CN102379935B - Composition for preventing or treating diseases caused by arteriosclerosis - Google Patents
Composition for preventing or treating diseases caused by arteriosclerosis Download PDFInfo
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Abstract
The invention provides a composition for preventing or treating diseases caused by arteriosclerosis. The composition comprises rhubarb, baikal skullcap root and coptis root. In the terms of the weights of dry medicinal materials, the ratio of the rhubarb to the baikal skullcap root to the coptis root is (1-3):(0.5-1.5):(0.5-1.5).
Description
Technical field
The present invention relates to a kind of compositions of the disease for preventing or treat arteriosclerosis to cause.
Background technology
Arteriosclerosis is the main cause of cardiovascular and cerebrovascular disease, these two kinds of main causes that disease is mortality rate in industrialized country.Arteriosclerosis is a progressive process, relate to many factors, as several media and information path, comprise ERK 1/2 signal (Millette E, Rauch BH, Defawe 0, Kenagy RD, Daum G, Clowes AW.Platelet-derived growth factor-bb-induced human smooth muscle cell proli feration depends on basic fgf release and fgfr-1 activation.Circ Res 2005; 96:172-179; Abraham ST, Benscoter HA, Schworer CM, Singer HA.A role for ca
2+/ calmodulin-dependent protein kinase ii in the mitogen-activated protein kinase signaling cascade of cultured rat aortic vascular smooth muscle cells.Circ Res 1997; 81:575-584; Libby P, Aikawa M.Stabilization of atherosclerotic plaques:New mechanisms and clinical targets.Nat Med 2002; 8:1257-1262), inflammatory response, somatomedin, cytohormone, chemotactic hormone and adhesion molecule (Libby P, Ridker PM, Maseri A.Inflammation and atherosclerosis.Circulation2002; 105:1135-1143; Pussinen PJ, Tuomisto K, Jousilahti P, Havulinna AS, Sundvall J, Salomaa V.Endotoxemia, immune response to periodontal pathogens, and systemic inflammation associate with incident cardiovascular disease events.Arterioscler Thromb Vasc Biol2007; 27:1433-1439).Except lipid, inspire the scorching factor (pro-inflammatory factor) and also play the part of important role (Wang JM in facilitating the pathological process of arteriosclerosis disease, Sica A, Peri G, Walter S, Padura IM, Libby P, Ceska M, Lindley I, Colotta F, Mantovani A.Expression of monocyte chemotactic protein and interleukin-8 bycytokine-activated human vascular smooth muscle cells.Arterioscler Thromb 1991; 11:1166-1174; Loppnow H, Libby P.Proliferating or interleukin 1-activated human vascular smooth muscle cells secrete copious interleukin 6.J Clin Invest 1990; 85:731-738; Dinarello CA.Biologic basis for interleukin-1 in disease.Blood 1996; 87:2095-2147).At lipid and inspire that under the stimulation of the scorching factor, arterial smooth muscle cell will move and breed, cause arteriosclerotic development (Schwartz SM.Smooth muscle migration in atherosclerosis and restenosis.J Clin Invest1997; 100:S87-89; Ross R.Atherosclerosis-an inflammatory disease.N Engl J Med 1999; 340:115-126).
The arteries chief component is by film in adventitia and the smooth muscle layer of controlling vasodilation, contraction, and the endothelial layer inner membrance consists of.Arteriosclerosis is that a kind of lipid and inflammatory cells are built up, and be accompanied by the degeneration of the caused tunica intima fibrosis of vascular smooth muscle cell curing and extracellular matrix liquid secretion, cause ductus arteriosus wall to thicken and follow the string, cause vessel lumen narrow and small, cause at last the diseases such as apoplexy, myocardial infarction or external perihaemal canal obstruction.Among Taiwan compatriots' that Department of Health issues the ten large causes of the death, disease by cardiovascular pathological changes causes as heart disease, cerebrovascular disease, hypertensive heart disease, comes second respectively, three, ten, so how the prophylactic treatment cardiovascular disease, become the task of top priority.
During the arteriosclerosis generation, vascular smooth muscle cell (VSMC) can convert to from the normal physiological state and a kind of synthesizing dedifferente attitude (synthetic dedifferentiated phenotype), and facilitates fibroplasia and speckle (plaque) to form.It should be noted that vascular smooth muscle cell can move to theca interna from the middle level, propagation, cause the formation of arteriosclerosis plaque subsequently.Many researchs have confirmed that various somatomedin and cytohormone (cytokine) playing the part of important role on arteriosclerotic initial sum progress.In addition, more and more many proof cytohormone IL-1 β and TNF-α can regulate arteriosclerotic pathological process, and in the generation of vascular smooth muscle cell moderate stimulation IL-6 and IL-8.
In addition, at present in the research of epigenetics (Epigenetic), find that the modification that methylates of cell DNA can participate in the expression of controlling gene, when presenting high methylation, the expression of gene is suppressed when the locational CpG of promoter (promoter) island (CpG island) of gene.Find that in cancer cell genosome DNA has phenomenon (the Park IY of comprehensive demethylation, Sohn BH, Choo J, Joe CO, Seong JK, Lee YI, Chung JH.Deregulation of DNA methyltransferases and loss of parental methylation at the insulin-like growth factor II (Igf2)/H19 loci in p53 knockout mice prior to tumor development.J.Cell.Biochem.2005; 94:585-596), this demethylation phenomenon also has relevant with the transfer of cell to tumor simultaneously.Find in arteriosclerosis plaque, ApolipoproteinE clpp gene deratization arteriosclerosis, the genosome DNA methylation expression of smooth muscle cell presents downward trend, and cause smooth muscle cell proliferation, promote progress (the DNA Methylation of arteriosclerotic lesion, Smooth Muscle Cells, and Atherogenesis.Hiltunen MO
-Herttuala S.Arterioscler Thromb Vasc Biol.2003; 23:1750-1753).
On the associated conditions drug application that treatment arteriosclerosis causes, HMG-CoA reductase inhibitor (being called for short statin) is at present clinical the most often the use and the most effective blood lipid-lowering medicine, by cholesterol biosynthesis and increase liver cell low density lipoprotein, LDL (the Low Density Lipoproteins that suppresses liver, LDL) accepter quantity with active, effectively reduces T-CHOL and low-density lipoprotein cholesterol.At present the trade name of common commercially available Statin has Lipitor (lipitor), Mevacor (U.S. weary fat), Mevalotin (Mevalotin), Lescol (beneficial fat can), Zocor (element fruit), Crestor (hat fat is appropriate).
The SANHUANG XIEXIN TANG is a kind of Chinese medicine formula, contains three kinds of compositions: the rhizome of Rhizoma Coptidis (rhizomes of Coptis chinesisFranch; CR), the root of Radix Scutellariae (roots of Scutellaria baicalensis Georgi; SR), reach rhizome (the rhizomes of Rheum officinale BAILL of Radix Et Rhizoma Rhei; RR).Traditionally, the SANHUANG XIEXIN TANG can be used for treating gastrointestinal tract inflammatory diseases (Lin WC, Tan TW.The role of gastric muscle relaxation in cytoprotection induced bysan-huang-xie-xin-tang in rats.J Ethnopharmacol 1994; 44:171-179; Saegusa Y, Sugiyama A, Takahara A, Nagasawa Y, Hashimoto K.Relationship between phosphodiesterase inhibition induced by several kampo medicines and smooth muscle relaxation of gastrointestinal tract tissues of rats.J Pharmacol Sci 2003; 93:62-68).Recently in vitro and in vivo research report SANHUANG XIEXIN TANG biological activity comprise antioxidation (Iijima OT, Takeda H, Matsumiya T.Effects of san ' o-shashin-to on the antioxidative mechanism in spontaneous familial hypercholesterolaemic rabbits.Pharmacol Res2000; 41:137-141), anti-inflammatory effect (Lo YC, Lin YL, Yu KL, Lai YH, Wu YC, Ann LM, Chen IJ.San-huang-xie-xin-tang attenuates inflammatory responses in lipopolysaccharide-exposed rat lungs.J Ethnopharmacol2005; 101:68-74; Lo YC, Tsai PL, Huang YB, Shen KP, Tsai YH, Wu YC, Lai YH, Chen IJ.San-huang-xie-xin-tang reduces lipopolysaccharides-induced hypotension and inflammatory mediators.J Ethnopharmacol 2005; 96:99-106; Shih YT, Wu DC, Liu CM, Yang YC, Chen IJ, Lo YC.San-huang-xie-xin-tang inhibits helicobacter pylori-induced inflammation in human gastric epithelial ags cells.J Ethnopharmacol 2007; 112:537-544), antihypertensive function (Tsai HH, Chen IJ, Lo YC.Effects of san-huang-xie-xin-tang on u46619-induced increase in pulmonary arterial blood pressure.J Ethnopharmacol 2008; 117:457-462; Chen HC, Hsieh MT, Tsai HY, Chang HH, Wang TF, Shibuya T.Studies on the " San-huang-hsieh-hsin-tang " In the treatment of essential hypertension.Taiwan Yi Xue Hui Za Zhi 1984; 83:340-346; Chen HC, Hsieh MT.Two-year experience with " San-huang-hsieh-hsin-tang " In essential hypertension.Am J Chin Med 1986; 14:51-58) and neurocyte protection (Shih YT, Chen IJ, Wu YC, Lo YC.San-huang-xie-xin-tang protects against activated microglia-and 6-ohda-induced toxicity in neuronal sh-sy5y cells.Evid Based Complement Alternat Med 2009; Doi:10.1093/ecam/nep 1025).Can infer that by these protective effects the SANHUANG XIEXIN TANG is to reduce inflammatory response by suppressing cytohormone/chemotactic hormone.
Although above-mentioned discovery to SANHUANG XIEXIN TANG anti-inflammatory effect is arranged, someone does not go to inquire into the arteriosclerosis effect of SANHUANG XIEXIN TANG yet at present.
Summary of the invention
The present invention studies the SANHUANG XIEXIN TANG for the arteriosclerosis impact of vascular smooth muscle cell.Use endotoxin (lipopolysaccharide is called for short LPS) to induce arteriosclerotic change, endotoxin is a kind of lipopolysaccharide, is the poisonous chemical substance from gram-negative bacteria cell wall.It is a kind of powerful bacterial virulence factors, can induce inflammatory response.Known this inflammation is an arteriosclerotic Major Risk Factors.Previous research points out that LPS can cause several main scorching factors that inspire, and causes vascular inflammatory and arteriosclerosis (Dinarello CA.Biologic basis for interleukin-1in disease.Blood 1996; 87:2095-2147).
The present invention finds that the SANHUANG XIEXIN TANG can suppress propagation and the migration of mankind's aortic smooth muscle cell (HASMC).The present invention confirms that also the arteriosclerosis effect of SANHUANG XIEXIN TANG also can inspire inflammatory cell hormone/chemotactic hormone (comprising IL-1 β, IL-6, IL-8 and MCP-1) and downgrade COX-2 by active oxygen (ROS), minimizing in the minimizing vascular smooth muscle cell.In addition, the SANHUANG XIEXIN TANG reduces the amount that LPS processes phosphorylation ERK 1/2 in rear HASMC, and it also helps to reduce arteriosclerotic carrying out.The present invention confirms that also the SANHUANG XIEXIN TANG can suppress the generation of smooth muscle cell genosome DNA demethylation phenomenon.Therefore the present invention finds that the SANHUANG XIEXIN TANG can be applicable to treat on arteriosclerosis and cardiovascular disease.
Cytohormone IL-1 β can mediate arteriosclerotic pathology process, and facilitate generation (the Wang JM of endogenous IL-6 and IL-8 in mankind's vascular smooth muscle cell, Sica A, Peri G, Walter S, Padura IM, Libby P, Ceska M, Lindley I, Colotta F, Mantovani A.Expression of monocyte chemotactic protein and interleukin-8 bycytokine-activated human vascular smooth muscle cells.Arterioscler Thromb 1991; 11:1166-1174; Loppnow H, Libby P.Proliferating or interleukin 1-activated human vascular smooth17muscle cells secrete copious interleukin 6.J Clin Invest 1990; 85:731-738.).IL-6 and IL-8 can promote synthetic cell proliferation (the Ikeda U that then causes of DNA in vascular smooth muscle cell, Ikeda M, Oohara T, Oguchi A, Kamitani T, Tsuruya Y, Kano S.Interleukin 6stimulates growth of vascular smooth muscle cells in a pdgf-dependent manner.Am J Physiol 191991; 260:H1713-1717; Selzman CH, Shames BD, Reznikov LL, Miller SA, Meng X, Barton HA, Werman A, Harken AH, Dinarello CA, Banerjee A.Liposomal delivery of purified inhibitory-κ b α inhibits tumor necrosis factor-α-induced human vascular smooth muscle proliferation.Circ Res1999; 84:867-875; Yue TL, Wang X, Sung CP, Olson B, McKenna PJ, Gu JL, Feuerstein GZ.Interleukin-8.A mitogen and chemoattractant for vascular smooth muscle cells.Circ Res 1994; 75:1-7).
MCP-1 can convene monocyte to enter the blood vessel of arteriosclerotic lesion.MCP-1 in blood also can be used as biomarker (the Ikeda U of assessment arteriosclerosis state and coronary artery disease, Matsui K, Murakami Y, Shimada K.Monocyte chemoattractant protein-1 andcoronary artery disease.Clin Cardiol 2002; 25:143-147).
COX-2 is an inflammation labelling, and has been considered to an arteriosclerotic risk factor (Hansson GK.Inflammation, atherosclerosis, and coronary artery disease.N Engl J Med2005; 352:1685-1695).
ROS be aerobic metabolism produce toxic byproduct arranged, but also can transmit molecule (Irani K.Oxidant signaling in vascular cell growth as message in cell in vascular cell, death, and survival:A review of theroles of reactive oxygen species in smooth muscle and endothelial cell mitogenic andapoptotic signaling.Circ Res 2000; 87:179-183).In vascular smooth muscle cell, ROS can affect different information paths, comprise ERK1/2, NF-κ B, Akt etc., these all with various cell phenotypes, associated (the Irani K.Oxidant signaling in vascular cell growth that similarly is that cell proliferation and apoptosis have, death, and survival:A review of theroles of reactive oxygen species in smooth muscle and endothelial cell mitogenic andapoptotic signaling.Circ Res 2000; 87:179-183; Gupta K, Kshirsagar S, Li W, Gui L, Ramakrishnan S, Gupta P, Law PY, Hebbel RP.Vegf prevents apoptosis of human microvascular endothelial cells via opposing effects onmapk/erk and sapk/jnk signaling.Exp Cell Res 1999; 247:495-504; Sundaresan M, Yu ZX, Ferrans VJ, Irani K, Finkel T.Requirement for generation of h2o2for platelet-derived growth factor signal transduction.Science 1995; 270:296-299).
Word used in the present invention " one " or " a kind of ", it can mean " one ", but its also the implication with " one or more ", " at least one " and " one or more " is consistent.
Word "or" used in the present invention in order to mean " and/or ".
Therefore, the invention provides a kind of compositions of the disease for preventing or treat arteriosclerosis to cause, and said composition is in the purposes for the preparation of the medicine of prevention or the disease that causes for the treatment of arteriosclerosis, wherein said composition comprises Radix Et Rhizoma Rhei, Radix Scutellariae and Rhizoma Coptidis, wherein Radix Et Rhizoma Rhei: Radix Scutellariae: the ratio of Rhizoma Coptidis is about 1-3 with the weighing scale of dried medical material: 0.5-1.5: 0.5-1.5.In embodiments of the present invention, Radix Et Rhizoma Rhei: Radix Scutellariae: the ratio of Rhizoma Coptidis is about 2: 1: 1 with the weighing scale of dried medical material.Better Radix Et Rhizoma Rhei is the dry rhizome of sorrel (Rheum palmatum L), Rheum tanguticum (Rheum tanguticum Maxim.) or Rheum officinale (Rheum officinale Baill.).Better Radix Scutellariae is the dry root of Radix Scutellariae (Scutellaria baicalensis Georgi), Sutellaria viscidula (Scutellaria viscidula Bge.) or Radix Scutellariae (Scutellaria amoena C.H.Wright).Better Rhizoma Coptidis connects the dry rhizome of (Coptis chinensis Franch.), refined company (Coptis deltoidea C.Y.Cheng et Hsiao), Coptis Teeta Wall (Coptis teeta Wall.) or Taiwan Rhizoma Coptidis (Coptis quinquefolia Miq.) for flavor.
In preferred embodiment, Radix Et Rhizoma Rhei, Radix Scutellariae and Rhizoma Coptidis are the extract form, are more preferred from the water extract form.
In preferred embodiment, the disease that arteriosclerosis causes includes but not limited to that arteriosclerosis, myocardial infarction, apoplexy or external perihaemal canal block.
In preferred embodiment, compositions provided by the invention can suppress propagation and the migration of vascular smooth muscle cell.
In another preferred embodiment, compositions provided by the invention can suppress the generation of active oxygen in vascular smooth muscle cell (ROS) and cytohormone IL-1 β, IL-6, IL-8 and MCP-1.
In another preferred embodiment, compositions provided by the invention can suppress the activation of ERK 1/2 in vascular smooth muscle cell.
In another preferred embodiment, compositions provided by the invention can suppress vascular smooth muscle cell genosome DNA demethylation.
Description of drawings
The inhibition that the HASMC that Fig. 1 demonstration is induced for LPS by MTT test method detection SANHUANG XIEXIN TANG breeds.Cell is exposed to LPS (100ng/ml) and processes the processing of SANHUANG XIEXIN TANG (or without) and cultivated 24 hours with SANHUANG XIEXIN TANG (0.2-0.6mg/ml).#p＜0.01 is with respect to control group,
*Graceful-Whitney U calibration calculation p value is used with respect to the LPS group in p＜0.01.Meansigma methods and SD repeat experiment from three.Each experiment is independent triplicate all.
Fig. 2 shows the inhibition of the cell migration that SANHUANG XIEXIN TANG in the analytical test of scratch wound repair is induced for LPS.The effect of representational Image Display SANHUANG XIEXIN TANG (0.2mg/ml) and Xin Weisiting (1nM) the 0th hour (the upper figure) or the 24th hour (figure below) after LPS (100ng/ml) processes to the HASMC migration.After LPS processes, cell migration increases, but the effect of LPS can be weakened by Xin Weisiting and SANHUANG XIEXIN TANG.White line represents the leading edge of migrating cell.
Fig. 3 shows that LPS is in the generation of HASMC moderate stimulation active oxygen (ROS).Generation by DCF fluorescent strength determining reactive oxygen species.HASMC was exposed to LPS (100ng/ml) after 24 hours, and the production of ROS significantly increases.SANHUANG XIEXIN TANG (0.2-0.6mg/ml) reduces significantly the ROS formation volume that LPS induces and has dose dependent (trend calibrating p=0.0036).#p＜0.01 is with respect to control group,
*Graceful-Whitney U calibration calculation p value is used with respect to the LPS group in p＜0.01.Meansigma methods and SD repeat experiment from three.Each experiment is independent triplicate all.
Fig. 4 A-4B shows in the HASMC that LPS induces the quantity of COX-2mRNA and albumen after SANHUANG XIEXIN TANG and Xin Weisiting process.Fig. 4 A represents COX-2mRNA is normalized to house-keeping gene GAPDH.Fig. 4 B represents the COX-2 protein expression analysis, reacts whole night under 4 ℃ with the specific antibody for COX-2, makes band as seen.#p＜0.05 is with respect to control group,
*Graceful-Whitney U calibration calculation p value is used with respect to the LPS group in p＜0.05.Meansigma methods and SD repeat experiment from three.Each experiment is independent triplicate all.
Fig. 5 shows that in HASMC, the SANHUANG XIEXIN TANG suppresses ERK 1/2 activation that LPS induces.Process HASMC with SANHUANG XIEXIN TANG (0.2mg/ml) and Xin Weisiting.Use western blotting, analyze ERK 1/2 and whole ERK 1/2 of phosphorylation in all cells lysate with specific antibody.(densitometry) quantizes each protein band with densitometry.#p＜0.01 is with respect to control group,
*P＜0.05,
*Graceful-Whitney U calibration calculation p value is used with respect to the LPS group in p＜0.001.Meansigma methods and SD repeat experiment from three.Each experiment is independent triplicate all.
Fig. 6 A-6B shows after the SANHUANG XIEXIN TANG is processed, the DNA demethylation that in HASMC, LPS induces has the trend of minimizing, and the SANHUANG XIEXIN TANG suppresses the effect of DNA demethylation, no matter at 24 hours (Fig. 6 A) or 48 hours (Fig. 6 B), the expression meeting of the genosome DNA demethylation dosage along with the SANHUANG XIEXIN TANG is increased.
The specific embodiment
The present invention may implement with different forms, is not limited in embodiment mentioned in following literary composition.The following example only as difference of the present invention towards and characteristics in representative.
Material and method:
1. the preparation of SANHUANG XIEXIN TANG
Below the SANHUANG XIEXIN TANG used of experiment is by the manufacturing of the village, Taiwan Song Rong pharmaceutical factory.The crude drug powder weighing ratio of 2: 1: 1 (360.0g: 180.0g: 180.0g) with the rhizome of the root of the rhizome of Radix Et Rhizoma Rhei, Radix Scutellariae and Rhizoma Coptidis.Every kind of medical material all adds the water of 10 times of volumes and boils.After extraction is completed, it is filtered with three kinds of extraction solutions of output.With three kinds of hot solution in conjunction with and then be concentrated to 1/20 of whole water volume of only being left to add originally three kinds of medical materials.With the concentrated solution spray drying that newly forms, draw the extract dry powder of 200.2g.The Production and quality control of raw material and authorizing product is all according to Pharmacopoea Chinensis and instruct rules to test.
2. cultivation and the processing of mankind's aortic smooth muscle cell (HAMSC)
Mankind's aortic smooth muscle cell is frozen third generation cultured cell available from Cascade Biologics (OR, USA).It is incubated in the culture bottle that contains Smooth Muscle Cell base (Medium 231, Cascade Biologics, Inc., OR, USA) makes its growth.(100 units/ml), streptomycin (100pg/ml) and antifungal (Fungizone (1.25mg/ml)) are formed the Smooth Muscle Cell base because of (3ng/ml), insulin (10mg/ml), penicillin by hyclone (FBS, 5%), human epidermal growth factor (10ng/ml), the growth of mankind's alkaline fiber blast cell.Cell culture is at 37 ℃, moist 5%CO
2In atmosphere.When cell aggregation approximately carries out the successive transfer culture of HAMSC during 70-80%.Use the HAMSC in successive transfer culture 4-9 generation to test.Use endotoxin (LPS) to cause arteriosclerotic change in HAMSC.Before LPS processes, first with cell culture in the culture medium that does not contain FBS 24 hours.Then under the existence of LPS (100ng/ml), cell was cultivated 24 hours at 37 ℃.In addition, use a kind of Statin " Xin Weisiting (Simvastatin) " that is widely used for reducing cholesterol (1nM) as the positive control medicine, to compare the arteriosclerosis effect of SANHUANG XIEXIN TANG in HAMSC.
3. the proliferation assay of one of arteriosclerosis estimating: HAMSC test
Process cell proliferation later in order to measure LPS, add the MTT (Sigma-Aldrich) of 0.5mg/ml in each grid of HAMSC micropore dish, and cultivated 2.5 hours under 37 ℃, afterwards again with the micropore dish in 37 ℃, 5%CO
2 Lower cultivation 24 hours.Then read the micropore dish to measure dyestuff to the absorbance (Benchmark PLUS Microplate Spectrophotometer, Bio-Rad) of wavelength 540nm and reference wavelength 630nm with spectrophotometer.All experiments are all carried out three times and are repeated.Absorbance represents mitochondrial enzymatic activity, namely means cell proliferation.
4. two of arteriosclerosis estimating: the migration analysis test of HAMSC
The standardization program of migration analysis test basis scratch wound repair analytical test is carried out.Merge the mechanical injuries of HAMSC and carrying out (Ashton AW described in other document of injury repairing analytical test, Yokota R, John G, Zhao S, Suadicani SO, Spray DC, Ware JA.Inhibition ofendothelial cell migration, intercellular communication, and vascular tube formation bythromboxane a2.J Biol Chem 1999; 274:35562-35570).With HAMSC with every lattice 1 * 10
6Cell culture forms in 6 porose discs and merges monolayer.Mark with 200-μ g standard micropipet tip the wound that crosses together grid on cell monolayer.With phosphate-buffered normal saline solution (PBS), injured cell monolayer is cleaned twice, and cultivate with the blood serum medium that contains that is supplemented with LPS.Measure the speed of wound closure and take pictures within the time of 24 hours.
5. three of arteriosclerosis estimating: the active oxygen of HAMSC (ROS) analytical test
Use fluorescence analysis test, with 2 ', 7 '-Fluorecein diacetate (2 ', 7 '-dichlorofluorescin diacetate (DCF-DA; Sigma-Aldrich)) measure endogenous ROS quantity.With the DCF-DA of 10 μ m in the oxidation (DCF-DA is oxidized to fluorescent chemicals DCF) with assessment ROS mediation in 30 minutes of 37 ℃ of lower cultured cells.By spectrophotometer (Bio-Rad), measure at excitation wavelength 485nm and emission wavelength 525nm the fluorescence that the DCF of oxidation sends.
6. COX-2 is made quantitative PCR in real time
Use quantitative PCR in real time assessment gene expression.Use Trizol reagent (Invitrogen), the operating process that provides according to manufacturer extracts its RNA from cultured cells.Use NanoPhotometer
TMSpectrophotometer (IMPLEN GmbH, Munich, Germany) is expressed concentration and the integrity of whole RNA.Use High-Capacity cDNA Archive Kit (Applied Biosystems) (capacity cDNA storehouse test kit greatly), the operating process that provides according to manufacturer becomes cDNA from whole rna transcriptions.The TaqMan real-time analysis of carrying out standard tests to detect the expression of target gene.Be used for the gene specific PCR primer of COX-2 and TaqMan probe from
ABI applying gene analytical test (production code member: Hs00153133_m1; Applied Biosystems).PCR uses Applied Biosystems 7500 PCR in real time instruments.Gene expression amount is normalized to house-keeping gene (housekeeping gene) glyceraldehyde phosphate dehydrogenase (GAPDH).
7. COX-2 and ERK 1/2 are carried out the protein blot analysis
In the protein extracting reagent (Thermo) of 100 μ l and protease inhibitor (Panomics), HAMSC is homogenized.Measure protein concentration with Pierce BCA Protein Assay Kit (Thermo).The protein of 20 μ g is added in perforation, separated 120 minutes under 100V with the mini colloid electrophoresis of NuPAGE Novex Bis-Tris 4-12% (Invitrogen) in Novex Xcell-II device, and be transferred on the Immbilon-PVDF transfer film in the immunoblotting mode.At room temperature block nonspecific in conjunction with 1 hour with 5% skim milk.Use for COX-2, all the specific antibody of ERK and p-ERK react whole night under 4 ℃ so that band as seen.Then, goat-anti rabbit (being used for p-ERK) or anti-mice (being used for COX-2 and whole ERK) secondary antibody are bonded to Radix Cochleariae officinalis and cross oxygenase (horseradish peroxidase), and be exposed to film for medical X-ray radiography (Fuiifilm) to measure the chemiluminescence (Immobilon Western, Millipore) that strengthens.
8. cytohormone/chemotactic hormone is made the ferment immunoassay
Before experiment, make HASMC grow to fusion in 6 porose discs, and then cultivated 24 hours in serum-free medium.HASMC is cultivated 24 hours also supernatant of collection condition culture medium, use ELISA cover group (BD Pharmingen
TM), measure IL-1 β, IL-6, IL-8 and MCP-1 according to the operating process that manufacturer provides.
9.HASMC DNA methylation is measured
Utilize fixedly smooth muscle cell after 15 minutes of 4% metaformaldehyde, clean and add 0.2%Triton X-100 reaction 30 minutes with 0.05%PBST.Afterwards again with 4N hydrochloric acid solution reaction 10 minutes, after cleaning, 0.05%PBST add 2% bovine serum albumin to reduce non-specific combination, recycling 5-methylcytosine one-level antibody and FITC secondary antibody are carried out immunofluorescence dyeing, utilize afterwards the intracellular fluorescence intensity of flow cytometry analysis, be the expression of 5-methylcytosine, calculate respectively cell genosome DNA methylation intensity of variation after 24 and 48 hours with this.
10. add up
Value in all literary composition neutralization figure all represents with meansigma methods ± SD.With graceful-Whitney U calibrating assessment statistical discrepancy, in all experiments, p value 0.05 is considered as significantly.With SigmaPlot software analysis data and drawing.
Result:
1.LPS the impact (processing with/without the SANHUANG XIEXIN TANG) on HASMC propagation
LPS (100ng/ml) stimulated rear 24 hours, and the multiplication effect that can cause HASMC is 1.5 times (Fig. 1) of control group.The SANHUANG XIEXIN TANG can reduce the speed (p＜0.01) of cell proliferation significantly in the HASMC that LPS induces.Simultaneously, positive control group Xin Weisiting (simvastatin) also reduces the speed (p＜0.01) of cell proliferation significantly in the HASMC that LPS induces.
2.HASMC the inhibition of migration
Use the analytical test of scratch wound repair, check the mobility whether the SANHUANG XIEXIN TANG can weaken the HASMC that processes through LPS.The 24 hour significantly increases of cell migration after LPS processes.And Xin Weisiting (1nM) can significantly reduce cell migration (Fig. 2) with SANHUANG XIEXIN TANG (0.2mg/ml).
3. suppress the analytical test of the ROS that LPS induces
When being exposed to LPS, the ROS in HASMC can increase (Fig. 3 significantly; P＜0.01).After processing with SANHUANG XIEXIN TANG (0.2-0.6mg/ml), can be observed the ROS that in HASMC, LPS induces and significantly reduce (p＜0.01), and the effect of SANHUANG XIEXIN TANG is on the affecting that meeting increases along with the dosage of SANHUANG XIEXIN TANG of ROS and the inhibition that presents ROS increases (trend calibrating p=0.0036).
4. the COX-2 after the SANHUANG XIEXIN TANG is processed expresses
LPS (100ng/ml) stimulates the COX-2mRNA quantity that causes after 24 hours in HASMC to increase by 6.9 times.Process with SANHUANG XIEXIN TANG (0.2mg/ml) by Xin Weisiting (1nM), can significantly reduce quantity (Fig. 4 A of COX-2mRNA; P＜0.01).Western blotting confirms that LPS can induce the manufacturing of COX-2 albumen, but SANHUANG XIEXIN TANG (0.2mg/ml) can lower respectively 20%COX-2 protein manufacture (p＜0.05) (Fig. 4 B) with Xin Weisiting (1nM).
5. the SANHUANG XIEXIN TANG suppresses ERK 1/2 activation that in HASMC, LPS induces
LPS stimulated rear 24 hours, and p-ERK 1/2 activation increases by 3 times of (Fig. 5; P＜0.01).SANHUANG XIEXIN TANG (0.2mg/ml) significantly reduces p-ERK 1/2 that LPS induces to original 30% (p＜0.01).Compare with the SANHUANG XIEXIN TANG, Xin Weisiting (1nM) on the contrary stimulates p-ERK 1/2 activation to make to increase by 31% (p＜0.05).
6. the SANHUANG XIEXIN TANG suppresses the cytohormone that in HASMC, LPS induces/chemotactic hormone expression
LPS stimulates the expression (table 1 that significantly increases IL-1 β, IL-6, IL-8 and MCP-1 after 24 hours; P＜0.01).The SANHUANG XIEXIN TANG significantly suppresses IL-1 β, IL-6, IL-8 and the MCP-1 expression (p＜0.01) that LPS induces.Xin Weisiting also reduces the expression degree that LPS processes rear IL-1 β, IL-6 and MCP-1, but can the slight expression that increases IL-8.
In the HASMC that table 1:LPS induces, the expression degree of IL-1 β, IL-6, IL-8 and MCP-1 after the SANHUANG XIEXIN TANG is processed
#p＜0.01 is with respect to control group,
*Graceful-Whitney U calibration calculation p value is used with respect to the LPS group in p＜0.01.Process HASMC with LPS (100ng/ml), then processed 24 hours with SANHUANG XIEXIN TANG (0.2-0.6mg/ml) or Xin Weisiting (1nM).Collecting cell culture medium after processing finishes.Use ELISA to measure the expression degree of IL-1 β, IL-6, IL-8 and CCL2.Numerical value is the meansigma methods ± SD of 3 independent experiments.
7. the SANHUANG XIEXIN TANG is for suppressing DNA demethylation effect
When cell is exposed in LPS, the DNA demethylation in HASMC can increase (Fig. 6 A and Fig. 6 B) significantly.After processing with SANHUANG XIEXIN TANG (0.1-0.6mg/ml), can be observed DNA demethylation that in HASMC, LPS induces has the trend of minimizing, and the SANHUANG XIEXIN TANG suppresses the effect of DNA demethylation, no matter at 24 or 48 hours, the expression meeting of the genosome DNA demethylation dosage along with the SANHUANG XIEXIN TANG is increased.
The person of ordinary skill in the field can realize the present invention very soon can be easy to reach target, and obtains result and the advantage mention, and those are present in thing wherein.Compositions in the present invention, mixture and fabrication schedule thereof and method are the representative of preferred embodiment, and it is exemplary and not only is confined to field of the present invention.The person of ordinary skill in the field will expect wherein can revising part and other purposes.These modifications all lie in spirit of the present invention, and limit in the claims.
Description of the present invention and embodiment are all in detail open, under making, any technical staff of technical field can make and use the present invention, even various change, modification are wherein arranged, reach progressive part, must be considered as without departing from the spirit or scope of the invention.
All patents and the publication mentioned in description are all to be as the criterion with the technical staff who invents relevant field.All patents and publication all are included into identical reference degree at this, just specifically and are individually pointed out to include in reference as each indivedual publication.
In this suitably illustrational invention, any important document can lacked, perhaps many important documents, restrictive condition or and nonspecific for implementing under limited case disclosed herein.The noun that uses and expression are the descriptions of book as an illustration and unrestricted, have no intent to simultaneously use this class to get rid of shown in any being equal to and the characteristics of explanation or noun and the expression of its part, but what need see clearly is various change might occur in claim scope of the present invention.Therefore, although should be appreciated that according to preferred embodiment and arbitrarily characteristics specifically disclose the present invention, but the person of ordinary skill in the field still can revise and change the content that wherein discloses, and suchlike modifications and variations are still in claim scope of the present invention.
Claims (10)
1. a compositions is in the purposes of the medicine of the disease that causes for the preparation of prevention or treatment arteriosclerosis, wherein said composition is comprised of Radix Et Rhizoma Rhei, Radix Scutellariae and Rhizoma Coptidis, wherein Radix Et Rhizoma Rhei: Radix Scutellariae: the ratio of Rhizoma Coptidis is counted 1-3 with the weight of dried medical material: 0.5-1.5: 0.5-1.5.
2. purposes according to claim 1, is characterized in that, Radix Et Rhizoma Rhei: Radix Scutellariae: the ratio of Rhizoma Coptidis is counted 2: 1: 1 with the weight of dried medical material.
3. purposes according to claim 1, is characterized in that, Radix Scutellariae is the dry root of Radix Scutellariae, Sutellaria viscidula or Radix Scutellariae.
4. purposes according to claim 1, is characterized in that, Rhizoma Coptidis is the dry rhizome of distinguish the flavor of company, refined company, Coptis Teeta Wall or Taiwan Rhizoma Coptidis.
5. purposes according to claim 1, is characterized in that, Radix Et Rhizoma Rhei is the dry rhizome of sorrel, Rheum tanguticum or Rheum officinale.
6. purposes according to claim 1, is characterized in that, this disease is that arteriosclerosis, myocardial infarction, apoplexy or external perihaemal canal block.
7. purposes according to claim 1, is characterized in that, said composition suppresses propagation and the migration of vascular smooth muscle cell.
8. purposes according to claim 1, is characterized in that, the generation of active oxygen and cytohormone IL-1 β, IL-6, IL-8 and MCP-1 in said composition inhibition vascular smooth muscle cell.
9. purposes according to claim 1, is characterized in that, said composition suppresses the activation of ERK 1/2 in vascular smooth muscle cell.
10. purposes according to claim 1, is characterized in that, said composition suppresses vascular smooth muscle cell genosome DNA demethylation.
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| 于洪炜.祛湿化痰排毒法治疗脑动脉硬化症124例.《辽宁中医药大学学报》.2007,第09卷(第03期),145-146. |
| 祛湿化痰排毒法治疗脑动脉硬化症124例;于洪炜;《辽宁中医药大学学报》;20070505;第09卷(第03期);145-146 * |
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