CN104546821A - Application of danshinolic acid B in preparation of medicine of treating scleroderma - Google Patents
Application of danshinolic acid B in preparation of medicine of treating scleroderma Download PDFInfo
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
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- A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
Abstract
The invention relates to the field of chemical engineering in a hospital, particularly relates to a new application of a small molecule compound, and further particularly relates to an application of a small molecule compound danshinolic acid B in preparation of a medicine of treating scleroderma. The invention provides an application of danshinolic acid B in preparation of a medicine of treating scleroderma, and further provides a corresponding medicine composition and a medicine preparation. The inventor of the invention makes an animal experiment, which shows that a compound SAB remarkably relieves the pulmonary fibrosis of a mice suffering from the scleroderma, and a further cell experiment indicates that the small molecule compound can inhibit cell inflammation induced by TNFalpha, and can inhibit the skin flbroblast of a patient suffering from the scleroderma cultured in vitro, human embryonic lung fibroblast proliferation and collagen expression, and inhibit transformation of pulmonary epithelial cells to mesenchymal cells. The application shows that the compound has a function of resisting fibrosis of the scleroderma, and has a good application prospect for preventing and treating the scleroderma.
Description
Technical field
The present invention relates to hospital's chemical field, especially relate to a kind of novelty teabag of micromolecular compound, relate to the purposes of micromolecular compound salvianolic acid B (also known as salvianolic acid B, salvianolic acid second) in the sclerodermatous medicine of preparation treatment particularly.
Background technology
Scleroderma (scleroderma), also known as systemic sclerosis (systemic sclerosis, SSc), one to thicken with limitation or diffuse cutaneous and turn to feature with fiber, and involve the autoimmune disease of the internal organs such as the heart, lung, kidney, digestive tract, be attended by the infringement of Microvasculature simultaneously.Collagen fiber hyperplasia in autoimmune response (there is multiple autoantibody in hyperimmunoglobulinemia and serum), vascular lesion (thin vessels inner film injury and acra, internal organs take place frequently Raynaud phenomenon) and skin, viscera tissue is the three large factors causing systemic sclerosis to produce Multisystem damage.
Systemic sclerosis pathological changes is distally expanded by trunk, and Raynaud phenomenon is few, state of an illness weight, and lesion growth is fast, poor prognosis, often invades one or comprise multiple organs of lung, heart, digestive tract etc.Patients with scleroderma's extremity near-end skin involvement, causes finger function obstacle, affects the work of patient's daily life, the caused death of severe symptoms.In the U.S., 5 years survival rates of systemic sclerosis are about 50%, suitable with tumor, and the fibrosis of internal organs is the most important factor causing Patients with scleroderma's high mortality, the person that involves internal organs, and five year survival rate is low.Lung is one of organ of often involving of scleroderma, and pulmonary fibrosis is scleroderma main causes of death.SSc sickness rate is only second to rheumatoid arthritis, systemic lupus erythematosus (sle) and occupy the 3rd in connective tissue disease.
Scleroderma study mechanism and current treatment status: now there are some researches show that sclerodermatous morbidity is on the basis of heredity, under environmental factors inspires, cause immune system disorder, vascular endothelial injury, fibroblasts activation, the multiple short Collagen Proliferation factor too much discharges, and the extracellular matrix composition and decompositions such as collagen are unbalance, thus cause tissue fibering.Scleroderma is a kind of chronic auto-immune disease, turns to feature with multi viscera inflammation and Diffuse fibrous.Scleroderma process can be divided into the fibrosis phase in early stage inflammatory phase and late period, and inflammation has important function in sclerodermatous development.The main place of human body synthesis collagen is fibroblast, therefore the synthesis of fibroblastic quantity and activity and collagen is closely related.Regulate extracellular matrix metabolism cytokine in, with extracellular matrix build-up relation the closest be transforming growth factor β (transforming growth factor-β, TGF-β).In addition EMT (epithelial-to mesenchymal transition, Epithelial-mesenchymal transition) play a significant role in pulmonary fibrosis generating process, can there is EMT in epithelial cell, produce fibroblast, finally cause tissue fibering.
The main remedy measures of current doctor trained in Western medicine has the following aspects: 1. for the medicine of autoimmune disorder as glucocorticoid, immunosuppressant CTX etc.; 2. regulate the medicine of vascular function exception as prostaglandin, calcium channel inhibitor, angiotensin-convertion enzyme inhibitor; 3. regulate the medicine of extracellular matrix metabolism and biological product as penicillamine, IFN-γ, relaxin (relaxin) etc.But, these remedy measures less effective, or side effect is large, as renal failure, bone marrow depression, hepatic injury, pulmonary fibrosis etc.At present, medicine safely and effectively or the intervening measure that can improve the SSc state of an illness is not had.Early stage at disease progression, immunosuppressant, anti-inflammatory drug or anti-fibrosis medicine have the potentiality of symptom management.And disease is once develop into fiberising stage, existing treatment means is just difficult to stop disease progression and tissue injury.For scleroderma, the remedy measures less effective based on Western medicine or side effect are comparatively large, so far without any the anti-scleroderma agent that FDA (Food and Drug Adminstration) (FDA) and State Food and Drug Administration of China (SFDA) ratify.Therefore Be very effective is found and the little scleroderma treatment medicine of side effect becomes the task of top priority.
Clinical treatment in recent years and research display, Chinese medicine has obvious curative effect to scleroderma, and side effect significantly reduces, and many scholars treat this sick acquisition good therapeutic effect by activating blood circulation to dissipate blood stasis method for many years.Radix Salviae Miltiorrhizae is clinical the most frequently used blood-activating and stasis-removing, for many years, domestic use contain Radix Salviae Miltiorrhizae compound Chinese medicinal preparation treatment SSc, achieve good curative effect: its skin sclerosis, Raynaud phenomenon, pulmonary fibrosis are all improved significantly, and untoward reaction comparatively Western medicine is low.
The important water soluble ingredient of Radix Salviae Miltiorrhizae is salvianolic acid B (Salvianolic acid B, SAB), and also known as salvianolic acid B, salvianolic acid second, be the unique a kind of monomer component extracted from Radix Salviae Miltiorrhizae of listing at present, it occupies critical role in effect of Radix Salviae Miltiorrhizae.SAB has antiplatelet aggregation, antithrombus formation, improves the effect such as microcirculation, anti-oxidative damage.Clinical in Treating Stable Angina Pectoris of Coronary Artery Disease, SAB is to the Delayed Protection of heart microvascular endothelial cell.During heart ischemia anoxia; first and most easy damaged heart microvascular endothelial cell; experimentation show salvianolic acid B pretreatment can suppress Calcium overload in myocardial ischemia-reperfusion injury process, reduce the release of endothelin-1 (ET-1) and tumor necrosis factor α (TNF-α), reduce anoxia/reoxygenation injury after the expression of endotheliocyte ICAIU, play the effect of protection endotheliocyte.
Scleroderma is that a kind of thickening with limitation or diffuse cutaneous turns to feature with fiber, and involves the autoimmune disease of the internal organs such as the heart, lung, kidney, digestive tract, is attended by the infringement of Microvasculature simultaneously.Its reason feature is mainly collagen deposition and blood vessel injury in tissue, in view of the correlation function of above-mentioned SAB, intends the effect of this micromolecular compound of research in alleviation scleroderma fibrosis, and this research clinically provides solid theoretical foundation by being applied to for it.
Summary of the invention
Prior art in view of the above, the object of the present invention is to provide the novelty teabag of a kind of salvianolic acid B (Salvianolic acid B, SAB, CAS No.115939-25-8), for solving the problems of the prior art.
Salvianolic acid B chemical formula C
36h
30o
16, relative molecular mass: 718.62.Salvianolic acid B pure product is faint yellow, Powdered, has special odor, mildly bitter flavor, puckery.Water soluble, ethanol, methanol, multiple phenolic hydroxyl group in this micromolecular compound structure, has stronger non-oxidizability, and concrete molecular structure is as follows:
In the prior art, it is Radix Salviae Miltiorrhizae extraction method that the source of salvianolic acid B mainly contains two kinds: one, and two is chemical synthesiss.The special separating and extracting process for salvianolic acid B has a lot at present, and salvianolic acid B is that three molecule danshensus and the condensation of a part caffeic acid form, and the root and rhizome for labiate Radix Salviae Miltiorrhizae extracts and obtains.The separation preparation of salvianolic acid B, technical process is the technique such as extracting and developing, purification.The extracting method of salvianolic acid B has: traditional water decoction method, ultrasonic extraction, reflux extraction, supercritical CO
2extractions etc., the separation method of use has silica gel column chromatography, macropore natural gum chromatography, high performance countercurrent chromatography method etc.For chemical synthesis process; with 3-hydroxyl-4-methoxybenzaldehyde (different 4-hydroxyl-3-methoxylbenxaldehyde) for raw material; through Claisen rearrangement, acetylation and oxidation reaction synthesis 4-hydroxy-5-methyl oxygen base chromen-2-one; with methoxy methoxy base different 4-hydroxyl-3-methoxylbenxaldehyde, Aldol condensation reaction occurs again, open lactonic ring and produce methylol, methylol oxidation generates aldehyde; under hydrobromic acid effect, cyclization generates trans 2; 3-Dihydrobenzofuranes, aldehyde radical and malonic acid condensation generate acrylic acid, obtain salvianolic acid B.Salvianolic acid B goes on the market at present, and there are sale in many companies, and as Sigma etc., its preparation method is also prior art.
First aspect present invention provides the purposes of salvianolic acid B in the medicine of preparation treatment scleroderma (systemic sclerosis).
Preferably, described scleroderma is mainly the fibrosis of skin and internal organs, and the fibrosis of internal organs is mainly the fibrosis of lung.
Due to early stage in scleroderma, inflammatory reaction is its main manifestations, is also to promote fibroblast to activate further and the principal element of synthetic cell epimatrix.Wherein, cytokine network is unbalance, as the multiple proinflammatory inflammation factors such as TNF-a and IL-1 β take part in fibroblastic activation.Therefore inventor is by studying compound salvianolic acid B to the effect of the cellular inflammation that TNF α induces, find the cellular inflammation that salvianolic acid B can suppress TNF α to induce, and the rise of inflammatory factor gene expression can be reduced, thus reach inflammation-inhibiting signal path, block the effect of sclerodermatous disease progression.In addition, inventor also finds that salvianolic acid B has the effect suppressing EMT from protein level, and can effectively alleviate or T suppression cell fibrosis, reaches the object for the treatment of or relief of symptoms further.
In described medicine, can using salvianolic acid B as the sclerodermatous sole active ingredient for the treatment of, also can using salvianolic acid B as treatment one of sclerodermatous effective ingredient.
The medicine of described treatment scleroderma (systemic sclerosis) is the medicine suppressing the cellular inflammation of TNF α induction to block scleroderma disease progression; Or, for alleviating or T suppression cell fibrosis and treat scleroderma or alleviate the medicine of Scleroderma symptoms.
The present invention further discloses salvianolic acid B suppresses in the preparation of the cellular inflammation of TNF α induction or alleviation or the Fibrotic preparation of T suppression cell purposes in preparation.
Second aspect present invention provides one to be used for the treatment of the pharmaceutical composition of scleroderma (systemic sclerosis), includes the salvianolic acid B of effective amount in described pharmaceutical composition.
In described pharmaceutical composition, active ingredient is the salvianolic acid B of single component.
In described pharmaceutical composition, can also comprise other pharmaceutically acceptable promoter, described promoter can improve salvianolic acid B further at the beneficial effect for the treatment of or alleviate in sclerodermatous process, and/or reduces the side effect of salvianolic acid B.
Third aspect present invention provides one to be used for the treatment of or alleviates the pharmaceutical preparation of scleroderma (systemic sclerosis), and described pharmaceutical preparation is made up of described pharmaceutical composition and pharmaceutically acceptable adjuvant.
Described pharmaceutical preparation can be various forms known in those skilled in the art, as injection, tablet, unguentum, decoction etc., and is obtained by the customary preparation methods of this area.
The invention provides the new purposes of salvianolic acid B, i.e. the use of salvianolic acid B in anti-scleroderma fibrosis.Inventor significantly alleviates scleroderma mouse pulmonary fibrosis by zoopery display compound S AB, further cell experiment shows, the cellular inflammation that this micromolecular compound can suppress TNF α to induce, and scleroderma patient's skin flbroblast of In vitro culture, the propagation of human embryonic lung fibroblast and collagen expression can be suppressed, suppress pulmonary epithelial cells to the conversion of mesenchymal cell.The present invention shows this compound and has the Fibrotic effect of anti-scleroderma, has a good application prospect for sclerodermatous Prevention and Curation.
Accompanying drawing explanation
Fig. 1 is the schematic diagram of the effect that in prevention group mice of the present invention, SAB has amelioration of inflammation and suppresses myofibroblast to be formed;
Fig. 2 is the schematic diagram that in precaution and treatment group mice of the present invention, SAB stops the generation of pulmonary fibrosis;
Fig. 3-1 reduces the schematic diagram (P group) of the expression of glue protogene and albumen in mouse lung tissue for SAB of the present invention;
Fig. 3-2 reduces the schematic diagram (P & T group) of the expression of glue protogene and albumen in mouse lung tissue for SAB of the present invention;
Fig. 4 is the schematic diagram that SAB of the present invention reduces the expression of glue protogene and albumen in mouse lung tissue;
Fig. 5 is the schematic diagram that TNF α of the present invention can induce inflammatory Cytokines Expression in MRC5 cell;
Fig. 6-1 can reduce the schematic diagram of the expression of the inflammatory factor IL-6 that TNF α induces in NIH3T3 cell for SAB process of the present invention;
Fig. 6-2 can reduce the schematic diagram of the expression of the inflammatory factor iNOS that TNF α induces in NIH3T3 cell for SAB process of the present invention;
Fig. 6-3 can reduce the schematic diagram of the expression of the inflammatory factor MMP9 that TNF α induces in NIH3T3 cell for SAB process of the present invention;
Fig. 6-4 can reduce the schematic diagram of the expression of the inflammatory factor COX-2 that TNF α induces in NIH3T3 cell for SAB process of the present invention;
Fig. 7 is the schematic diagram that SAB process of the present invention can reduce the expression of the inflammatory factor that TNF α induces in MRC5 cell;
Fig. 8-1 can suppress scleroderma patient's skin flbroblast to breed schematic diagram for the present invention is based on xCELLigence systematic study result display SAB;
Fig. 8-2 can reduce collagen gene expression schematic diagram for SAB of the present invention;
Fig. 8-3 is secreted into the collagen content schematic diagram in cell conditioned medium for SAB of the present invention can reduce;
Fig. 9-1 is SAB antiproliferative effect schematic diagram in MRC5 cell of the present invention;
Fig. 9-2 is SAB reduction collagen gene expression schematic diagram in MRC5 cell of the present invention;
Fig. 9-3 is that in MRC5 cell of the present invention, SAB reduces the collagen content schematic diagram be secreted in cell conditioned medium;
Figure 10 is that SAB process of the present invention can the schematic diagram of conversion of T suppression cell mesenchyme sample;
Figure 11 is the expression that SAB of the present invention raises the epithelial marker protein E-cadherin that TGF-β associating TNF α suppresses, and reduces the schematic diagram of the expression of a marker protein α-SMA of myofibroblast.
Detailed description of the invention
Below by way of specific instantiation, embodiments of the present invention are described, those skilled in the art the content disclosed by this description can understand other advantages of the present invention and effect easily.The present invention can also be implemented or be applied by detailed description of the invention different in addition, and the every details in this description also can based on different viewpoints and application, carries out various modification or change not deviating under spirit of the present invention.
Before further describing the specific embodiment of the invention, should be understood that protection scope of the present invention is not limited to following specific specific embodiments; It is also understood that the term used in the embodiment of the present invention is to describe specific specific embodiments, instead of in order to limit the scope of the invention; In description of the present invention and claims, unless explicitly pointed out in addition in literary composition, singulative " ", " one " and " this " comprise plural form.
When embodiment provides numerical range, should be understood that except non-invention is otherwise noted, between two end points of each numerical range and two end points, any one numerical value all can be selected.Unless otherwise defined, the same meaning that all technology used in the present invention and scientific terminology and those skilled in the art of the present technique understand usually.Except the concrete grammar used in embodiment, equipment, material, according to those skilled in the art to the grasp of prior art and record of the present invention, any method of prior art that is similar with the method described in the embodiment of the present invention, equipment, material or that be equal to, equipment and material can also be used to realize the present invention.
Unless otherwise indicated, disclosed in the present invention experimental technique, detection method, preparation method all adopt the routine techniques of the molecular biology of the art routine, biochemistry, chromatin Structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and association area.These technology existing improving in existing document illustrates, specifically can see Sambrook etc.
MOLECULAR CLONING:A LABORATORY MANUAL, Second edition, Cold Spring HarborLaboratory Press, 1989and Third edition, 2001; Ausubel etc., CURRENT PROTOCOLS INMOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; The seriesMETHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATINSTRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODSIN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), AcademicPress, San Diego, 1999; With METHODS IN MOLECULAR BIOLOGY, Vol.119, ChromatinProtocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc.
Embodiment 1.
The compound salvianolic acid B used in the present invention is all obtained by commercially available approach.
Compound salvianolic acid B alleviates scleroderma mouse lung fiber:
Subjects: 7 week age C57BL/6 female mice, purchased from Fudan University's Experimental Animal Center
Test method:
1) foundation of scleroderma pulmonary fibrosis mice model: bleomycin induction scleroderma mouse model be ripe, by the Fibrotic good tool of the confessed research of various countries scientist.Bleomycin tracheal intubation imports pulmonary, can cause pulmonary fibrosis.Adopt C57BL/6 female mice in 7 week age, employing tracheal intubation administering mode sets up the scleroderma pulmonary fibrosis model that bleomycin (bleomycin, BLM) is induced.Bleomycin consumption, for changing to 9mg/kg, disposablely to give.[note: mice incubation time is as 2) as described in]
2) compound S AB process is to the Fibrotic effect of scleroderma: be divided into two parts:
I. first three sky of mice Intratracheal instillation bleomycin starts to give compound salvianolic acid B, and sclerotic model continues to give SAB mono-week, consumption 1.14mg/ pcs/day after setting up again, to stop Fibrotic formation, is called prevention group (Prevention, P);
Ii. first three sky of mice Intratracheal instillation bleomycin gives SAB, sclerotic model continues to give three weeks after setting up, consumption 1.14mg/ pcs/day, fibrosis is formed to alleviate, be called prevention+processed group (Prevention & treatment, P & T); Control group mice intraperitoneal injection of saline.
3) detection of the ECM gene such as the detection of scleroderma fibrosis mouse tissue and collagen and protein expression: the fibroid lung tissue obtaining above-mentioned mice, a frozen, for making paraffin section after a injection formalin solution fixes one week in addition, section is carried out HE dyeing and Masson dyeing, light Microscopic observation lung tissue inflammation and pulmonary fibrosis situation, compare to each group of mouse pulmonary fibrosis degree.In addition, utilize frozen tissue by the expression of Real-time PCR detection fibers related gene, comprise collagen protein, FN1, CTGF etc., detect the expression of collagen protein further by Sircol assay, and compared with control group mice.
Result of the test: lumbar injection micromolecular compound SAB can significantly alleviate scleroderma pulmonary fibrosis: the effect (Fig. 1) that in prevention group, SAB has amelioration of inflammation and suppresses myofibroblast to be formed; In prevention & processed group, SAB stops the generation (Fig. 2) of pulmonary fibrosis; SAB reduces the expression (Fig. 3-1, Fig. 3-2,4) of glue protogene and albumen in lung tissue.In addition, do not affect the physiological situations such as the body weight of mice compared to model group lumbar injection SAB, visible medicine does not produce toxic and side effects to mice.As shown in Figure 1, in prevention group, the mouse lung of Intratracheal instillation bleomycin there occurs obvious inflammatory reaction and fibrosis, and perfusion SAB then can significantly alleviate these inflammation and Fibrosis parameters, and suppresses myofibroblast to be formed.As shown in Figure 2, SAB can be observed in prevention+processed group equally there is the effect stoping fibrosis to occur.As shown in Fig. 3-1 and Fig. 3-2, the mouse lung tissue collagen gene C ol1a1 of Intratracheal instillation bleomycin, Col1a2 and Col3a1 up-regulated, in prevention group (P) and prevention & processed group (P & T), lumbar injection micromolecular compound SAB obviously can reduce the expression of these genes.As shown in Figure 4, in prevention group and prevention & processed group, lumbar injection compound S AB obviously can reduce the mouse lung tissue collagen protein content of Intratracheal instillation bleomycin.
Embodiment 2.
Compound salvianolic acid B suppresses the generation of the cellular inflammation of TNF α induction:
Subjects: NIH3T3 cell (mouse embryo fibroblasts), MRC5 cell (human embryonic lung fibroblast) test method:
1) apply TNF α irritation cell and set up external inflammatory cell model (Zhu X; Liu Q; Wang M; Liang M; Yang X; Xu X, Zou H, Qiu J.Activation of Sirt1by resveratrol inhibits TNF-α induced inflammation in fibroblasts.PLoS One.2011; 6 (11): e27081), the expression of the inflammatory factors such as IL-1 β, IL-6, COX-2, iNOS in TNF α different time points is detected by Real-time PCR;
2) cell through SAB or TNF α different disposal group is obtained, for SAB and TNF α processed group simultaneously, NIH3T3 cell DMEM culture fluid is cultivated, MRC5 cell MEM culture fluid is cultivated, 24 orifice plates about spread 5 powers of 8*105(10) individual cell, after cultivating 24h, the SAB of variable concentrations is added in cell culture fluid that (final concentration is 50 μ g/ml, 100 μ g/ml, 150 μ g/ml) after cultured cell 24h, add TNF α (final concentration is 10ng/ml), Real-time PCR detects the gene expression dose of relevant inflammatory factors, inquire into SAB anti-inflammatory effect in NIH3T3 and MRC5 cell.
Result of the test: In vitro cell experiment shows that SAB suppresses the cellular inflammation of TNF α induction, and inflammation-inhibiting effect should realize by suppressing NF-κ B signal path, concrete outcome is as follows:
1) TNF α stimulation establishes external inflammatory cell model: as relevant report, can induce inflammatory Cytokines Expression in MRC5 cell as Fig. 5 shows TNF α.The effect being established as follow-up study SAB inflammation-inhibiting of external inflammatory cell model provides the foundation.In Fig. 5, TNF-α (10ng/ml) processes MRC-5 different time, detects the expression about inflammatory factor mRNA, and result shows: TNF-α can the expression of the incite inflammation factor, and tool time effect; 1-36h all has inflammatory effector, and during 8h, inflammatory Cytokines Expression is relatively strong, therefore selects this time point to carry out subsequent detection.
2) inflammation gene expression that SAB suppresses TNF α to raise is expressed: SAB process can reduce the expression of the inflammatory factor that TNF α induces in NIH3T3 cell (Fig. 6-1 is to Fig. 6-4) and MRC5 cell (Fig. 7).As shown in figure Fig. 6-1 to Fig. 6-4, TNF α significantly can raise inflammatory factor: IL-6, iNOS, MMP9, COX-2 mrna expression; And SAB process can reduce the rise of inflammatory factor gene expression, and inhibitory action have dose dependent (
##p<0.001vs.Control; * P<0.05, * * P<0.001vs.TNF α); As shown in Figure 7, in MRC5 cell, same discovery TNF α significantly can raise inflammatory factor: IL-1B, IL-6, MMP9, COX-2 mrna expression; And SAB process can reduce inflammatory factor gene expression rise (
##p<0.001vs.Control; * P<0.05, * * P<0.001vs.TNF α).
Embodiment 3.
Compound salvianolic acid B plays the effect of fibrosis in fibroblast:
Subjects: scleroderma patient's skin flbroblast test method of MRC5 cell (human embryonic lung fibroblast), original cuiture:
1) based on the cyto-dynamics Model analysis system of xCELLIgence technology, SAB is on the impact of fibroblast proliferation in research.In the hole of each E-Plates plate, add 1000 cells, after cultivating 24h, replacing does not contain or contains the culture fluid of 100ug/mlSAB in cell, and Real Time Observation cell proliferation curve, cell index shows that more greatly cell is more.[illustrating: xCELLigence system, is the cold real-time cell analytical system of one that Roche is released, and utilizes biologic resistance reading to carry out non-subjectivity ground real-time quantization cell state.This system can monitor cellular activity in real time, and need not add labelling.This system can detect the electrical impedance on the interdigital microelectrode that is integrated in bottom tissue culturing E-Plates.Cell on E-Plates is more, and electrical impedance is larger, and cell index is larger, thus can showed cell number and cell proliferative conditions.】
2) cell through SAB or TGF β different disposal group is obtained, namely after first cultivating attached cell 24h with serum-free medium, 500ul1%FBS DMEM culture medium containing 100 μ g/ml SAB, 10ng/mlTGF β or 100 μ g/ml SAB+10ng/mlTGF β is added in 24 orifice plate cells, cultured cell 24h, Real-time PCR detects the expression change of glue protogene.Collagen is detected on protein level by Sircol assay collagen detection kit;
Result of the test: vitro in fibroblast experiment shows that SAB suppresses scleroderma human skin fibroblast (Fig. 8) and MRC5 cell (Fig. 9) propagation, but does not produce toxic action to cell; SAB can suppress the expression of glue protogene and albumen in scleroderma skin flbroblast and the beta induced lung fibroblast MRC5 of TGF-.As shown in Figure 8, scleroderma patient's skin flbroblast can be suppressed to breed (Fig. 8-1) based on xCELLigence systematic study result display SAB; In addition SAB can reduce collagen gene expression (Fig. 8-2, Real-time PCR) and be secreted into the collagen content (Fig. 8-3, sircol assay) in cell conditioned medium.As shown in Figure 9, the same antiproliferative effect of SAB (Fig. 9-1) in MRC5 cell; Reduce collagen gene expression (Fig. 9-2, Real-time PCR), and be secreted into the collagen content (Fig. 9-3, sircol assay) in cell conditioned medium.
Embodiment 4.
Salvianolic acid B suppresses pulmonary epithelial cells to the conversion of mesenchymal cell:
Subjects: A549 cell (Lu-csf-1 is lung II type epithelial cell)
Test method:
1) A549 cell is carried out different disposal: untreated fish group, TGF-β+TNF α Combined Treatment group, SAB processed group is added on basis to TGF-β+TNF α Combined Treatment.Namely bed board A549 cell the previous day is tested, within second day, change serum-free culture and cell is carried out Nature enemy 24h, then (24 orifice plates add 500ulDEME culture medium in cell culture fluid to add different reagent by above grouping, wherein each Reagents Final Concentration is 200 μ g/ml SAB, 10ng/mlTGF-β, 10ng/ml TNF α), after cultivating 48h, basis of microscopic observation cellular morphology change observation of cell metamorphosis, inquires into the impact that SAB occurs EMT.
2) detected the expression change of EMT related gene and albumen by Real-time PCR and Western blot, further clear and definite SAB is on the impact of EMT.
Result of the test: application A549 cell carries out EMT experiment in vitro and shows that SAB can suppress TGF-β to combine the conversion of A549 cell as derived mesenchymal-like cells of TNF α induction: under microscope, visible TGF-β associating TNF α can obviously induce A549 iuntercellular mesenchymal sample to transform, and SAB process can the conversion (Figure 10) of T suppression cell mesenchyme sample, further Western blot also shows the expression that SAB can raise the epithelial marker protein E-cadherin that TGF-β associating TNF α suppresses, reduce the expression (Figure 11) of a marker protein α-SMA of myofibroblast.As shown in Figure 10, have or without the condition of SAB under apply 10ng/ml TGF-β+10ng/ml TNF α process A549 cell, after 48h basis of microscopic observation cellular morphology change.Result display is compared to Control group (A), and in cellular morphology, visible TGF-β+TNA α processed group cell EMT occurs obviously (B), and SAB process can suppress the generation (C) of EMT.A:Control; B:TGF-β+TNF α; C:SAB+ (TGF-β+TNF α); As shown in figure 11, Western blot result display TGF-β combines the expression that TNF α obviously reduces epithelial marker protein E-cadherin, raises the expression of a marker protein α-SMA of myofibroblast, promotes the generation of EMT.And SAB process can raise the expression of E-cadherin, reduce the expression of derivative α-SMA, reflect the effect of SAB suppression EMT from protein level.
In sum, the present invention effectively overcomes various shortcoming of the prior art and tool high industrial utilization.
Above-described embodiment is illustrative principle of the present invention and effect thereof only, but not for limiting the present invention.Any person skilled in the art scholar all without prejudice under spirit of the present invention and category, can modify above-described embodiment or changes.Therefore, such as have in art usually know the knowledgeable do not depart from complete under disclosed spirit and technological thought all equivalence modify or change, must be contained by claim of the present invention.
Claims (4)
1. the purposes of salvianolic acid B in the sclerodermatous medicine of preparation treatment.
2. be used for the treatment of a sclerodermatous pharmaceutical composition, in described pharmaceutical composition, include the salvianolic acid B of effective amount.
3. pharmaceutical composition as claimed in claim 2, also comprises pharmaceutically acceptable promoter in described pharmaceutical composition.
4. be used for the treatment of a sclerodermatous pharmaceutical preparation, described pharmaceutical preparation is made up of the pharmaceutical composition described in the arbitrary claim of claim 2-3 and pharmaceutically acceptable adjuvant.
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| CN201310493321.5A Pending CN104546821A (en) | 2013-10-18 | 2013-10-18 | Application of danshinolic acid B in preparation of medicine of treating scleroderma |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104926764A (en) * | 2015-07-13 | 2015-09-23 | 广东环球制药有限公司 | Method for preparing dimethyl lithospermate B |
| CN111803486A (en) * | 2020-09-03 | 2020-10-23 | 上海交通大学医学院附属仁济医院 | Application of dihydroartemisinin in preparation of medicine for treating scleroderma |
| CN112138159A (en) * | 2019-06-28 | 2020-12-29 | 复旦大学 | Use of lactate dehydrogenase in the treatment of tissue inflammation and fibrosis |
| CN114081877A (en) * | 2020-08-24 | 2022-02-25 | 中国科学院上海药物研究所 | Application of magnesium salvianolate in preparation of anti-pulmonary fibrosis drugs |
| CN114870019A (en) * | 2022-05-13 | 2022-08-09 | 温州医科大学 | Application of STAP2 as target in preparation of medicine for treating scleroderma |
| CN115737622A (en) * | 2022-09-28 | 2023-03-07 | 中国人民解放军海军军医大学 | Application of salvianolic acid B in preparing medicine for preventing or relieving jellyfish sting |
-
2013
- 2013-10-18 CN CN201310493321.5A patent/CN104546821A/en active Pending
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| KOCA S. S.等: "Effectiveness of etanercept in bleomycin-induced experimental scleroderma", 《RHEUMATOLOGY》 * |
| QINGMEI LIU等: "The Effects of Salvianolic Acid B in Fibrotic Models in Vivo and in Vitro", 《2012 ACR/ARHP ANNUAL MEETING》 * |
| 朱鹭冰等: "系统性硬化病成纤维细胞单克隆Ⅲ型前胶原基因转录特性及丹参对其的调控研究", 《中华皮肤科杂志》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104926764A (en) * | 2015-07-13 | 2015-09-23 | 广东环球制药有限公司 | Method for preparing dimethyl lithospermate B |
| CN112138159A (en) * | 2019-06-28 | 2020-12-29 | 复旦大学 | Use of lactate dehydrogenase in the treatment of tissue inflammation and fibrosis |
| CN112138159B (en) * | 2019-06-28 | 2022-07-12 | 复旦大学 | Use of lactate dehydrogenase in the treatment of tissue inflammation and fibrosis |
| CN114081877A (en) * | 2020-08-24 | 2022-02-25 | 中国科学院上海药物研究所 | Application of magnesium salvianolate in preparation of anti-pulmonary fibrosis drugs |
| CN111803486A (en) * | 2020-09-03 | 2020-10-23 | 上海交通大学医学院附属仁济医院 | Application of dihydroartemisinin in preparation of medicine for treating scleroderma |
| CN114870019A (en) * | 2022-05-13 | 2022-08-09 | 温州医科大学 | Application of STAP2 as target in preparation of medicine for treating scleroderma |
| CN115737622A (en) * | 2022-09-28 | 2023-03-07 | 中国人民解放军海军军医大学 | Application of salvianolic acid B in preparing medicine for preventing or relieving jellyfish sting |
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