischemia-reperfusion injury has protective effect.
Fig. 5 is the positive cell expression figure that chrysin (Chrysin) reduces iNOS; Illustrate that chrysin has neuroprotective.
Fig. 6 is the positive cell expression figure that chrysin (Chrysin) reduces COX-2; Illustrate that chrysin has neuroprotective.
Fig. 7 is the positive cell expression figure that chrysin (Chrysin) reduces NF-κ B; Illustrate that chrysin has neuroprotective.
Fig. 8 is relative expression's spirogram that chrysin (Chrysin) reduces iNOS, COX-2, NF-κ B mRNA; Illustrate that chrysin has neuroprotective.
Fig. 9 is the positive cell expression effect figure that chrysin (Chrysin) reduces GFAP; Illustrate that chrysin has neuroprotective.
Figure 10 is the positive cell expression effect figure that chrysin (Chrysin) reduces Iba-1; Illustrate that chrysin has neuroprotective.
Figure 11 is mice Brain Tissue SOD Vigor after chrysin (Chrysin) raising ischemia-reperfusion, reduces the design sketch that its MDA expresses; Illustrate that chrysin has the brain protective effect.
Figure 12 is the expression effect figure of the inflammatory factors such as IL-1 β, IL-6 after chrysin (Chrysin) reduction cerebral tissue ischemia-reperfusion, IL-12, IL-1A, IL-17A, TNF-α, IFN-γ; Illustrate that chrysin has the brain protective effect.
The specific embodiment
Embodiment 1
1.1MCAO the foundation of mouse model
(1) laboratory animal and source
72 of male wild type C57/BL6 pathogen-free domestic (SPF level) mices, age in 6-8 week, body weight 25-30g, purchased from Test Animal Centre, Academy of Military Medical Sciences, P.L.A, raise and Medical University Of Tianjin's Experimental Animal Center, the feeding environment of mice is room temperature 20-25 ℃, relative humidity 40%-60%.
(2) experiment grouping
For the drug effect of research chrysin in mice MCAO animal model, mice is divided into three groups at random, 24 every group, is respectively sham operated rats; Ischemia-reperfusion injury model group (model group); Chrysin treatment group (treatment group).Treatment group gave chrysin 50mg/Kg/ days gavage in first 7 days with preparation MCAO model.Sham operated rats and model group prepare the normal saline gavage given in first 7 days with the treatment group equal volume in the MCAO model.Model group and treatment group mice adopt Longa line bolt method to set up the MCAO model, ischemia 1h, then pour into 24h.
(3) experimental procedure
A. mice is weighed, and 10% chloral hydrate (3ml/Kg) intraperitoneal injection of anesthesia, be fixed on its dorsal position on dissecting operation table, and the left side middle cerebral artery of take is operation side, cervical region preserved skin, iodophor disinfection;
B. median incision before capable cervical region, cut off skin, separates subcutaneous tissue, exposes the bubbling gland and also push it against both sides, and the blunt separation tissue, expose common carotid sheath;
C. blunt separation common carotid artery (common carotid artery, CCA), internal carotid artery (intemal carotid artery, ICA), external carotid artery (external carotid artery, ECA) and the Main Branches tremulous pulse occipital artery of ECA, superior thyroid artery etc., the protection peripheral nerve is as injury-free as vagus nerve;
D. the Main Branches tremulous pulse of first ligation ECA, ligation CCA proximal part, put a standby silk thread at distal end and slightly do ligation, gives over to static line bolt use, puts a standby silk thread at the ICA proximal part, giving over to hemostasis and using;
E. on CCA, cutting a v-notch between two ligatures, the line bolt of having got ready is inserted in CCA, slightly tighten up the distal end ligature, the crotch of the line bolt being crossed to ECA and ICA enters the ICA tube chamber, slowly send into intracranial, while running into slight resistance, arrive the initial part (mean depth of insertion for apart from the 9-10mm of aortic bifurcation place inside and outside neck) of middle cerebral artery (middle cerebral artery, MCA), tighten up and ligation CCA on ligature;
F. confirm that wound is without oozing of blood, layer-by-layer suture muscle and skin, the iodophor disinfection operative incision, line bolt ends exposed is in skin outside, and by its labelling;
G. after ischemia 1h, the line bolt is exited to about 5mm, but line bolt head is retained in still in the ECA broken ends of fractured bone, thereby makes mice realize pouring into again, cut off the redundance of line bolt;
H. the nursing of postoperative attention insulation and otch, the operation animal rear single cage of reviving is raised, and keeps the mouse cage dry cleansing, supply liquid, close observation.
I. the sham operated rats model prepares the samely, but inserts only about 5mm of line bolt through the ECA sidewall, and namely the line bolt does not insert the ICA intracranial segment.
(4) inclusion criteria of MCAO model
With reference to the method for Zea Longa, after the mice surgery anesthesia is clear-headed, carry out system scoring in 5 minutes:
0 minute: impassivity function damage symptom;
1 minute: while carrying tail, thromboembolism tremulous pulse offside forelimb can not stretch;
2 minutes: during walking to the thromboembolism tremulous pulse to sideway swivel;
3 minutes: to thromboembolism tremulous pulse offside, topple over during walking;
4 minutes: can not spontaneously walk, loss of consciousness.
Scoring enters group the 1-3 person of dividing.
(5) exclusion standard of MCAO model
A. according to Zea Longa point system, function of nervous system marks lower than 1 minute person;
While b. getting brain, find concurrent subarachnoid hemorrhage person;
C.TTC dyeing has no ischemic focus person;
D. died in ischemia-reperfusion 24h.
Because above-mentioned factor causes each experimental group number of animals deficiency person, polishing number of animals modeling again at any time.
1.2 neurological deficits score
After the mice surgery anesthesia is clear-headed, adopt neurological deficits score (the modified neurological severity scores of improvement, mNSS) motion, sensation, equilibrium function and the reflection of mice are estimated, the scoring scope is that 0-18 divides, and score value is higher, its nervous lesion is more serious, wherein, 13-18 is divided into major injury, and 7-12 is divided into moderate lesion, 1-6 is divided into the minor injury, and 0 is divided into not damaged.
Table 1: the neurological deficits score of improvement (mNSS)
1.3 the evaluation of mouse brain Infarction volume
Application TTC dyeing is estimated cerebral infarction volume.
After MCAO mice ischemia-reperfusion 24h, fix with dorsal position after the excessive anesthesia of 10% chloral hydrate, the fast open breast exposes heart, by apex, No. 16 syringe needles is inserted to aortic root, and the blood vessel clamp closes ventral aorta, cuts off simultaneously the right auricle blood-letting.Through 4 ℃ of normal saline 100ml left and right of heart quick filling, until effluent becomes clear.After perfusion, break end and get brain immediately, remove olfactory bulb, cerebellum and low brain stem, after freezing 20min, take out in-20 ℃ of refrigerators, from the forebrain antinion, cut out continuously backward the Coronal brain section of 5 about 2mm of thickness.The brain sheet is put in advance to the 1%TTC solution of preparation, 37 ℃ of lucifuges are hatched about 30min, and during 15min, brain sheet turn-over once, makes itself and TTC even contact.Then the brain sheet is placed in to fixedly 24h of freshly prepared 4% paraformaldehyde solution, observes and take pictures, normal cerebral tissue is cerise, does not dye and be pale asphyxia in necrotic area.
Analyze: application Image J image (Media Cybernetics, Inc) process software calculates the percentage ratio that Infarction volume accounts for the homonymy brain volume.Consider the impact of cerebral edema, adopt the Infarction volume computing formula after proofreading and correct: the infarct size of the measurement infarct size of correction=measurement * { 1 one [(a pair of side hemisphere of homonymy hemisphere area area)/homonymy hemisphere area] }.
1.4 the mouse brain tissue water content is measured
Adopt dry weight in wet base method, after the removal antinion, get the thick cerebral tissue of the about 2mm of pathological changes side and measure for brain water content.
(1) cerebral tissue is put in advance to the tinfoil of weigh (A), weigh immediately (B), B-A namely obtains weight in wet base;
(2) with tinfoil, wrap up cerebral tissue, put into 95 ℃ of baking boxs and dry after 24h to take out and return to room temperature, weigh (C), C-A namely obtains dry weight, and weighing is to constant weight repeatedly;
(3) the substitution formula calculates brain water content: (cerebral tissue weight in wet base-cerebral tissue dry weight)/weight in wet base * 100%, i.e. (B-C)/(B-A) * 100%.
Result is as follows:
Table 2: mice neurological deficits score and cerebral infarction volume are relatively
With model group, compare,
aP<0.05
1.5 the preparation of mouse brain tissue paraffin section de
(1) application 10% chloral hydrate anesthesia mice, give lumbar injection by 3ml/Kg;
(2) after anesthesia is satisfied, does an anterior midline and be about the 3cm thoraco-abdominal incision, fully expose thoracic cavity and abdominal viscera;
(3) free heart, find right auricle and cut off, open vein (it is fast that action is wanted);
(4) from left ventricle, insert the scalp acupuncture (the scalp acupuncture blunt, from long axis of body miter angle direction inserting needle, in needle point insertion ascending aorta, moves and wants soft) that is connected with the 20ml syringe;
(5) push the 20ml normal saline, the organs such as visible liver, intestinal bleach, and after having pushed away, change rapidly 4 ℃ of paraformaldehyde 20ml, and push fast;
(6) the successful sign of perfusion: while just starting to pour into, the mice whole body is acutely twitched, and whole body is stiff afterwards;
(7) application Mus broken end cutter. mouse head is taken off;
(8) the application shears is cut off skin along a median line, and rejects muscle of head, fully exposes skull;
(9) with small size mosquito forceps (directly), break except skull, remove meninges, fully expose cerebral tissue, and mosquito forceps is goed deep into to basis cranii, take out cerebral tissue;
(10) cerebral tissue is placed in to fixedly 24h of 4% paraformaldehyde;
(11) from the low-concentration ethanol to the alcohol in high concentration, dewater successively: (note: the alcohol in high concentration time can not be oversize, otherwise organize frangible.) 70% ethanol can spend the night, 80% ethanol 1h, 95% twice of ethanol 1h(), twice of dehydrated alcohol 1h();
(12) transparent: as to apply isopyknic ethanol and dimethylbenzene transparent, generally soak 30-45min and get final product (dimethylbenzene is transparent is 30min * 2 time);
(13) waxdip: tissue is first put into soft wax (fusing point the is 52-54 ℃) 1h of fusing after transparent; Then enter hard wax (fusing point 56-58 ℃) 1h;
(14) embedding: tissue is put into to the imbedded mold of preheating, the embedded box top set, continue to add paraffin, until at the bottom of embedding, box section is immersed in paraffin;
(15) cooling: mould is placed on cooling stage, fully cooling after, take out the wax stone storage at normal temperature standby.
1.6 mouse brain tissue paraffin section de HE dyeing
(1) the brain tissue slice routine dewaxes with dimethylbenzene, extremely washing after ethanol dehydrations at different levels: dimethylbenzene (I) 5min, dimethylbenzene (II) 5min, 100% ethanol 2min, 95% ethanol 1min, 80% ethanol 1min+75% ethanol 1min, distillation washing 2min;
(2) haematoxylin dyeing 5min, tap water rinses 2min;
(3) acidic alcohol acidify 30s(carry insert several under);
(4) tap water soaks 15min or warm water (approximately 50 ℃) 5min;
(5) put Yihong liquid dyeing 2min;
(6) conventional dehydration, transparent, mounting: 80% ethanol (I) 1min, 95% ethanol (II) 1min, 100% ethanol (I) 1min, 100% ethanol (II) 1min, dimethylbenzene carbolic acid (3:1) 1min, dimethylbenzene (I) 1min, dimethylbenzene (II) 1min, resinene sealing.
1.7 mouse brain tissue paraffin section de immunofluorescence dyeing
(1) dewaxing: tissue slice is placed in to 60 ℃ of 2h of baking box;
Aquation: tissue slice is placed in successively:
(2) on shaking table, wash paraffin section with PBS, 5min * 2 time;
(3) antigen retrieval: electric furnace or water-bath heating 0.01M sodium citrate buffer solution (pH6.0), to 95 ℃ of left and right, are put into brain tissue slice heating 20min;
(4) slowly cool to room temperature;
(5), on shaking table, wash 5min * 3 time with PBS;
(6) add 3%BSA, 37 ℃ of constant temperature incubation 30min, dry, and do not wash;
(7) drip primary antibodie (anti-iNOS, anti-COX-2, anti-NF-κ B, anti-GFAP), 4 ℃ are spent the night; From after 4 ℃ of taking-ups, room temperature is placed rewarming 30min;
(8), on shaking table, wash 5min * 3 time with PBS;
(9) drip fluorescence two and resist, the room temperature lucifuge is hatched 1h;
(10) on shaking table, with PBS, wash 5min * 3 time, note lucifuge;
(11) on shaking table, with PBS, wash 5min * 3 time, note lucifuge;
(12) drip DAPI, the room temperature lucifuge is hatched 5min;
(13) on shaking table, with PBS, wash 5min * 3 time, note lucifuge;
(14) with anti-fluorescent quenching mountant mounting;
(15) fluorescence microscope Microscopic observation.
Result is observed: under optical microscope, get infarction tissue's periphery 2mm scope and observe, 6 visuals field (200 * or 400 *) statistics positive cell number is got in every section, averages as measured value.
1.8 mouse brain tissue paraffin section de immunohistochemical staining
(1) dewaxing, aquation (with 1.7);
(2) on shaking table, wash paraffin section with PBS, 5min * 2 time;
(3) antigen retrieval: electric furnace or water-bath heating 0.01M sodium citrate buffer solution (pH6.0), to 95 ℃ of left and right, are put into brain tissue slice heating 20min;
(4) slowly cool to room temperature;
(5) adding concentration is 3% H
2O
2Hatch the 30min(room temperature);
(6), on shaking table, wash 5min * 3 time with PBS;
(7) add 3%BSA, 37 ℃ of constant temperature incubation 30min, dry, and do not wash;
(8) drip primary antibodie, 4 ℃ are spent the night; From after 4 ℃ of taking-ups, room temperature is placed rewarming 30min;
(9), on shaking table, wash 5min * 3 time with PBS;
(10) drip the polymer adjuvant work of the super quick two-step method two of rabbit in anti-also, 37 ℃ of incubation 15min;
(11), on shaking table, wash 5min * 3 time with PBS;
(12) drip the horseradish peroxidase labelling goat-anti rabbit polyclonal IgG II anti-working solution (1:200) of the super quick two-step method two of rabbit in anti-, 37 ℃ of incubation 20min;
(13), on shaking table, wash 5min * 3 time with PBS;
(14) in the use, the DAB of mountain company colour developing suit develops the color, micro-Microscopic observation colour developing situation, distilled water cessation reaction (each of 1ml distilled water+A, B, C liquid);
(15) haematoxylin is redyed, about 5min;
(16) hydrochloride alcohol differentiation, depending on redying the depth; Tap water rinses, and 1% ammonia returns indigo plant;
(17) dehydration: 80% ethanol, 90% ethanol, dehydrated alcohol, dimethylbenzene respectively once, need 5min at every turn;
(18) mounting: drip resinene, add coverslip (tissue site is sure not residual minute bubbles), naturally dry;
(19) observed result under optical microscope.
Result is observed: under optical microscope, get infarction tissue's periphery 2mm scope and observe, 6 visuals field (200 * or 400 *) statistics positive cell number is got in every section, averages as measured value.
1.9 mouse brain tissue homogenate superoxide dismutase (SOD) vigor detects
(1) preparation of brain tissue homogenate: accurately take tissue weight, volume ratio adds the normal saline of 9 times to make tissue homogenate by weight, and 2500 rev/mins, centrifugal 10min, get i.e. 10% tissue homogenate of supernatant;
(2) supernatant preparation: get 10% tissue homogenate 0.1ml, add 0.1ml reagent one and mix, 4000/ minute centrifugal 10min, get supernatant to be measured;
(3), after above-mentioned sample is ready to, measure protein concentration;
(4) according to following table, carry out application of sample;
Table 3: mouse brain tissue homogenate superoxide dismutase (SOD) vigor detects and adds quadrat method
(5) computing formula
(6) application SPSS17.0 software carries out statistical analysis.Measurement data is with mean ± standard deviation
Mean, the relatively employing one factor analysis of variance between a plurality of sample averages, the SNK method of relatively going in twos between group is checked.There is statistical significance P<0.05 for difference.
2.0 mouse brain tissue homogenate malonaldehyde (MDA) assay
(1) according to 1.9, prepare brain tissue homogenate's supernatant;
(2) according to following table, carry out application of sample;
Table 4: mouse brain tissue homogenate malonaldehyde (MDA) assay adds quadrat method
(3) whirlpool mixes, the test tube mouth is tightened with antistaling film, with syringe needle thorn one aperture, 95 ℃ of water-bath 40min, after taking out, flowing water is cooling, then 4000 rev/mins, centrifugal 10min, get supernatant and be added in cuvette, at visible spectrophotometer 532nm place, the 1cm optical path, the distilled water zeroing, measure and respectively manage absorbance;
(4) computing formula is: MDA content (nmol/mgprot) in tissue=(measuring pipe absorbance-mensuration blank tube absorbance)/(standard pipe absorbance-standard blank tube absorbance) * standard substance concentration (10nmol/ml) ÷ sample to be tested protein concentration (mgprot/ml);
(5) application SPSS17.0 software carries out statistical analysis.Measurement data is with mean ± standard deviation
Mean, the relatively employing one factor analysis of variance between a plurality of sample averages, the SNK method of relatively going in twos between group is checked.There is statistical significance P<0.05 for difference.
Result:
Table 5: the impact of chrysin on MCAO/R mice Brain Tissue SOD Vigor, MDA content
With sham operated rats, compare,
aP<0.05; With model group, compare,
bP<0.05
2.1 mouse brain organizes real-time quantitative PCR to detect
(1) primer sequence
(2) specimen leaves and takes
After each experimental mice sacrificed by decapitation, under sterile working, get infarction side cortex cerebral tissue, take fast 100mg, put in cryopreservation tube put into liquid nitrogen preserve stand-by, for extracted total RNA.
(3) total tissue RNA is extracted
A. get and organize 50mg, by follow procedure, extract total RNA and in ice bathing homogenizer, add 1ml Trizol and 50mg tissue, grind rapidly homogenate is transferred in the aseptic centrifuge tube of DEPC processing, 4 ℃ of centrifugal 10min of 12000rpm.
B. supernatant is transferred in another centrifuge tube, after incubated at room 5min, adds the 200ml chloroform, and concussion mixes, more at room temperature hatches 3min, acutely jolts 30s, places 5-10min on ice
C.4 ℃, 12000rpm, centrifugal 20min
D. get the upper water phase transfer to another EP pipe, add isopyknic isopropyl alcohol, more than mixing gently-20 ℃ of placement 2h
e.4℃，12000rpm，20min
F. abandon gently supernatant, add 1ml75% ethanol and mix gently
G.4 ℃, 12000rpm, 10min, abandon supernatant gently, and dry in the sun 5min in air adds the deionized water dissolving precipitation that 20 μ l DEPC process, and RNA are dissolved in 58 ℃ of water-baths, and-80 ℃ save backup
(4) extract the detection of RNA integrity
Get RNA solution 4 μ l, detect through 1% agarose gel electrophoresis, obviously visible 28S, 18S band, and 28S is the twice of 18S band, 5S band a little less than, and disperse, show that the RNA degraded is less, integrity is good.With 756 type ultraviolet spectrophotometers, measure respectively OD
260With OD
280, OD
260/ OD
280Ratio, between 1.8-2.0, shows the RNA protein contamination of extraction seldom, therefore can be in follow-up reverse transcription reaction.
(5) extract the quantitative of RNA
With 756 type ultraviolet spectrophotometers, measure OD
260/ OD
280Ratio, can detect purity and the content of RNA, selects OD
260/ OD
280Ratio is that the RNA of 1.8-2.0 is used as reverse transcription.Rna content adopts the UV spectrophotometer measuring method.
(6) cDNA the first chain is synthetic
Table 6:cDNA the first chain synthesis reaction liquid forms
PCR thermal circulation parameters: 96 ℃ of 4min, then three-step reactions: 94 ℃ of 30S, 8 ℃ of 30s, 2 ℃ of 30s, 40 circulations of row, 72 ℃ of 30s of the 3rd step of each circulation collect fluorescence signal.
(8) real-time fluorescence quantitative PCR interpretation of result
After amplification, enter the interpretation of result interface, the β-actin of take is the internal reference gene, compares with matched group, obtains the relative quantification value (RQ value) of destination gene expression, and the RQ Data-Statistics is analyzed.
Table 8: chrysin is organized the relative expression quantity of NF-κ B, iNOS, COX-2 gene to the MCAO/R mouse brain
With sham operated rats, compare,
aP<0.05; With model group, compare,
bP<0.05
2.2 the content that mouse brain is organized inflammatory factor in supernatant is measured in elisa (enzyme-linked immunosorbent assay, ELISA)
(1) in advance 20min from refrigerator, taking out test kit, with balance to room temperature;
(2) Wash buffer diluent: with distilled water, dilute with 1:10,50ml wash buffer adds the 450ml distilled water to 500ml;
(3) Assay buffer diluent: with distilled water, dilute with 1:20,5ml assay buffer adds the 95ml distilled water to 100ml;
(4) Sample Dilution Buffer diluent: 2ml10%BSA adds Sample Dilution Buffer to 20ml;
(5) preparation of HRP junctional complex (Avidin-HRP): with the Assay buffer liquid of 1 times, dilute with 1:1000, at the bottom of 11 μ l HRP junctional complexs, liquid joins in 11ml Assay buffer.
(6) preparation of Antigen Standard Cocktail: 12 kinds of antigen standard substance are respectively drawn to the Sample Dilution Buffer that 10 μ l put into 880 μ l dilutions, be mixed with concentrated Antigen Standard Cocktail, then by 1:4, be mixed with Final Antigen Standard Cocktail. with the Sample Dilution Buffer of dilution
(7) Dection Antibodies diluent: add 855 μ l Assay Buffer to mix in 12 kinds of Detection Antibodies respectively.
(8) from balance to the sealing bag of room temperature, take out the required lath of test;
(9) with multichannel pipettor, add 50 μ l Assay Buffer to wash plate to each hole:
(10) application of sample: respectively specimen and 12 kinds of standard substance are added to (50ul/ hole) in respective aperture, seal reacting hole with the shrouding gummed paper, incubated at room 2h;
(11) wash plate 3 times:
(12) add the Dection Antibody Solution of 100 μ l dilutions, incubated at room 1h;
(13) wash plate 4 times;
(14) add 100 μ l Avidin-HRP, incubated at room 30min;
(15) wash plate 4 times;
(16) add 100 μ l Development Solution, the room temperature lucifuge is hatched 15min;
(17) add 100 μ l Stop Solution, microplate reader 450nm reads at place the OD value.
Chrysin 50g is mixed homogeneously with 280g starch, with starch slurry (get starch 220g water and make starch slurry) granule processed, sieves, and drying, encapsulated.
Chrysin 80g is mixed homogeneously with starch 340g, with starch slurry (get starch 210g water and make starch slurry) granule processed, sieves, and drying, add 6% magnesium stearate, mixes, and in flakes, film coating gets final product in compacting.
Chrysin 100g, starch 100g and cellulose sieve in right amount, and fully mix, appropriate polyvinylpyrrolidonesolution solution is mixed with above-mentioned powder, sieve, make wet granular and 60 ℃ of dryings, by the hydroxymethyl starch sodium salt, 6% magnesium stearate and Pulvis Talci sieve in advance, then join tabletting in above-mentioned granule.
Above embodiments of the invention are had been described in detail, but described content is only preferred embodiment of the present invention, not in order to limit the present invention.All any modifications of making in application range of the present invention, be equal to and replace and improvement etc., within all should being included in protection scope of the present invention.
Claims (4)
1. the application of chrysin aspect preparation treatment ischemic cerebral apoplexy Chinese medicine, the structure of described chrysin be as the formula (1):
2. application according to claim 1, is characterized in that the dosage form of described medicine comprises oral agents and injection.
3. application according to claim 1, is characterized in that effective dose every day of described chrysin is the 47-53mg/kg body weight.
4. application according to claim 1, is characterized in that effective dose every day of described chrysin is the 50mg/kg body weight.
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Cited By (4)
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| CN105726537A (en) * | 2016-03-11 | 2016-07-06 | 李春华 | Application of domperidone in preparation of therapeutics for cerebral ischemia-reperfusion injury |
| CN109432081A (en) * | 2018-12-07 | 2019-03-08 | 西南交通大学 | A kind of compound is preparing the application in neuroprotection and/or neural restoration Related product |
| CN109966291A (en) * | 2019-04-16 | 2019-07-05 | 苏州大学 | Application and its pharmaceutical composition of the compound SB216763 in the drug of preparation prevention and/or treatment cranial vascular disease |
| WO2021115412A1 (en) * | 2019-12-12 | 2021-06-17 | 南方医科大学珠江医院 | Use of nitric oxide synthase pathway inhibitor in preparation of medicine |
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| CN109061139A (en) * | 2018-06-19 | 2018-12-21 | 温州医科大学附属第医院 | Application of the serum inflammatory biomarker in prevention and treatment acute cerebral ischemic infarction |
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