CN101125146B - Medicine target for preventing and treating cardiovascular and cerebrovascular diseases associated with inflammation and its inhibitor - Google Patents
Medicine target for preventing and treating cardiovascular and cerebrovascular diseases associated with inflammation and its inhibitor Download PDFInfo
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- CN101125146B CN101125146B CN2007100248100A CN200710024810A CN101125146B CN 101125146 B CN101125146 B CN 101125146B CN 2007100248100 A CN2007100248100 A CN 2007100248100A CN 200710024810 A CN200710024810 A CN 200710024810A CN 101125146 B CN101125146 B CN 101125146B
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Abstract
The present invention relates to a drug target for the prevention and the treatment of cerebrovascular diseases and the medical usage of the inhibitors thereof, more particularly, the present invention relates to the nonmuscle myosin heavy chain 9(MyH9) used as a new drug target for the prevention and the treatment of inflammation-related cerebrovascular diseases and the usage of the inhibitors ruscogenin and the genin thereof in the prevention and the treatment of the tumor differentiation and metastasis, glaucoma and other diseases.
Description
Technical field
The present invention relates to a kind of novel drug target of cardiovascular and cerebrovascular diseases associated with inflammation and medical usage of inhibitor thereof prevented and treated, be specially non-muscle myoglobulin heavy chain 9[nonmuscle myosin heavy chain9 (MyH9) as the control novel drug target of cardiovascular and cerebrovascular diseases associated with inflammation and inhibitor ruscogenin thereof and glycosides thereof in the purposes of control with diseases such as tumor transdifferentation, glaucomas.
Technical background
The medicine innovation research mainly comprises two aspect contents, the one, and the chemical compound innovation is promptly found new construction type reactive compound according to existing target; The 2nd, the drug targets innovation, i.e. methods such as integrated use chemical biology, molecular biology, pharmacology, bioinformatics are found drug effect new target drone and new role mechanism and relevant a series of signal transduction path.From the innovation of source, the discovery of medicine new target drone has more scientific value and practical significance than the chemical compound innovation.At present, along with developing rapidly of genomics, proteomics, bioinformatics, with existing medicine or reactive compound, particularly Chinese herbal medicine effective ingredients or bioactive natural product are probe, searching is another approach of the genome times afterwards comprehensively finding drug targets with albumen or other biomacromolecule of its specific bond, is the developing direction and the focus of new drug development in recent years.Discovery and biology and pharmaceutical research as the anticancer natural drug paclitaxel cause the discovery of cancer therapy drug new target drone tubulin, cause new round screening anticancer medicine climax; With the capsaicin is probe, filters out non-addicted analgesics thing target, for non-addicted analgesics thing has been opened up new direction.
China has abundant Chinese herbal medicine resource, Chinese medicine is as the important means of the traditional Chinese medical science in order to prevent and cure diseases, containing abundant theory of Chinese medical science, with the traditional Chinese medical science rule of treatment close ties are arranged, its effect characteristics and the mechanism of action may be different from based on the chemical medicine of modern medical theory and west plant amedica, active ingredient of Chinese herbs is the material base of its performance unique effect of relying, therefore utilize modern multidisciplinary interleaving techniques, seek and find its direct acting specificity target spot, to be expected to really explain the mechanism of action of Chinese medicine, and provide new target drone for the original new drug development of reflection Chinese medicine effect characteristic.
Be representative YIN nourishing Chinese medicine commonly used Radix Ophiopogonis, sweet, the little hardship of the property of medicine, is slightly cold, and returns stomach, lung heart channel, effect with YIN nourishing and the production of body fluid promoting, clearing away heart-fire and moistening the lung, clinical practice is very extensive, as one of main composition medicine of famous ancient prescription SHENGMAI SAN, zhigancao decoction etc., and treatment cardiovascular disease determined curative effect.The inventor finds by long-term a large amount of early-stage Study; the ophiopogonin constituents; the glycosides that promptly mainly with the ruscogenin is parent nucleus comprises that C1 or C3 position connect monoglycosides, bioside, three glucosides that glucose, rhamnose, fucose or arabinose are formed; it is active all to have the protection of significant antiinflammatory and cardiovascular and cerebrovascular vessel; the sapogenin that it is main---ruscogenin partly is its active function center parent nucleus; has remarkable antiinflammatory antithrombotic acitivity; intensity is similar to dexamethasone, warfarin etc., but does not influence PGE
2Release, COX-2 expression etc., no warfarin easily causes the hemorrhage side reaction of Denging, points out its effect to be different from glucocorticoid medicine and oral anticoagulant commonly used.Therefore, fully utilize multidisciplinary technological means, seek and find the binding proteins specific of ruscogenin in cardiovascular and cerebrovascular disease linked groups cell, and verify this protein function, be expected to find and the relevant novel drug target of major disease such as cardiovascular and cerebrovascular disease, and find the ruscogenin novel medical use relevant with target protein.Have not yet to see the pertinent literature report.
The method of seeking drug target protein at present mainly contains affinity chromatography, phage display clone, yeast three-hybrid system, medicine trace, DNA chip technology, magnetic Nano probe technique etc., and pluses and minuses are respectively arranged.Wherein affinity chromatography is the most classical a kind of method.Be successfully applied to the search of multiple drug target proteins such as colchicine, cytochalasin B, FK506, cyclosporin, E3330 so far.Common affinity chromatography has solid phase specificity elution method and medicine competition law.But solid phase specificity elution method is subject to the dissociate influence of speed of " affinity column-target protein " complex, but also contrast post that must preparation non-activity similar medicine; The medicine competition law is because albumen can be with insoluble drug precipitation, and is very high to the requirement of medicine dissolution.The series affinity chromatography is a kind of new affinity chromatography that can overcome above shortcoming of report in 2006, and the target that is not applied to native compound is so far as yet found.
Summary of the invention
The present invention is under national sub-problem of 11th Five-Year science and technology support plan (2006BAI08BO5) and project of national nature science fund project (30672603) subsidy, utilize serial affinity chromatography to find the binding proteins specific of the sweet active component of soap Radix Ophiopogonis center parent nucleus ruscogenin in cardiovascular and cerebrovascular disease relevant cell, tissue, and be accredited as non-muscle myoglobulin heavy chain 9 (nonmuscle myosin heavy chain9) (being called for short MyH9) by ESI-MS and MALDI-TOF-TOF-MS.MyH9 has another name called Nonmuscle myosin heavy chain II A (be called for short NMHC-IIA), is by the MyH9 gene code on No. 22 chromosome of people.Be distributed widely in the various kinds of cell such as vascular endothelial cell, macrophage, fibroblast, T cell, neutrophilic granulocyte.Have now found that it can combine with multiple protein (as: Menin, angiotensin converting enzyme, paranuclein, β-actin, NMHC-IIB) in born of the same parents.This illustrates that non-muscle myoglobulin heavy chain 9 is a kind of and the closely-related functional protein of physiological and pathological process.
At present, research has confirmed that the transhipment, neoplasm metastasis, deafness of MyH9 and cell adhesion and migration, cytokinesis, organelle and microgranule etc. is closely related, but does not see that it is as the report of preventing and treating the cardiovascular and cerebrovascular diseases medicament target.Blebbistatin is the specific inhibitor of a kind of MyH9 of just finding in recent years, found that blebbistatin can reversibly block cell and bubble, dose dependent ground blocking-up cell moves, cytokinesis, and the metastatic capacity and the aggressivity of energy anticancer, reduces glaucomatous generation.The present invention finds that first blebbistatin and ruscogenin have similar activity, the activity that can significantly suppress activatory ECV304 cell of tumor necrosis factor and HL60 cell adhesion and downward modulation tissue factor, but and the active performance of antagonism ruscogenin, so can think that MyH9 is the function target protein of ruscogenin and glycosides thereof.According to the remarkable activity of ruscogenin to cardiovascular and cerebrovascular diseases associated with inflammation, MyH9 promptly can become a kind of drug targets of new control cardiovascular and cerebrovascular disease; Ruscogenin and glycosides thereof are the MyH9 inhibitor of new construction type, point out it to have broad prospect of application with the MyH9 diseases associated preventing and treating cardiovascular and cerebrovascular vessel and neoplasm metastasis, glaucoma etc.
The cardiovascular and cerebrovascular disease that above-mentioned described inflammation is relevant can be an atherosclerosis, coronary heart disease, angina pectoris, arrhythmia, cerebral infarction, cerebral ischemia or thrombosis inflammation.
The glycosides that above-mentioned described ruscogenin is a parent nucleus can be that C1 or C3 position connect monoglycosides, bioside or three glucosides that glucose, rhamnose, fucose or arabinose are formed.
Description of drawings
Fig. 1 be the influence of sticking effect of RUS-2HS and Rus pair cell (
##Compare with matched group P＜0.01;
*P＜0.05,
*Compare with model group P＜0.01)
Fig. 2 be RUS-2HS and Ruscogenin to the inductive macrophage of Lps discharge NO influence (
##Compare with matched group P＜0.01,
*P＜0.05,
*Compare with model group P＜0.01)
Fig. 3 be RUS-2HS and Ruscogenin to the influence of chmice acute cerebral anoxia (
*P＜0.05,
*Compare with matched group P＜0.01)
A figure is SDS-PAGE-coomassie brilliant blue staining collection of illustrative plates (M, the molecular weight of albumen labelling (KD) of the binding proteins specific of ruscogenin in normal and activatory ECV304 cell of inflammatory factor and Human umbilical vein endothelial cells among Fig. 4; A1, B1, C1 act on for the first time conjugated protein on the post material; A2, B2, C2 act on for the second time conjugated protein on the post material; A1, A2 are normal ECV304 cell, and B1, B2 are inflammatory conditions ECV304 cell, and C1, C2 are Human umbilical vein endothelial cells; Arrow indication MyH9); B figure is that the antibody with MyH9 carries out to respective markers band among the A figure that A figure is binding proteins specific (M, the molecular weight of albumen labelling (KD) of ruscogenin in cardiovascular and cerebrovascular disease linked groups in the Western-Blotting proof diagram 5; A1, A2, heart tissue protein series affinity chromatography result; B1, B2, cerebral tissue protein series affinity chromatography result; C1, C2, the series affinity chromatography result of vascular tissue; Arrow is designated as binding proteins specific); B figure carries out the Western-Blotting checking with the antibody of MyH9 to respective markers band among the A figure
Fig. 6 be blebbistatin to Ruscogenin performance anti-cell stick effect influence (
##Compare with matched group P＜0.01,
*P＜0.05,
*Compare with model group P＜0.01)
Fig. 7 be Blebbistatin and Ruscogenin influence that ECV304 cell TF is expressed (
#Compare with matched group #P＜0.01,
*Compare with model group P＜0.05, and compare with RUS (0.1 μ M) group Δ P＜0.05
Fig. 8 be blebbistatin to Ruscogenin bring into play the influence that anti-macrophage discharges the NO effect (
##Compare with matched group P＜0.01,
*Compare with model group P＜0.01, and compare with RUS (1 μ M) group Δ P＜0.05, and Δ Δ P＜0.01 is organized relatively with RUS (1 μ M)
The specific embodiment
The preparation of EAH Sepharose4B-RUS-2HS affinity column and active checking
1. experiment material
1.1 cell and cell strain
The ECV304 cell strain, the HL-60 cell strain, former generation macrophage take from the healthy mice peritoneal fluid.
1.2 medicine and reagent
Ruscogenin (purity is greater than 98%); Succinic anhydride; EAH Sepharose4B agarose gel (AmershamBiosciences); 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC, Sigma); Dexamethasone sodium phosphate injection; Nimotop; Recombination human tumor necrosis factor-alpha; Hydrocortisone; Lipopolysaccharide; Tetrazolium bromide.
2 experimental techniques
The preparation of the synthetic and EAH Sepharose4B-RUS-2HS affinity column of ruscogenin succinate monoester (RUS-2HS), preparation method sees that the inventor has delivered document: the preparation and the application in antibody purification that with the ruscogenin are the affinity column of aglucon, China's natural drug, 2006,4 (5): 363-367.The external anti-cell of RUS-2HS derivant sticks, anti-macrophage NO discharges active the detection, and the determination of activity of anti-mouse brain anoxia is carried out with reference to following document respectively in the body:
[1] ruscogenin is to the influence of HL-60 and EC V304 cell adhesion, and Chinese Pharmacological is circulated a notice of, and 2006,22 (6): 706-709;
[2] triptolide active and excretory influence of NO to mouse macrophage, Zhejiang combination of Chinese and Western medicine magazine, 2006,16 (2): 70-72;
[3] Xu Shuyun. pharmacological experimental methodology [M] .2 version. Beijing: People's Health Publisher, 1991:948.
3. experimental result
3.1 successfully prepared EAH Sepharose4B-RUS-2HS affinity column, the coupling amount of affinity column is that 7.11mg/mL, coupling rate are 71.1%.
3.2RUS-2HS the specific activity of derivant and Ruscogenin
Accompanying drawing 1 and accompanying drawing 2 results show, 0.1,1.0 μ mol.L
-1The external pretreatment ECV304 of Ruscogenin, RUS-2HS cell after all significantly or highly significant reduce activatory ECV304 of TNF-α and HL-60 cell adhesion, significantly suppress the NO release of the inductive Turnover of Mouse Peritoneal Macrophages of Lps.When concentration was 1 μ M, 0.1 μ M, RUS-2HS and Ruscogenin inhibitory action did not all have and do not have significant difference, showed that anti-inflammatory activity and the Ruscogenin of derivant RUS-2HS is suitable; Accompanying drawing 3 results show in addition, but the equal significant prolongation mice broken end breathing time of Ruscogenin, RUS-2HS8,24 μ mol/kg vivo medicine-feedings points out the two anti-cerebral anoxia effect suitable, shows that its avtive spot is not sealed by deriveding group.
Seek the binding proteins specific of Ruscogenin in cardiovascular and cerebrovascular disease relevant cell, tissue with serial affinity chromatography
1. experiment material
1.1 cell and cell strain
The ECV304 cell strain, former generation Human umbilical vein endothelial cells.
1.2 animal
Male SD rat, 250-340g.
1.3 medicine and reagent
Cell pyrolysis liquid and histone lysate are universal non-denatured protein lysate, with preceding adding PMSF, leupeptin, aprotinin, trypsin inhibitor; BeadBuffer; BCA determination of protein concentration test kit (the green skies).
2. experimental technique
2.1 the preparation of cell, histone liquid
2.1.1 the preparation of cell protein liquid
Discard cell culture fluid, (contain 0.05mmolL with PBS
-1PMSF) wash twice.With scraper plate cell is scraped, be collected in the proper volume centrifuge tube, with a small amount of PBS flushing plate, suck centrifuge tube (operation on ice), 4 ℃ of centrifugal 5min of 1000rpm make cell precipitation.Abandon supernatant, sedimentary cell is hanged with the PBS that contains PMSF, 1000rpm is centrifugal again, repeats twice, whole cell culture fluids of flush away and serum composition.Abandon supernatant, add the cell pyrolysis liquid of an amount of pre-cooling, firmly blow and beat cell precipitation, ice bath concussion 10min, the centrifugal 15min of 12000g, supernatant are total protein of cell liquid.
2.1.2 the preparation of histone liquid
The rat sacrificed by decapitation is got its internal organs and is cleaned in the normal saline of pre-cooling.Add 1:4 (w/v) histone lysate, ice bath homogenate, the centrifugal 10min of 9500rpm, supernatant be again with 12, the centrifugal 30min of 000rpm, with plain white on the histone lysate dilution gained to 7mg/ml, store in-70 ℃ stand-by.
2.2 serial affinity chromatography
Method reference literature: A versatile method of identifying specific binding proteins on affinity resins, Analytical Biochemistry, 2006 (352): 15-23.
Get 0.5～1ml protein liquid (containing 1.5～3mg total protein approximately) in the 1.5ml centrifuge tube, mend to 1ml with Bead buffer, add 15 μ l affinity column materials (A1), incubate slow stirring 40～60min altogether for 4 ℃, 8000rpm is instantaneous centrifugal, supernatant is transferred to another centrifuge tube, adds the same affinity column material (A2) of 15 μ l more in addition, incubate slow stirring altogether for 4 ℃ and spend the night, 8000rpm is instantaneous centrifugal, abandon supernatant, the careful washing of Bead Buffer 3 times adds 30 μ l LoadingBuffer in the post material after two secondary actions, boil 5min in the boiling water, the centrifugal 1min of 8000rpm is with supernatant SDS-PAGE electrophoresis detection, coomassie brilliant blue staining.The band of Duoing on the bonded post material than effect for the second time on the post material of effect is the binding proteins specific of aglucon for the first time.Same test repeats 3 times.
3. experimental result
3.1 the conjugated protein (see figure 4) of ruscogenin in endotheliocyte
Experiment shows, ruscogenin energy specific binding molecules amount in normal and activatory ECV304 cell of inflammatory factor and normal former generation Human umbilical vein endothelial cells be the albumen of 210KD.Cut albumen in the glue, enzymolysis, digestion, ESI-MS (LCQ-DECA-XP plus, Finnigan) or MALDI-TOF-TOF-MS (AutoFlex MALDI-TOF-TOF-MS, Bruker) order-checking, peptide section sequence is compared in IPI human protein pool and is accredited as respectively: the albumen of 210KD is nonmusclemyosin heavy chain9 (MyH9), a kind of myosin that is expressed in endotheliocyte, and utilize this protein antibodies to carry out western blot experiment conclusive evidence.
3.2 the conjugated protein (see figure 5) of ruscogenin in cardiovascular and cerebrovascular disease linked groups
Utilize serial affinity chromatography to seek the binding proteins specific of ruscogenin in tissues such as rat heart, brain.The result shows that binding proteins specific is all arranged in heart, brain, vascular tissue, wherein molecular weight is that the protein protomer about 210KD is MyH9, and utilizes this protein antibodies to carry out western blot experiment conclusive evidence.
The functional verification of Ruscogenin binding proteins specific MyH9
Studies show that in a large number Ruscogenin has significant antiinflammatory antithrombotic acitivity.The present invention passes through:
Activatory ECV304 cell sticking of TNF-α to the HL-60 cell;
Activatory ECV304 cellular expression of TNF-α and the closely-related tissue factor of cardiovascular and cerebrovascular disease (TF) are measured; 3. lipopolysaccharide-induced macrophage discharges the NO experiment, and whether checking MyH9 is that Ruscogenin brings into play active target proteins.
1. experiment material
Human Factor's (magnificent blue biological engineering joint-stock company), Factor Xa Chromogenic substrate (Sigma), other is with embodiment 1.
2. experimental technique and result
2.1MyH9 specific inhibitor blebbistatin and Ruscogenin to the influence of activatory ECV304 of TNF-α and the effect of HL-60 cell adhesion.
Behind the drug effect ECV304 cell to the experimental technique of inductive HL-60 cell of TNF-α and ECV304 cell adhesion function influence with embodiment 1.The result as seen from Figure 6,0.1,1.0 μ molL
-1Ruscogenin pretreatment ECV304 cell after, all can significantly reduce activatory ECV304 of TNF-α and HL-60 cell adhesion.0.2 the blebbistatin of μ M, 2 μ M also can be significantly and is reduced activatory ECV304 of TNF-α and HL-60 cell adhesion extremely significantly.Illustrate that Ruscogenin and blebbistatin have similar effect, the anti-cell of Ruscogenin sticks activity may be with the target protein of blebbistatin---MyH9 be relevant.Simultaneously, have the leukocyte of inhibition and endothelial cell adhesion effect, for the inventor discloses first about MyH9 inhibitor blebbistatin.
2.2blebbistatin Ruscogenin is brought into play the influence of anti-TNF-α inductive ECV304 cellular expression tissue factor (TF).
Experiment is divided into blank group, model group, medicine group, incubates after 5 hours cracking after the administration altogether and respectively organizes cell.
Cell pyrolysis liquid and prothrombin complex solution are hatched 15min jointly under 37 ℃, every hole adds FXa Chromogenicsubstrate (containing 25mmol/L EDTA), and 37 ℃ of incubation 3min measure the 405nm absorbance, with reflection TF procoagulant activity.The result as seen from Figure 7, TNF-α effect ECV304 cell can make the active of TF significantly raise after 5 hours, and all can significantly reduce the rising of its TF during the independent function cells of the blebbistatin of the Ruscogenin of 0.1 μ M, 0.2 μ M and 2 μ M.But when Ruscogenin and blebbistatin combined effect cell, the expression of TF does not only reduce, raise during on the contrary than independent medication, prompting Ruscogenin and blebbistatin are not synergism influencing aspect the TF activity, but antagonism, blebbistatin2 μ M can significantly resist the inhibitory action that Ruscogenin0.1 μ M expresses TF, shows that Ruscogenin is active by suppressing the MyH9 performance, and MyH9 is its effect target protein.Simultaneously, has the effect of the important drugs target spot TF that suppresses cardiovascular and cerebrovascular disease, for the inventor confirms and open first about MyH9 inhibitor blebbistatin.
2.3MyH9 specific inhibitor blebbistatin Ruscogenin is brought into play the influence that anti-macrophage discharges the NO effect
Experimental technique is with embodiment 1.
The result as seen from Figure 8, LPS effect macrophage can promote NO to discharge by highly significant after 48 hours, Ruscogeninl μ M is individually dosed can be suppressed the inductive NO of LPS extremely significantly and discharges, and blebbistatin1,5,10,20 μ M individually dosed then do not have obvious inhibitory action; But when Blebbistatin and Ruscogenin combined effect cell, the inhibitory action that the antagonism Ruscogenin of blebbistatin energy dose dependent discharges NO, confirm that further Ruscogenin plays a role by its binding proteins specific MyH9, its active specific inhibitor blebbistatin with MyH9 has certain difference.
In sum; the inventor utilizes the affinity column that does not influence Chinese medicine active component Radix Ophiopogonis Ruscogenin avtive spot; separate the binding proteins specific of Ruscogenin in the relevant cell of cardiovascular and cerebrovascular disease, tissue by serial affinity chromatography; and find that by pharmacological evaluation checking MyH9 is its function target protein; realization with have the protection of remarkable antiinflammatory, cardiovascular and cerebrovascular vessel active in pharmaceutically active ingredient be probe; find the novel drug target of control cardiovascular and cerebrovascular disease; and provide the inhibitor of this target new construction type, thereby finish the present invention.
Claims (3)
1. non-muscle myoglobulin heavy chain 9 is prevented and treated the purposes of the medicine of the relevant cardiovascular and cerebrovascular disease of inflammation at in-vitro screening as target.
2.Blebbistatin prevent and treat the purposes of the medicine of cardiovascular and cerebrovascular diseases associated with inflammation as preparation.
3. ruscogenin and glycosides thereof are as the purposes of non-muscle myoglobulin heavy chain 9 inhibitor of preparation.
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| CN101481415B (en) * | 2009-02-04 | 2010-05-12 | 山东农业大学 | Novel PRRS virus receptor and block inhibitor thereof |
| ES2602078T3 (en) * | 2013-03-15 | 2017-02-17 | Lonza Cologne Gmbh | Compositions and methods to improve the therapeutic potential of stem cells |
| KR101858791B1 (en) * | 2015-06-15 | 2018-05-17 | 이화여자대학교 산학협력단 | Diagnostic and treating method for cardiovascular diseases using NMHC IIA |
| CN108700597A (en) * | 2016-02-16 | 2018-10-23 | 公立大学法人横浜市立大学 | Biomarker in blood for detecting artery sclerosis |
| CN111471653B (en) * | 2017-03-01 | 2022-11-25 | 中国科学院动物研究所 | Method for converting non-neuron cells into neuron cells |
| CN106727613A (en) * | 2017-03-20 | 2017-05-31 | 中国药科大学 | Ophiopogonin unit is used to prepare the application of the disease mediated medicine of tissue factor |
| CN110441537B (en) * | 2019-08-23 | 2022-09-13 | 北京丹大生物技术有限公司 | Method for measuring content of 25-hydroxy vitamin D |
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