CN107260715A - A kind of method and its application of the Smads paths of regulation and control TGF β 1 - Google Patents

Abstract

The invention provides a kind of method and its application of the Smads paths of regulation and control TGF β 1, specifically the invention provides a kind of purposes of RXR α receptor stimulating agents, for preparing medicine or reagent, the medicine or reagent are used to suppress rats with acute myocardial infarction cardiac muscle I, the synthesis of III Collagen Type VI, and improve heart function etc..

Description

A kind of method and its application of regulation and control TGF-β 1-Smads paths

Technical field

The present invention relates to biomedicine field, in particular it relates to it is a kind of regulate and control TGF-β 1-Smads paths method and its Using.

Background technology

Ventricular remodeling after myocardial infarction mainly includes necrosis, apoptosis, hypertrophy and the fibrosis of cardiac muscle cell, and its Myocardial is thin The fibrosis of matrix is the main pathological change remolded after myocardial infarction, including the increase of Cardiac Fibroblasts activity and cardiac muscle Between extracellular matrix increase, finally result in arrhythmia cordis and heart failure.

Nuclear receptor is the superfamily member of a class ligand-dependent type transcription factor, retinol X receptor (Retinoid X Receptor, RXR) it is nuclear receptor superfamily important member, participate in differentiation and hyperplasia, the regulating cell energy generation of regulation cell Thank, lipid synthesis and immunologic function, played a significant role in the Physiopathologic proceeding of regulation and control angiocardiopathy.RXR can and its His a variety of nuclear receptors crosslink effect, such as vitamin D receptor (vitamin D receptor), Thyroid Hormone Receptors (TR), peroxisome Proliferators acceptor (peroxisome proliferators activated receptor α, β, γ, PPAR α, beta, gamma) and liver X receptor (liver X receptor, LXR) etc., all can be with RXR formation heterodimer (such as VDR/RXR, TR/RXR, PPAR/RXR, LXR/RXR etc.), improve the efficiency that they are combined with DNA.

The content of the invention

It is an object of the invention to provide a kind of method and its application of regulation and control TGF-β 1-Smads paths.

The first aspect of the present invention is there is provided a kind of purposes of RXR α receptor stimulating agents, for preparing medicine or reagent, institute State one or more purposes that medicine or reagent are used to be selected from the group：

(1) reduction TGF-β 1, and/or P-Smad3 level；

(2) P-Smad2, and/or Smad7 levels are improved；

(3) myocardium I, and/or III Collagen Type VI synthesis is suppressed；

(4) apoptosis of cardiac muscle cell is suppressed；

(5) the related disease for the treatment of TGF-β 1.

In another preference, the purposes includes suppressing acute myocardial infarction AMI object volume cardiac muscle I and III Collagen Type VI is closed Into.

In another preference, the purposes includes reduction myocardial infarction junctional area TGF-β 1, and/or P-Smad3 water It is flat.

In another preference, the purposes includes the level for improving myocardial infarction junctional area P-Smad2 and/or Smad7.

In another preference, the purposes includes reduction P-Smad3 levels, and improves P-Smad2 level.

In another preference, the purposes includes suppressing collage synthesis after myocardial infarction, so as to improve remodeling ventricle.

In another preference, the purposes includes improving the apoptosis of cardiac muscle cell after myocardial infarction.

In another preference, the RXR α receptor stimulating agents are Bexarotene, its analog, its derivative or its pharmacy Upper acceptable salt.

In another preference, " the related disease of TGF-β 1 " is myocardial infarction.

TGF-β 1 and/or P-Smad3 levels are reduced there is provided a kind of external non-therapeutic in the second aspect of the present invention Method, including step：By RXR α receptor stimulating agents and cells contacting, so as to reduce cell TGF-β 1 and/or P-Smad3 water It is flat.

In another preference, described contact is in the presence of RXR α receptor stimulating agents, to cultivate the cell.

In another preference, described cell includes cardiac muscle cell.

In another preference, methods described is additionally operable to a kind of at least following application：

Improve P-Smad2, and/or Smad7 levels external non-therapeutic；

Suppress myocardium I, and/or III Collagen Type VI synthesis external non-therapeutic；With

Suppress the apoptosis of cardiac muscle cell external non-therapeutic.

Prevent and/or treatment TGF-β 1, and/or Smads relevant diseases there is provided a kind of in the third aspect of the present invention Method, wherein the disease is relevant with TGF-β 1 and/or P-Smad3 overexpression or hyperactivity, methods described includes step： Object to needs applies RXR α receptor stimulating agents, or applies the pharmaceutical composition of the receptor stimulating agents of α containing RXR.

In another preference, described TGF-β 1 and/or Smads relevant diseases include myocardial infarction.

In another preference, described administration includes：Intravenous injection, oral tablet, intramuscular injection etc..

In another preference, described object is mammal.

In the fourth aspect of the present invention there is provided a kind of method, methods described be used for inner or in vitro reduction TGF-β 1 and/ Or P-Smad3 level or improve P-Smad2, and/or Smad7 levels, methods described include step：Object to needs Using RXR α receptor stimulating agents.

It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment) It can be combined with each other between each technical characteristic of body description, so as to constitute new or preferred technical scheme.As space is limited, exist This no longer tires out one by one states.

Brief description of the drawings

Fig. 1 shows ECG Change after the ligation of SD rat coronary arterys left anterior descending branch.10% chloraldurate intraperitoneal anesthesia, 4-5 Intercostal, which exposes, ligatures left anterior descending branch at heart, left auricle of heart lower edge 2-3mm.A. limb leads II before ligaturing, chart speed is 50mm/s；B. After ligation；Compared with before ligation, it is seen that II ST-Segments are substantially raised.

Fig. 2 shows the heart general form of different group rats.

Fig. 3 shows each experimental group echocardiography image.

Fig. 4 shows each experimental group HE dye images (× 200 times).Control groups and the myocardium marshalling of Sham groups, knot Structure is clear, accidental inflammatory cell；MI groups cardiac muscle arrangement disorder quantity is reduced, inflammatory cell infiltration, the visible a large amount of hyperplasia groups of interstitial Knit；MI+Bex10 and MI+Bex30 treatment groups structure is more complete, and the arrangement of cardiac muscle cell is relatively neat, interstitial proliferation tissue compared with It is few.

Fig. 5 shows each experimental group sirius red dye image (× 200 times).Control groups and Sham groups cardiac muscular tissue In visible a small amount of red dye filamentary fibers；Diffusivity is distributed the red dye fiber showed increased of MI groups in the form of sheets；MI+Bex10 and MI+ Bex30 treatment groups collagen is distributed in pencil shape current wellability.

Fig. 6 shows each experimental group I types, III Collagen Type VI SABC image (× 200 times).Control groups and Sham groups Without the expression of obvious collagen；Visible I type of MI groups, the obvious increase of III collagen type expression；MI+Bex10 and MI+Bex30 are controlled The type for the treatment of group I, the expression of III collagen type are reduced.

Fig. 7 A- Fig. 7 D show each group rat heart muscle tissue T GF- β 1, Smad2, P-Smad2, Smad3, P-Smad3 and The comparison of Smad7 protein expressions.Each group gel electrophoresis exposure figure and corresponding exposed plate photodensitometry figure：Fig. 7 A： TGF-β1、β-actin；Fig. 7 B：Smad7、β-actin；Fig. 7 C：P-Smad2, Smad2 and β-actin；Fig. 7 D：P-Smad3、 Smad3 and β-actin.Each experiment is repeated 3 times, and numerical value is expressed as mean ± standard deviation (n=3).＄ p<0.05, VS Sham Group；+p<0.05, VS MI groups；#p<0.05, VS MI+Bex10 groups.

Fig. 8 shows each group rat heart muscle Tunel stained apoptotics situation (× 200 times).Control groups, Sham groups, MI Group, MI+Bex10 groups and MI+Bex30 group rat myocardial infarction models area, infarct border area and non-infarcted region Tunel stained apoptotic figures Picture.

Fig. 9 shows different group myocardial infarction marginal zone tissue hyper-microstructure images (× 8K times).Control groups, Sham groups, MI groups, MI+Bex10 groups and cardiac muscular tissue of MI+Bex30 group rat infarct borders area ultra microstructure image.

Embodiment

The present inventor is surprised to find that RXR alfa agonists Bex can suppress acute first by in-depth study extensively Rat of Myocardial Infarction cardiac muscle I, the synthesis of III Collagen Type VI, and improve heart function；RXR alfa agonists Bex can significantly reduce myocardial infarction The level of junctional area TGF-β 1, P-Smad3, and improve P-Smad2 and Smad7 levels；RXR alfa agonists Bex may pass through TGF- β 1/Smads signal paths suppress collage synthesis after myocardial infarction, so as to improve remodeling ventricle；RXR alfa agonists Bex can improve the heart The apoptosis of cardiac muscle cell after flesh infarct.On this basis, the present invention is completed.

Transforminggrowthfactor-β1

Transforming growth factor-β (transforming growth factor- β, TGF-β) belong to one group it is newly discovered Adjust the TGF-β superfamily of cell growth and differentiation.TGF-β be a multi-functional pleiotropic growth factor cell proliferation, Migration, differentiation and apoptosis have wide influence.In mammal, TGF-β has three hypotypes (TGF-β 1,2 and 3), by three Individual different gene code.The biological function research of TGF-β is main in terms of inflammation, tissue repair and embryonic development, closely Find that growth, differentiation and immunologic function of the TGF-β to cell have important adjustment effect over year.TGF-β 1, β 2 and the functions of β 3 Similar, in general, TGF-β acts as a spur to the cell that mesenchyma originates from, and to the thin of epithelium or neuroectodermal origin Born of the same parents play inhibitory action.The major function of TGF-β includes：(1) propagation of immunocompetent cell is suppressed；(2) to the tune of cell phenotype Section；(3) differentiation of lymphocyte is suppressed；(4) cell factor generation etc. is suppressed.

Although the signal of TGF-β hypotype is by identical cell surface receptor and common target cell, they have not Same expression pattern.TGF-β 1 is main hypotype in cardiovascular system and wide expression, and other hypotypes are limited thin The spectrum of born of the same parents and tissue is found.Although the function of three kinds of hypotypes in vivo may be different, it is acted in myocardial fibrosis at present Most of knowledge is only limitted to TGF-β 1.In the molecular mechanism of myocardial fibrosis, TGF-β may is that topmost fibrosis life The long factor, what TGF-β can be notable and lasting in myocardial fibrosis experimental animal model and human heart fibrosis is activated.

TGF-β 1 is to be present in a kind of dormancy compound in normal heart, it is impossible to contacted with its acceptor.Heart injury Afterwards, the TGF-β of the resting state of extracellular aggregation is quickly converted to activity form；The dormancy compound TGF-β of relatively small amount Activation be enough to cause huge cell effect.Extensive medium has been described as TGF-β " activation " thing, in activation not Played a role with the stage.Protease, such as fibrinolysin, metalloproteinases 2 (MMP-2) and MMP-9 (MMP-9), The anakmetomeres of substrate degradation are connected, the integrality and stability of matrix is kept.In cardiac remodeling, extracellular matrix protein TSP-1 plays an important roll to TGF-β activation.In addition, the generation of active oxygen contributes to activation and the acidity of myocardial infarction TGF-β Environment also contributes to the activation of myocardial infarction TGF-β.The specific signals that TGF-β is activated in cardiac fibrosis are possibly relied on just The cardiac muscle of beginning is damaged to be proven by the mouse of the gene overexpression of TGF-β 1, the class of the confirmed myocardial such as Rosenkranz TGF wounds Type and intensity.

The rush Fibrosis parameters expression of TGF-β can aggravate collagen deposition and suppression interstitial collagenase causes myocardial fibrosis. Show that TGF-β take part in the fibrosis of remodeling ventricle using the method for afunction by different experiments.TGF-β 1+/- heterozygosis lacks The mouse of mistake shows the mitigation of age-related fibrosis and the improvement of left room compliance compared with wild type animal.This Outside, TGF-β retarding agent can prevent the fibrosis of cardiac pressure excess load rat heart muscle.

The synthesis of heart collagen is mainly by the signal cascade enlarge-effect of TGF-β, and the signal reaction is by TGF-β I types Initiated with the different aggressiveness that II types subunit is constituted.There is abundant evidence that Smad is the effect protein in the signal path downstream of TGF-β 1, Collagen deposition is caused to increase after heart injury, the combination of TGF-β and its acceptor promotes in R-Smad2 and/R-Smad3 in silk ammonia Sour threonine kinase activity causes phosphorylation.The R-Smads and Co-Smad of phosphorylation are combined, and are formed Smad2/3-smad4 and are combined Body dystopy, as transcription factor, drives the expression such as I-type collagen of target gene into nucleus.The expression of matrix gene is needed Smad3 induction is wanted, when Smad3 gene delections, fibrosis associated genes such as I-type collagen and CTGF (CTGF/CCN2) expression is interrupted.Inhibition Smads, I-Smad6 and I-smad7 by competition binding TGF-β I receptors and Increase receptor degradation, so as to block Smad2 and Smad3 activation.Effect of the TGF-β downstream signal in myocardial fibrosis by Confirm extensively.

The mechanism of myocardial fibrosis includes transforminggrowthfactor-β1 (transforming growth factor β 1, TGF-β 1), the activation of endothelin -1 and Angiotensin II etc. and extracellular Matrix albumen increase (such as NTx, III type glue Original etc.).Smads family proteins are the crucial effectors of TGF-β 1 intracellular one, the part of TGF-β 1 and the type of 1 II type of TGF-β I by Body is combined, and promotes Smad2/3 phosphorylations, and the Smad2/3 and Smad4 of phosphorylation, which are combined into after complex, enters regulation cell in core Consideration convey is recorded and then promotes the expression of extracellular matrix, and such as α-SMA, NTx, the expression of III Collagen Type VI increase, and Smad7 is TGF- The suppression albumen of β 1/Smad type signal paths, Smad7 can competitively be combined the TGF-β 1 I of activation with Smad2, Smad3 Receptor, blocks Smad2, Smad3 phosphorylation, and hinders the formation of Smad2, Smad3 and Smad4 compound of activation, from And inhibitory action is produced to TGF-β/Smad signals.

Bexarotene (Bexarotene, Bex)

Bexarotene (Bexarotene, Bex) is a kind of retinoid X receptor (RXR) activator, is used as the work of the present invention Property composition, its molecular formula be C24H28O2, chemical entitled 4- [1- (5,6,7,8- tetrahydrochysenes -3,5,5,8,8- pentamethyl -2- naphthyls) Vinyl] benzoic acid, its structural formula is as follows：

Inventive compound can also with as pharmaceutically or physiology it is acceptable acid or alkali derived from salt form use. These salt include but is not limited to the salt formed with following acid：Hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, breast Acid, pyruvic acid, acetic acid, butanedioic acid, oxalic acid, fumaric acid, maleic acid, oxaloacetic acid, methanesulfonic acid, ethyl sulfonic acid, benzene sulfonic acid or hydroxyl second Sulfonic acid.Other salt include：The salt formed with alkali metal or alkaline-earth metal (such as sodium, potassium, calcium or magnesium), and with ester, carbamic acid The form of ester or other conventional " pro-drugs ".

Term " treatment " refers to based on healing, alleviation, improvement, mitigation, influence treatment target disease, symptom, disease constitution (predisposition) purpose and PAQR3, its activator or the antagonist for giving the object present invention for needing to treat.

Term " object " refers to mouse, people and other mammals.

Term " therapeutically effective amount " be refer to reach in treatment target body therapeutic purposes inventive compound (including Its pharmaceutically acceptable salt) amount.It should be appreciated by those skilled in the art that " therapeutically effective amount " can be with the present invention The method of administration of active component, excipient substance used and different and different from other drugs drug combination situation.

Pharmaceutical composition and application process

The pharmaceutical composition of the present invention includes the inventive compound or its analog and medicine in the range of safely, effectively amount Acceptable excipient or carrier in reason.Wherein " safely, effectively measure " and refer to：The amount of active component is enough to be obviously improved disease Feelings, and be unlikely to produce serious side effect.Generally, pharmaceutical composition contains 0.001-1000mg active component/agent, preferably Ground 0.05-300mg active component/agent, more preferably, active component/agent containing 0.5-200mg.

The present invention active component and its pharmacologically acceptable salt can be made into various preparations, wherein comprising safely, effectively Active component of the invention or its pharmacologically acceptable salt and pharmacologically acceptable excipient or carrier in the range of amount. Wherein " safely, effectively measure " and refer to：The amount of active component is enough to be obviously improved the state of an illness, and is unlikely to produce serious secondary work With.Active component is safely, effectively measured according to concrete conditions such as the age for the treatment of target, the state of an illness, the courses for the treatment of to determine.

" pharmacologically acceptable excipient or carrier " is referred to：One or more biocompatible solids or liquid filler or Gelatinous mass, they are suitable for people and used and it is necessary to have enough purity and sufficiently low toxicity." compatibility " herein means Be in composition each component energy and the compound of the present invention and they between mutually admix, and significantly reduce the medicine of compound Effect.Pharmacologically acceptable excipient or carrier part example have cellulose and its derivates (such as sodium carboxymethylcellulose, second Base sodium cellulosate, cellulose ethanoate etc.), gelatin, talcum, kollag (such as stearic acid, magnesium stearate), calcium sulfate, plant Thing oil (such as soya-bean oil, sesame oil, peanut oil, olive oil), polyalcohol (such as propane diols, glycerine, mannitol, sorbierite), breast Agent (such as tween), wetting agent (such as lauryl sodium sulfate), colouring agent, flavor enhancement, stabilizer, antioxidant, preservative, Apirogen water etc..

During using the present composition, can orally, rectum, parenteral (intravenous, intramuscular is subcutaneous), local give Medicine.

The present composition can be administered alone, or with other pharmaceutically acceptable compound administering drug combinations.

The microcapsules of composition containing the present invention can be used for the sustained-release administration of the active component of the present invention.Recombinant protein Microcapsule controlled-release medicine-feeding technology has been successfully applied to human growth hormone recombinant (rhGH), recombinant human interferon alpha 2 (rhIFN), proleulzin With MNrgp120 (Johnson et al., Nat.Med., 2:795-799(1996)；Yasuda,Biomed.Ther27:1221- 1223(1993)；WO 97/03692,WO 96/40072,WO 96/07399；U.S.Pat.No.5654010.

The sustained release preparation of the active component of the present invention can use with the compatible and wide in range biodegradability of good biological It is prepared by lactic-glycolic acid high polymer (PLGA).PLGA catabolite, lactic acid and hydroxyacetic acid can quickly be removed by human body.And And, the degradation capability of the high polymer can with its molecular weight and composition difference, from some months extend to several years (Lewis, “Controlled release of bioactive agents form lactide/glycolide polymer,”in: M.Chasin and R.Langer(Eds.),Biodegradable Polymers as Drug Delivery Systems (MarcelDekker:New York,1990),pp.1-41))。

It is the lactation that the active component of the invention of safe and effective amount is applicable to treatment during using pharmaceutical composition Animal (such as people), wherein dosage is the effective dosage pharmaceutically thought when applying, for the people of 60kg body weight, every time Dosage is usually 0.01~300mg, preferably 0.5~100mg.Certainly, specific dosage is also contemplated that method of administration, Bing Renjian The factors such as health situation, within the scope of these are all skilled practitioners technical ability.

Beneficial effect of the present invention：

RXR alfa agonists Bex can significantly reduce myocardial infarction junctional area TGF-β 1, P-Smad3 (Smad3 of phosphorylation), P-Smad2 (Smad2 of phosphorylation) and Smad7 levels are improved, suppresses rats with acute myocardial infarction cardiac muscle I, the synthesis of III Collagen Type VI, So as to improve remodeling ventricle, and improve heart function, and the apoptosis of cardiac muscle cell after myocardial infarction can be improved.It therefore, it can For treating the diseases such as myocardial infarction.

With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention Rather than limitation the scope of the present invention.The experimental method of unreceipted actual conditions in the following example, generally according to conventional strip Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold SpringHarbor Laboratory Press, 1989) described in condition, or according to the condition proposed by manufacturer.Unless otherwise indicated, otherwise percentage and Number is percentage by weight and parts by weight.

Material

1.1 animal

12 week old male SD rats, body weight (250 ± 10g) (Shanghai Slac Experimental Animal Co., Ltd.)；

1.2 main agents and kit

10% chloraldurate, Benzylpenicillin sodium salt (The First Affiliated Hospital, Fujian Medical University medicine Drug Manufacturing Room)；RXR α receptor agonisms Agent Bexarotene (Bex) (Eisai companies)；Rabbit-anti rat Smad2 polyclonal antibodies, rabbit-anti P of Rats-Smad2 Anti-TNF-αs Body, rabbit-anti rat Smad3 polyclonal antibodies, rabbit-anti P of Rats-Smad3 polyclonal antibodies, rabbit-anti rat Smad7 polyclonal antibodies (Sigma companies)；Rabbit-anti rat β-actin monoclonal antibodies (Santa Cruz companies)；SABC secondary antibody kit, DAB Colour reagent box (Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge)；Rat TGF-β 1, NTx, III Collagen Type VI ELISA reagents Box (bioengineering Co., Ltd is built up in Nanjing)；The small Collagen Type VI monoclonal antibody of anti-rat III (Abcam companies)；Rabbit-anti rat NTx polyclonal antibody (Sigma companies)；X-ray film (Japanese Kodak)；Protein Molecular Weight Marker (10wer) (Thermo companies)；Pvdf membrane (Millipore companies).

1.3 capital equipments and apparatus

1.3.1 disposable syringe, immobilizing bandage, small animal surgical platform, operating scissors, knife blade, No. 7 band sheath veins are stayed Put pin, vessel forceps, ophthalmic tweezers, eye speculum, cotton swab, beauty treatment suturing needlework；

1.3.2 TKR-200 toys lung ventilator (special power anesthesia respiration machine equipment Co., Ltd, Jiangxi)；

1.3.3 BS110S precision electronic balances (Sartorius companies, Germany)；

1.3.4 4 DEG C of refrigerator SC-278A (company of Haier, Qingdao)；

1.3.5-20 DEG C of refrigerator BCD-208K (company of Haier, Qingdao)；

1.3.6-70 DEG C of ultra low temperature freezer DW-86L386 (company of Haier, Qingdao)；

1.3.7 stainless steel vertical electric steam pressure sterilization pot (three Shen Medical Devices Co., Ltd.s, Shanghai)；

1.3.8 DYY-III-6B types electrophoresis apparatus (Liuyi Instruments Plant, Beijing)；

1.3.9 Powerlab rats physiograph (Ai De companies, Australia)

1.3.10 decolorization swinging table (TS-100 types) (its woods Bel's instrument manufacturing Co., Ltd, Jiangsu)；

1.3.11 510 type pH analyzers (Cyberscan companies, Germany)；

1.3.12 iases of Image Pro Plus Version 4.5；

1.3.13 GE VIVID E9 ultrasonic systems (GE companies Japan)；

1.3.14 light microscope OLYMPUS CX31；

1.3.15 Philip EM208 type transmission electron microscopes

The preparation of 1.4 various buffer solutions and other solution

1.4.1 l×PBS：NaCl 8g, KCl 0.2g, Na₂HPO₄1.38g, KH₂PO₄0.2g, is dissolved in 1L distilled waters In, set up pH to 7.2~7.3.

1.4.5 5 × Tris- glycine running buffers：Tris alkali l5.1g, glycine 94g, SDS 5g, adjustment pH is extremely 8.3, plus distilled water is to 1000ml, room temperature preservation.

1.4.8 10 × TBS buffer solutions：NaCl 80g, Tris 30g, KCl 2g, after addition distilled water fully dissolves, 35% hydrochloric acid adjusts pH value to 7.2~7.3, and distilled water is settled to 1000ml, and 4 DEG C store for future use.

1.4.9 1 × TBST washes film buffer solution：L0 × TBS buffer solutions 100ml, Tween-20 1ml, distilled water is settled to 1000ml, room temperature preservation is standby.

1.4.10 3% skimmed milk power confining liquid：L × TBST buffer solution 20ml, skimmed milk power 0.6g.

1.4.11 30% acrylamide/N, N '-methylene bisacrylamide gel solution：Acrylamide 29g, N, N '-methylene Bisacrylamide 1g, distilled water is settled to 100ml, and room temperature preservation is standby.

1.4.12 1% Ponceaux solution：Glacial acetic acid 5ml, Ponceaux powder 1g, distilled water is settled to 100ml, completely molten Filter paper filtering room temperature is saved backup after solution.

1.4.13 10% ammonium persulfate solution：Ammonium persulfate 1g, deionized water is settled to 10ml, brown after being completely dissolved 4 DEG C of bottle is kept in dark place standby.

1.4.14 6 × DNA agarose gel electrophoresis sample-loading buffer：0.25% bromjophenol blue, 30% glycerine water solution.

1.4.15 50×TAE：2.0M Tris, 1.0M NaAc, 50mM EDTA, pH8.0.

1.4.16 5×TBE：Tris 54.45g, boric acid 27.9g, EDTA 3.72g, are completely dissolved with 1000ml distilled waters Adjust pH value to 8.3 afterwards, room temperature preservation is standby.

1.4.17 RIPA Tissue Lysis Buffers：50mM Tris-HCl (pH 6.8), 150mM NaCl, 1%Triton EDTA, 0.1%SDS, 4 DEG C of X-100, l%Sodium deoxycholate, 1mM is standby.

1.4.18 histone lysate：H₂The 200 10 μ L of μ L, 100 × PMSF of μ L, 5 × DTT of O 800, albumen enzyme level The μ L of agent 10.

1.4.19 10% separation gel 10ml：H₂O 4ml, 30% acrylamide solution 3.3ml, 1.5mol/l Tris (pH8.8) 2.5ml, 10%SDS 0.1ml, 10% ammonium persulfate 0.1ml, TEMED 0.004ml.

1.4.20 5% layer glue 5ml is moved：H₂O 3.4ml, 30% acrylamide solution 0.83ml, 1.0mol/l Tris (pH6.8) 0.63ml, 10%SDS 0.05ml, 10% ammonium persulfate 0.05ml, TEMED 0.005ml.

Method

2.1 experimental animals and packet：

12 week old male SD rats 40, body weight (220 ± 10g), purchased from this Leco Corp. of Shanghai.Rearing conditions：4-5 Only/cage, constant humidity 55 ± 5%, 22 ± 2 DEG C of constant temperature, each 12 hour/day of artificial lighting light and shade and takes food 24 hours free waters, raises Material is provided by this Leco Corp. of Shanghai.40 SD are randomly divided into 5 groups：1) A groups, control group (Control, n=8)；2) B groups, it is false Operation group (Sham, n=8)；3) C groups, heart infarction group (MI, n=8)；4) D groups, heart infarction 10mg/kg/d Bex administration groups (MI+ Bex10, n=8)；5) E groups, heart infarction 30mg/kg/d Bex administration groups (MI+Bex30, n=8).It is subjected to postoperative survival acute Heart infarction rat is randomized into C groups, D groups and E groups.Bexarotene is dissolved in physiological saline in D groups, E groups, given using administration by gavage Medicine, A groups, B groups and C groups then use normal saline gavage, and three groups once a day, continue 4 weeks.This animal experiment is via good fortune Build the license of experimental animal Ethics Committee of medical university.

The foundation of 2.2 acute myocardial infarction rat models：

1) reference literature Olivetti methods set up heart infarction model, 10% chloraldurate 0.2-0.3ml/100g dosage abdominal cavity note Penetrate anesthesia；

2) after the completion of 3-5min rat anesthesias, lie on the back in toy operating desk, fix its head and four limbs, double upper limbs and the right side Lower limb connect polygraph, record preoperative electrocardiogram；

3) pareordia and neck mouse hair are shaved, Iodophor wipes neck and tracheae is exposed in midsection separation, and No. 7 veins are stayed Pin is put in inserting tracheae 2-3cm under thyroid cartilage, nook closing member is exited, is positioned over before needle tubing whether check tracheae ventilation with thin cotton balls silk Smooth, fixed remaining needle connects lung ventilator, and parameter setting is respiratory rate 60 times/min, throughput 20-25ml/min；

4) skin is cut off through the intercostal of left side the 4th, blunt separation musculature opens thoracic cavity, cuts off pericardium, squeeze out the heart It is dirty；

5) No. 7-0 noninvasive suture ligature rat is used close at aortic root 2mm between left auricle of heart and pulmonary conus Left coronary artery descending anterior branch, is observed rat left chamber's antetheca ligature and is lost color with lower portion, heartbeat is slack-off, connects rat electrocardio after ligation Figure instrument, electrocardiogram shows lift prompting successful surgery on the ST sections of back of a bow；

6) it is rapid that heart is put back into thoracic cavity, exclude layer-by-layer suture after Thoracic gas and close thoracic cavity, suture chest and neck flesh Meat and skin, after extubation after rat autonomous respiration recovery.

Sham-operation group only in coronary artery anterior descending branch same area threading but is not ligatured, and other operations are with modeling group.It is postoperative The injection of the U hindlimb muscles of penicillin 80,000 is given, puts back in cage and continues to feed.To carrying out heart function and other indexs of correlation after 4 weeks Detection.

2.3 Colour Doppler echocardiography instrument survey rat heart structure and function

1) intraperitoneal injection of anesthesia is carried out to rat using 10% chloraldurate, the rat dorsal position anaesthetized is fixed on hand On art platform；

2) the preceding regio pectoris mouse hair of rat is rejected；

3) using GE VIVID E9 Doppler's echocardiography instrument, 12S probes (frequency 4-12MHz, equipped with EchoPAC113.1.3 work stations) collection M type ultrasonoscopy is seen in parasternal long axis of left ventricle, in chordae tendineae of mitral valve level acquisition Blood flow pulse Doppler image, the record organization doppler image at mitral anterior lobe annulus, synchronous recording heart rate；

4) measure and calculate between left ventricular end diastolic dimension (LVEDd), left ventricular end-systolic dimension (LVEDs), room Every end-diastolicthickness (IVSd), interventricular septum end-systole thickness (IVSs), LVPW end-diastolicthickness (LVPWd), a left side Room rear wall end-systole thickness (LVPWs), tissue Doppler imaging (TDI) detection aortic blood flow diastole early stage move with annulus Peak velocity ratio (E/e'), left LVSF (FS), by improvement Simpson methods calculate LVEF；

5) data above measured for 5 cardiac cycle, averaged.

It is prepared by 2.4 rat blood serum samples

1) 3-5ml rat cardiac ventricular blood is extracted after the completion of rat anesthesia to be placed in liquaemin anticoagulant tube；

2) 3500rpm centrifuges 5min, draws supernatant；

3) another EP pipes are moved into, -80 DEG C of refrigerators are standby.

2.5 rat weights, heart general form, cardiac mass, left ventricular mass and left ventricular mass index are determined

1) all rats are before the test (fasting can drink water)；

2) claim the weight of animals (BW) before putting to death, 10% chloraldurate (0.2ml-0.3ml/100g is injected intraperitoneally；)

3) 3-5min quickly cuts off thoracic cavity with scissors after anaesthetizing, and takes out heart, is positioned over culture dish normal saline flushing 2-3 times；

4) heart surface moisture is blotted, acquiring cardiac general form photo cuts off blood vessel and heart surface tissue, weighs the heart Dirty quality, calculates Heart quality index (HWI)；

5) eye scissors removes heart lung arteriovenous, superior and inferior vena cava and peripheral adipose tissue, filter paper along bilateral auricle upper limb Weighed after suck dry moisture；

6) atrium and ventricle are separated along mitral valve plane, then right ventricle are wiped out along interventricular septum, retain left ventricle and interventricular septum, Weigh, i.e. left ventricular mass (LVW)；

7) it is left ventricular mass index according to LVW/BW calculated values.

2.6 ventricular homogenate

1) rat left chamber antetheca cardiac muscular tissue 50mg plus cold saline 1ml are taken (containing protease suppression in per ml physiological saline Preparation 10ul), homogenizer is fully homogenized；

2) homogenate is placed into -80 DEG C of refrigerator multigelations 2 times；

3) Ultrasound Instrument concussion 10min, 3500rpm centrifugation 10min, move supernatant and are managed in another EP, -80 DEG C of refrigerators are standby.

2.7 cardiac muscular tissue HE are dyed

1) remove rat heart to rinse 2-3 times with PBS, cut the about 0.5cm apexes of the heart and atrial tissue, what input was prepared in advance 10% formalin fixes 48h；

2) heart tissue is taken out from formalin solution, running water rinses 30min；

3) transparent 30min in each 30min of 70%-100% alcohol serial dehydration, dimethylbenzene；

4) transparent cardiac muscular tissue is placed in the paraffin dissolved, is put into wax-dissolving box insulation, treats that paraffin is completely immersed in group Knit and embedded after block；

5) embedded wax stone is fixed on slicer, is cut into 4-6um thin slices, the thin slice cut is put into heating 40-50 Plate, then be attached on slide in DEG C hot water, put 60 DEG C of baking box 30min；

6) slide is taken out from baking box and is put into dimethylbenzene 15min, dimethylbenzene 25min, 100%-75% alcohol gradient water Change, often walk 3-5min, be put into running water washing 10min；

7) slide is taken out in water, the haematoxylin 1-2min of instant is added dropwise；

8) color separation, each several seconds in sour water and ammoniacal liquor；

9) flowing water rinses 1-2min and is put into distilled water a moment；

10) it is dehydrated each 5min in 75% and 95% alcohol；

11) alcohol eosin stains liquid dyeing 2-3min is entered；

12) section after dyeing is dried through 95% alcohol 30s；

13) neutral gum is added dropwise in section, and covered is dried, labelled.

2.8 cardiac muscular tissue's sirius red stains

1) draw materials and fixed with HE staining procedures 1-6 1) slide is taken out in water, 0.1% picric acid Picro-Sirius red is added dropwise Dye 1-2min；

2) flowing water enters distilled water a moment after rinsing；

3) each 5min is dehydrated in 75% and 95% alcohol, dried；

4) neutral gum is added dropwise in section, and covered is dried, labelled.

2.9 cardiac muscular tissue's immunohistochemical stainings

1) it is used for Collagen I and Collagen III detection, paraffin section de-waxing aquation, phosphate buffer (phosphate buffer saline, PBS) is rinsed；

2) 3% hydrogen peroxide lucifuge is incubated 15min, and PBS is rinsed；

3) will section immersion 0.01mol/L citrate buffers (ph 6.0), high fire 5min in Microwave method, in low fire 10min, liquid to be repaired naturally cools to room temperature；

4) PBS is rinsed；Normal Goat Serum confining liquid is added dropwise, room temperature 20min gets rid of surplus liquid；

5) (primary antibody concentration is respectively Collagen I 1 to dropwise addition primary antibody:100, Collagen III 1:50), in wet box 4 DEG C incubate Educate 16h；

6) room temperature rewarming 30min, PBS are rinsed；DAB is developed the color, and developing time is controlled under mirror, and color development stopping, flowing water is rinsed 10min；

7) haematoxylin dyeing 5min, flowing water rinses 10min；

8) 1% hydrochloride alcohol differentiation 4s, flowing water rinses 10min, dehydration, transparent, mounting；

9) × 200 times of images are gathered with light microscope OLYMPUS CX31.

2.10 Western-blotting detections cardiac muscular tissue Smad2, P-Smad2, Smad3, P-Smad3 and Smad7 Protein expression

2.10.1 rat heart muscle histone is extracted

1) antetheca cardiac muscular tissue of rat left chamber 50mg is taken to add 1ml RIPA Tissue lysates, homogenizer is homogenized 2- on ice 3min；

2) homogenate is moved in EP pipes, -80 DEG C of placement refrigerator multigelation 2-3 times；

3) volume 1 is pressed:1 adds 2 × SDS, and 10min is shaken with Ultrasound Instrument after forced oscillation 15s, and boiling water places 10min, 12000g centrifuges 10min, takes supernatant to move into another EP pipes, -80 DEG C of refrigerators are saved backup.

2.10.2 Coomassie Brilliant Blue cardiac muscular tissue determination of protein concentration

1) analysis of protein standard curve is drawn, 1g/ml bovine serum albumin(BSA)s (BSA) solution is prepared；

2) 96 orifice plates, plus 200 μ l/well G250 Coomassie brilliant blue dye liquors are taken, are added with concentration 0,2,4,6,8,10mg/ml Enter BSA, and take multiple holes, place 15min；

3) ELIASA 595nm wavelength detectings absorbance, makes standard regression curve and curve equation；

4) 96 orifice plates, plus 200 μ l/well G250 Coomassie brilliant blues dye liquors and 1 μ l protein samples, blank control group separately are taken Plus the μ l of cell lysis buffer solution 1, it is stored at room temperature 15min after mixing；

5) the OD values of ELIASA 595nm wavelength detectings sample, are subtracted after blank control, and it is dense to calculate albumen according to standard curve Degree.

2.10.3 protein electrophoresis

2.10.3.1 encapsulating

1) glass plate is cleaned, distilled water is rinsed 1 time, and 95% alcohol is cleaned, clamping plate is installed in drying；

2) 10%SDS-PAGE separation gels 10ml, is mixed rapidly after adding TEMED；

3) 10% separation gel is injected into double glazing unit with pipettor, glue liquid surface is away from plastic comb lower edge line about lcm, then 95% alcohol about 0.5ml is uniformly added into, about 30min is stood at room temperature, the non-agglomeration liquid in upper strata is discarded, is rinsed and separated with distilled water The upper glue surface of glue 3 times, raffinate is sucked with filter paper；

4) 5%SDS-PAGE spacer gels 5ml (dd H2O 3.4ml, 30% acrylamide 0.83ml, 1.0M PH6.8Tris 0.63ml, 10%SDS 50ul, 10% ammonium persulfate 50ul, TEMED 8ul), mixed immediately after adding TEMED, by spacer gel Directly it is filled on the separation gel having polymerize, is immediately inserted into Teflon combs (being eluted with water before use, ethanol is dried), notes not It is mixed into bubble；

5) 30min is stood at room temperature, is gently extracted, is steamed with double straight up after the both sides that comb is pinched after spacer gel solidification Water rinses loading slot, to remove the acrylamide of residual, if necessary with the well of sample injector amendment spacer gel.

2.10.3.2 albumen loading

1) protein sample to be measured is taken out from -80 DEG C of refrigerator, is placed in 100 DEG C of water-bath 3-5min；

2) pipettor draws 15ul samples and adds loading groove in a predetermined order.

2.10.3.3 electrophoresis

1) PAGE gel after sample-adding is put into electrophoresis tank (inwardly, high glass-board surface is outside for short glass-board surface)；

2) electrophoretic buffer is added in electrophoresis tank, electrophoresis liquid need to cover groove under inner glass plate, electrophoresis and add running buffer Liquid does not descend groove electrode excessively；

3) switch on power, positive pole connects lower groove, adjust 80-100V electrophoresis to bromjophenol blue forward position to enter separation gel interface, electricity Pressure adds to 120V, treats that bromjophenol blue band electrophoresis, to separation gel bottom, closes power supply.

2.10.4 transferring film

1) 5 × 2cm Whatman filter paper 4 is taken, onesize pvdf membrane is cut, pvdf membrane soaks 1-2min in methyl alcohol, Immersed after distilled water rinsing together with filter paper in transferring film buffer solution；

2) glass plate is gently pried open, spacer gel is scraped off, notes avoiding damage to separation gel, mesh is recognized according to albumen Marker Molecular weight of albumen size is marked, cutting separation gel, with big, immerse transferring film electricity and turn liquid 5min with pvdf membrane；

3) to it is lower and on according to carbon anode plate, filter paper, gel, pvdf membrane, filter paper, negative electrode carbon plate order align place, often One layer is stacked to be intended to remove bubble removing with glass bar；

4) add about 3-5ml transferring film electricity and turn liquid, cover electroplax, switch on power, electric current is 2m A/cm2, electric transferring film electricity turns 1.5h；

5) pvdf membrane is taken out, distilled water is rinsed 2 times, immerses 0.1% Ponceaux dye liquor pre-dyed, judges the albumen one after electric turn Band distribution, the film according to where albumen Marker clip target proteins, size about 1-2cm is wide.

2.10.5 closing

Pvdf membrane after trimming is moved in 3% skimmed milk power confining liquid, shaking table is incubated 1h at room temperature.

2.10.6 primary antibody, secondary antibody are incubated

1) primary antibody：3% skimmed milk power is 1:1000, together it is put into closed poly-bag with pvdf membrane, pvdf membrane is totally submerged In primary antibody dilution (3% skimmed milk power)；

2) 4 DEG C of incubation 12h；

3) pvdf membrane is taken out, 1 × TBST is rinsed 3 times at room temperature, each 5min；

4) secondary antibody：3% skimmed milk power is 1:5000, together it is put into closed poly-bag with pvdf membrane, film is totally submerged in two In anti-dilution (3% skimmed milk power)；

5) 37 DEG C of incubation 1-1.5h；

6) pvdf membrane is taken out, 1 × TBST is rinsed 3 times at room temperature, each 5min.

2.10.7 Protein Detection

1) pvdf membrane is lain on preservative film, A, B developer manage medium volume mixture in EP in ECL kits, uniformly Drop on pvdf membrane (protein powder), be incubated at room temperature 1min；

2) ECL working solutions unnecessary on pvdf membrane are discarded, pvdf membrane is wrapped up with preservative film；

3) by memebrane protein facing to the film placed in exposure box, 1-2min is exposed；

4) 2-3min in film, immersion developer solution is taken out in darkroom；

5) film is taken out from developer solution, immersion fixing solution about 5min after clear water rinsing；

6) film is taken out from fixing solution, clear water washing 3-5min dries；

7) scanning of film strip band, the Reinhoit Zahl of target protein and β-actin is analyzed with gel images processing system, Represent the expression of target protein.

2.11 cardiac muscular tissue Elisa are detected

1) BNP contents in TGF-β 1, Collagenl and the contents of Collagen III, serum in thought-read muscular tissue, according to ELISA kit illustrates to be operated, 450nm wavelength measurement OD values；

2) regression equation of standard curve is calculated according to normal concentration and OD values, and calculates sample concentration.

2.12 paraffin section Tunel are detected

1) paraffin section dimethylbenzene embathes 2 times, each 5min；Respectively soaked with graded ethanol (100,95,90,80,70%) Wash 1 time, each 3min；

2) PBS rinses 2 times × 5min；With Proteinase K working solutions processing tissue 15-30min in 21-37 DEG C of (temperature Degree, time, concentration are both needed to grope)；

3) PBS rinses 2 times × 5min；

4) liquid unnecessary around tissue on sheet glass is sucked with filter paper, 50 μ l tunnel reaction mixtures are added dropwise, put 37 DEG C are reacted 1 hour in wet box；

5) PBS rinses 3 times × 5min；

6) POD is added dropwise, 37 DEG C × 30min in wet box is put；PBS rinses 3 times × 5min；

7) colour developing of DAB solution, micro- Microscopic observation is added dropwise；

8) haematoxylin is redyed, and gradient alcohol dehydration, dimethylbenzene are transparent, neutral gum mounting.Brown color or brown cytochrome Core is apoptotic cell, and bluish violet is non-apoptotic cell, and negative control piece is without brownish discoloration；

9) light Microscopic observation and piece is taken the photograph, 5 visuals field is randomly choosed respectively in infarcted region, infarct border area and non-infarcted region, To have muscle fibre to confirm as cardiac muscle cell around nucleus, the apoptotic cell dyed using TUNEL uses Image- as positive cell The softwares of Pro-Plus 6.0 count each visual field Apoptosis and account for the percentage of sum as apoptotic index respectively (Apoptosis Index, AI).

2.13 transmission electron microscope

1) draw materials：(on demand and to ask for sample)；

2) it is fixed before：3% glutaraldehyde -1.5%, 4 DEG C of a couple of days (more than 2h) of paraformaldehyde -0.1M PBS (pH7.2)；

3) rinse:0.1M PBS (pH7.2) 3 times；

4) fixed after：4 DEG C of 1.5h of -1.5% potassium ferrocyanide of 1% osmic acid；

5) rinse:0.1M PBS (pH7.2) 3 times；

6) it is dehydrated：50% alcohol 10min → 4 DEG C of 70% alcoholization acetic acid uranium dye liquor stay overnight → 90% alcohol 10min → The acetone 10min of 90% alcohol-acetone 10min → 90% → anhydrous propanone 10min X 3 times；

7) it is impregnated with：(1) embedding medium (1 of anhydrous propanone+epoxy resin 618:1) 1.5h,

(2) pure 618 embedding medium, 35 DEG C of 3h；

8) embed, polymerize：35 DEG C of 12h, 45 DEG C of 12h, 60 DEG C of 3d.

9) fast, semithin section, positioning are repaiied

10) cut into slices, dye：Leica UC-6 type ultra-thin section machine-cuts 100nm ultra-thin section；Through acetic acid uranium, citric acid Lead dyes 5-15min respectively, and distilled water is washed.

11) observed under PHILIPS EM208 type transmission electron microscopes and take the photograph piece.

The Rat of Myocardial Infarction of embodiment 1. is modeled and survival condition

Coronary artery anterior descending branch is tied in art, it is seen that tie the following regional myocardial hypomotility in position, and color is pale, with art Before compare, electrocardio illustrate ST sections substantially raise (Fig. 1).Operated rats 45, postoperative 8 rats of sham-operation group all survive, and deposit Motility rate 100%；Operation group survival of rats 26, survival rate 70%.Overdose of anesthesia 1 is died from, heart massive haemorrhage 2 is died from, extremely In pneumothorax 3, ventricular fibrillation and cardiac arrest 5 are died from.Each group rat ensures that experiment survives 8 only for materials after terminating.Do evil through another person Art group rat, growth conditions are good, and the closely knit light of hair is smooth, and activity is big, agile.The postoperative Hair of Rats of myocardial infarction Sparse and mixed and disorderly dimness, activity is less, is short of breath, and acts slower.

The Bexarotene of embodiment 2. treat 4 weeks after to heart infarction SD rats TNF-α, IL-10, myocardium enzyme, blood fat, renal function And the influence of liver function

Sham groups, MI groups, the TNF-α of MI+Bex10 groups and MI+Bex30 groups, BUN levels are without significant difference, MI groups, MI+ TG, VLDL-C level of Bex10 groups and MI+Bex30 groups are without significant difference, MI+Bex10 groups, MI+Bex30 groups and Sham groups HDL-C levels are without significant difference, and Sham groups, the NEFA of MI groups level are without significant difference.

TG, VLDL-C level of MI groups significantly reduce (P compared with Sham groups<0.05)；MI+Bex10 groups and MI+Bex30 groups IL-10, VLDL-C, TG, NEFA expression are remarkably decreased (P compared with Sham groups<0.05)；MI+Bex10 groups, MI+Bex30 groups NEFA levels compared with MI groups substantially reduction (P<0.05).

MI groups Scr level is compared with Sham groups significantly rise (P<0.05)；The TCHO of MI+Bex10 groups and MI+Bex30 groups, HDL-C, LDH, LDL-C level are compared with MI groups significantly rise (P<0.05)；MI+Bex30 group CK-MB levels compared with Sham groups, MI groups and MI+Bex10 group groups significantly raise (P<0.05)；LDH, LDL-C, ALT, Scr level of MI+Bex10 groups and MI+Bex30 groups with Sham groups are than more significant rise (P<0.05)；MI+Bex30 groups Scr level is compared with MI groups significantly rise (P<0.05)；MI+Bex10 Group ALT level is compared with MI groups significantly rise (P<0.05).(being shown in Table 1)

The Bexarotene of table 1 treat 4 weeks after to heart infarction SD rats TNF-α, IL-10, myocardium enzyme, blood fat and hepatic and renal function Influence

Note：Numerical value is expressed as mean ± standard deviation (n=8).^＄p<0.05, VS Sham groups；⁺p<0.05, VS MI groups；^#p< 0.05, VS MI+Bex10 groups.

Rat heart general form is observed after the myocardial infarction of embodiment 3.

Sham groups：Heart surface is smooth, and geometric shape is regular, and profile is in turbination；Heart general form, cardiac chamber volume Normally, ventricle thickness uniformity, elasticity and toughness are good；It is inflammatory hyperplasia under the external membrane of heart at threading, it is seen that slight pale, it is remaining Heart surface color is in light red color.

MI groups：Heart surface is rough, and infarcted region myocardial fibrosis forms cicatricial tissue, and profile is in the general large-scale heart, is lost Original geometric shape；Heart size and left chamber substantially expand, and the thinning outside bulge of infarcted region cardiac muscle, elastic and first property disappears Lose；Infarcted region cardiac muscle is in pale asphyxia, and notable difference is formed with non-infarcted region, and non-infarcted region locular wall is substantially thickened；Most mouse stalks Dead band cardiac muscle has formed aneurysm, and in body, bulging is in spherical, and in vitro rear shrinkage is recessed into ventricular chamber.

MI+Bex10 and MI+Bex30 treatment groups：Cardiac shape increases, and colour cast is dark red, ventricular wall thickness increase, infarcted region Elasticity and paving property weaken, and surface owes smooth.Compared with MI groups, infarcted region range shorter, it is seen that pale asphyxia；Heart can be maintained substantially Normal geometry, volume-diminished；Infarcted region still surviving cardiac muscle tissue, elasticity and first property are preferable；Cardiac chamber dilation degree mitigates.

The Bexarotene of embodiment 4. treat 4 weeks after to heart infarction SD rat body weights, whole-heartedly performance figure and left ventricular mass index

4 weeks model groups after heart infarction, the weight loss (P compared with Sham groups<0.05), and whole-heartedly performance figure, Zuo Xinzhi Volume index increases (P<0.05).4 weeks Bex treatment groups of MI+Bex10 and MI+Bex30 are compared with MI groups, body weight, whole-heartedly matter Volume index and left heart performance figure situation are made moderate progress, and the above-mentioned situation of MI+Bex30 treatment groups is treated better than MI+Bex10 Group.(being shown in Table 2)

The Bexarotene of table 2 treat 4 weeks after to heart infarction SD rat body weights, whole-heartedly quality, left ventricular mass, whole-heartedly performance figure and The influence of left ventricular mass index

Note：Numerical value is expressed as mean ± standard deviation (n=8).^＄p<0.05, VS Sham groups；⁺p<0.05, VS MI groups；^#p< 0.05, VS MI+Bex10 groups.

Influence of the Bexarotene intervention of embodiment 5. to heart infarction rat heart structure and function

Compared with Sham groups, MI groups left ventricular end diastolic dimension (LVEDd), left ventricular end-systolic dimension (LVEDs), LVPW end-diastolicthickness (LVPWd), LVPW end-systole thickness (LVPWs) and tissue Doppler imaging (TDI) Detection aortic blood flow diastole early stage significantly increases (P with annulus motion peak velocity ratio (E/e')<0.05), left room short axle contracting Short rate (FS), Left Ventricular Ejection Fraction (LVEF) significantly reduce (P<0.05), interventricular septum end-diastolicthickness (IVSd), interventricular septum End-systole thickness (IVSs) no significant difference；MI+Bex10 and MI+Bex30 treatment groups left ventricular end diastolic dimension (LVEDd), LVPW end-diastolicthickness (LVPWd), LVPW end-systole thickness (LVPWs), tissue Doppler show As (TDI) detection aortic blood flow diastole early stage and annulus motion peak velocity ratio (E/e') and Left Ventricular Ejection Fraction (LVEF) Make moderate progress (P<0.05).(being shown in Table 3, Fig. 3)

Compared with Control groups, Sham groups, MI groups ELISA method surveys Plasma brain natriuretic peptide and significantly raises (BNP Control291.59±17.08pg/ml vs Sham 304.30±11.96pg/ml vs MI 471.90±97.19pg/ Ml, P<0.05)；Compared with MI groups, MI+Bex10 groups and MI+Bex30 group Plasma brain natriuretic peptides significantly reduce (BNP MI471.90 ± 97.19pg/ml vs 354.81 ± 68.88pg/ml of MI+Bex10 vs MI+Bex30309.51 ± 87.68pg/ml, P ＜ 0.05)；MI+Bex10 groups are with MI+Bex30 group Plasma brain natriuretic peptides without significant difference.(being shown in Table 3)

Influence to heart infarction SD rat hearts structure and function after the Bexarotene of table 3 is treated 4 weeks

Note：Numerical value is expressed as mean ± standard deviation (n=8).^＄p<0.05, VS Sham groups；⁺p<0.05, VS MI groups；^#p< 0.05, VS MI+Bex10 groups.

The rats with myocardial infarction tissue HE coloration results of embodiment 6.

Compared with Control groups and Sham groups, MI groups infarcted region locular wall is thinning, and cardiac muscle cell's arrangement disorder quantity is reduced, Inflammatory cell infiltration, the visible a large amount of hyperplastic tissues of extracellular matrix；Compared with MI groups, left room antetheca cell quantity is more, MI+ Bex10 and MI+Bex30 treatment groups myocardial structural is more complete, and the arrangement of cardiac muscle cell is relatively neat, and cardiac muscle cell's external series gap increases Raw tissue is less；Compared with MI+Bex10 groups, MI+Bex30 treatment groups myocardial structural is more complete, arranges more neat, cardiac muscle Extracellular matrix hyperplasia is less.(see Fig. 4).

Influence of the Bexarotene of embodiment 7. to fibrosis area after rat myocardial infarction model

Picro-Sirius red picric acid coloration result is shown：Visible a small amount of red dye silk in Control groups and Sham groups cardiac muscular tissue Shape fiber；MI groups：Diffusivity is distributed the red dye fiber showed increased in myocardial infarction area and infarct border area in the form of sheets, and locular wall is thinning； MI+Bex10 and MI+Bex30 treatment groups：The visible collagen in myocardial infarction area and infarct border area in pencil shape current wellability distribution (see Fig. 4).Myocardial collagen analysis result is shown：Compared with Control groups and Sham groups, the horizontal showed increased (P of MI group myocardial collagens< 0.05)；Compared with MI groups, MI+Bex10 and MI+Bex30 treatment groups collagen level decreases (P<, and MI+ 0.05) Bex30 treatment groups are compared with the MI+Bex10 treatment groups lower (P of fibrosis area numerical value<0.05) (4 are shown in Table).

Each experimental group Rat Myocardial Fibrosis Area comparison of table 4

Note：Numerical value is expressed as mean ± standard deviation (n=8).^＄p<0.05, VS Sham groups；⁺p<0.05, VS MI groups；^#p< 0.05, VS MI+Bex10 groups.

Embodiment 8.ELISA methods and Immunohistochemical Method detect the albumen table of myocardial infarction area type i collagen, III Collagen Type VI respectively Reach

Compared with Control groups and Sham groups, MI groups, the type of infarct border area of MI+Bex10 and MI+Bex30 treatment groups I, III Collagen type expression dramatically increases (P<0.05)；Compared with MI groups, the type of MI+Bex10 and MI+Bex30 treatment groups I, III type glue Former protein expression is significantly reduced, and the type of MI+Bex30 treatment groups I, III collagen type expression quantity are substantially less than MI+Bex10 treatments Group (P<0.05) (it is shown in Table 5, table 6, Fig. 6).

The ELISA method of table 5 surveys each group rat heart muscle tissue Collagen I, the levels of Collagen III

Note：Numerical value is expressed as mean ± standard deviation (n=8).^＄p<0.05, VS Sham groups；⁺p<0.05, VS MI groups；^#p< 0.05, VS MI+Bex10 groups.

The expression of the cardiac muscular tissue's collagen of table 6 and the ratio result of I/III collagen

Note：Numerical value is expressed as mean ± standard deviation (n=8).^＄p<0.05, VS Sham groups；⁺p<0.05, VS MI groups；^#p< 0.05, VS MI+Bex10 groups.

The Bexarotene of embodiment 9. is to heart infarction rat heart infarction marginal zone cardiac muscular tissue TGF-β 1, P-Smad2, Smad2, P- The influence of Smad3, Smad3 and Smad7 level

Compared with Control groups and Sham groups, the left room anterior infarcts marginal zone cardiac muscular tissue TGF-β 1 of MI groups, P-Smad2, P-Smad3 and Smad7 dramatically increase (P<0.05)；Compared with MI groups, MI+Bex10 groups and MI+Bex30 groups TGF-β 1, P- Smad3 significantly reduces (P<0.05), and P-Smad2, Smad7 level dramatically increase (P<0.05), and MI+Bex30 groups TGF-β 1, P-Smad3 is less than MI+Bex10 groups (P<0.05), and P-Smad2, Smad7 level be higher than MI+Bex10 groups (P<0.05)；Each group Between Smad2, Smad3 level without significant difference.(being shown in Table 7, Fig. 7 A, B, C, D)

The ELISA method of table 7 surveys the level of each group rat myocardial infarction model marginal zone TGF-β 1

Note：Numerical value is expressed as mean ± standard deviation (n=8).^＄p<0.05, VS Sham groups；⁺p<0.05, VS MI groups；^#p< 0.05, VS MI+Bex10 groups.

The Bexarotene of embodiment 10. intervenes the influence to cardiac muscle cell apoptosis after heart infarction rat

Compared with Control groups and Sham groups, MI groups infarcted region, infarct border area and non-infarcted region cardiomyocyte apoptosis index are aobvious Write increase (P ＜ 0.05)；Compared with MI groups, MI+Bex10, MI+Bex30 group infarcted region, infarct border area and the non-infarcted region heart Flesh apoptotic index is significantly reduced (P ＜ 0.05)；Compared with MI+Bex10 groups, in infarct border area, MI+Bex30 apoptotic indexs are notable Reduce (P ＜ 0.05)；In infarcted region and non-infarcted region MI+Bex10, MI+Bex30 group apoptotic index no significant difference.(8 are shown in Table, Fig. 8)

The Bexarotene of table 8 is thin to heart infarction SD rat myocardial infarction models area, infarct border area and non-infarcted region cardiac muscle after treating 4 weeks The influence of born of the same parents' apoptotic index

Note：Numerical value is expressed as mean ± standard deviation (n=5).Compared with Sham groups,^＄p<0.05；Compared with MI groups,⁺p< 0.05；；Compared with MI+Bex10 groups,^#p<0.05。

The Bexarotene of embodiment 11. treat 4 weeks after to the shadow of heart infarction SD rat myocardial infarction models marginal zone tissue hyper-microstructure Ring

Compared with Control groups and Sham groups, MI groups cardiac muscle fibre arranges sparse, disorder, and part cardiac muscle cell's plasma membrane is not Intact mitochondria is spilt and in swelling state, it is seen that autophagy lysosome increasing；With myocyte's focal necrosis and atrophy, interstitial can See a large amount of collagen fiber hyperplasias；Compared with MI groups, MI+Bex10 groups and MI+Bex30 groups, most of muscle fibre arrangement are close, Cardiac muscle cell's plasma membrane is relatively complete, has no that mitochondria is spilt, it is seen that a small amount of autophagy lysosome, interstitial Collagen Proliferation is not obvious； Both MI+Bex10 and MI+Bex30 groups are compared to each other, and the arrangement of MI+Bex30 groups muscle fibre is more close, and cardiac muscle cell's plasma membrane is more To be complete, and interstitial Collagen Proliferation is less.(see Fig. 9)

All documents referred in the present invention are all incorporated as reference in this application, independent just as each document It is incorporated as with reference to such.In addition, it is to be understood that after the above-mentioned instruction content of the present invention has been read, those skilled in the art can To be made various changes or modifications to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited Enclose.

Claims (10)

1. a kind of purposes of RXR α receptor stimulating agents, it is characterised in that for preparing medicine or reagent, the medicine or reagent are used In the one or more purposes being selected from the group：

(1) reduction TGF-β 1, and/or P-Smad3 level；

(2) P-Smad2, and/or Smad7 levels are improved；With

(3) myocardium I, and/or III Collagen Type VI synthesis is suppressed.

2. the method as described in claim 1, it is characterised in that the purposes includes suppressing the cardiac muscle of acute myocardial infarction AMI object I and III Collagen Type VI is synthesized.

3. the method as described in claim 1, it is characterised in that the purposes include reduction myocardial infarction junctional area TGF-β 1, And/or P-Smad3 level.

4. the method as described in claim 1, it is characterised in that the purposes includes improving myocardial infarction junctional area P-Smad2 And/or Smad7 level.

5. the method as described in claim 1, it is characterised in that the purposes also includes suppressing collage synthesis after myocardial infarction, So as to improve remodeling ventricle.

6. the method as described in claim 1, it is characterised in that the purposes also includes suppressing cardiac muscle cell after myocardial infarction Apoptosis.

7. the method for reducing the level of TGF-β 1, it is characterised in that including step a kind of external non-therapeutic：RXR α acceptors are swashed Dynamic agent and cells contacting, so as to reduce the level of cell TGF-β 1.

8. the method for reducing P-Smad3 levels, it is characterised in that including step a kind of external non-therapeutic：By RXR α acceptors Activator and cells contacting, so as to reduce cell P-Smad3 level.

9. a kind of method prevented and/or treat TGF-β 1, and/or Smads relevant diseases, wherein the disease and TGF-β 1 And/or P-Smad3 overexpression or hyperactivity are relevant, methods described includes step：Object to needs applies RXR α acceptors Activator, or apply the pharmaceutical composition of the receptor stimulating agents of α containing RXR.

10. a kind of method, it is characterised in that methods described is used for inner or in vitro reduction TGF-β 1 and/or P-Smad3 water Flat or raising P-Smad2, and/or Smad7 levels, methods described includes step：Object to needs swashs using RXR α acceptors Dynamic agent.