CN107375821A - A kind of Chinese medicine composition is preparing the application in protecting vascular endothelial cell medicine - Google Patents
A kind of Chinese medicine composition is preparing the application in protecting vascular endothelial cell medicine Download PDFInfo
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Abstract
The invention discloses a kind of application of Chinese medicine composition in the medicine for preparing protection endothelial cell, experiment is confirmed after being handled by carcinogenic chemical and/or mechanical damage Human Umbilical Vein Endothelial Cells, can Human Umbilical Vein Endothelial Cells produce infringement, cell chemotactic factor and expression of receptor is caused to lack of proper care, endothelial cell after being anticipated using the Chinese medicine composition, handled again by carcinogenic chemical and/or mechanical damage Human Umbilical Vein Endothelial Cells, it was found that Chinese medicine composition of the present invention can significantly improve the expression imbalance of the endothelial cell chemotactic factor and acceptor, endothelial cell existence activity is improved simultaneously, promote growing multiplication, increase nitric oxide (NO) secretion, reduce Endothelin receptor A (ET 1) expression, improve endothelial cells secrete function.Therefore it can be confirmed that Chinese medicine composition of the present invention can effectively protect endothelial cell.
Description
Technical field
The present invention relates to a kind of pharmaceutical applications of Chinese medicine composition, specifically, being related to the Chinese medicine composition is preparing protection
Application in endothelial cell medicine.
Background technology
Endothelial cell, some being distributed across in brain, lymph node, lung, liver, spleen etc. organ-tissue have common feature
Phagocyte general name, they swallow foreign matter, bacterium, necrosis and the tissue of aging, also participate in collective Immunization Activities.Endothelium is thin
Born of the same parents can synthesize and secrete various bioactivators, adjust body function.
Chemotactic factor (CF) is the small cytokines for being capable of the movement of chemotactic cell directional, and first chemotactic was found from 1988
Since factor CCL2 (MCP21), the structure of chemotactic factor (CF) and its acceptor, function and effect in vivo have become numerous
The focus of person's research.The interaction of chemotactic factor (CF) and its acceptor participates in growth, development, differentiation, apoptosis and distribution of cell etc.
Different physiological roles, and being played a significant role in a variety of pathologic processes, as inflammatory reaction, pathogenic infection, wound repair and
Tumour formation and transfer etc..
Chemotactic factor (CF) is small molecule basic protein, is made up of 70-125 amino acid, molecular weight 6-14kD.So far
At least have found that more than 50 plant chemotactic factor (CF) untill the present, can be by chemotactic factor (CF) according to the position of its N-terminal cysteine residues and number
It is divided into 4 subtribes:C, CC, CXC and CX3C.CC, CXC and CX3C chemotactic factor (CF) have 4 conservative cysteines, and C chemotactics because
Son only has 2 cysteines, equivalent to former three the 2nd and the position of the 4th cysteine.2 half Guang ammonia before CC chemotactic factor (CF)s
Sour neighbour, and 1 and 3 other amino acid are respectively separated before CXC and CX3C chemotactic factor (CF)s between 2 cysteines.It is most
Chemotactic factor (CF) belongs to CC and the races of CXC two, and the former has 28 members, and the latter includes 16 member's chemotactic factor (CF)s.
Chemotactic factor (CF) with the specific receptor of cell surface by combining the corresponding biological action of performance.Chemotactic factor (CF) by
Body category g protein coupled receptor, there are 7 membrane spanning segments, be mainly expressed in each WBC sub-population of derived from bone marrow, while also express
In on the cell of the types such as epithelial cell, vascular endothelial cell, nerve cell.The part that chemokine receptors is combined according to it is not
It is same to be also classified into 4 subfamilies:C chemokine receptors (XCR), CC chemokine receptors (CCR), Gro-beta-T
Acceptor (CXCR) and CX3C chemokine receptors (CX3CR).At least clone 18 kinds of chemokine receptors, wherein XCR at present
There is a kind, CCR10 kinds, CXCR6 kinds and CX3CR1 kinds.Some chemotactic factor (CF)s are specifically combined with a kind of acceptor, such as
CXCL12 (SDF21) is only combined with CXCR4;And some chemotactic factor (CF)s can be combined with several acceptors, such as CCL5
(RAN2TES) can be combined with CCR1, CCR3 and CCR5;It is equally sometimes a kind of to be combined by physical efficiency with several chemotactic factor (CF)s, such as,
CCR3 can combine a variety of chemotactic factor (CF)s such as CCL5, CCL7, CCL8, CCL24 and CCL26, this result cause a kind of chemotactic because
Son can express the immunocyte directional migration of different chemokine receptors with chemotactic, and a kind of immunocyte can also be it is a variety of become
Change the factor to be recruited.
Endothelin (ET) is the 21 amino acid groups of one kind isolated and purified out from the aortic endothelial cell of pig for 1988
Into vasoactive polypeptides.It is distributed mainly in kidney, liver, the heart, brain, sustainer and vein endothelial cell, is a kind of endogenous
Property often effect and potent vasoconstrictor, vessel retraction, platelet aggregation and smooth muscle cell proliferation can be caused.1980 first
It was found that endothelium-derived relaxing factor (EDRF) turned out to be nitric oxide (NO).It is thin from endothelial cell, cranial nerve
Born of the same parents, non-adrenal gland non-cholinergic neuronal epithelial cell (NANC) and by toxin, the macrophage and smooth muscle of cytokine activation
Cell.On the one hand NO has vasodilator effect, on the other hand as cell interior and the courier of intercellular trafficking biological information
Played a role under the pathological conditions such as ischemic.Endothelin and nitric oxide are all important endothelial factors.Research prompting blood plasma ET
The horizontal reacting condition coincident with severity degree of condition of ICVD.
The content of the invention
The present invention relates to a kind of new application of Chinese medicine composition, in particular it relates to which a kind of Chinese medicine composition is preparing protection
Application in endothelial cell medicine.
Chinese medicine of the present invention can be according to《National Chinese medicine preparation specification》Or《Dictionary of medicinal plant》Processed.The present invention
Medicine is made of the bulk drug of following weight ratio:
Radix Astragali 120-360, fruit of glossy privet 100-300, ginseng 30-95, ganoderma lucidum 30-95, curcuma zedoary 65-195, bighead atractylodes rhizome 30-90, Sculellaria barbata
65-195, gynostemma pentaphylla 120-360, Poria cocos 30-95, the membrane of a chicken's gizzard 15-45, mock-strawberry 65-195, bittersweet 65-195, oriental wormwood 65-195,
Paniculate swallowwort 65-195, ground bettle 10-30, oldenlandia diffusa 65-195.
Preferably, the Chinese medicine composition is made up of the bulk drug of following parts by weight:
The Radix Astragali 120, the fruit of glossy privet 300, ginseng 30, ganoderma lucidum 95, curcuma zedoary 65, the bighead atractylodes rhizome 90, Sculellaria barbata 65, gynostemma pentaphylla 360, Poria cocos 30,
The membrane of a chicken's gizzard 45, mock-strawberry 65, bittersweet 195, oriental wormwood 65, paniculate swallowwort 195, ground bettle 10, oldenlandia diffusa 195.
Or
The Radix Astragali 360, the fruit of glossy privet 100, ginseng 95, ganoderma lucidum 30, curcuma zedoary 195, the bighead atractylodes rhizome 30, Sculellaria barbata 195, gynostemma pentaphylla 120, Poria cocos
95th, the membrane of a chicken's gizzard 15, mock-strawberry 195, bittersweet 65, oriental wormwood 195, paniculate swallowwort 65, ground bettle 30, oldenlandia diffusa 65.
Or
The Radix Astragali 250, the fruit of glossy privet 200, ginseng 65, ganoderma lucidum 65, curcuma zedoary 132, the bighead atractylodes rhizome 64, Sculellaria barbata 128, gynostemma pentaphylla 256, Poria cocos
65th, the membrane of a chicken's gizzard 30, mock-strawberry 128, bittersweet 128, oriental wormwood 128, paniculate swallowwort 128, ground bettle 20, oldenlandia diffusa 128.
Preferably, in the raw materials used medicine of the Chinese medicine composition, the bighead atractylodes rhizome is rhizoma atractylodis macrocephalae.
Present invention also offers the active component of the Chinese medicine composition to be made up of following steps:
(1) Chinese medicine, is weighed according to bulk drug part by weight, is cleaned;
(2), the fruit of glossy privet, people participate in 6-10 times and measure 50-90% ethanol extraction 1-3 times, each 1-4 hours, merge extract solution, mistake
Filter, filtrate recycling ethanol are extremely standby without alcohol taste, filtrate and the dregs of a decoction;
(3), curcuma zedoary, the bighead atractylodes rhizome, paniculate swallowwort add 4-8 times to measure water extraction volatile oil, collect volatile oil, and another device is collected, residue and water-soluble
Liquid is standby;
(4), ground bettle, the membrane of a chicken's gizzard, Poria cocos are ground into fine powder;
(5) income earner in, the Radix Astragali, ganoderma lucidum, oldenlandia diffusa, Sculellaria barbata, gynostemma pentaphylla, mock-strawberry, bittersweet, oriental wormwood, with step (2)
Join, residue after the alcohol extracting of the fruit of glossy privet, and the middle gained curcuma zedoary of step (3), the bighead atractylodes rhizome, paniculate swallowwort propose residue merging after oil, add 7-10 times
Water is measured, heating decocts 1-3 times, each 1-3 hours, merges decoction liquor, adds gained ginseng in step (2), fruit of glossy privet alcohol extracting filter
Liquid and step(3)In the aqueous solution, be concentrated into decoction 65 DEG C survey when relative density be 1.15-1.30, dry, pulverize, it is standby;
Step(4)Gained comminuted powder, step(3)Gained volatile oil and step(5)The dried cream powder of gained collectively forms the Chinese medicine group
The active component of compound.
The formulation of medicine of the present invention is capsule, tablet, powder, oral liquid, soft capsule, pill, tincture, syrup, bolt
Agent, gel, spray or injection.
The wherein preparation method of capsule, is made up of following steps:
(1) Chinese medicine, is weighed according to bulk drug part by weight, is cleaned;
(2), ginseng, the fruit of glossy privet, 8 times of 70% alcohol refluxs of amount are added to extract 2 times, 3 hours for the first time, second 2 hours, merging carried
Liquid is taken, is filtered, recovery ethanol is extremely standby without alcohol taste, filtrate and ginseng, fruit of glossy privet residue;
(3), curcuma zedoary, the bighead atractylodes rhizome, paniculate swallowwort, merging extraction volatile oil, carry the oily time no less than 8 hours, the another device of volatile oil is collected,
Residue and the aqueous solution are standby;
(4), ground bettle, the taste animal drugs of the membrane of a chicken's gizzard two, washing, 60 DEG C of drying, merge with Poria cocos, be ground into 100 mesh powder, sterilize
It is standby afterwards;
(5) income earner in, the Radix Astragali, ganoderma lucidum, oldenlandia diffusa, Sculellaria barbata, gynostemma pentaphylla, mock-strawberry, bittersweet, oriental wormwood, with step (2)
Join, residue after the alcohol extracting of the fruit of glossy privet, and the middle gained curcuma zedoary of step (3), the bighead atractylodes rhizome, paniculate swallowwort propose residue merging after oil, add 9 times of amounts
Water, heating decoct 2 times, 2 hours every time, merge decoction liquor, add gained ginseng, fruit of glossy privet alcohol extracting filtrate and step in step (2)
Suddenly(3)In the aqueous solution, be condensed into relative density 1.20-1.25 clear cream, dry, pulverize, get dry extract powder;By gained dried cream powder
Add appropriate pharmaceutically acceptable auxiliary material granulation;
(6), gained volatile oil in step (3) is sprayed into the fine powder of the middle gained Poria cocos of step (4), ground bettle, the membrane of a chicken's gizzard, mixed
It is even, with step(5)The particle of middle gained mixes, closed half an hour, encapsulated to produce.
After other formulations of medicine of the present invention weigh bulk drug in proportion, prepared using the preparation method of routine, for example, model
Bi Ting《Pharmacy of Chinese materia medica》(Shanghai Science Press December the 1st edition in 1997)The preparation technology of record, pharmacy, which is made, to be connect
The regular dosage form received.
To enable above-mentioned formulation to realize, pharmaceutically acceptable auxiliary material need to be added when preparing these formulations, such as:Filling
Agent, disintegrant, lubricant, suspending agent, adhesive, sweetener, flavouring, preservative, matrix etc..Filler includes:It is starch, pre-
Gelling starch, lactose, mannitol, chitin, microcrystalline cellulose, sucrose etc.;Disintegrant includes:Starch, pregelatinized starch, crystallite
Cellulose, sodium carboxymethyl starch, PVPP, low-substituted hydroxypropyl cellulose, Ac-Di-Sol etc.;
Lubricant includes:Magnesium stearate, lauryl sodium sulfate, talcum powder, silica etc.;Suspending agent includes:Polyvinylpyrrolidine
Ketone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methyl cellulose etc.;Adhesive includes, starch slurry, polyvinylpyrrolidone,
Hydroxypropyl methyl cellulose etc.;Sweetener includes:Saccharin sodium, aspartame, sucrose, honey element, enoxolone etc.;Flavouring bag
Include:Sweetener and various essence;Preservative includes:Parabens, benzoic acid, sodium benzoate, sorbic acid and its esters, benzene prick bromine
Ammonium, acetic acid chloroethene are fixed, eucalyptus oil etc.;Matrix includes:PEG6000, PEG4000, insect wax etc..In above-mentioned formulation is realized
Medicine pharmacy, pharmaceutically acceptable other auxiliary materials need to be added when preparing these formulations(Fan Biting《Pharmacy of Chinese materia medica》, Shanghai section
Learn the auxiliary material that each formulation is recorded in publishing house December the 1st edition in 1997).
Embodiment
Embodiment 1:
Raw material medicine composition is:
Radix Astragali 250g, fruit of glossy privet 200g, ginseng 65g, ganoderma lucidum 65g, curcuma zedoary 132g, rhizoma atractylodis macrocephalae 64g, Sculellaria barbata 128g, gynostemma pentaphylla
256g, Poria cocos 65g, the membrane of a chicken's gizzard 30g, mock-strawberry 128g, bittersweet 128g, oriental wormwood 128g, paniculate swallowwort 128g, ground bettle 20g, long-noded pit viper
Tongue grass 128g.
Preparation method is:
(1) Chinese medicine, is weighed according to bulk drug part by weight, is cleaned;
(2), ginseng, the fruit of glossy privet, 8 times of 70% alcohol refluxs of amount are added to extract 2 times, 3 hours for the first time, second 2 hours, merging carried
Liquid is taken, is filtered, recovery ethanol is extremely standby without alcohol taste, alcohol extracting filtrate and ginseng, fruit of glossy privet residue;
(3), curcuma zedoary, rhizoma atractylodis macrocephalae, paniculate swallowwort, merge extraction volatile oil, carry the oily 8 hours time, the another device of volatile oil is collected, residue and
The aqueous solution is standby;
(4), ground bettle, the membrane of a chicken's gizzard, washing, 60 DEG C of drying, merge with Poria cocos, be ground into 100 mesh powder, radiated in 3KGY60CO-r
It is standby after sterilizing;
(5) income earner in, the Radix Astragali, ganoderma lucidum, oldenlandia diffusa, Sculellaria barbata, gynostemma pentaphylla, mock-strawberry, bittersweet, oriental wormwood, with step (2)
Join, residue after the alcohol extracting of the fruit of glossy privet, and the middle gained curcuma zedoary of step (3), rhizoma atractylodis macrocephalae, paniculate swallowwort propose the residue merging after oil, add 9 times
Measure water, heating decocts 2 times, 2 hours every time, merges decoction liquor, add gained ginseng in step (2), fruit of glossy privet alcohol extracting filtrate and
Step(3)In the aqueous solution, be condensed into the clear cream of relative density 1.25, dry, pulverize, it is standby;
(6) dried cream powder obtained by step (5), is added into 134 grams of starch, with 85% alcohol granulation;
(7), gained volatile oil in step (3) dissolve with 85% ethanol, spray into step (4) it is middle obtained by Poria cocos, ground bettle, in chicken
In the fine powder of gold, mix, with step(6)The particle of middle gained mixes, closed half an hour, loads 1000 capsules and produces.
Embodiment 2:
Raw material medicine composition is:
Radix Astragali 360g, fruit of glossy privet 100g, ginseng 95g, ganoderma lucidum 30g, curcuma zedoary 195g, rhizoma atractylodis macrocephalae 30g, Sculellaria barbata 195g, gynostemma pentaphylla
120g, Poria cocos 95g, the membrane of a chicken's gizzard 15g, mock-strawberry 195g, bittersweet 65g, oriental wormwood 195g, paniculate swallowwort 65g, ground bettle 30g, oldenlandia
Careless 65g.
Preparation method is:
(1) Chinese medicine, is weighed according to bulk drug part by weight, is cleaned;
(2), ginseng, the fruit of glossy privet, 6 times of 90% alcohol refluxs of amount are added to extract 4 hours, extract solution filtering, extract solution reclaims ethanol to nothing
Alcohol taste, alcohol extracting filtrate and ginseng, fruit of glossy privet residue are standby;
(3), curcuma zedoary, rhizoma atractylodis macrocephalae, paniculate swallowwort merge, and add 4 times of amount water extraction volatile oil, carry oily 10 hours time, the another device of volatile oil
Collect, residue and the aqueous solution are standby;
(4), ground bettle, the membrane of a chicken's gizzard, washing, 60 DEG C of drying, merge with Poria cocos, be ground into 100 mesh powder, radiated in 3KGY60CO-r
It is standby after sterilizing;
(5) income earner in, the Radix Astragali, ganoderma lucidum, oldenlandia diffusa, Sculellaria barbata, gynostemma pentaphylla, mock-strawberry, bittersweet, oriental wormwood, with step (2)
Join, residue after the alcohol extracting of the fruit of glossy privet, and the middle gained curcuma zedoary of step (3), rhizoma atractylodis macrocephalae, paniculate swallowwort propose the residue merging after oil, add 7 times
Water is measured, heating decocts 3 times, 1 hour for the first time, second 2 hours, third time 3 hours, merges decoction liquor, adds in step (2)
Gained ginseng, fruit of glossy privet alcohol extracting filtrate and step(3)In the aqueous solution, be condensed into the clear cream of relative density 1.15, dry, powder
It is broken, it is standby;
(6) dried cream powder obtained by step (5), is added into 112 grams of starch, with 78% alcohol granulation;
(7), gained volatile oil in step (3) dissolve with 85% ethanol, spray into step (4) it is middle obtained by Poria cocos, ground bettle, in chicken
In the fine powder of gold, mix, with step(6)The particle of middle gained mixes, and routinely 1000 tablets are made in formulation method.
Embodiment 3:
Raw material medicine composition is:
Radix Astragali 120g, fruit of glossy privet 300g, ginseng 30g, ganoderma lucidum 95g, curcuma zedoary 65g, bighead atractylodes rhizome 90g, Sculellaria barbata 65g, gynostemma pentaphylla 360g,
Poria cocos 30g, the membrane of a chicken's gizzard 45g, mock-strawberry 65g, bittersweet 195g, oriental wormwood 65g, paniculate swallowwort 195g, ground bettle 10g, oldenlandia diffusa
195g。
Preparation method is:
(1) Chinese medicine, is weighed according to bulk drug part by weight, is cleaned;
(2), ginseng, the fruit of glossy privet, 10 times of 50% alcohol refluxs of amount are added to extract 2 times, 3 hours for the first time, second 2 hours, merging carried
Liquid is taken, is filtered, recovery ethanol is extremely standby without alcohol taste, alcohol extracting filtrate and ginseng, fruit of glossy privet residue;
(3), curcuma zedoary, the bighead atractylodes rhizome, paniculate swallowwort merge, and add 10 times of amount water extraction volatile oil, carry oily 6 hours time, the another device of volatile oil
Collect, residue and the aqueous solution are standby;
(4), ground bettle, the membrane of a chicken's gizzard, washing, 60 DEG C of drying, merge with Poria cocos, be ground into 100 mesh powder, radiated in 3KGY60CO-r
It is standby after sterilizing;
(5) income earner in, the Radix Astragali, ganoderma lucidum, oldenlandia diffusa, Sculellaria barbata, gynostemma pentaphylla, mock-strawberry, bittersweet, oriental wormwood, with step (2)
Join, residue after the alcohol extracting of the fruit of glossy privet, and the middle gained curcuma zedoary of step (3), the bighead atractylodes rhizome, paniculate swallowwort propose the residue merging after oil, add 10 times
Water is measured, heating decocts 3 hours, merges decoction liquor, adds gained ginseng, fruit of glossy privet alcohol extracting filtrate and step in step (2)(3)In
The aqueous solution, be condensed into the clear cream of relative density 1.30, dry, pulverize, it is standby;
(6), gained volatile oil in step (3) dissolve with 80% ethanol, spray into step (4) it is middle obtained by Poria cocos, ground bettle, in chicken
In the fine powder of gold, mix, with step(5)The dried cream powder of middle gained, routinely formulation method 1000 pills are made.
Experimental example:
To confirm the therapeutic effect of medicine of the present invention, with the Capsule content extract solution as made from embodiment 1(Hereinafter referred to as
DME25), carried out tests below research:
1. material and method:
1.1 DME25 preparation:
The Capsule content as made from embodiment 1 is added to DMSO(Dimethyl sulfoxide (DMSO))In solution, 4 C, 100rpm, rotation
Instrument concussion processing 12 hours;Solution centrifuges 20 minutes under the conditions of 4 C, rotating speed 15000g, then takes supernatant, 0.20 μm of filter
DME25 is made after membrane filtration, 4 C are saved backup.
The foundation of 1.2 chemicals endothelial cell injury models and pharmaceutical intervention experiment:
By the endothelial cell of identical quantity(Human umbilical vein endothelial cell line HUVEC, human vascular endothelial system HECV)Connect respectively
Kind into 25 square centimeters or so of Tissue Culture Dish, be placed in 37 DEG C, in 5% CO2 incubators culture until cell fusion 80% is left
The right side, reject nutrient solution, experiment packet are as follows:1. blank control group:With normal nutrient solution culture HUVEC or HECV;2. DIET is damaged
Hinder group:With containing chemicals(DIET)Nutrient solution culture HUVEC or HECV;3. pharmaceutical intervention group:First with the training containing DME25
Nutrient solution preincubate 8 hours, discards nutrient solution, with containing chemicals(DIET)With DME25 nutrient solution culture HUVEC or
HECV.Each group is incubated 8 hours.
The foundation of 1.3 mechanical damage endothelial cell models and pharmaceutical intervention experiment:
By the endothelial cell of identical quantity(Human umbilical vein endothelial cell line HUVEC, human vascular endothelial system HECV)Connect respectively
Kind into 25 square centimeters or so of Tissue Culture Dish, be placed in 37 DEG C, in 5% CO2 incubators culture until cell fusion 80% is left
It is right.Reject nutrient solution, experiment packet are as follows:1. blank control group:With normal nutrient solution culture HUVEC or HECV;2. machinery damage
Hinder group:After damaging monolayer endothelial cell using more pipette stations, with normal nutrient solution culture HUVEC or HECV;3. medicine is done
Pre- group:First with the nutrient solution preincubate 8 hours containing DME25, nutrient solution is discarded, it is thin to damage individual layer endothelium using more pipette stations
After born of the same parents, with nutrient solution culture HUVEC or HECV containing DME25.Each group is incubated 8 hours.
2. Testing index
The observation of 2.1 each group endothelial cell morphologies
The change of each group endothelial cell morphology after chemicals and mechanical damage endothelial cell is observed using inverted microscope.
2.2 methods detection each group cells survival activity
After experiment terminates, cell culture supernatant is removed, adds the μ L of serum-free basic culture solution 100 containing CCK-8(CCK-8
Solution:Serum-free basic culture solution=1:10), 37 DEG C are placed in, continues to be incubated 1-4h, ELIASA detection in 5% CO2 incubators
OD values at 450 nm wavelength.Using blank control group OD values as reference, cell survival rate is with each group OD values and blank control group OD
The ratio of value represents.
2.3 each group cell culture supernatant nitric oxides(Nitric oxide, NO)And endothelin -1(endothelin-
1, ET-1)The change of content
After experiment terminates, each group cell culture supernatant is collected, after 1000 rpm centrifuge 2 min, cell fragment is removed, takes supernatant
Liquid.Using nitrate reductase method and enzyme linked immunosorbent assay(Enzyme linked immunosorbent assay, ELISA
Method)NO and ET-1 contents in cell supernatant are detected respectively, and laboratory operating procedures are carried out in strict accordance with specification.
2.4 genechip detection
RNA extraction:After experiment terminates, culture medium is absorbed, PBS 2 times, then add Trizol lysates(5x 105~
10 cells of x 105 add 1mL lysates)Cell lysis, gently rubs shake, cracks 5 min at room temperature, lysate is transferred to
In 1.5 mL EP pipes, add 0.2 mL chloroforms and shaken on turbula shaker 30 seconds, place 5 min on ice.Refrigerated centrifuge 4
DEG C, 12000 rpm centrifuge 15 min, and careful upper strata supernatant of drawing is moved in another 1.5 mL EP pipe, added isometric
Isopropanol mix, 2 h are stood in -20 DEG C of refrigerators.4 DEG C of refrigerated centrifuge again, 12000 rpm, 15 min are centrifuged, are discarded
Clearly, after adding the mixing of 1/3 volume absolute ethyl alcohol, 4 DEG C of refrigerated centrifuge, 12000 rpm, centrifuge 15 min again, absorb clean
After supernatant, precipitation is washed 3 times with RNA Wash Buffer, is added the treated deionized water dissolvings of 20 μ L DEPC, is obtained
Precipitated to total serum IgE.Pass through the purity and quantity of gel imaging and spectrophotometric determination nucleic acid.
MRNA separation and cDNA amplifications:Using micro- magnetic bead mRNA extracts kits, the mRNA in total serum IgE is separated, is used
bioanalyser 2100(Agilent)Detect ribosomes degraded.Pass through ion total serum IgE kit V2(Life
Technologies)Generate RNA fragment libraries(About 150~200bp), these fragments be incorporated into cDNA synthesis joint on.Profit
CDNA amplifications are carried out with the random RNA5' and 3' primers of ionic, pass through 2100 analysing amplified cDNA of bioanalyser piece
Duan great little and peak concentration.
The preparation of template and RNA sequencings:Utilize the kit v3 (Life of Ion PI template OT2 200
Technologies) kit is carried out further clone's amplification to cDNA fragment libraries by suspension PCR and forms ion microballoon
Particle (ISPs).Reclaimed and be enriched with by the Ion OneTouch ES systems ISP positive to template after PCR, and go unless
The positive ISP of template.The ISP of enrichment is sequenced using kit specification.In Torrent Suite version after sequencing
Base determination, joint finishing, bar code deconvolution and calibration are carried out by STAR RNA-seq calibration inserts on 3.6, and according to people
Genoid group(hg19)Form export data as BAM files.
3rd, statistical analysis
3.1 use the statistical analysis softwares of SPSS 19.0, and all continuous datas use mean ± standard deviation(±s)Represent.Examine first
Test whether data meet normality distribution, meet normal distribution uses one-way analysis of variance (One-Way ANOVA), if side
It is poor neat, compare two-by-two using least significant difference between group(Least significant difference, LSD)If variance is not
Qi Ze is examined using Dunnett`s T3, P<0.05 is that difference is statistically significant, P<0.01 is that difference has conspicuousness statistics
Meaning.
The result of 3.2 gene expressions is represented with mean ± standard deviation, statistical analysis is examined with t between group.
4th, result
The form of 4.1 each group endothelial cells compares
Blank control group HUVEC/HECV karyomorphisms are regular, edge clear, it is seen that cell division is bred;DIET damages group and
Mechanical damage group karyopyknosis, marginalisation, nucleus volume reduce, and karyorrhexis, produce apoptotic body;It is interior after pharmaceutical intervention
Chrotoplast edge is more visible, and karyopycnosis, marginalisation phenomenon postpone, lesser extent, and visible cell division growth.
4.2 chemicals DIET are damaged and the change of mechanical damage endothelial cell each group existence activity
4.2.1 the change of DIET endothelial cell injuries each group existence activity
Compared with blank control group, DIET damage group HUVEC/HECV endothelial cells existence activity reduces(P<0.01);With DIET
Damage group is compared, the significantly rise of pharmaceutical intervention group endothelial cell existence activity(P<0.01).(It is shown in Table 1)
4.2.2 the change of mechanical damage endothelial cell each group existence activity
Compared with blank control group, after mechanical damage HUVEC/HECV endothelial cells, endothelial cell existence activity is obvious to be reduced(P<
0.01);Compared with mechanical damage group, pharmaceutical intervention group endothelial cell existence activity is significantly raised(P<0.01).(It is shown in Table 2)
4.3 chemicals DIET and mechanical damage endothelial cell each group NO and ET-1 level change
4.3.1 the change of DIET endothelial cell injuries each group supernatant NO and ET-1 content
Compared with blank control group, DIET damage group supernatants NO is horizontal to be reduced(P<0.05), ET-1 levels are significantly raised(P<
0.01);Compared with DIET damage groups, pharmaceutical intervention group supernatant NO contents significantly raise(P<0.01), ET-1 contents significantly drop
It is low(P<0.01).(It is shown in Table 3)
4.3.2 the change of mechanical damage endothelial cell each group supernatant NO and ET-1 content
Compared with blank control group, after mechanical damage endothelial cell, NO is horizontal to be reduced(P<0.05, P<0.01), ET-1 is horizontal to be risen
It is high(P<0.05, P<0.01);Compared with mechanical damage group, pharmaceutical intervention group supernatant NO contents are significantly raised(P<0.01), ET-
1 content significantly reduces(P<0.01).(It is shown in Table 4)
4.4 genechip detection results
4.4.1 HUVEC/HECV cells CCL families factor expression situation
As shown in table 5, CCL28, CCL3, CCL5, CCL21, CCL23, CCL27 high level expression in HUVEC cells, CCL8,
CCL13 also has higher level expression;CCL3L3, CCL28 high level expression in HECV cells, CCL1, CCL3, CCL5, CCL8,
CCL23 also has higher level expression.
4.4.2 chemicals DIET/ mechanical damages HECV endothelial cells each group CCL families factor expression
As shown in table 6, chemicals DIET processing can induce HECV endothelial cells CXCL1, CCL2, CCL3, CCL5, CCL7,
CCL27, CXCL12 abnormal expression reduce, the rise of CCL14, CCL15, CCL21, CCL23 abnormal expression(P<0.05, P<0.01);
Medicine DME25 intervenes the expression that can improve CXCL1, CCL2, CCL3, CCL5, CCL7, CCL27, CXCL12(P<0.05, P<
0.01), reduce CCL14, CCL15, CCL21, CCL23 expression(P<0.05, P<0.01).
As shown in table 7, mechanical damage can cause CXCL1, CXCL2, CXCL6, CXCL16, CSCL6, CCL2 abnormal expression to raise(P
<0.05, P<0.01), the reduction of CCL14, CCL19, CCL28, CCL3, CCL5, CCL23 abnormal expression(P<0.05, P<0.01);Medicine
Thing DME25 intervenes the expression that can significantly reduce CXCL1, CXCL2, CXCL6, CXCL16, CSCL6, CCL2(P<0.05, P<
0.01), raising CCL14, CCL19, CCL28, CCL3, CCL5, CCL23 expression(P<0.05, P<0.01).
4.4.3 chemicals DIET/ mechanical damages HECV endothelial cells each group chemokine receptor expression
As shown in table 8, chemicals DIET can increase HECV endothelial cells CCR2 expression, reduce CCR7, CCRL1,
CXCR4, CXCR6, CXCR7 and CCR5 are expressed(P<0.05, P<0.01).Medicine DME25, which intervenes, can significantly reduce CCR2 expression,
Increase CCR5, CCRL1, CXCR4, CXCR7, CCR6 expression(P<0.05, P<0.01).
As shown in table 9, in HECV cells, mechanical damage causes CXCR4, CCRL1 to lower, CCR2 up-regulations(P<0.05).
DME25 lowers CXCR4, CCR2 expression, up-regulation CCRL1 expression after intervening(P<0.05).
5th, conclusion
Chemical irritants diethylstilbestrol(DIET)With mechanical damage method can endothelial cell injury, destroy endothelial cell shape
State, endothelial cell existence activity is reduced, causes endothelial cell karyopyknosis, apoptosis, suppresses endothelial cell growth propagation.Simultaneously
DIET and mechanical damage endothelial cell method can reduce endothelial cell NO secretions, increase ET-1 expression, endothelial cells secrete function
Unbalance, endothelial cell correlation factor CCL family's factors and chemokine receptors are not normal.After DME25 intervenes, endothelial cell can be protected
Form, endothelial cell existence activity is improved, promotion endothelial cell growth propagation, increase NO secretions, ET-1 is reduced and expresses, in improvement
Skin cell secretion function, adjust CCL family's factors and chemokine receptor expression.
Claims (10)
1. application of a kind of Chinese medicine composition in the medicine for preparing protection endothelial cell, it is characterised in that be following weight part ratio
Made of the bulk drug of example:
Radix Astragali 120-360, fruit of glossy privet 100-300, ginseng 30-95, ganoderma lucidum 30-95, curcuma zedoary 65-195, bighead atractylodes rhizome 30-90, half
Lotus 65-195, gynostemma pentaphylla 120-360, Poria cocos 30-95, the membrane of a chicken's gizzard 15-45, mock-strawberry 65-195, bittersweet 65-195, oriental wormwood 65-195
Paniculate swallowwort 65-195 ground bettle 10-30 oldenlandia diffusas 65-195.
2. application according to claim 1, it is characterised in that be made up of the bulk drug of following parts by weight:
The Radix Astragali 120, the fruit of glossy privet 300, ginseng 30, ganoderma lucidum 95, curcuma zedoary 65, the bighead atractylodes rhizome 90, Sculellaria barbata 65, gynostemma pentaphylla 360, Poria cocos 30,
The membrane of a chicken's gizzard 45, mock-strawberry 65, bittersweet 195, oriental wormwood 65, paniculate swallowwort 195, ground bettle 10, oldenlandia diffusa 195.
3. application according to claim 1, it is characterised in that be made up of the bulk drug of following parts by weight:
The Radix Astragali 360, the fruit of glossy privet 100, ginseng 95, ganoderma lucidum 30, curcuma zedoary 195, the bighead atractylodes rhizome 30, Sculellaria barbata 195, gynostemma pentaphylla 120, Poria cocos
95th, the membrane of a chicken's gizzard 15, mock-strawberry 195, bittersweet 65, oriental wormwood 195, paniculate swallowwort 65, ground bettle 30, oldenlandia diffusa 65.
4. application according to claim 1, it is characterised in that be made up of the bulk drug of following parts by weight:
The Radix Astragali 250, the fruit of glossy privet 200, ginseng 65, ganoderma lucidum 65, curcuma zedoary 132, the bighead atractylodes rhizome 64, Sculellaria barbata 128, gynostemma pentaphylla 256, Poria cocos
65th, the membrane of a chicken's gizzard 30, mock-strawberry 128, bittersweet 128, oriental wormwood 128, paniculate swallowwort 128, ground bettle 20, oldenlandia diffusa 128.
5. according to the application described in claim any one of 1-4, it is characterised in that the active component of the Chinese medicine composition by with
Lower step is made:
(1) Chinese medicine, is weighed according to bulk drug part by weight, is cleaned;
(2), the fruit of glossy privet, people participate in 6-10 times and measure 50-90% ethanol extraction 1-3 times, each 1-4 hours, merge extract solution, filter,
Filtrate recycling ethanol is extremely standby without alcohol taste, filtrate and the dregs of a decoction;
(3), curcuma zedoary, the bighead atractylodes rhizome, paniculate swallowwort add 4-8 times to measure water extraction volatile oil, collect volatile oil, and another device is collected, residue and water-soluble
Liquid is standby;
(4), ground bettle, the membrane of a chicken's gizzard, Poria cocos are ground into fine powder;
(5) income earner in, the Radix Astragali, ganoderma lucidum, oldenlandia diffusa, Sculellaria barbata, gynostemma pentaphylla, mock-strawberry, bittersweet, oriental wormwood, with step (2)
Join, residue after the alcohol extracting of the fruit of glossy privet, and the middle gained curcuma zedoary of step (3), the bighead atractylodes rhizome, paniculate swallowwort propose residue merging after oil, add 7-10 times
Water is measured, heating decocts 1-3 times, each 1-3 hours, merges decoction liquor, adds gained ginseng in step (2), fruit of glossy privet alcohol extracting filter
Liquid and step(3)In the aqueous solution, be concentrated into decoction 65 DEG C survey when relative density be 1.15-1.30, dry, pulverize, it is standby;
Step(4)Gained comminuted powder, step(3)Gained volatile oil and step(5)The dried cream powder of gained collectively forms the Chinese medicine group
The active component of compound.
6. according to the application described in claim any one of 1-4, it is characterised in that the pharmaceutical dosage form is capsule, tablet, dissipated
Agent, oral liquid, pill, tincture, syrup, suppository, gel, spray or injection.
7. application according to claim 6, it is characterised in that the preparation method of the medicine capsule is by following steps
It is made:
(1) Chinese medicine, is weighed according to bulk drug part by weight, is cleaned;
(2), ginseng, the fruit of glossy privet, 8 times of 70% alcohol refluxs of amount are added to extract 2 times, 3 hours for the first time, second 2 hours, merging carried
Liquid is taken, is filtered, recovery ethanol is extremely standby without alcohol taste, filtrate and ginseng fruit of glossy privet residue;
(3), curcuma zedoary, the bighead atractylodes rhizome, paniculate swallowwort, merge extraction volatile oil, carry oily time 6-12 hours, the another device of volatile oil is collected, residue
And the aqueous solution is standby;
(4), ground bettle, the taste animal drugs of the membrane of a chicken's gizzard two, washing, 60 DEG C of drying, merge with Poria cocos, 100 mesh powder are ground into, after sterilizing
It is standby;
(5) income earner in, the Radix Astragali, ganoderma lucidum, oldenlandia diffusa, Sculellaria barbata, gynostemma pentaphylla, mock-strawberry, bittersweet, oriental wormwood, with step (2)
Join, residue after the alcohol extracting of the fruit of glossy privet, and the middle gained curcuma zedoary of step (3), the bighead atractylodes rhizome, paniculate swallowwort propose residue merging after oil, add 9 times of amounts
Water, heating decoct 2 times, 2 hours every time, merge decoction liquor, add gained ginseng, fruit of glossy privet alcohol extracting filtrate and step in step (2)
Suddenly(3)Obtained aqueous solution, relative density 1.20-1.25 clear cream is condensed into, dry, pulverize, get dry extract powder;By gained dried cream powder
Add appropriate pharmaceutically acceptable auxiliary material granulation;
(6), gained volatile oil in step (3) is sprayed into the fine powder of the middle gained Poria cocos of step (4), ground bettle, the membrane of a chicken's gizzard, mixed
It is even, with step(5)The particle of middle gained mixes, closed half an hour, encapsulated to produce.
8. according to the application described in claim any one of 1-4, it is characterised in that it is described protection endothelium for confrontation endothelial cell by
The reduction of Secretion of Nitric Oxide after damage, the rise of endothelin -1 after confrontation endothelial cell is impaired.
9. according to the application described in claim 1-4, it is characterised in that the protection endothelial cell is regulation damaged endothelial cells
Cell chemotactic factor and acceptor expression.
10. according to the application described in claim 1-4, it is characterised in that the endothelial cell be human umbilical vein endothelial cell line or
Human vascular endothelial system.
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